studies on soluble and immobilized plant ...sodium dodecyl sulphate and surf excel caused activation...
TRANSCRIPT
STUDIES ON SOLUBLE AND IMMOBILIZED PLANT PEROXIDASES AND THEIR APPLICATIONS
STJJ^IAJLTtie
THESIS SUBMITTED FOR THE AWARD OF THE DEGREE OF
Boctor of ^Ijilos^oplip IN
BIOCHEMISTRY / /
BY
MAHREEN MATTO
DEPARTMENT OF BIOCHEMISTRY
FACULTY OF LIFE SCIENCES
AL IGARH M U S L I M UNIVERSITY
AL IGARH ( INDIA)
2008
I Enzymologists are searching for new advances in environmental fields, from
enzymatic bioremediation to the development of renewable methods for the
biochemical cleaning of 'wastewater'. Peroxidases in particular have been underlined
for this purpose due to their low energy requirements, operation over a wide range of
conditions and having minimal environmental impact. A tailoring tool for this
achievement is enzyme immobilization with two main benefits, enhancement of
storage/operational stability and reusability. Meeting the demand for "green
biotechnology", turnip and bitter gourd peroxidase have enormous potential for
replacing conventional wastewater treatment processes.
The present study aimed to woric out an inexpensive, simple and high yield
procedure for the immobilization of turnip peroxidase, which would be of extreme
interest in the remediation of several types of aromatic compounds present in polluted
water. Turnip peroxidase was fractionated from the buffer extract by 10-90%
ammonium sulphate and was insolubilized by using jack bean extract, a source of
concanavalin A. Soluble and concanavalin A complex of turnip peroxidase were
entrapped into calcium alginate-pectin beads and these entrapped enzyme
preparations retained 52% and 63% of the original activity, respectively. The stability
against various denaturing agents is an important factor when selecting an appropriate
enzymatic system for any application. Calcium alginate-pectin entrapped
concanavalin A-tumip peroxidase showed impressive gains in resistance to
inactivation induced by pH, heat, urea, detergents and organic solvents. The exposure
of soluble and immobilized turnip peroxidase to low concentrations of detergents;
Sodium dodecyl sulphate and Surf Excel caused activation in enzyme activity.
However, the immobilized enzyme preparations exhibited further activation in turnip
peroxidase activity at higher concentrations of detergent as compared to soluble
counterpart.
Another promising plant peroxidase involved in environmental application is
bitter gourd peroxidase. In order to increase its potential use, calcium alginate-starch
hybrid gel was employed as an enzyme carrier both for surface immobilization and
entrapment of bitter gourd peroxidase. The insoluble concanavalin A-bitter gourd
peroxidase complex retained 70% of the initial activity. In order to prevent the
dissociation of concanavalin A-bitter gourd peroxidase complex, this preparation was
crosslinked by 0.5% glutaraldehyde. Entrapped crosslinked concanavalin A-bitter
gourd peroxidase retained 52% of the initial activity while surface immobilized and
glutaraldehyde crosslinked enzyme showed 63% activity. Crosslinking of bitter gourd
peroxidase resulted in small loss of enzyme activity but glutaraldehyde based
chemistry is an effective method for increasing mechanical and operational support of
enzymes.
A comparative stability of both forms of immobilized bitter gourd peroxidase
was investigated against pH, temperature, chaotropic agent; like urea, heavy metals,
water-mlscible organic solvents, detergent and inhibitors. The pH and temperature-
optima for both immobilized preparations was the same as for soluble counterpart at
pH 5.0 and 40 "C. Entrapped bitter gourd peroxidase was remarkably more stable than
surface immobilized and soluble peroxidase when incubated for different times at 60
°C. Entrapped peroxidase was significantly more stable as compared to surface
immobilized enzyme followed by soluble form of enzyme under various physical and
chemical denaturing conditions. Enzyme reuse provides a number of cost effective
advantages that are often an essential pre-requisite for establishing an economically
viable enzyme catalyzed process. Entrapped crosslinked concanavalin A-bitter gourd
peroxidase showed 75% of the initial activity while the surface immobilized and
crosslinked bitter gourd peroxidase retained 69% activity after their seventh repeated
uses.
The ability of partially purified peroxidases to treat direct dyes used in textile
industries was also reviewed. Dye solutions (50-100 mg L'') were treated with 0.094
U mL"' of turnip peroxidase under various experimental parameters. Direct dyes were
recalcitrant to the action of turnip peroxidase, however the rate and extent of
decolorization of direct dyes by turnip peroxidase was significantly enhanced in the
presence of different kinds of redox mediators. Six out often investigated compounds
showed their potential in enhancing the decolorization of direct dyes. Various
parameters such as pH, temperature, enzyme and redox mediator concentrations were
standardized in order to obtain maximum rate of decolorization of direct dyes.
Maximum decolorization of dyes over 60% occurred in the presence of 0.6 mM 1-
hydroxybenzotriazole/violuric acid in sodium acetate buffer, pH 5.5 at 30 °C. In order
to prove the capability of plant peroxidase in the treatment of industrial effluents, the
treatment of mixtures of direct dyes was also investigated. Complex mixtures of dyes
were also maximally decolorized in the presence of 0.6 mM redox mediator (1-
hydroxybenzotriazole/violuric acid). In order to examine the operational stability of
the enzyme, the enzyme was exploited for the decolorization of mixtures of dyes for
different times in a stirred batch process. More than 80% of the dye mixtures were
decolorized within first hour of incubation with turnip peroxidase. However, the
treatment of direct dyes/mixtures in the presence of redox mediators by turnip
peroxidase caused the formation of insoluble precipitate, which could be removed by
the process of centrifugation, filtration or adsorption. Thus indicating the removal of
end products of the enzyme mediated dye decolorization. Total organic carbon
analysis of treated dyes or their mixtures showed that these results were quite
comparable to the loss of color from solutions. The results suggested that catalyzed
oxidative coupling reactions might be important for natural transformation pathways
for dyes and indicated their potential use as an efficient means for removal of dyes
color from waters and wastewaters.
Surface immobilized bitter gourd peroxidase has been used for the effective
decolorization of textile industrial effluent. Effluent was recalcitrant to the action of
bitter gourd peroxidase, however in the presence of some redox mediators, it was
successfully decolorized. Effluent decolorization was maximum upto 70% in the
presence of 1.0 mM 1-hydroxybenzotriazole within 1 h of incubation. However,
immobilized bitter gourd peroxidase had optimum decolorization at pH 5.0 and 40 °C.
Immobilized bitter gourd peroxidase decolorized more than 90% effluent color after 3
h of incubation in a batch process. In order to evaluate the efficiency of salt
fractionated plant peroxidase for the decolorization of textile effluent, it was
necessary to decolorize textile effluent in a continuous mode. A two-reactor system,
one reactor containing immobilized peroxidase and the other had adsorbent; activated
silica was used for the effective decolorization of textile effluent. The system was
capable of decolorizing 40% effluent even after 2 months of continuous operation
with a flow rate of 16 mL h''. The absorption spectra of the untreated and treated
effluent exhibited a marked difference in absorbance at various wavelengths. Thus
immobilized peroxidase/1-hydroxybenzotriazole system has been successfully
employed for the treatment of a large volume of effluent in a continuous reactor.
Further an attempt has been made to use a simple, inexpensive concanavalin
A-wood shaving bound turnip peroxidase for the decolorization of a direct dye and
mixture of direct dyes in batch processes and continuous reactors. The study has
shown that wood shaving could be exploited as an inexpensive material for the
preparation of bioafflnity support due to its high affinity for concanavalin A; being
the natural source of cellulose. Wood shaving (1.0 g) adsorbed nearly 22 mg of
concanavalin A from jack bean extract. Wood shaving adsorbed concanavalin A was
used for the immobilization of peroxidase directly from ammonium sulphate
fractionated proteins of turnip. Concanavalin A-wood shaving bound turnip
peroxidase showed very high effectiveness factor 'T|' of 0.67. The comparative results
between soluble and immobilized turnip peroxidase obtained from the batch process
revealed the ability of immobilized turnip peroxidase, to decolorized Direct Red 23
and mixture of dyes up to 92% and 83% after 1 h of incubation in the presence of 0.6
mM 1-hydroxybenzotriazole, respectively. Enzymes used for the treatment of
wastewater contaminated with aromatic pollutants could also be affected by the
presence of water-miscible organic solvents/salts/heavy metals. Therefore, the
decolorization of direct dye and mixture of direct dyes by turnip peroxidase was
carried in the presence of water-miscible organic solvent, salt and heavy metals. The
immobilized turnip peroxidase could effectively remove more than 70% of color in
the presence of metals/salt. The dye decolorizing reusability was gradually decreased
upto 8"' repeated uses. Immobilized peroxidase could decolorize 47% and 30% of the
initial color from Direct Red 23 and mixture of direct dyes even after 8* repeated use,
respectively. Decolorization of Direct Red 23/mixture of direct dyes carried in a
continuous reactor containing a cheap biocatalyst, immobilized enzyme with a flow
rate of 7.2 mL h"' was highly efficient even till 4 and 3 months of operation, removing
64% and 50% of color from the solution of Direct Red 23 and mixture of direct dyes,
respectively. Total organic carbon analysis of treated dye or mixture of dyes exhibited
that these results were quite comparable to the loss of color fi-om solutions. Thus, this
work may provide a reasonable basis for development of biotechnological processes
for continuous color and aromatic compounds removal from various industrial
effluents at large scale.
STUDIES ON SOLUBLE AND IMMOBILIZED PLANT PEROXIDASES AND THEIR APPLICATIONS
THESIS SUBMITTED FOR THE AWARD OF THE DEGREE OF
fi ©octor of ^l)iIogopI)p •<? / X IN
BIOCHEMISTRY
BY
MAHREEN MATTO
DEPARTMENT OF BIOCHEMISTRY
FACULTY OF LIFE SCIENCES
AL IGARH M U S L I M UNIVERSITY
AL IGARH l I N D I A ) .
2008
T6789
dedicated to my
bving parents
Unrversity Exchange Nos 0571-2700920, 2700916 Exf, (INT)Office - 3720 Chairman's Chamber - 3721 Ext (0571)2700741 Fax. No. (0571)2706002
D E P A R T M E N T O F B I O C H E M I S T R Y FACULTV OF LIFE SCIENCES
ALIGARH MUSLIM UNIVERSITY ALIGARH-202002 (INDIA)
ReJ.So DatedJi/lJ^^l
Certificate
This is to certify that the thesis entitted "Studies on sofuBie and
immoBiGzed pCant perojQdases and their appRcations" Being suBmitted By
Mahreen Matto to JA[igarh 'MusCim Vniversity, JlCigarhfor the award of the
degree of (Doctor of (Phiibsophy in (Biochemistry is a record of Bonafide research
wor^ carried out By her. Mahreen 9datto has wor^d under my guidance and
supervision and has fuCflCCed the requirements for the suBmission of this thesis.
The resuCts contained in this dissertation have not Been suBmitted in
part or infuCCto any other Vniversity or Institute for the award of any degree.
<ProfQgyyum Husain
ACKNOWLEDGEMENT
Foremost I would like to thank my research supervisor Prof. Qayyum
Husain, Department of Biochemistry, AMU, Aligarh not only for sharing with me
his experience and knowledge, but also for his guidance, perpetual support and faith
in me, which helped me to begin and to complete this work. It's his hard work,
sincerity, dedication, perseverance and passion which have been put in shaping this
thesis. He made my research work easier since the time I joined his lab whether it
was with regard to drafting of papers or formatting of thesis. I will never forget...
I also express my genuine thanks to my Chairman, Prof S. M. Hadi
Department of Biochemistry, AMU, Aligarh for all his support. I personally thank
all the teachers of my department for their encouragement.
As fire makes fine steel and heat makes gold, so did this university mould me
into a better person. I am endlessly gratefiil to all the people who encouraged and
prodded me when I bogged down.
I would like to personally thank my seniors, Amj'ad bhai, Suhail bhai who
encouraged me to get the help I needed. I extend my gratitude to Yasha apafor being
the sparkling senior of our lab, Aiman apa for her advices. Their assistance and
support is beyond appreciation.
Special mention of our T-6 group (Toshiba, Zoheb, Rukshana, Humaira,
Skakeel, Mahreen). It has always been wonderful to be part of this group. I have
spent some cherishable moments of my life in this group. I have known Toshiba
since more than 8 years now we have shared together highs and lows of our lives at
personal and professional front. Admire her for the hard core person she is. I am
indebted to Rukhsanafor all the support and frank advice over the years. It feels
pleasure to express my extended acknowledgement to Zoheb for his superfluous help
and being so caring and supportive. Shakeel being the vivacious junior has always
been the mood freshening centre thanks for updating me to new english words, I am
grateful to Humaira for being like the caring younger sister. My deepest thanks will
always go to all of them for their support at every step of my journey all through
these years; they acted as redox mediators in enhancing my moral. I truly will miss
them all
Thanks Rose for being such a persistent friend, you have always been so
supportive. My unlimited thanks and acknowledgement to all the people that I met
during my stay in aligarh for their pleasant company, conversations, help and
friendship, which are listed in a random order: Nida, Deeba, Nishtha, Sabika, Am
bhaiya, Shamila apa, Deeba apa, Sadia apa, Shuba apa, Jeelani, Iram apa,
Medha, Sara, Aliya, Faisal, Shanawaz, Shams, Sheeba, Sarmad, Uzma, Fahad,
Atif Rayees, Ashraf Mohd, Ausaf Wasim, Badar, Saima, Aiman, Gulshan,
Sadia, Roshan, Iram, Nargis, Aabro and many more.
My family has always have been a bedrock of support for me. My
unspeakable gratefulness to my parents for their unconditional love and patience.
Knowing the days I barely achieved anything yet encouraging me to persist and to
finish. lean write a thesis on them but I think for a life time one thesis is enough. No
word can express my feeling for them. It's their hard work, sacrifice, prayers which is
bound in this thesis. Special recognition must be made of my doting sisters for their
proximity in spite of such long distance. Nazia is the most caring sister one could
ever have in the whole universe who called every single day to hear whether my day
was productive or not. Tazeen the most adorable, pampered, lovable yet sensible
child of our family always gave me the moral boost to drive my research journey, in
these years I missed her a lot.
Above all, I want to acknowledge and thank Almighty Allah. Without his
transforming power, I would never have begun this Journey. Without the strength,
courage, blessing and favour he bestowed, I would never have finished this work.
(Mahreen Matto)
LIST OF CONTENTS
Page No.
List of Abbreviations
List of Figures
List of Tables
1. Chapter I
2. Chapter II
3. Chapter III
4. Chapter IV
5. Chapter V
Review of Literature
Entrapment of concanavalin A-peroxidase complex into hybrid calcium alginate-pectin gel.
2.1. Introduction
2.2. Materials & Methods
2.3. Results
2.4. Discussion
Calcium alginate-starch hybrid support for both surface immobilization and entrapment of bitter gourd peroxidase.
3.1. Introduction
3.2. Materials & Methods
3.3. Results
3.4. Discussion
Decolorization of direct dyes by salt fractionated turnip proteins in the presence of H2O2 and redox mediators.
4.1. Introduction
4.2. Materials & Methods
4.3. Results
4.4. Discussion
Decolorization of textile effluent by bitter gourd peroxidase immobilized on concanavalin A layered calcium alginate-starch beads.
1
iii
V
1-36
37-54
37
38
41
52
55-78
55
55
60
73
79-97
79
79
84
94
98-119
6. Chapter VI
5.1. Introduction
5.2. Materials & Methods
5.3. Results
5.4. Discussion
Decolorization of direct dyes by immobilized turnip peroxidase in batch and continuous processes.
6.1. Introduction
6.2. Materials & Methods
6.3. Results
6.4. Discussion
7. Summary
8. Bibliography
9. List of Publications and Presentations
98
98
102
115
120-138
120
120
124
136
139-142
143-171
172-174
LIST OF ABBREVIATIONS
ABTS
AOT
BGP
BPB
CdCl2
COD
Con A
d
DB80
DMF
DR23
DR239
DY4
DyP
E-BGP
EDTA
Fig.
HgCl2
HOBT
HRP
I-BGP
I-TP
LiP
min
Mixture 1
Mixture 2
Mixture 3
Mixture 4
mM
2,2'-azino-bis-(3-ethyIbenzthiazoline-6- sulfonic acid)
bis(2-ethylhexyl) sulphosuccinate sodium salt
Bitter gourd peroxidase
Bromophenol Blue
Cadmium chloride
Chemical oxygen demand
Concanavalin A
Days
Direct Blue 80
Dimethyl formamide
Direct Red 23
Direct Red 239
Direct Yellow 4
Dye decolorizing peroxidase
Calcium alginate-starch entrapped crosslinked Con A-BGP
Ethylenediamine tetracetic acid
Figure
Mercuric chloride
1 -hydroxybenzotriazole
Horseradish peroxidase
Immobilized bitter gourd peroxidase
Immobilized turnip peroxidase
Lignin peroxidase
Minutes
Direct Blue 80+ Direct Yellow 4+Direct Red 23
Direct Blue 80+ Direct Yellow 4+Direct Red 239
Direct Red 23+ Direct Yellow 4+Direct Red 239
Direct Red 23+Direct Blue 80+Direct Red 239 milli Molar
MnP
MP 11
M,
nm
NNDS
;?-HBA
RBBR
rDyP
S-BGP
SEP
SDS
Sl-BGP
SP
S-TP
TCVPl
TEMPO
TMP
TOC
TP
v/v
VA
VHP
VLA
VN
VP
w/v
Wo
WRF
WS
Manganese peroxidase
Microperoxidase 11
Molecular weight
Nanometer
1 -nitroso-2-naphthol-3,6-disulfonic acid
para-hydro\yhenzoic acid
Remazol Brilliant Blue R
Recombinant dye decolorizing peroxidase
Soluble bitter gourd peroxidase
Soybean peroxidase
Sodium dodecyl sulphate
BGP immobilized on the surface of concanavalin A layered
calcium alginate-starch beads and crosslinked by glutaraldehyde
Sulfonaphthalein
Soluble turnip peroxidase
Thanatephorus cucumeris Dec 1
2,2',6,6'-tetramethylpiperidine-Ar-oxyl
Tomato peroxidase
Total organic carbon
Turnip peroxidase
Volume by volume
Veratryl alcohol
Vanadium haloperoxidase
Violuric acid
Vanillin
Versatile peroxidase
Weight by volume
Hydration degree
White rot fungi
Wood shaving
LIST OF FIGURES
Figure No. Page No.
1 Redox cycle for mediating substrate oxidation by 29
oxidoreductases.
2 InsolubilzationofTPusingjack bean extract. 42
3 pH-activity profiles of soluble and immobilized 45
TP.
4 Temperature-activity profiles of soluble and 46
immobilized TP.
5 Thermal denaturationofsoluble and immobilized 47
TP.
6 Effect ofurea on soluble and immobilized TP. 48
7 Schematic diagram of (a) E-BGP and (b) SI-BGP. 61
8 pH-activity profiles of soluble and immobilized 64
BGP.
9 Temperature-activity profiles for soluble and 65
immobilized BGP.
10 Thermal denaturationofsoluble and immobilized 66
BGP.
11 Effect of 4.0 M urea on soluble and inunobilized 67
BGP.
12 Effect of Tween 20 on soluble and immobilized 70
BGP.
13 Effect of NaCl on soluble and immobilized BGP. 74
14 Reusability of immobilized preparations of BGP. 75
15 Chemical structures of investigated direct dyes. 81
16 Decolorization of mixture 3 and 4 by TP in the 93
presence of HOBT/VLA in stirred batch processes.
17 Absorption spectra (a) DB 80 and (b) mixture 4 in 95
the presence of HOBTAT^A.
18 UV-visible spectra for textile effluent. 103
ni
19 Treatment of textile effluent by BGP in the 104
presence of various redox mediators.
20 Effect of HOBT concentration on BGP-catalyzed 106
effluent decolorization.
21 Textile effluent decolorization reusability of 112
immobilized BGP.
22 Schematicrepresentationof a two-reactor system 113
used for decolorization and removal of colored
compounds from textile effluent.
23 Evaluation of decolorized textile effluent collected 114
from two-reactor system.
24 UV-visible spectra of the textile effluent. 116
25 Effect of pH on the decolorization of direct dyes 127
by soluble and immobilized TP.
26 Effect of temperature on the decolorization of 128
direct dyes by soluble and immobilized TP.
27 Dye/dyes mixture decolorization reusability of 133
immobilized TP.
28 Absorption spectra of DR 23 and a mixture of 135
direct dyes treated in a continuous two-reactor
system.
IV
LIST OF TABLES
Table No. Page No.
1 Physical and chemical methods for dye removal from 3-4
polluted water.
2 Dye removal by immobilized peroxidases. 27
3 Applications of redox mediators in dye removal. 34
4 Immobilization of TP by using jack bean extract and 44
calcium alginate-pectin matrix.
5 Effect of detergents on soluble and immobilized TP. 50
6 Effect of organic solvents on soluble and immobilized 51
TP.
7 Immobilization of BGP into and on calcium alginate- 62
starch beads.
8 Effect of organic solvents on soluble and immobilized 68
BGP.
9 Effect of sodium azide/EDTA on soluble and 71
immobilized BGP.
10 Effect of HgCb/CdCb on soluble and immobilized BGP. 72
11 Compounds investigated as redox mediators for TP- 85
catalyzed dye decolorization.
12 Effect of various concentrations of HOBTAT^AAnST on 86
dye decolorization by TP.
13 Effect of TP concentration on decolorization of direct 87
dyes in the presence of HOBTAHLAA^.
14 Effect of pH on decolorization of direct dyes by TP in 89
the presence of HOBTA^LAATvJ.
15 Effect of temperature on decolorization of direct dyes by 90
TP in the presence of HOBTA^LA.
16 TOC content of direct dyes after treatment with TP. 91
17 Treatment of mixtures of direct dyes by TP in the 92
presence of HOBTAT.A.
18 Effect of BGP concentration on effluent decolorization. 107
19 Effect of pH on BGP-catalyzed decolorization of textile 108
effluent.
20 Effect of temperature on BGP-catalyzed decolorization 109
of textile effluent.
21 Decolorization of textile effluent by BGP in batch 111
processes.
22 Immobilization of TP on Con A-WS. 125
23 Effect of NaCl on TP-catalyzed dye decolorization. 129
24 Effect of heavy metals on TP-catalyzed dye 130
decolorization.
25 Effect of dioxane on TP-catalyzed dye decolorization. 132
26 Continuous removal of color and TOC from DR 23 and a 134
mixture of direct dyes by I-TP in a two-reactor system.
VI
1.1. INDUSTRIAL DYEING EFFLUENTS AS ENVIRONMENTAL
CONCERN
Intensive industrial and agricultural activities during 20th century have led to a
considerable contamination of soil and water by toxic organic pollutants, which may
have catastrophic impact on human health and environment (Torres et al., 2003;
Bhole et al., 2004). Like the climate change issues, biodiversity and increasing
municipal waste generation, these "red lights" signal pressures on the environment
for which urgent actions or measures are needed. Among the industrial effluents,
wastewater from textile and dyestuff industries is one of the most difficult to treat.
This is because dyes usually have synthetic and complex aromatic molecular
structures, which make them more stable and difficult to degrade (Padmesh et al.,
2005).
Since 1856, when the first synthetic dye was reported, the use of dye in
industries and house-hold has increased remarkably. There are more than 10,000 dyes
available commercially, and about 7x10^ tons of dyestuffs are produced annually
(Zollinger, 1991; Aksu and Tezer, 2005). The main consumers of dyes are the textile,
tannery, paper and pulp and electroplating industries (Rai et al., 2005). Dyes are also
used as additives in petroleum products. In addition, a number of dyes and dyestuffs
are widely used in the food, pharmaceutical and cosmetic industries (Harazono and
Nakamura, 2005). This huge growth in the textile dyeing and dyestuff manufacturing
industries has resulted in an immense increase in the volume and complexity of the
wastewater discharged to the environment. During textile processing, inefficiencies in
dyeing results in a large amount of dyestuff being directly lost in wastewater, which
ultimately finds way into the environment. About 15-20% of the used dyes are lost in
the effluent during the dyeing process (Mahmoud et al., 2007) while in the case of
reactive dyes, as much as 50% of the initial dye load is present in the dye bath effluent
(Azmi and Banerjee, 2001). The presence of even trace concentrations of dyes in
effluent is highly visible and undesirable (Hao et al., 2000).
Dye effluent usually contains chemicals, including dye itself that may be toxic,
carcinogenic and mutagenic to various microbiological and aquatic animals. Concern
arises, as several dyes are made of known carcinogens such as benzidine and other
aromatic compounds (Robinson et al., 2001). It has been reported that azo and nitro
1
compounds have reduced in sediments of aquatic bodies, consequently yielding
potentially carcinogenic amines that spread in the ecosystem (Verma et al., 2003). The
presence of dyes or their degraded products in water can also cause human health
disorders such as nausea, hemorrhage, ulceration of skin and mucous membranes, and
can even cause severe damage to the kidney, reproductive system, liver, brain and
central nervous system. These concerns have led to new and/or stricter regulations
concerning colored wastewater discharges, compelling the dye manufacturers and
users to adopt "cleaner technology" approaches, for instance, development of new
lines of ecologically safe dyeing auxiliaries and improvement of exhaustion of dyes on
to fiber (Hao et al., 2000; Rott, 2003; Hai et al., 2007).
1.2. ENVIRONMENTAL CONCERN
1.2.1. Chemical and Physical methods for dye decolorization
Water pollution control is presently one of the major thrust areas of scientific
research. However, the colored organic compounds generally impart only a minor
fraction of the organic load to wastewaters but their color renders them aesthetically
unacceptable (Anjaneyulu et al., 2005). Stringent regulations are forcing industries to
treat their waste effluents prior to their final discharge to increasingly high standards.
Several decolorization techniques have been reported during past two decades, few of
them have been accepted by some industries. The advantages and disadvantages of
various physical and chemical methods used for decolorization of dyes are listed in
Table 1.
1.3. BIOLOGICAL TECHNIQUES
As a viable alternative, biological processes have received increasing interest
owing to their cost, effectiveness and environmental benignity (McMullan et al., 2001;
Chen et al., 2003). Biological processes have potential to mineralize dyes to harmless
inorganic compounds like carbon dioxide, water and the formation of a lesser quantity
of relatively harmless sludge (Mohan et al., 2002). Many researchers have
demonstrated partial or complete biodegradation of dyes by pure or mixed cultures of
Table 1: Physical and chemical methods for dye removal from polluted water
Physical/Chemi -cal methods Fentons reagent
Ozonation
Photochemical
NaOCl
Cucurbituril
Electrochemical destruction Activated carbon
Peat
Wood chips
Silica gel Ion exchange
Irradiation
Membrane filtration Photocatalysis
Sonolysis
UV/O3
Advantages
Effective decolorization of both soluble and insoluble dyes Applied in gaseous state: Alteration of volume
No sludge production
Initiates and accelerates azo-bond cleavage Good sorption capacity for various dyes Break dovm compounds are non-hazardous Good removal of wide variety of dyes
Good adsorbent due to cellular structure
Good sorption capacity for acid dyes Effective for basic dyes Regeneration, no adsorbent loss Effective oxidation at laboratory scale Appropriate membrane may remove all dyes No sludge production, considerable reduction of COD, solar light utilization
No chemical additives required Applied in gaseous state, good removal of all types of dyes
Disadvantages
Sludge formation
Short half life (20 min)
Formation of byproducts Release of aromatic amines High cost
High cost of electricity Very expensive
Lower surface area of adsorption than activated carbon Long retention time
Side reactions Not effective for all dyes Requires a lot of dissolved O2 High cost
Light penetration limitation, fouling of catalysts, problem of fine catalyst separation Requires a lot of dissolved oxygen Poor removal of disperse dyes, impose additional loading of water with ozone, no COD removal
References
Hao et al., 2000; Alaton and Teksoy, 2007
Koch et al., 2002; Oguz et al., 2005; Simdrarajan et al., 2007 Yang etal., 1998; Coute and Sanroman, 2002 Kao et al., 2001; Forgacs et al., 2004 Karcher etal., 2001
Chen, 2004
Fu and Viraraghavan, 2001; Santhy and Selvapathy, 2006 Nigam et al., 2000; Sun and Yang, 2003
Nigam et al., 2000
Idris and Saed, 2003 Robinson et al., 2001; Liu et al., 2007 Keharia and Madamvar, 2003; Liu and Liang, 2004 Chakraborty et al., 2003
Konstantinou and Albanis, 2004
Arslan-Alaton, 2003
Hao et al., 2000; Fu and Viraraghavan, 2001
UV/H202
Wet air oxidation
Coagulation
Chemical oxidation
No sludge formation, short reaction times and reduction of COD to some extent
Well-established technology
Economically feasible; satisfactory removal of disperse, sulphur and vat dyes
Initiates and accelerates azo-bond cleavage
Not applicable for all types of dyes, requires separation of suspended solid and suffers from UV light penetration Not complete mineralization, high costs pH dependent removal, produces large quantity of sludge, not suitable for soluble dyes Thermodynamic and kinetic limitations, secondary pollution, Not applicable for disperse dyes
Gogate and Pandit, 2004; Shu and Chang, 2006
Arslan-Alaton, 2003
Fu and Viraraghavan et al., 2001; Robinson et al.. 2001; Shi etal., 2007
Robinson et al., 2001
bacteria, fungi and algae. Several types of microorganisms have been isolated in recent
years that are able to degrade dyes previously considered non-degradable (Stolz, 2001;
Nyanhongo et al., 2002). Biological treatment for degradation of textile effluents may
be aerobic, anaerobic or combination of both depending on the type of microorganism
being employed (Keharia and Madamwar, 2003).
1.3.1. Bacterial biodegradation
Efforts to isolate bacterial cultures capable of degrading azo dyes started in the
1970's with reports of a Bacillus subtilis (Chung et al., 1978), followed by different
other bacteria. Several investigators demonstrated that mixtures of dyes were
decolorized by anaerobic bacteria in 24-30 h, using free growing cells or in the form of
biofilms on various support materials (Robinson et al., 2001). Pearce et al. (2003)
represented the concern to use whole bacterial cells for the reduction of water-soluble
dyes present in textile effluents. Moreover, bacteria including Citrobacter sp., Kurthia
sp., Corneybacterium and Mycobacterium sp. and a mixed culture consisting of
Pseudomonas mendocina and Pseudomonas alcaligenes could also successfully
degrade triphenylmethane dyes from solution (Rai et al., 2005).
Parshetti et al. (2006) described that Malachite Green (50 mg L"') was
completely decolorized under static anoxic conditions within 5 h by bacteria Kocuria
rosea MTCC 153. Kocuria rosea also showed decolorization of azo, triphenylmethane
and other industrial dyes (Cotton Blue, Methyl Orange, Reactive Blue 25, Direct Blue
6, Reactive Yellow 81 and Red HE4B).
Sulfonated azo dyes were decolorized by two wald type photosynthetic
bacterial strains {Rhodobacter sphaeroides AS 1.173 7 and Rhodopseudomonas
palustris AS 1.2352) and a recombinant strain {Escherichia coli YB). All the strains
could decolorize azo dye up to 900 mg L"' (Liu et al., 2007).
1.3.2. Fungal treatment
The majority of studies on biological decolorization have been focused on
fungal strains. Decolorization of the azo dyes; Orange 11, Tropeolin O, Congo Red,
Acid Red 114, Acid Red 88, Biebrich Scarlet, Direct Blue 15, Chrysopheninetetrazine
and Yellow 9 and the triphenylmethane dyes; Basic Green 4, Crystal Violet, Brilliant
5
Green, Cresol Red, Bromophenol Blue (BPB) and Para Rosanilines by various fungi
have been reported (Selvam et al., 2003). The degradation of azo, anthraquinone,
heterocyclic, triphenylmethane and polymeric dyes by Phanerochaete chrysosporium
has been extensively studied (Pointing, 2001). Trametes versicolor, Bjerkandera
adusta and Phanerochaete chrysosporium were able to decolorize commercially used
reactive textile dyes; Reactive Orange 96, Reactive Violet 5 and Reactive Black 5 and
two phthalocyanine dyes; Reactive Blue 15 and Reactive Blue 38 (Heinfling et al.,
1997). Matthew and Bumpus (1998) observed the degradation of Congo Red by
Phanerochaete chrysosporium in agitated liquid cultures.
Aspergillus foetidus (Sumathi and Manju, 2000), Phanerochaete
chrysosporium (Mielgo et al., 2001), Trametes versicolor (Borchert and Libra, 2001),
Trametes hirsuta (Abadulla et al., 2000) Coriolus versicolor (Kapdan and Kargi,
2002), Cunninghamella polymorpha (Sugimori et al., 1999), Geotrichum candidum
and Rhizopus arrhizus (Aksu and Tezer, 2005) are the major ftmgal strains used for
decolorization purposes. Moreover, there are various ftmgi other than white rot fungi
(WRF), such as Umbelopsis isabellina and Penicillium geastrivous which could also
decolorize or biosorb diverse dyes (Yang et al., 2003).
Some studies have even reported decolorization of sulfur containing dyes by
using WRF, Dichomitus squalens and Coriolus versicolor (Eichlerova et al., 2006a;
Sanghi et al., 2006).
1.3.3. Algal biodegradation
Degradation of a number of azo dyes by algae has been described in few
studies (Semple et al., 1999). It was reported that more than 30 azo compounds were
decolorized and biodegraded into simpler aromatic amines by Chlorella pyrenoidosa,
Chlorella vulgaris and Oscillateria tenuis (Yan and Pan, 2004).
Macro alga, Azolla filiculoides was able to cause biosorption of Acid Red 88,
Acid Green 3 and Acid Orange 7 (Padmesh et al., 2005). The potential of Cosmarium
species, belonging to green algae, was investigated as a viable biomaterial for
biological treatment of triphenylmethane dye; Malachite Green (Daneshvar et al,
2007).
1.3.4. Limitations of biological treatment
Dye effluents are poorly decolorized by conventional biological treatments and
may be toxic for the microorganisms present in the treated effluent plants due to their
complex aromatic structures. Furthermore, following anaerobic digestion, nitrogen-
containing dyes are transformed into aromatic amines that are more toxic and
mutagenic than the parent molecules (Gottlieb et al., 2003; Zouari-Mechichi et al.,
2006). Biological degradation of dyes includes properties such as water solubility,
large molecular weight and fused aromatic ring structures, which inhibits permeation
through biological cell membranes. Other limitations of using microbes for treating
pollutants are; high costs of production of microbial culture, process of
decolorization/degradation of dyes is quite slow requiring several days (d) to months
and metabolic inhibition (Duran and Esposito, 2000; Husain and Jan, 2000; Nazari et
al., 2007). Various organisms have been used for the complete degradation of aromatic
compounds but much success has not been achieved yet.
1.4. ENZYMATIC APPROACH
The implementation of increasingly stringent standards for the discharge of
wastes into the environment has necessitated the need for the development of
altemative waste treatment processes. A large number of enzymes from a variety of
different plants and microorganisms have been reported to play an important role in an
array of waste treatment applications (AbaduUa et al., 2000; Husain, 2006). This is
mainly because, unlike the chemical catalysts, the enzymatic systems have the
advantages of accomplishing complex chemical conversions under mild environmental
conditions with high efficiency (Husain 2006; Husain and Husain, 2008; Michniewicz
et al., 2008). The variety of chemical transformations catalyzed by enzymes has made
these catalysts a prime target of exploitation by the emerging biotechnological
industries. Enzymes can act on specific recalcitrant pollutants to remove them by
precipitation or transformation to other products (Akhtar ad Husain, 2006; Rojas-
Melgarejo et al., 2006). They also can change the characteristics of a given waste to
render it more amenable for the treatment or aid in converting waste material to value-
added products (O'Nicell et al., 1999).
Enzymatic systems fall between the two traditional categories of chemical and
biological processes, since they involve chemical reactions based on the action of
biological catalysts (Anjaneyulu et al., 2005). Enzymes that have been isolated from
their parent organisms are often preferred over intact organisms containing the enzyme
because the isolated enzymes act with greater specificity, their activity can be better
standardized, they are easier to handle and store, and enzyme concentration is not
dependent on bacterial growth rates (Wagner and Nicell, 2003; Husain and Husain,
2008).
Due to their high specificity to individual species or classes of compounds,
enzymatic processes can be developed to specifically target selected compounds that
are detrimental to the enviroiunent (Ryan et al., 2003). Compounds that are candidates
for this type of treatment are usually those that cannot be treated effectively or reliably
using traditional techniques (Kadhim et al., 1999; Couto et al., 2006). Alternatively,
enzymatic treatment can be used as a pretreatment step to remove one or-more
compounds that can interfere with subsequent downstream treatment processes. For
example, if inhibitory or toxic compounds can be removed selectively, the bulk of the
organic material could be treated biologically, thereby minimizing the cost of
treatment (Gianfreda and Rao, 2004). Due to susceptibility of enzymes to inactivation
by the presence of other chemicals, it is likely that enzymatic treatment will be most
effective in those streams where the highest concentration of target contaminants and
the lowest concentration of other contaminants that may tend to interfere with
enzymatic treatment are present (Ghioureliotis, 1997). It is suggested that the
following situations are those where the use of enzymes might be most beneficial:
• Removal of specific chemicals from a complex industrial waste mixture
prior to on-site or off-site biological treatment.
• Removal of specific chemicals from dilute mixtures, for which
conventional mixed culture biological treatment might not be feasible.
• Polishing of a treated wastewater or groundwater to meet limitations on
specific pollutants or to meet whole effluent toxicity criteria.
• Treatment of wastes generated infrequently or in isolated locations,
including spill sites and abandoned waste disposal sites.
• Treatment of low-volume, highly-concentrated wastewater at the point of
generation to permit reuse of the treated process wastewater, to facilitate
8
recovery of soluble products, or to remove pollutants known to cause
problems downstream when mixed with other wastes from the plant
(Karam and Nicell, 1997).
Some potential applications of enzymes that have been identified for the
improvement of waste quality include the transformation of aromatic compounds,
cyanide, color-causing compounds, pesticides, surfactants and heavy metals (Karam
and Nicell, 1997; Duran and Esposito, 2000).
Before the full potential of enzymes may be realized, a number of significant
issues remain to be addressed. These include: development of low-cost sources of
enzymes in quantities that are required at the industrial scale, demonstration of the
feasibility of utilizing the enzymes efficiently under the conditions encountered during
wastewater treatment, characterization of reaction products and assessment of their
impact on downstream processes or on the environment into which they are released
and identification of methods for the disposal of solid residues (Lopez et al., 2002;
Akhtar et al., 2005a; Alcalde et al., 2006; Mao et al., 2006). Current research is
focusing on addressing these issues, particularly for the development of enzymatic
treatment systems that target the removal of aromatic compounds fi-om industrial
wastewaters. The main challenge for enzymatic decolorization of industrial dyes is the
large diversity of chemical structures among the synthetic dyes, which include
anthraquinones, azo nitro, nitroso and polyenes compounds (Wesenberg et al., 2003;
Akhtar et al., 2005b).
1.5. IMMOBILIZED ENZYMES
The two major driving concerns in an industrial scale enzymatic process are
how to lower the unit cost and to increase production per fixed time; the first step
towards achieving these goals is enzyme immobilization (David et al., 2006). In
particular, the use of soluble enzymes necessitates regular replenishment of the
enzymes, lost with the product at the conclusion of a reaction process. Also the
recovery of enzymes from reactor, at the end of the catalytic process is difficult and
expensive (Gomez et al., 2006). These disadvantages restrict the use of enzymes to
batch operations. The single use is obviously quite wasteful when the cost of enzymes
is considered. Thus, there is an incentive to use enzymes in an immobilized or
insolubilized form so that they may be retained in a biochemical reactor to catalyze
further the subsequent feed and offer more economical exploitation of biocatalysts in
industry; waste treatment and in the development of bioprocess monitoring devices
like the biosensor (Husain and Jan, 2000; Rojas-Melgarejo et al., 2004).
To improve their economic feasibility in industrial processes, immobilization
of enzyme is a common choice. A number of different organic and inorganic support
matrices and enzyme immobilization techniques have been tried with a view to
achieve high level of enzyme uptake with a minimum of enzyme degradation or
inactivation (Liang et al., 2000; Roy et al., 2004). Different methods exist for enzyme
immobilization, sometimes referred to as enzyme insolubilization. The overwhelming
majority of the methods can be classified into six main categories: entrapment,
microencapsulation, adsorption, covalent bonding, chemical aggregation and
bioaffinity based immobilization.
1.5.1. Entrapment
Among the various immobilization techniques one such approach is the
immobilization of an enzyme by physical entrapment. While entrapment is a simple
process and generally affords a high uptake of enzyme without appreciable
inactivation during the immobilization process, the enzyme once entrapped is
surrounded by a matrix imposing a mass transfer barrier (Kierstan and Bucke, 2000).
The entrapment method may be a good choice for enzyme immobilization as the
process of entrapment is mild and causes relatively less damage to the native structure
of the enzyme (Duran et al., 2002). Different types of polysaccharides such as agar,
alginate, carrageenan, chitin, chitosan and polyacrylamide have been used as
immobilization matrices (Lu et al., 2007). Huang et al. (2003) obtained a highly
catalytic microperoxidase 11 (MP 11) biosensor for H2O2 detection, by entrapping MP
11 into didodecyldimethylammonium bromide lipid membrane. Musthapa et al. (2004)
have entrapped glutaraldehyde ciosslinked turnip peroxidase (TP) in calcium alginate
gel for high yield inmiobilization and stabilization of enzyme. Pezzotti et al. (2004)
immobilized Coprinus cinereus peroxidase by entrapment in a polyacrylamide matrix
and this preparation was used for the degradation of 2,6-dichlorophenol.
One of the major limitations of entrapment technique is the diffusion as well as
the steric hindrance, especially when the macromolecular substrates are used (Liang et
10
al, 2000). Diffusion problem can be minimized by entrapping enzymes in fine fibers of
cellulose acetate or other synthetic material or by using an open pore matrix. Recently,
the development of some hydrogels has attracted attention in the field of
biotechnology (Sakai and Kawakami, 2007). However, such gels with a water content
of about 96% provide a microenvironment for the immobilized enzyme with minimal
diffusion restrictions (Hermink and van Nostrum, 2002). Alcohol oxidase and
horseradish peroxidase (HRP) can be co-immobilized in a spongiform hydrogel matrix
of hydroxyethyl carboxymethyl cellulose (Wu and Choi, 2004).
1.5.2. Microencapsulation
Microencapsulation can be achieved by interfacial polymerization (a chemical
process) or by co-acervation (a physical phenomena) leading to immobilization of
enzymes, which remain chemically unmodified. The technique was introduced by
Chang (1964) and further developed by several other workers (Founlds and Lowe,
1988; Chang and Prakash, 2001). The encapsulated enzyme carmot leach out or diffuse
fi-om the membrane bag but substrate can be dialyzed easily across the membrane. The
significance of encapsulation is that enzyme remains chemically unmodified and
hence catalytically active. Kadnikova and Kostic (2003) immobilized MP 11 by
encapsulation into sol-gel silica glass and by physisorption, chemisorption and
covalent attachment to silica gel. A comparison between immobilized MP 11 with one
another and with dissolved MP 11 as catalysts was made for the sulfoxidation of
methyl phenyl sulfide by hydrogen peroxide.
1.5.3. Adsorption
Direct physical adsorption of an enzyme on to a support, without any
entrapment, is generally characterized by relatively weak binding between enzyme
and support, leading to significant enzyme desorption (Norouzian, 2003; Pisklak et
al., 2006). This is perhaps the simplest of all the techniques and one which does not
grossly alter the activity of the bound enzyme. Ease of enzyme immobilization,
regeneration and reuse of the support are the important advantages of adsorption.
Moreover, a dynamic equilibrium is normally observed between the adsorbed enzyme
and the support which is often .affected by pH as well as the ionic strength of the
II
surrounding medium. This property of reversible binding has often been used for the
economic recovery of the support (Yang et al., 2003; Zhang et al., 2003). The various
types of carriers have been used for this purpose and these include alumina, clay,
glass, cellulose, acrylic composite, etc. (Tomotani and Vitolo, 2006; Mahmoud,
2007).
Ferapontova and Dominguez (2002) immobilized differently charged forms of
HRP by adsorption on metal electrodes of different nature. HRP was immobilized by
adsorption on totally cirmamoylated derivates of d-glucosone or d-maimitol. The
results showed that cirmamic carbohydrate esters represented an appropriate support
for peroxidase immobilization and could be used for several peroxidase based
applications (Rojas-Melgarejo et al., 2004).
1.5.4. Covalent bonding
Covalent binding is an extensively used technique for the immobilization of
enzymes (Leckband and Langer, 1991), though it is not a good technique for the
inrmiobilization of cells. Enzymes like glucose oxidase, peroxidase, invertase, etc. have
been immobilized using this technique (D'Urso and Fortier, 1996).
Covalent binding has been extensively investigated using inorganic supports.
Nahar et al. (2001) demonstrated the immobilization of HRP on an activated
polystyrene surface. Glucose oxidase, HRP and lactate oxidase have been covalently
immobilized on NH2- functionalized glass and a NH2- cellulose film via 13 different
coupling reagents (Tiller et al., 2002). More recently chloroperoxidase was bound on a
mesoporous sol-gel glass possessing a highly ordered porous structure via a
bifunctional ligand, trimethoxysilylpropanol (Borole et al , 2004). HRP covalently
bound to inorganic supports could be used for wastewater treatment (Rojas-Melgarejo
et al., 2004).
1.5.5. Chemical aggregation
Enzyme immobilization can be achieved by either chemical aggregation or
insolubilization with bifunctional or polyfunctional agents. These agents are used for
the insolubilization of active protein molecules or for binding these molecules onto
specific suitable supports. Crosslinking reagents have been used for introducing
12
intermolecular crosslinking in homogenous solution of protein or between protein and
other molecules. Among the numerous crosslinkers available; glutaraldehyde,
hexamethylene diisocyanate, l,5-difluoro-2,4-dinitrobenzene, diazobenzidine-3,3-
dicarboxylic acid, 2,4-diisocyantate toluene, N,N-hexamethylene bisiodoacetamide,
2,4-dimethyl adipimate are more widely used (Goldstein and Manecke, 1976). Some
reports on crosslinking of enzymes, adsorbed onto solid supports are also available
(Carvalho et al., 2000).
Biocatalysts can also be immobilized through chemical cross-linking using
homo as well as hetero-bifimctional cross-linking agents. Among these,
glutaraldehyde which interacts with amino groups has been extensively used due to
its; low cost, high efficiency and stability. The enzymes or the cells have been
normally crosslinked in the presence of an inert protein like gelatin, albumin and
collagen. Adsorption followed by crosslinking has also been used for the
immobilization of enzymes (Migneault et al., 2004).
The stability of manganese peroxidase (MnP) can be improved by
immobilization in sodium alginate, gelatin or chitosan as carriers, respectively and
glutaraldehyde as the cross-linking agent. The immobilized MnP showed significant
enhancement in azo dyes decolorization as compared to its soluble counterpart (Xiao-
Bin et al., 2007). The immobilization of HRP on composite membrane prepared by
coating nonwoven polyester fabric v^th chitosan glutamate in the presence of
glutraldehyde as a cross-linking agent has been investigated. The immobilized HRP
showed very high yield of immobilization and markedly high stabilization against
several forms of denaturants, thus offering potential for several applications
(Mohamed et al , 2008).
1.5.6. Bioaffinity based immobilization
Among the different techniques used for the immobilization of enzymes,
bioaffinity supports have attracted much attention of enzymologist due to its merits
over the other known classical methods (Khan et al., 2005). This procedure of
immobilization; does not disturb the native conformation of proteins, has least effect
on the active site of the enzyme, permits a gentle way of immobilizing enzymes by
exploiting bioaffinity of the enzyme towards an affinity support, allows oriented
immobilization of the enzyme via structural components which are far away from
13
active site and permits free accessibility to the incoming substrate and outgoing
products (Turkova, 1999; Mislovicova et al., 2000; Kulshrestha and Husain, 2006a;
Khan and Husain, 2007).
The immobilization of bitter gourd peroxidase (BGP) on concanavalin A (Con
A)-Sephadex, bioaffmity support showed very high yield of immobilization and
markedly high stabilization against several forms of denaturants as compared to its
free form (Akhtar et al., 2005c). This immobilized preparation showed high potential
in the decolorization/degradation of reactive dyes and removal of phenols from
polluted water (Akhtar et al., 2005a; Akhtar and Husain, 2006).
A more recent version of bioaffinity based immobilization is affmity layering
which allows deposition of alternate layers of affmity ligand and enzyme on the matrix
(Jan et al., 2001; Jan and Husain, 2004; Sardar and Gupta, 2005). The approach has
been so far mostly used to immobilize enzymes for biosensor design, bioanalytical
formats and biotransformation/bioconversion of various usefiil compounds (Gemeiner
et al., 1998; Jan et al., 2001; Dalai and Gupta, 2007).
1.6. OXIDATIVE ENZYMES IN DYE REMOVAL
In the early 1980 s, researchers developed an idea of using oxidoreductases for
treating wastewater contaminated with aromatic pollutants (Duran and Esposito,
2000; Husain and Jan, 2000; Duran et al., 2002). An advantage of this approach is that
these enzymes can react with a broad range of substrates, like phenols, chlorophenols,
methylated phenols, bisphenols, anilines, benzidines and other heterocyclic aromatic
compounds under dilute conditions and are less sensitive to operational upsets than
the microbial populations (Held et al., 2005; Gonzalez et al, 2006; Husain and
Husain, 2008).
The major oxidoreducatases; laccases and peroxidases have great potential in
targeting wide spectrum of colored compounds (Bhunia et al., 2001; Yang et al., 2003;
Akhtar et al., 2005a; 2005b). These enzymes can act on a broad range of substrates
and convert them into less toxic insoluble compounds, which can be easily removed
out of waste by a mechanism involving free radicals and show low substrate
specificity (Torres et al., 2003; Husain, 2006).
14
1.6.1. Peroxidases
Peroxidase (E.C. 1.11.1.7) is a heme-containing enzyme that is widely
distributed in plants, microorganisms and animals (Duarte-Vazquez et al., 2003a).
Heme is a complex between an iron ion (Fe" ) and the molecule protoporphyrin IX.
Peroxidases are classified into two superfamilies, animal and plant enzymes having a
molecular weight (Mr) ranging from 30 to 150 KDa (Regalado et al., 2004). The plant
peroxidase superfamily is further categorized into three classes according to its origin.
Class 1 the intracellular peroxidases, include yeast cytochrome c peroxidase,
ascorbate peroxidase and bacterial catalase peroxidases (Passardi et al., 2007).
Class II consists of secretory fungal peroxidases: ligninases, or lignin
peroxidase (LiP), and MnP. These are monomeric glycoproteins involved in the
degradation of lignin. The peroxidases most commonly studied for dye decolorization
are fimgal LiP and MnP.
Class III consists of secretory plant peroxidases, which have multiple tissue
specific functions: e.g. removal of H2O2 from chloroplasts and cytosol, oxidation of
toxic compounds, biosynthesis of the cell wall, defence responses towards wounding,
indole-3-acetic acid catabolism, ethylene biosynthesis, etc. Some of the well known
peroxidases of this class are HRP, TP, BGP and soybean peroxidase (SBP). Class III
peroxidases are also monomeric glycoproteins, containing four conserved disulphide
bridges and required calcium ions (SchuUer et al., 1996).
In recent years a great deal of research has been focused towards developing
processes in which peroxidases from plants and fungi were employed for wastewater
treatment (Bhunia et al., 2001; Wesenberg et al., 2003; Akhtar et al., 2005a; 2005b;
Mohan et al., 2005; Husain, 2006; Husain and Husain, 2008).
1.7. MICROBIAL PEROXIDASES
1.7.1. Manganese peroxidase (MnP)
The catalytic cycle of MnP proceeds through an initial oxidation by H2O2 to an
intermediary compound that in turn promotes the oxidation of Mn' ^ to Mn' '' (Gold et
al., 2000; Hofrichter, 2002). Mn"*" is stabilized by organic acids such as oxalic acid
15
and the Mn''" -organic acid complex formed, which acts as an active oxidant (Schlosser
and Hofer, 2002). Thus, MnP was able to oxidize their natural substrate, i.e. lignin as
well as textile dyes (Heinfling et al., 1998a). MnP isoenzymes could efficiently
decolorize azo dyes and phthalocyanine complexes in a Mn ^ independent manner.
Moreira et al. (2001) have evaluated an enzymatic action of the ligninolytic enzyme,
MnP as a feasible system for in vitro degradation of highly recalcitrant polymeric dye
(Poly R-478). The enzymatic treatment provoked not only the destruction of the
chromophoric groups but also a noticeable breakdown of chemical structure of the
dye. MnP was reported as the main enzyme involved in dye decolorization by
Phanerochaete chrysosporium (Chagas and Durrant, 2001). A novel dye-decolorizing
strain of the bacterium Serratia marcescens efficiently decolorized two chemically
different dyes; Ranocid Fast Blue and Procion Brilliant Blue-H-GR belonging to the
azo and anthraquinone groups, respectively. However, an involvement of MnP was
found in the decolorization of both dyes (Verma and Madamwar, 2002a).
MnP was detected during dye decolorization by culture of Phlebia tremellosa
when the culture medium was supplemented with MnCb (Kirby et al., 2000). MnP
from Clitocybula dusenii was involved in the breakdown of dyes in the real dye-
containing effluent (Wesenberg et al., 2002). Some investigators have developed
enzymatic membrane reactors for the oxidation of azo dyes by MnP (Lopez et al.,
2002; 2004; 2007). Under the best conditions, continuous operation with a dye
decolorization higher than 85% and minimal enzymatic deactivation was feasible for
18 d, attauiing an efficiency of 42.5 mg Orange II oxidized/MnP unit consumed
(Lopez et al., 2004). Yang et al. (2003) have shown that the two yeasts; Debaryomyces
polymorphus, Candida tropicalis and filamentous fungi; Umbelopsis isabellina could
completely decolorize 100 mg Reactive Black 5 within 16-48 h. MnP activity was
detected in culture supernatants of these organisms. Selvam et al. (2003) reported that
an azo dye. Orange G was decolorized 10.8% by 15 U mL"' of MnP present in WRF,
Thelephora sp. Mielgo et al. (2003) described a MnP based azo dyes degradation. The
WRF, Irpex lacteus decolorized the textile industry wastewater efficiently without
adding any chemicals. The degree of decolorization of the dye effluent by shaking or
stationary cultures was 59% and 93%, respectively, on 8'*' day. Higher decolorization
was related to the level of MnP which was detected in stationary cultures than in the
cultures shaken (Shin, 2004).
Partial decolorization was observed in cultures containing 200 ppm of Brilliant
16
Cresyl Blue and Methylene Blue. High MnP activity but very low LiP and laccase
activities were detected in the culture of the WRF, Lentinula edodes. Experimental
data suggested that MnP appeared to be the main enzyme responsible for the
capability of Lentinula edodes to decolorize synthetic dyes (Boer et al., 2004).
Verma and Madamwar (2005) found that Basidiomycete PV002, a white-rot
strain efficiently decolorized Ranocid Fast Blue (96%) and Acid Black 210 (70%) on
5" and 9" day under static conditions, respectively. The degradation of azo dyes
under different conditions was strongly correlated with high MnP activity. Machado
et al. (2005) used Remazol Brilliant Blue R (RBBR) dye as substrate to evaluate
ligninolytic activity in 125 basidiomycetous fungi isolated from tropical ecosystems.
Extracellular extracts of 30 selected fungi grown on solid medium with sugar can
bagasse showed RBBR decolorization and peroxidase activity. Eight fungi produced
MnP activity which had RBBR decolorization capacity.
Decolorization of sulfonaphthalein (SP) dyes by MnP was investigated and
abnost all dyes were decolorized at pH 4.0 (Christian et al., 2003). The WRF,
Pleurotus ostreatus produced MnP which decolorized most of the SP dyes at pH 4.0.
The order of preference for SP dyes as substrate for the MnP-catalyzed decolorizing
activity was Phenol Red>o-cresol Red>m-cresol Purple > Bromophenol
Red > Bromocresol Purple > BPB > Bromocresol Green (Shrivastava et al., 2005).
Harazono and Nakamura (2005) decolorized mixtures of four reactive textile dyes,
including azo and anthraquinone dyes, by a white-rot basidiomycete Phanerochaete
sordida. Phanerochaete sordida decolorized dye mixtures (200 mg L"') by 90%
within 48 h in nitrogen-limited glucose-ammonium media. MnP played a major role
in dye decolorization by Phanerochaete sordida.
Eichlerova et al. (2006b) tested eight different Pleurotus species for their
Orange G and RBBR decolorization capacity and their ligninolytic properties. Strain
CCBAS 461 of species Pleurotus calyptratus produced a relatively high amount of
MnP, laccase and aryl-alcohol oxidase activity. Within 14 d the strain decolorized
upto 91% Orange G and 85% RBBR in liquid culture and more than 50%> of these
dyes on agar plates. Pleurotus calyptratus was able to decolorize efficiently also other
azo and phthalocyanine dyes, but only a limited decolorization capacity was found in
the case of polyaromatic and triphenylmethane dyes.
Litter-decomposing basidiomycete fungi including environmental isolates
from oak forest soil were compared with WRF for ligninolytic enzymes production
17
and decolorization of synthetic dyes; Poly B-411, Reactive Black 5, Reactive Orange
16 and RBBR. The highest activity of MnP was detected in the culture of Collybia
dryophila with the activity over 30 U L~'. The fastest degradation of Poly B-411 was
performed by the strains with high levels of MnP and laccase while the decolorization
of other dyes did not depend so strictly on enzyme activities (Baldrian and Snajdr,
2006). The decolorization of 12 different azo, diazo and anthraquinone dyes was
carried out using a new isolate WRF, strain L-25. A decolorization efficiency of 84.9-
99.6% was achieved by cuUivation in 14 d using an initial dye concentration of
40 mg L~'. MnP produced by strain L-25 was used for the enzymatic decolorization of
dyes thus confirming the capability of enzyme for this purpose (Kariminiaae-
Hamedaani et al., 2007).
The production of MnP by Phanerochaete chrysosporium and the level of
decolorization of 13 dyes were evaluated using static, agitated batch and continuous
cultures. For concentrations of 100 mg L"' of Acid Black 1, Reactive Black 5,
Reactive Orange 16 and Acid Red 27, the decolorization efficiency was over 90%. In
batch cultures with Acid Black 1 and Reactive Black 5 a significant increment in
priinary post-metabolism biomass was observed. For Acid Black 1 and Reactive
Black 5, it was possible to explore the response of the continuous system during 32 to
47 d, with concentrations between 25 to 400 mg L"', obtaining decolorization greater
than 70% for 400 mg L'' (Urra et al., 2006). A partially purified MnP from
Bjerkandera adusta was tested for the decolorization of several artificial dye baths.
The most efficient decolorization was observed in dye bath of anthraquinone dyes;
Reactive Blue 19, diazo dyes; Reactive Black 5 and Acid Orange 7 (Mohorcic et al.,
2006). The azo dyes; Congo Red, Orange G and Orange IV were efficiently
decolorized by MnP isolated and purified from Schizophyllum sp. (Cheng et al.,
2007). Purified MnP from Ischnoderma resinosum could decolorize textile dyes;
Reactive Black 5, Reactive Blue 19, Reactive Red 22 and Reactive Yellow 15. The
highest decolorization was seen at acidic pH (Kokol et al., 2007).
Park et al. (2007) described the decolorization of six commercial dyes by 10
fungal strains. Under the experimental conditions, extracellular laccase and MnP were
detected. The decolorization mechanisms by Funalia trogii ATCC 200800 involved a
complex interaction of enzyme activity and biosorption. This study suggested that it
was possible to decolorize a high concentration of commercial dyes, which could be a
great advancement in the treatment of dye containing wastewater.
18
Pricelius et al. (2007) investigated the conversion of azo dyes; Flame Orange
and Ruby Red, into their N-demethylated form and accompanying polymerization by
different oxidoreductases. Laccase from Pycnoporus cinnabarinus, MnP from
Nematoloma frowardii and the novel Agrocybe aegerita peroxidase, used a similar
mechanism to decolorize/degrade azo dyes. On the other hand the mechanism for
cleavage of the azo bond by azo-reductases of Bacillus cereus and Bacillus subtilis
was based on reduction of azo bond at the expense of NAD(P)H. Thus establishing a
new ecofriendly method for dye decoloration.
1.7.2. Lignin peroxidase (LiP)
LiP also known as ligninase or diaryl propane oxygenase was first reported in
1983. LiP catalyzes the oxidation of non-phenolic aromatic lignin moieties and similar
compounds. LiP catalyzes several oxidations in the side chains of lignin and related
compounds (Tien and Kirk, 1983). LiP has been used to mineralize a variety of
recalcitrant aromatic compounds (Duran and Esposito, 2000), polychlorinated
biphenyls (Krcmar and Ulrich, 1998) and dyes (AbaduUa et al., 2000; Husain, 2006).
Heinfling et al. (1998b) have described the transformation of six industrial azo and
phthalocyanine dyes by ligninolytic peroxidases from Bjerkandera adusta and other
WRF. Phanerochaete chrysosporium cultures, extracellular fluid and purified LiP
were able to degrade Crystal Violet and six other triphenylmethane dyes by sequential
N-demethylations (Bumpus and Brock, 1988). Pointing and Vrijmoed (2000) have
shown that LiP played a major role in the decolorization of azo, triphenylmethane,
heterocyclic and polymeric dyes by Phanerochaete chrysosporium. LiP was reported
as the main enzyme involved in dye decolorization by Bjerkandera adusta (Robinson
et al., 2001). Verma and Madamwar (2002b) demonstrated that more than 50%
decolorization of Procion Brilliant Blue HGR, Ranocid Fast Blue, Acid Red 119 and
Navidol Fast Black was catalyzed by partially purified LiP from Phanerochaete
chrysosporium grown on neem hull waste.
Ferreira-Leitao et al. (2003) compared the oxidation of Methylene Blue and
Azure B dyes by plant HRP and LiP from Phanerochaete chrysosporium. Results
showed HRP was able to N-demethylate both dyes, but exhibited much slower
reaction kinetics than LiP and required higher H2O2 concentrations. Product yields
were also different for HRP, and contrary to LiP, HRP was xmable to achieve aromatic
19
ring cleavage. Furthermore, Ferreira-Leitao et al. (2007) in a recent work has
compared the usefulness of fungal LiP with plant HRP concerning the degradation of
Methylene Blue and its demethylated derivatives. It has been shown that although
both enzymes were able to oxidize Methylene Blue and its derivatives, HRP reactions
required higher H2O2 concentrations and presenting a considerably lower reaction
rate, contrary to LiP. However, HRP was unable to achieve aromatic ring cleavage.
The oxidation potential of LiP was roughly double than that of less effective HRP and
it explained relative efficacy. Thus, LiP could be more suitable for
decolorization/degradation of phenothiazine dyes from waste streams.
Lan et al. (2006) has made an effort to couple a H2O2 producing enzymatic
reaction to the LiP catalyzed oxidation of dyes. H2O2 was produced by glucose
oxidase and its substrate glucose. Due to controlled release of H2O2, a sustainable
constant activity of LiP was observed. Degradation of three dyes; Xylene Cyanol,
Fuchsine and Rhodamine B by LiP coupled with glucose oxidase indicated that H2O2
was very effective for improvement of efficiency of the decolorization of dyes.
Pseudomonas desmolyticum NCIM 2112 was able to degrade a diazo dye.
Direct Blue 6 (100 mgL'') completely within 72 h of incubation with 88.95%
reduction in chemical oxygen demand (COD) in static anoxic condition.
Decolorization of Direct Blue 6 in batch culture represented the role of oxidative
enzymes; LiP, laccase and tyrosinase in the degradation of dye (Kalme et al., 2007).
1.7.3. Dye decoloring peroxidase (DyP)
The potential of a newly isolated fungus, Geotrichum candidum Dec 1
peroxidase, a glycoprotein was investigated for the degradation and decolorization of
many xenobiotic compounds such as synthetic dyes, food coloring agents, molasses,
organic halogens, lignin and kraft pulp effluents (Kim and Shoda, 1999). Sugano et al.
(2000) isolated a new DyP from Trametes cucumeris Dec 1, which decolorized more
than 30 types of synthetic dyes. DyP is a key enzyme that degrades azo and
anthraquinone dyes (Sato et al., 2004). Further, these workers have reported that DyP
from Trametes cucumeris Dec 1 was able to decolorize a representative anthraquinone
dye. Reactive Blue 5 to light red-brown compounds (Sugano et al., 2006).
Decolorization of anthraquinone dye, RBBR was performed by crude
recombinant dye decolorizing peroxidase (rDyP) obtained from Aspergillus oryzae. In
20
batch culture, equimolar batch addition of H2O2 and RBBR produced complete
decolorization of RBBR by rDyP, with a turnover capacity of 4,75. In stepwise fed-
batch addition of H2O2 and enzyme, the turnover capacity increased to 5.76 and 14.3,
respectively. When H2O2 was added in continuous fed-batch and 1.6 mM dye was
added in stepwise fed-batch mode, 102 g of RBBR was decolorized by 5000 U of
crude rDyP in 650 min increasing the turnover capacity to 20.4 (Shakeri and Shoda,
2007).
1.7.4. Versatile peroxidase (VP)
VP has been recently described as a new family of ligninolytic peroxidases,
together with LiP and MnP obtained from for Phanerochaete chrysosporium
(Martinez, 2002). Interestingly, these enzymes possessed both LiP and MnP-like
characteristics; they were able to oxidize Mn " to Mn " at around pH 5.0 while also 9+
oxidizing aromatic compounds at around pH 3.0, regardless of the presence of Mn
(Heinfling et al., 1998b; Ruiz-Duenas et al., 2001). These enzymes were thus termed
MnP-LiP hybrid peroxidases, or VP. VPs from various sources Pleurotus
pulmonarius (Camarero et al., 1996), Pleurotus ostreatus (Cohen et al., 2002),
Bjerkandera adusta (Heinfling et al., 1997; 1998a; 1998b; 1998c), Pleurotus eryngii
(Gomez-Toribio et al., 2001) and Bjerkandera sp. (Master and Field, 1998; Moreira et
al., 2005) have shovm their azo dye degradation potential.
From the extracellular fluid of a novel strain of Bjerkandera sp. VP was
isolated, purified and identified as the main enzyme responsible for RBBR dye
decolorization (Moreira et al., 2006). Sugano et al. (2006) reported the purification
and characterization of a new VP from the decolorizing microbe, Thanatephorus
cucumeris Dec l(TcVPl). TcVPl exhibited particularly high decolorizing activity
towards azo dyes. Furthermore, co-application of TcVPl and the DyP from
Thanatephorus cucumeris Dec 1 was able to completely decolorize a representative of
anthraquinone dye, Reactive Blue 5. This decolorization proceeded sequentially, DyP
decolorized Reactive Blue 5 to light red-brown compounds and then TcVPl
decolorized these colored intermediates to colorless products. These findings provide
important new insights into microbial decolorizing mechanisms and may facilitate the
future development of treatment strategies for dye wastewater.
21
1.7.5. Vanadium haloperoxidase (VHP)
VHP have been reported to mediate oxidation of halides to hypohalous acid
and the sulfoxidation of organic sulfides to the corresponding sulfoxides in the
presence of H2O2. However, traditional heme peroxidase substrates were reported not
to be oxidized by VHP. It was found that the recombinant vanadium chloroperoxidase
from the fungus Curvularia inaequalis catalyzed the bleaching of an industrial
sulfonated azo dye, Chicago Sky Blue 6B in the presence of H2O2 (ten Brink et al.,
2000).
1.8. PLANT PEROXIDASES
1.8.1. Horseradish peroxidase (HRP)
Bhunia et al. (2001) have shown that HRP can catalyze an effective
degradation and precipitation of industrially important azo dyes. They evaluated
specificity of HRP toward different dyes, such as Remjizol Blue and Cibacron Red.
For Remazol Blue, the enzyme activity was found to be far better at pH 2.5 than at
neutral pH. In addition, Remazol Blue acted as a strong competitive inhibitor of HRP
at neutral pH. HRP showed broad substrate specificity towards a variety of azo dyes.
Mohan et al. (2005) showed the significance of HRP catalyzed enzymatic reaction in
the treatment of an acid azo dye. Acid Black 10 BX. However, the performance of
HRP catalyzed reaction was found to be dependent upon the aqueous phase pH,
contact time, H2O2, dye and HRP concentrations. Standfird plating studies performed
with Pseudomonas putida showed enhanced degradation of HRP catalyzed dye
compared to control.
The oxidation of Direct Yellow 12 dye was tested as a fiinction of HRP at
fixed concentrations of H2O2 and HRP. The effective degradation of azo dye by HRP
proved the use of an enzymatic treatment process as a viable approach for the
degradation of azo dyes from aqueous solutions (Maddhinni et al., 2006). Ulson de
Souza et al. (2007) investigated the potential of HRP in the decolorization of textile
dyes and effluents. For the dyes treated, results indicated that the decolorization of the
dye, Remazol Turquoise Blue G 133% was approximately 59% and 94%) for the
22
Lanaset Blue 2R; for the textile effluent, the decolorization was 52%. The tests for
toxicity towards Daphnia magna showed that there was a reduction in toxicity after
enzymatic treatment.
1.8.2. Turnip peroxidase (TP)
Turnip roots, which are readily grown in several countries, are a good source of
peroxidase and because of their kinetic and biochemical properties they have a high
potential as an economic alternative to HRP (Duarte-Vazquez et al., 2002).
Peroxidases from turnip roots were highly effective in decolorizing acid dyes
having vdde spectrum chemical groups. Dye solutions, containing 40-170 mg dye L'
in the presence of 2.0 mM 1-hydroxybenzatriazole (HOBT) were successfully treated
by TP at pH 5.0 and 40 °C. Complex mixtures of acid dyes were also significantly
decolorized by TP in the presence of HOBT (Kulshrestha and Husain, 2007).
1.8.3. Bitter gourd peroxidase (BGP)
Akhtar et al. (2005b) have investigated the potential of peroxidases from
Momordica charantia in decolorizing industrially important dyes. Momordica
charantia peroxidase was able to decolorize 21 dyes, being used by the textile and
other important industries by forming an insoluble precipitate. The greater fraction of
the color was removed when the textile dyes were treated with increasing
concentrations of enzyme but four out of eight reactive dyes were recalcitrant to
decolorization by BGP. The rate of decolorization was enhanced when the dyes were
incubated with fixed quantity of enzyme for increasing times. Decolorization of non-
textile dyes resulted in the degradation and removal of dyes from the solution without
any precipitate formation. Thus indicating an effective biocatalyst for the treatment of
effluents containing recalcitrant dyes from textiles, dye manufacturing and printing
industries.
1.8.4. Tomato peroxidase (TMP)
Multiple efforts have been directed towards optimized processes in which
peroxidase from cheap plant sources were used to remove dyes from polluted water
23
(Husain, 2006). Matto and Husain (2008) described the role of partially purified TMP
in decolorizing direct dyes; Direct Red 23 (DR 23) and Direct Blue 80 (DB 80). These
dyes were maximally decolorized by TMP at pH 6.0 and 40 °C. The absorption spectra
of the untreated and treated dyes exhibited marked differences in the absorption at
various wavelengths.
Decolorization and decontamination of two textile carpet industrial effluents
was carried out by using TMP. Textile carpet effluent red and blue were decolorized
69% and 59% by 0.705 U mL"' TMP at pH 6.0 and 40 °C, respectively. The TMP
treated effluents exhibited significant loss of total orgcinic carbon (TOC) fi-om the
effluent (Husain and Kulshrestha, 2008).
1.9. MICROPEROXIDASE 11 (MP 11)
MP 11, a heme containing undecapeptide derived fi^om horse heart cytochrome
C, was utilized as a peroxidative catalyst. The decolorization of water insoluble
synthetic dyes by MP 11 in 90% methanol was attempted. MP 11 exhibited effective
decolorization activity against azo or anthraquinone types of dyes. The degradation
pathway for Solvent Orange 7 was investigated, showing that MP 11 catalyzed the
oxidative cleavage of azo linkage to generate 1,2-naphthoquinone and 2,4-
dimethylphenol as key intermediates (Wariishi et al., 2002).
2.1. DYE REMOVAL BY IMMOBILIZED PEROXIDASES
In general, it is accepted that in order to increase the potential use of enzymes
in wastewater treatment processes, immobilization of enzymes is absolutely necessary
for their stability and reuse (Akthar et al., 2005a; Liu et al., 2006). Various
applications of peroxidases immobilized on different supports have been reviewed
and one such advantage is of dye removal from various dyeing effluents (Duran and
Esposito, 2000; Torres et al., 2003; Husain, 2006; Husain and Husain, 2008).
MnP oxidizes a wide range of substrates, rendering it an interesting enzyme
for potential applications. The significant decolorization of azo dyes in static and
shaky situation by gelatin-immobilized MnP was studied, and there was no loss of
immobilized enzyme activity after two repeated uses in batch process (Xiao-Bin et al.,
2007).
24
MP 11 entrapped in the system of bis(2-ethylhexyl) sulphosuccinate sodium
salt (AOT)-reversed micelles exhibited peroxidase activity in the presence of H2O2. It
effectively catalyzed the oxidative decolorization of an azo dye; Solvent Yellow 7 and
an anthraquinone dye, Solvent Blue 11 in the hydrophobic organic solvent. To
optimize the reversed micellar system, the effect of pH and molar ratio of H2O/AOT
hydration degree (Wo) was examined, indicating that MP 11 exhibited a maximal
decolorization activity at pH 8.0-10 with the WQ value of 20 (Okazaki et al., 2002).
MP 11 was immobilized in hybrid periodic mesoporous organosilica materials and in
a nano-crystalline metal organic [Cu(OOC-C6H4-C6H4-COO).«/2 C6H12N2]n
framework. The conversion of Amplex-Ultra Red and Methylene Blue to their
respective oxidation products occurred successfully in the presence of immobilized
MP 11 (Pisklak et al., 2006).
Shakeri and Shoda (2008) carried the immobilization of rDyP produced from
Aspergillus oryzae using silica-based mesoporous materials, FSM-16 and AlSBA-15.
rDyP immobilized on FSM-16 at pH 4.0, decolorized eight sequential batches of an
anthraquinone dye, RBBR in repeated-batch decolorization process.
Fruhwirth et al. (2002) used a catalase peroxidase from the newly isolated
Bacillus SF to treat textile-bleaching effluents. The enzyme was immobilized on
various alumina-based carriers of different shapes. Bleaching effluent was treated in a
horizontal packed-bed reactor containing 10 kg of the immobilized catalase
peroxidase at a textile-finishing company. The treated liquid (500 L) was reused
within the company for dyeing fabrics with various dyes, resulting in acceptable color
differences of below Delta E*=l .0 for all dyes.
Hydrophobic matrix bound Saccharum peroxidase was used for the
degradation of four textile dyes; Procion Navy Blue HER, Procion Brilliant Blue H-
7G, Procion Green HE-4 BD and Supranol Green. These dyes at an initial
concentration of 50 mg L"' were completely degraded within 8 h by the peroxidase
immobilized on modified polyethylene matrix. The immobilized peroxidase was used
in a batch reactor for the degradation of Procion Green HE-4BD and its reusability
was studied for 15 cycles and the half-life was found to be 60 h (Shaffiqu et al., 2002).
Acrylamide gel immobilized HRP showed effective performance compared to
alginate entrapped and free HRP in the removal of Acid Black 10 BX. Alginate
entrapped HRP showed inferior performance over the free enzyme due to the
consequence of non-availability of the enzyme to the dye molecule (Mohan et al.,
25
2005). Maddhinni et al. (2006) studied the decolorization of Direct Yellow 12 dye by
soluble and immobilized HRP under various experimental parameters; pH, H2O2, dye
and enzyme concentrations. The efficiency of polyacrylamide entrapped HRP for the
oxidation of Direct Yellow 12 dye was higher followed by alginate entrapped HRP
and then by free HRP. The alginate and polyacrylamide immobilized HRP
preparations were further used two-three times for removal of same dye with lower
efficiency, respectively.
An electroenzymatic process is an interesting approach that combines enzyme
catalysis and electrode reactions. Kim et al. (2005) studied an electroenzymatic
method that used an immobilized HRP to degrade Orange II (azo dye) within a two-
compartment packed-bed flow reactor. To evaluate the electroenzymatic degradation
of Orange II, electrolytic experiments were carried out with 0.42 U mL'* HRP at
0.5 V. The overall application of the electroenzymatic approach led to a greater
degradation rate than the use of electrolysis alone. Also the by-products formed were
found to consist primarily of an aromatic amine, sulfanilic acid and unknown
compounds. Degradation of Orange II by an electroenzymatic method using HRP
boimd on an inexpensive and stable inorganic Celite®R-646 beads bound with 2%
aqueous glutaraldehyde was studied in a continuous electrochemical reactor with in
situ generation of H2O2. Based on the parametric studies, over 90% degradation of
Orange II occurred during continuous operation for 36 h. From the results of GC/MS
analysis, degradation products were identified and a possible breakdown pathway for
Orange II was proposed (Shim et al., 2007).
BPB and Methyl Orange removal capability of citraconic anhydride-modified
HRP was compared with those of native HRP. Upon chemical modification, the
decolorization efficiency was increased by 1.8% and 12.4% for BPB and Methyl
Orange, respectively. Lower dose of citraconic anhydride-modified HRP was required
than that of native enzyme for the decolorization of both dyes to obtain the same
decolorization efficiency. Citraconic anhydride-modified HRP showed a good
decolorization of dye over a wide range of dye concentrations from 8 to 24 or
32 ^mol L~ at 300 nmol L~' H2O2, which would meet industrial expectations (Liu et
al., 2006).
Con A-Sephadex immobilized BGP was exploited for the decolorization of
industrially important dyes from polluted water. The dye decolorization by
BGP was maximum at pH 3.0 and 40 °C. This enzyme was repeatedly used for the
26
Table 2: Dye removal by immobilized peroxidases
Enzyme MnP
Plant peroxidase
MP 11
rDyP
Catalase peroxidase
HRP
BGP
TP,TMP
Source Schizophyllum sp. F17 Ipomea palmate, Saccharum spontaneum
horse heart cytochrome c
Aspergillus oryzae
Bacillus SF
type Vl-A
Horseradish roots
Type IV-A Momordica charantia Brassica rapa, Lycopersicon esculentum Lycopersicon esculentum
Support Gelatin
Polyethylene matrix
AOT-reversed micelles
Periodic mesoporous organosilica
Silica-based mesoporous materials, FSM-16 andAlSBA-15 Alumina-based carriers
Graphite felt Alginate and acrylamide polymeric matrix Citraconic anhydride
Alginate and acrylamide polymeric matrix Celite®R-646 Con A-Sephadex
Con A-cellulose
Dye Azo dyes
Textile dyes
Solvent Yellov^ 7 and Solvent Blue 11 Amplex-Ultra Red and Methylene Blue RBBR
Textile-bleaching effluents Orange II Acid Black 10 BX
BPBand Methyl Orange Direct Yellow 12
Orange II Reactive dyes Textile carpet industrial effluents Direct dyes
References Xiao-Bin et al., 2007 Shaffiqu et al., 2002
Okazaki et al., 2002
Pisklak et al., 2006
Shakeri and Shoda, 2008
Fruhwirth et al., 2002
Kim et al., 2005 Mohan et al., 2005
Liu et al., 2006
Maddhinni et al., 2006
Shim et al., 2007 Akhtar et al., 2005a
Husain and Kulshrestha, 2008
Matto and Husain, 2008 1
27
decolorization of eight reactive textile dyes and after lO"' repeated use the immobilized
enzyme retained nearly 50% of its initial activity. The mixtures of dyes were also
successfully decolorized by immobilized BGP (Akhtar et al., 2005a). Decolorization
and decontamination of two textile carpet industrial effluents was carried by using Con
A-cellulose bound turnip and tomato peroxidases. Textile carpet effluent red and blue
were decolorized 78% and 84% by immobilized TP (0.423 U mL''), respectively.
However, 0.705 U mL"' of immobilized tomato peroxidases decolorized effluent red
73% and blue 74%. The immobilized TP treated effluent even exhibited significant
loss of TOC from the solution (Husain and Kulshrestha, 2008). TMP immobilized on a
bioaffinity support, Con A-cellulose was highly effective in decolorizing direct dyes as
compared to free TMP. More than 70% of DR 23 and DB 80 were decolorized by
TMP at pH 6.0 and 40 °C. Immobilized TMP showed lower Michaelis constant, Km
than the free enzyme for the direct dyes (Matto and Husain, 2008).
3.1. REDOX MEDIATORS
Redox mediators are compounds that speed up the reaction rate by shuttling
electrons from the biological oxidation of primary electron donors or from bulk
electron donors to the electron-accepting aromatic compounds (Fabbrini et al., 2002a).
Redox mediators provide high redox potentials (>900 mV) to attack on recalcitrant
structural analogs and are able to migrate into aromatic structure of the compound.
The mechanism of action of laccase-mediator system has been extensively studied
(Couto et al., 2005). However, it was shown that the presence of small molecules
capable to act as electron transfer mediators were able to oxidize non-phenolic
compounds (Crestini et al., 2003), thus expanding the range of compounds that can be
oxidized by enzymes. Several organic and inorganic compounds have been reported as
effective redox mediators. These include thiol and phenol aromatic derivatives, N-
hydroxy compounds and ferrocyanides (Husain and Husain, 2008).
The role of redox mediators in the laccase oxidation reaction is well
characterized and also can be applied for other enzymes. When a substrate is oxidized
by a laccase, the redox mediator forms cation radicals, short-lived intermediates that
co-oxidize non-substrates. These cation radicals can be formed by two mechanisms: (i)
the redox mediator can perform either one-electron oxidation of the substrate to a
28
radical cation (Xu et al., 2000; 2001); or (ii) the redox mediator cai\ abstract a proton
from the substrate, converting it into a radical (Fabbrini et al., 2002a). For example,
2,2'-azino-bis-(3-ethyl-benzothiazolinsulfonic acid) (ABTS) acts by the first
mechanism (Potthast et al., 2001), whereas HOBT acts by the second (Fabbrini et al.,
2002b; Hirai et al., 2006).
A redox mediator should have three properties: (i) it should be able to be
oxidized by one-electron directly from the enzyme to produce a free radical, (ii)
produces radical which should be stable enough to diffuse and react with the target
compound, and (iii) it should have an appropriate redox potential. The presence of
oxidizing mediators enhance the decolorization of dyes showing low decoloration
rates, while no effect is reported for dyes that are highly decolorized in the absence of
mediator (Astolfi et al., 2005; Tinoco et al., 2007).
Substrate ^ ^m Mediatorox ^ ^ A Enzyme ^ ^ ^m H2O2 (Peroxidase) (Dye) ^ ^ ^ ^ ^ % ^ T ^ ^ ^ ^ O2 (Laccase)
Substrateox^ f Mediator ^ ^ Enzymeox^ ^ H2O
Fig. 1: Redox cycle for mediating substrate oxidation by oxidoreductases
It has been speculated that laccase may react indirectly with non-phenolic
lignin components through mediation by phenolic species present in its natural
environment. These phenolic compounds perform as natural mediators of laccase in
depolymerization processes (Calcaterra et al., 2008), or act as lignin fragments
generated by other ligninolitic enzymes (Thurston, 1994), and open possible radical
routes to the oxidation of non-phenolic components (ten Have and Teunissen, 2001).
A number of phenolic compounds such as phenol red, catechol, hydroquinone, etc.
when oxidized by laccase to resonance-stabilized aryloxyl radicals, become able to
catalyze oxidation of non-phenolic compounds before being depleted in coupling or
O2- induced ring-cleavage reactions, have been proposed as natural mediators for
laccases (Calcaterra et al., 2008).
d'Acunzo and Galli (2003) reported that phenol red, which can be oxidatively
converted into a resonance-stabilized phenoxy radical, worked as a mediator in the
laccase-catalyzed oxidation of a non-phenolic substrate (4-methoxybenzyl alcohol)
29
and also of a phenol (2,4,6-ti-tert-butylphenol). In particular, phenol red was found to
be at least 10 times more efficient than 3-hydroxyanthranilate (a reported natural
phenolic mediator of laccase) in the oxidation of 4-methoxybenzyl alcohol. Camarero
et al. (2005) selected 10 phenols as natural mediators after screening 44 different
compounds to mediate the oxidation reaction catalyzed by laccase with the aim of
indentifying cheap, more effective and eco-friendly mediators for the decolorization of
recalcitrant dyes and for other industrial and environmental applications.
Among the mediators, those presenting the >N-OH moiety; HOBT, N-
hydroxyphthalimide and violuric acid (VLA) proved very efficient towards benzylic
substrates, through a radical H-abstraction route of oxidation involving an aminoxyl
radical (>N-0') intermediate. TEMPO (2,2',6,6'-tetramethyl piperidine-A -oxyl), a
stable >N-0" specie proved the most efficient mediator towards benzyl alcohols
(d'Acunzo et al., 2006).
Purified VP from Bjerkandera Adusta was tested for the industrial dye
decolorization. The presence of redox mediator veratryl alcohol (VA), acetosyringone
or TEMPO as oxidizing mediators in the reaction mixtvire generally enhanced the rate
of dye decolorization (Tinoco et al., 2007). A pure fiingal laccase, obtained from a
commercial formulation used in the textile industry, did not decolorize RBBR.
Decolorization was only observed when a small Mr redox mediator was added
together with the laccase. Under the conditions specified, VLA (5.7 mM) was the
most effective mediator studied and almost caused complete decolorization within 20
minutes (min). In contrast, HOBT (11 mM) decolorized RBBR at about a two-fold
slower rate and to a lesser extent. Also, higher concentrations of HOBT were
inhibitory which could be due to inactivation of laccase by the toxic HOBT radical.
The commercial laccase formulation that contained phenothiazine-10-propionic acid
as the mediator was least effective, giving 30% decolorization under equivalent
conditions. Thus suggesting that similar laccase plus mediator systems could be used
for the detoxification of related anthraquinone textile dyes (Soares et al., 2001a).
Knutson et al. (2005) demonstrated effective enzymatic decolorization of two
recalcitrant dyes; stilbene dye, Direct Yellow and Methine dye, Basazol 46L by HRP,
SBP and laccase in the presence of ABTS as a mediator. The stilbene dye, Direct
Yellow 11 responded to both SBP and laccase/ABTS. SBP was more effective in the
oxidative removal of methine dye, Basazol 46L as compared to the other peroxidases.
30
Akhtar et al. (2005b) have demonstrated the decolorization of 21 different
reactive textile and other industrially important dyes by using BGP in sodium acetate
buffer, pH 5.6 in the presence of 0.75 mM H2O2 and 1.0 mM HOBT. Decolorization
was drastically increased when dyes were treated with BGP in the presence of 1.0 mM
HOBT. Complex mixtures of dyes were also significantly decolorized by BGP in the
presence of 1.0 mM HOBT. The decolorization of acid dyes by TP was significantly
enhanced in the presence of 2.0 mM HOBT. The maximum decolorization of dyes was
obtained in 1 h. The decolorization of dyes was maximum at pH 5.0 and 40 °C.
Phytotoxicity test based on Allium cepa root growth inhibition has shown that majority
of the TP-treated dye product were less toxic than their parent dye. The polluted
wastewater contaminated with single dye or mixtures of dyes were treated with
enzyme and it resulted in a remarkable loss of TOC (Kulshrestha and Husain, 2007).
Three plant phenols, namely acetosyringone, syringaldehyde and /7-coumaric
acid, were selected as laccase redox mediators to investigate the enzymatic
delignification of paper pulp (obtained from kraft cooking of eucalyptwood) in
combination with peroxide bleaching, p-coumaric acid only caused minor increase of
pulp brightness and did not lower its kappa number (a rough estimation of the lignin
content), whereas, the use of acetosyringone or syringaldehyde as laccase mediators
enabled over 15% increase of final brightness and a similar decrease of final kappa
number (Camarero et al., 2007).
The degradation of Phenol Red dye by laccase in the presence or absence of
HOBT, was investigated. Decolorization of Phenol Red with laccase alone was
limited with a maximum decolorization degree of around 40% after 48 h of
incubation. However, in the presence of laccase/HOBT system coupled with high
redox potential complete degradation of Phenol Red was obtained (Moldes et al.,
2004).
Couto et al. (2005) demonstrated the effect of redox mediators on the synthetic
acid dye decolorization (Sella Solid Red and Luganil Green) by laccase from
Trametes hirsuta cultures. All the redox mediators tested; ABTS, HOBT and RBBR
led to higher activities than those obtained without mediator addition showing the
suitability of the laccase/mediator system in the decolorization of acid dyes. HOBT
was by far the most effective mediator, showing a decolorization of 88% in 10 min for
Sella Solid Red and of 49% in 20 min for Luganil Green.
Couto and Sanroman (2007) showed the effect of a redox mediator, VLA on
31
the decolorization of two recalcitrant acid dyes; Acid Red 97 and Acid Green 26 by
crude laccase obtained from Trametes hirsuta cultures. The laccase-mediator system
led to a higher extent of decolorization in shorter time than that obtained without
mediator addition, especially for Acid Red 97 the dye was 90% decolorized in only
3 min.
The effect of redox mediators in the decolorization of Indigo Carmine and
Phenol Red by two laccase isoenzymes from Trametes versicolor has been
investigated. All the redox mediators tested; HOBT, promazine, para-hydroxybenzoic
acid (p-HBA) and l-nitroso-2-naphthol-3,6-disulfonic acid (NNDS), led to higher dye
decolorization than those obtained without mediator. Among the different tested
mediators, promazine was the most effective redox mediator tested at a low range of
concentration (0.5-50 iM). The two laccase isoenzymes, Lac I and Lac II, showed
different decolorization capability depending on the mediator used. No significant
differences were detected for NNDS, however Lac II was more effective than Lac I in
the presence of promazine, while in the presence of HOBT Lac I was the fastest and
the most effective isoenzyme (Moldes and Sanroman, 2006).
Baysolex VP SP 20020 an enzymatic cocktail containing laccase, catechol
oxidase, bilirubin oxidase and peroxidase was used for the decolorization of process
wastewater, containing mainly three reactive azo dyes, from a textile dyeing and
printing company. The corresponding co-factors such as H2O2 and a redox mediator
were also added to the enzymatic cocktail. The decolorization was greatest for
Reactive Black 5, followed by Reactive Red 158, whereas Reactive Yellow 27 was
the least decolorized dyes (Soares et al., 2006).
Yu et al. (2006) have demonstrated in vitro decolorization of an industrial azo
dye, Reactive Brilliant Red K-2BP, by crude LiP and MnP obtained from
Phanerochaete chrysosporium. Decolorization by LiP was enhanced to the greatest
extent (89%) with higher addition of H2O2 and VA. It was suggested that the
optimization of H2O2 and VA was responsible for a high efficiency in continuous dye
degradation by crude enzyme.
Response surface methodology was applied for the decolorization of an azo
dye. Reactive Black 5 using purified laccase from a WRF Pleurotus sajor-caju. It was
observed that the presence of HOBT was essential for the decolorization of Reactive
Black 5 by purified laccase from Pleurotus sajor-caju. The optimum concentrations
of dye, enzyme, HOBT and time were found to be 62.5 mg L"', 2.5 U mL"\ 1.5 mM
32
and 36 h, respectively, for 84.4% decolorization of Reactive Black 5 (Murugesan et
al., 2007).
Ischnoderma resinosum produced extracellular ligninolytic enzymes, laccase
and MnP. Purified MnP performed decolorization of textile dyes; Reactive Black 5,
Reactive Blue 19, Reactive Red 22 and Reactive Yellow 15. Laccase was inactive
with Reactive Black 5 and Reactive Red 22, while all the other dyes were decolorized
by laccase after addition of the redox mediators VLA and HOBT (Kokol et al., 2007).
A comparative study for the decolorization and removal of two textile carpet
industrial effluents by soluble and immobilized turnip and tomato peroxidases was
studied. However, the decolorization of effluents was enhanced in the presence of 2.0
mM HOBT. Industrial effluents; textile carpet effluent red and textile carpet effluent
blue were decolorized 75% and 80% by soluble TP (0.423 U mL''), respectively.
However, the decolorization of these effluents was 69% and 59% by soluble TMP
(0.705 U mL"'), respectively. Both effluents were maximally decolorized at pH 5.0 by
soluble and immobilized TP whereas the maximum decolorization was at pH 6.0 by
soluble and immobilized TMP. These effluents were decolorized maximally at 40 °C
by soluble and immobilized peroxidases. Effluent treated by immobilized TP
exhibited significant loss of TOC (Husain and Kulshrestha, 2008).
Matto and Husain (2008) demonstrated the effect of various redox mediators
on the salt fractionated tomato (Lycopersicon esculentum) proteins decolorization of
direct dyes, used in textile industry. The rate and extent of decolorization of dyes was
significantly enhanced by the presence of different types of redox mediators. Six
compounds showed their potential in enhancing the decolorization of direct dyes. The
performance was evaluated at different concentrations of mediator and enzyme. The
decolorization of all tested direct dyes was maximum in the presence of 1.0 mM
HOBT at pH 6.0 and 40 °C.
33
Table 3: Applications of redox mediators in dye removal
Enzyme
LiP
MnP
Laccase
SBP
VP
BGP
TP
TMP
Redox Mediator
VA
VLA, HOBT VLA, phenothiazine-10-propionic
HOBT
HOBT, promazine, p-HBA and NNDS Acetosyringone, syringaldehyde and /7-coumaric acid VLA
ABTS HOBT VA
VA, acetosyringone, TEMPO
HOBT
HOBT
HOBT
HOBT, phenol, VN, VLA, VA, syringaldehyde
Dyes
Six industrial azo and phthalocyanine dyes Reactive Brilliant Red K-2BP Reactive dyes RBBR
Phenol Red
Sella Solid Red and Luganil Green Indigo Cannine and Phenol Red Delignification of paper pulp
Acid Red 97 and Acid Green 26 Alizarin Red Reactive Black 5 Direct Yellow and Basazol 46L Direct Yellow 58, Disperse Red 60, Vat Blue 7 and Reactive Yellow 2 21 different dyes Acid dyes
Textile carpet effluent
Textile carpet effluent
DR 23 and DB 80
Fold/% Enhance ment 8-100%
56%
80-90% 90%, 30%
60%
2 fold and 50% 10-15 fold 15%
3 fold
66% 84% 49-87%
390-2500%
7-100% 24-100%
3-7%
15-25%
30-90%
References
Heinflingetal., 1998b
Yu et al., 2006
Kokol et al., 2007 Soaresetal., 2001a
Moldes et al., 2004
Couto et al., 2005
Moldes and Sanroman, 2006 Camarero et al., 2007
Couto and Sanroman, 2007 Lu et al., 2007 Murugesan et al., 2007 Knutson et al., 2005
Tinoco et al., 2007
Akhtar et al., 2005b Kulshrestha and Husain, 2007
Husain and Kulshrestha, 2008 Husain and Kulshrestha, 2008 Matto and Husain, 2008
34
OBJECTIVE OF THE PRESENT WORK
Peroxidases are an important group of enzymes, which have assumed
importance in recent years in view of their various applications. Our objective
revolves around studying the important characteristic of plant peroxidases
from turnip {Brassica rapa) and bitter gourd {Momordica charantia) with
regard to their immobilization and treatment of polluted water.
In the first part (Chapter II) of the study, we have investigated the
entrapment of partially purified TP and Con A-TP complex in calcium
alginate-pectin beads. Entrapped preparations have been compared for
their stability with its soluble counter part against various physical and
chemical denaturants.
In the second part (Chapter III) of the work, an effort has been made to
immobilize BGP inside and on the surface of calcium alginate-starch beads.
Calciimi alginate-starch beads have been layered with Con A from the jack
bean extract. Con A-BGP complex has been entrapped within the polymeric
matrices of calcium alginate-starch beads. These immobilized preparations
have been compared for their stability against several parameters which can
affect the stability of enzyme during its use in wastewater treatment. The
reusability of both types of immobilized BGP preparations was also evaluated.
In view of the recalcitrant nature of several dyes an effort has been
made to use some redox mediators for the enhancement of TP
catalyzed decolorization of few direct dyes (Chapter IV). TP catalyzed
and redox mediators stimulated decolorization of direct dyes was
optimized under varying experimental conditions such pH,
temperature, concentration of redox mediators, TP etc.
In this section (Chapter V) of the study an attempt has been made to
exploit the use of surface immobilized BGP preparation for the
decolorization/degradation of textile effluent in the presence of redox
mediator, HOBT. A comparative stud> between soluble and
immobilized BGP for their ability to decolorize textile effluent under
various experimental conditions has been standardized.
35
• In the last part of the study (Chapter VI) focus has been laid on the use of an
inexpensive bioaffinity based support; Con A-WS for the immobilization of
TP. Con A-WS bound TP preparation was used for the decolorization of direct
dye and a mixture of direct dyes in batch processes and continuous reactors.
Dye removal was monitored by using UV-visible spectrophotometer and TOC
analysis.
36
TMbmpmmt qf cmtcmuwalm Ji-idase compiex into
2.1. INTRODUCTION
Immobilized enzymes have received a lot of attention for their use as
biocatalysts in the removal/biotransformation of toxic aromatic organic compounds
present in wastewater (Tatsumi et al., 1996; Akhtar et al., 2005b). However, the high
cost and low yield of immobilized enzymes are two important limitations in their
application for the treatment of industrial effluents (Tischer and Kasche, 1999; Duran
et al., 2002). Thus immobilized enzymes must be obtained by a cost effective and
technologically convenient method.
Among the different techniques used for immobilization, entrapment in natural
biopolymers is favoured due to non-toxicity of the matrix, variation in bead size of the
gel and high yield of immobilization (Smidsr^d and Skjdk-BraeK, 1990; Kierstan and
Bucke, 2000). Calcium alginate mediated entrapment has attracted much attention in
the detoxification of phenolic compounds present in industrial effluents (Davis and
Bums, 1990). However, recently some workers have reported that pectin beads are
significantly more stable than alginate beads (Kurillova et al., 2000). One of the
limitations in case of entrapped enzyme is leaching of enzymes from polymeric
matrices. In order to prevent the leaching of enzymes from physically entrapped gels,
a number of attempts have been made to increase molecular dimension of the
enzymes prior to their entrapment. The leakage could be avoided by entrapping
crosslinked or pre-immobilized enzyme into calcium alginate gel (Husain et al., 1985;
Musthapa et al., 2004).
In this work, an effort has been made to present an inexpensive, simple and
high yield procedure for immobilization of glycosylated turnip peroxidase. Insoluble
Con A-TP complex was entrapped in hybrid beads of calcium alginate-pectin. A
comparative stability study of soluble TP (S-TP), Con A-TP complex, entrapped S-TP
and entrapped Con A-TP complex has been performed against various forms of
denaturants; pH, heat, urea, detergents and water-miscible organic solvents.
37
2.2. MATERIALS AND METHODS
2.2.1. Materials
Sodium alginate was the product of Koch-Light, England. Bovine serum
albumin was obtained from Sigma Chemical Co. (St. Louis, MO) USA. Jack bean
meal was procured from DIFCO, Detroit, USA. o-dianisidine HCl was purchased
from the Centre for Biochemical Technology, CSIR, India. Dioxane, dimethyl
formamide (DMF), sodium dodecyl sulphate (SDS) and pectin were obtained from
SRL Chemicals Pvt. Ltd. Mumbai, India. Surf Excel and turnip were purchased from
the local market. Other chemicals and reagents employed were of analytical grade and
were used without any further purification.
2.2.2. Ammonium sulphate fractionation of turnip proteins
Turnip (250 g) was homogenized in 250 mL of 100 mM sodium acetate
buffer, pH 5.6. Homogenate was filtered through four layers of cheese-cloth. The
filtrate was then centrifuged at a speed of 10,000 g for 20 min at 4 °C on a Remi C-24
Cooling Centrifuge. The obtained clear solution was subjected to salt fractionation by
adding 10-90% (w/v) ammonium sulphate. The solution was continuously stirred
overnight at 4 °C. Precipitate was collected by centrifligation at 10,000 g on a Remi
C-24 Cooling Centrifuge for 20 min at 4 °C. The obtained precipitate was redissolved
in 100 mM sodium acetate buffer, pH 5.6 and dialyzed against the assay buffer.
2.2.3. Preparation of jack bean extract
Jack bean extract (10%, w/v) was prepared by adding 5.0 g of crude jack bean
meal to 50.0 mL of 100 mM sodium phosphate buffer, pH 6.2. The mixture was kept
overnight on a magnetic stirrer at room temperature. The clear jack bean extract
obtained after centrifugation at 3000 g for 20 min was used for precipitation of
peroxidases from ammonium sulphate fractionated turnip proteins.
38
2.2.4. Preparation of insoluble Con A-TP complex
To a series of tubes, TP (200 U) was mixed with increasing concentrations of
10% jack bean extract (Jan et al., 2006). The final volume was adjusted to 2.0 mL
with 100 mM sodium phosphate buffer, pH 6.2. The mixtures were incubated at 37 °C
for 12 h. Each precipitate was collected after centrifligation at 3000 g for 20 min at
room temperature and washed thrice with the same buffer. Finally the precipitates
were suspended in 2.0 mL of assay buffer. Each precipitate was analyzed for enzyme
activity. The precipitate exhibiting maximum activity was used for further studies.
2.2.5. Entrapment of soluble and Con A-TP complex in calcium alginate-pectin
beads
Soluble peroxidase (250 U) and Con A-TP complex (260 U) were mixed
independently with sodium alginate (2.5%) and pectin (2.5%) in 10.0 mL of sodium
acetate buffer, pH 5.6. The resulting mixture was slowly extruded as droplets through
a 5.0 mL syringe with attached needle No. 20 into 200 mM calcium chloride solution.
The formation of calcium alginate-pectin beads was instantaneous and the solution
was further gently stirred for 2 h (Husain et al., 1985; Kurillova et al., 2000). The
beads were then washed and stored in 100 mM sodium acetate buffer, pH 5.6 at 4 "C
for further use.
2.2.6. Measurement of peroxidase activity
Peroxidase activity was estimated from the change in the optical density (A460
nm) in 100 mM sodium acetate buffer, pH 5.6 at 37 °C by measuring the initial rate of
oxidation of o-dianisidine-HCl (6.0 mM) by hydrogen peroxide (18 mM) using the
two substrates in saturating concentrations (Akhtar et al., 2005b).
The assay mixtures with immobilized TP preparations were continuously
stirred for the entire duration of the assay. The assay was highly reproducible with
immobilized enzyme.
One unit (1.0 U) of peroxidase activity is defined as the amount of enzyme
protein that catalyzes the oxidation of 1.0 pmol of o-dianisidine HCl per min at 37 °C
39
into colored product (8m at 460 nm = 30,000 M"'cm-').
2.2.7. Protein estimation
The protein concentration was determined by the method of Lowry et al.
(1951). A suitable aliquot of the protein sample was diluted to 1.0 mL with distilled
water. To this, 5.0 mL of freshly prepared alkaline copper reagent was added. The
alkaline copper reagent was prepared by mixing copper sulphate (1%, w/v), sodium
potassium tartarate (2%, w/v) and sodium carbonate (2%, w/v) in 0.1 N NaOH in the
ratio of 1:1:100. After incubation for 10 min at room temperature, 0.5 mL of 1.0 N
Folin's reagent was added. The contents were mixed and color intensity was read after
30 min against the reagent blank at 660 nm. Bovine serum albumin was used as
standard protein.
2.2.8. Effect of pH on soluble and immobilized TP
The activity of S-TP, Con A-TP complex and calcium alginate-pectin
entrapped TP preparations (1.25 U) was measured in the buffers of various pH. The
molarity of each buffer was 100 mM. The remaining percent activity was calculated
by taking activity at optimum-pH as control (100%).
2.2.9. Effect of temperature on soluble and immobilized TP
The enzyme activity of S-TP, Con A-TP complex and alginate-pectin
entrapped TP preparations was measured at various temperatures under standard assay
conditions. The activity obtained at 30 °C was taken as control (100%) for the
calculation of percent activity.
S-TP, Con A-TP complex and calcium alginate-pectin entrapped TP
preparations (1.25 U) were incubated at 60 °C in 100 mM sodium acetate buffer, pH
5.6. Aliquots of each preparation were removed at indicated time intervals and chilled
quickly in crushed ice for 5 min. After chilling, all the tubes were brought to room
temperature and activity was measured. The activity obtained without incubation at 60
°C was taken as control (100%) for the calculation of remaining percent activity.
40
2.2.10. Effect of urea on soluble and immobilized TP
Soluble and immobilized TP preparations (1.25 U) were incubated with 4.0 M
urea dissolved in 100 mM sodium acetate buffer, pH 5.6 at 30 °C for 1 h. Aliquots
were removed at various time intervals and activity was determined. The activity of
urea untreated TP was considered as control (100%) for the calculation of remaining
percent activity.
2.2.11. Effect of detergents on soluble and immobilized TP
SDS and Surf Excel (0.1-1.0%, w/v) were used to investigate the effect of
detergents on the activity of TP. Soluble and immobilized TP preparations (1.25 U)
were incubated with increasing concentrations of detergents in 100 mM sodium
acetate buffer, pH 5.6 at 30 °C for 1 h. Peroxidase activity was determined at all the
indicated detergent concentrations. The activity obtained without exposure to
detergent was taken as control (100%) for the calculation of remaining percent
activity.
2.2.12. Effect of water-miscible organic solvents on soluble and immobilized TP
Soluble and immobilized TP preparations (1.25 U) were incubated wi h 10-
60% (v/v) of water-miscible organic solvents; DMF/dioxane prepared in 100 mM
sodium acetate buffer, pH 5.6 at 30 °C for 1 h. Peroxidase activity was determined at
all the indicated organic solvent concentrations. The activity of soluble and
immobilized TP preparations in assay buffer without exposure to water-miscible
organic solvent was taken as control (100%) for the calculation of remaining percent
activity.
2.3. RESULTS
2.3.1. Entrapment of TP into calcium alginate-pectin gel
Fig. 2 demonstrates the precipitation of glycosylated turnip proteins by
41
100 n
!
0.4 0.6
Jack bean extract (mL) 1,0
Fig. 2: Insolubilzation of TP using jack bean extract
S-TP (200 U) was incubated witli increasing concentrations of 10% jaclc bean
extract (0.1 to 1.0 mL) prepared in 100 mM sodium phosphate buffer, pH 6.2
in a total volume of 2.0 mL for 12 h at 37 °C. Precipitate of each preparation
was collected, washed thrice and activity was determined as described in the
text.
42
increasing concentration of jack bean extract. The maximum precipitation of
peroxidase activity was 70% which was obtained by adding 0.7 mL of 10% jacic bean
extract. This observation suggested that the major peroxidases from turnip roots are
glycosylated. Further, S-TP and Con A-TP complex were entrapped into calcium
alginate-pectin beads and entrapped preparations retained 63% and 52% of the
original activity, respectively (Table 4).
2.3.2. Effect of pH
Effect of pH on the activity of soluble and immobilized TP is shown in Fig. 3.
The pH-activity profile of the immobilized enzyme exhibited same pH-optima as that
of S-TP at pH 5.6. The immobilized enzyme preparations showed significant
broadening in pH-activity profiles. However, entrapped Con A-TP exhibited the
maximum stability at the suggested pH.
2.3.3. Thermal stability
Soluble and immobilized TP showed maximum activity at 30 °C (Fig. 4). Con
A-TP, entrapped S-TP and entrapped Con A-TP preparations retained greater
fractions of catalytic activity at higher temperatures as compared to soluble TP.
Soluble enzyme retained marginal enzyme activity between 60 and 80 °C, whereas the
alginate-pectin entrapped Con A-TP complex retained more than 40% of its original
activity at 60 °C.
S-TP, Con A-TP complex, calcium alginate-pectin-entrapped; S-TP and Con
A-TP complex were incubated at 60 °C for various time intervals. Incubation of
soluble enzyme at this temperature for 2 h resulted in a loss of nearly 92% activity,
whereas the entrapped S-TP retained 22% of the initial activity (Fig. 5). Moreover, the
entrapped Con A-TP complex exhibited markedly very high stability against thermal
denaturation and it retained more than 40% of the initial activity.
2.3.4. Effect of urea
The urea induced denaturation of TP preparations is shown in Fig. 6. S-TP lost
58% of its initial activity after 2 h incubation in 4.0 M urea while Con A-TP complex,
43
Table 4: Immobilization of TP by using jack bean extract and calcium alginate-
pectin matrix
Metliods of
immobilization
Con A-TP complex
Calcium alginate-pectin-
entrapped S-TP
Calcium alginate-pectin-
entrapped Con A-TP
Activity
expressed
(%)
70
63
52
Each value represents the mean for three-independent experiments performed in
duplicates, with average standard deviation, <5%.
44
100
80
2:-'> 5 60
o CO
O) c 'c ro E 0)
§ 40
20 -
6
PH
10
Fig. 3: pH-activity profiles of soluble and immobilized TP
The enzyme activity of soluble and immobilized TP was measured in the
buffers of various pH. The molarity of each buffer was 100 mM. The activity
at pH 5.6 for all the preparations was taken as control (100%) for the
calculation of percent activity. Symbols indicate, S-TP (•), Con A-TP
complex (o), calcium alginate-pectin entrapped S-TP (T) and calcium
alginate-pectin entrapped Con A-TP complex (V).
45
100
80 -
S 60 5" '5 '•? o CO
c g 40
2 0 -
10 20 30 40 50
Temperature (°C)
60 70 80
Fig. 4: Temperature-activity profiles of soluble and immobilized TP
The enzyme activity of soluble and immobilized TP was measured at various
temperatures under other standard assay conditions as described in text. The
activity obtained at 30 "C was taken as control (100%) for the calculation of
percent activity. Symbols indicate, S-TP (•), Con A-TP complex (o), calcium
alginate-pectin entrapped S-TP (T) and calcium alginate-pectin entrapped
Con A-TP complex (A).
46
20 40 60
Time (min)
80
Fig. 5: Thermal denaturation of soluble and immobilized TP
S-TP, Con A-TP complex, entrapped S-TP and entrapped Con A-TP complex
were incubated at 60 "C for various times in 100 mM sodium acetate buffer,
pH 5.6. Aliquots of each preparation were removed at different time intervals
and activity was measured according to the procedure described in the text.
Enzyme preparation without incubation at 60 "C was taken as control (100%)
for the calculation of remaining percent activity. Symbols indicate, S-TP (•),
Con A-TP complex (o), calcium alginate-pectin entrapped S-TP (T) and
calcium alginate-pectin entrapped Con A-TP complex (A).
47
100
TO
D>
c 'c 'S E 0) 0^
20 40 60
Time (min)
120
Fig. 6: Effect of urea on soluble and immobilized TP
Soluble and immobilized TP were incubated in 4.0 mM urea dissolved in 100
mM sodium acetate buffer, pH 5.6. Aliquots were removed at various time
intervals and activity was determined. The activity of urea untreated samples
was considered as control (100%) for the calculation of remaining percent
activity after urea exposure. Symbols indicate, S-TP (•), Con A-TP complex
(o), calcium alginate-pectin entrapped S-TP (T) and calcium alginate-pectin
entrapped Con A-TP complex (A).
48
calcium alginate-pectin entrapped S-TP and calcium alginate-pectin entrapped Con A-
TP complex retained 56%, 60% and 65% activity, respectively.
2.3.5. Effect of detergents
Table 5 demonstrates the effect of increasing concentrations of SDS (0.1-
1.0%, w/v) on the activity of TP. Pre-incubation of S-TP, entrapped S-TP, Con A-TP
complex and entrapped Con A-TP complex with 1.0% (w/v) SDS for 1 h resulted in
the loss of 66%, 60%, 56%) and 53% of the original enzyme activity, respectively. The
exposure of entrapped; S-TP and Con A-TP complex preparations to 0.1% SDS
showed an enhancement in enzyme activity and these preparations exhibited 153%
and 190% of original activity, respectively.
Pre-incubation of TP preparations with Surf Excel is also shown in Table 5.
Both soluble and immobilized enzyme preparations were activated by low
concentrations of Surf Excel. S-TP was more sensitive to Surf Excel exposure and lost
nearly 85%) of its enzyme activity after 1 h of incubation with 1.0% (w/v) detergent.
However, entrapped S-TP, Con A-TP complex and entrapped Con A-TP complex
preparations were more resistant to inactivation induced by 1.0% Surf Excel and
retained 24%, 29%) and 38%) of the initial activity, respectively.
2.3.6. Effect of organic solvents
The exposure of soluble enzyme to varying concentrations of DMF (10-60%,
v/v) resulted in a loss of greater fraction of catalytic activity while the immobilized
enzyme was quite resistant to inactivation induced by DMF. Entrapped Con A-TP
preparation exposed to 30% (v/v) DMF for 1 h retained half of the enzyme activity,
while soluble enzyme lost nearly 70% of its original activity under similar treatment
(Table 6).
The incubation of soluble and immobilized TP with increasing concentrations
of dioxane resulted in a continuous loss of enzyme activity. However, the
immobilized enzyme was more resistant to inactivation mediated by dioxane. The
treatment of S-TP with 60%) (v/v) dioxane for 1 h resulted in a loss of 86%) of the
initial activity while the Con A-TP complex and entrapped Con A-TP complex
retained 27%) and 33%) activity, respectively (Table 6).
49
Table 5: Effect of detergents on soluble and immobilized TP
Detergent
(%, w/v)
0.1
0.2
0.4
0.6
0.8
1.0
Remaining activity (%)
SDS
S-TP
80
65
50
41
38
34
Entrapped
S-TP
153
70
55
50
45
40
Con
A-TP
170
78
67
56
50
44
Entrapped
Con A-TP
190
94
74
62
58
47
Surf Excel
S-TP
210
50
40
30
26
15
Entrapped
S-TP
230
70
52
42
30
24
Con
A-TP
250
90
70
57
44
29
Entrapped
Con A-TP
260
160
80
70
60
38
Soluble and immobilized TP (1.25 U) were incubated with SDS/Surf Excel (0.1-
1.0%, w/v) prepared in 100 mM sodium acetate buffer, pH 5.6 at 30 °C for 1 h.
Peroxidase activity was assayed at all the indicated detergents concentrations. The
activity of soluble and immobilized TP in assay buffer without any detergent was
taken as control (100%) for the calculation of remaining percent activity. Each value
represents the mean for three independent experiments performed in duplicates, with
average standard deviation, <5%.
50
Table 6: Effect of organic solvents on soluble and immobilized TP
Organic
Solvent
(%, v/v)
10
20
30
40
50
60
S-TP
70
44
31
18
13
5
DMF
Entrapped
S-TP
82
62
38
21
16
12
Con
A-TP
90
77
45
28
22
18
Remaining activity (%)
Entrapped
Con A-TP
93
84
50
34
28
22
Dioxane
S-TP
54
48
39
23
17
14
Entrapped
S-TP
77
67
42
26
19
17
Con
A-TP
82
77
63
54
43
27
Entrapped
Con A-TP
93
86
69
63
54
33
Soluble and immobilized TP (1.25 U) were incubated with increasing concentrations
of DMF/dioxane (10-60%, v/v) in 100 mM sodium acetate buffer, pH 5.6 at 30 "C for
1 h. Peroxidase activity was assayed at all the indicated organic solvent
concentrations. The activity of soluble and immobilized TP in assay buffer without
any organic solvent was taken as control (100%) for the calculation of remaining
percent activity. Each value represents the mean for three-independent experiments
performed in duplicates, with average standard deviation, <5%.
51
2.4. DISCUSSION
Immobilization by means of entrapment is a very rapid and simple technique.
It has earlier been described that soluble enzyme could be leached out of gel beads on
long standing or use (Davis and Bums, 1990; Blandino et al., 2000; Veronese et al.,
2001; Przybyt and Bialkowska, 2002; Musthapa et al., 2004). In order to prevent the
leaching of enzymes out of porous gel beads, insoluble complex of TP was prepared
by using Con A from jack bean extract and subsequently entrapped into calcium
alginate-pectin gel. It was observed that a very low quantity of jack bean extract was
required to form the insoluble complex of TP (Fig. 2).
Several earlier reports indicated that crosslinked or pre-immobilized enzymes
could retain inside the polymeric matrices for longer duration than soluble enzymes
(Musthapa et al., 2004; Wilson et al., 2004; Betancor et al., 2005). The entrapped Con
A-peroxidase complex was quite active and retained 52% of the original activity
(Table 4). This evidence is in agreement with an earlier report which showed that
glutaraldehyde crosslinked peroxidase entrapped into calcium alginate gel retained a
moderate enzyme activity (Musthapa et al., 2004).
Enhancement in stability appeared to be another attractive feature of Con A-
TP and entrapped Con A-TP complex. The marked stability exhibited by these
immobilized preparations (Fig. 3-6, Table 5-6) goes in agreement with an earlier
report on the stabilization of glycoenzymes as a result of binding to Con A (Jan et al.,
2006).
Soluble and immobilized TP showed maximum activity at pH 5.6 and 30 °C
(Fig. 3, 4). However, it has been demonstrated that entrapment of enzymes in gel
beads provides a microenvironment for the enzyme, which plays an important role in
the state of protonation of the protein molecules (Musthapa et al., 2004; Akthar et al.,
2005c). Thus the formation of Con A-TP complex further confers additional
resistance to the enzyme against extreme conditions of pH.
The calcium alginate-pectin entrapped Con A-TP complex retained its
structure and remarkably high activity at elevated temperatures (Fig. 4, 5).
Improvement in the thermal stability of calcium alginate*pectin entrapped Con A
complex may come from multipoint complexing of peroxidase with Con A. However,
some earlier workers have described that the complexing of enzymes with lectins
52
enhanced its thermal stability. This enhancement in thermal stability was due to
several interactions between enzyme and Con A (Husain et al., 1985; Mislovicova et
al., 2000; Jan et al., 2006). Therefore, such an enzyme preparation could be highly
useful at relatively high temperatures.
Urea at a concentration of 4.0 M is a strong denaturant of some proteins and it
irreversibly denatures the soluble enzyme. The entrapped Con A-TP was found to be
more stable as compared to other preparations (Fig. 6). It is therefore obvious that the
free enzyme in beads was completely accessible to urea as it is already known that
urea can significantly affect the ionic interaction of entrapped soluble enzyme (Xu et
al., 1998). Although the action mechanism of urea on the protein structures is not yet
completely understood, several earlier studies have proposed that protein is unfolded
by the direct interaction of urea molecules with the peptide backbone via hydrogen
bonding/hydrophobic interaction, which contributes to the maintenance of protein
conformation (Makhatadze and Privalor, 1992). However, the complexing of glyco-
enzymes with Con A has been shown to result in an enhancement of their resistance
to denaturation (Akhtar et al., 2005a; Kulshrestha and Husain, 2006a). These
observations clearly indicate that the complexing of TP with Con A protects it from
urea-induced inactivation.
Wastewater from various elimination sites contains several types of
denaturants, including detergents, which can strongly denature the enzymes used for
the treatment of polluted water. In order to use such enzymes for the removal of
aromatic pollutants from wastewater it becomes necessary to monitor the stability of
enzymes in the presence of denaturants. Here, two different detergents have been
selected for comparative stability of soluble and immobilized TP (Table 5). The
results indicated that the presence of lower concentrations of SDS and Surf Excel was
not harmful to the enzyme structure and thus they can work more efficiently on the
industrial effluents containing lower concentrations of compounds like soap and
detergents (Kulshrestha and Husain, 2006b).
Industrial effluents and municipal wastewater sometimes also contain organic
solvents together with other aromatic pollutants. Therefore, it is of utmost importance
to investigate the role of some water-miscible organic solvents on the activity of
immobilized enzymes. Immobilized TP preparations showed higher resistance to
inactivation in the presence of organic solvents; DMF and dioxane (Table 6). In an
earlier study it has been reported that BGP immobilized on Con A and other supports
53
was significantly more stable against the denaturation induced by water-miscible
organic solvents (Akthar et al., 2005c; Kulshrestha and Husain, 2006b).
Calcium alginate-pectin entrapped Con A-TP exhibited significantly higher
stability against various physical and chemical denaturants as compared to S-TP and
Con A-TP complex. Thus, immobilized TP preparations could be exploited for
developing bioreactors for the treatment of phenolic and other aromatic pollutants
present in industrial wastewaters.
54
CMaptm'III Cdfcium afginate-starch fiyBrid
support for Sotfi surface
immoSifization and entrapment
of Sitter gourdperooddase
3.1. INTRODUCTION
Among the various techniques employed for the ifnmobilizationjrf'enzyraes,
entrapment may be a good choice owing to an inert aqueous environment Within the
matrix and causing relatively little damage to the structure of the native enzyme
(Musthapa et al., 2004). Alginate appears to be one of the most suitable polymers for
the entrapment of enzymes, cell organelles and even cells because of the following
advantages: hydrophilic nature, presence of carboxylic groups, natural origin,
mechanical stability and stability over extreme experimental conditions (Arica et al.,
2001; Zhu et al., 2005; Lu et al., 2007).
However, it has been reported that because of the porous nature of sodium
alginate, most of the entrapped material is released from the gel beads during its
application. In order to optimize the encapsulation efficiency and controlled release of
enzyme from the gel matrix, entrapment of crosslinked or pre-immobilized enzymes
has been done (Lee et al., 1993; Musthapa et al., 2004). However, the major limitation
of physical entrapment is that large molecular size substrates/products cannot easily
be diffuse in and out of the gel (Liang et al., 2000).
In this work an effort has been made to prepare a hybrid gel of alginate and
starch which could be exploited for the entrapment of enzymes as well as bioaflfinity
attachment of glycosylated enzymes on the surface of gel beads. These calcium
alginate-starch beads were layered with Con A. Con A layered calcium alginate-starch
beads were used for the surface immobilization of glycosylated peroxidases from
bitter gourd. Con A-BGP complex was also entrapped inside the calcium alginate-
starch beads. A comparative stability study of entrapped and surface immobilized
peroxidase has been carried out against various physical and chemical denaturants.
Immobilized BGP preparations have also been studied for their reusability.
3.2. MATERIALS AND METHODS
3.2.1. Materials
Jack bean meal was procured from DIFCO, Detroit, USA. o-dianisidine HCl
was obtained from the Centre for Biochemical Technology, CSIR, India. Cadmium
55
chloride (CdCb), dioxane, «-propanol, mercuric chloride (HgCb), starch and Tween
20 were obtained from the SRL Chemicals Pvt. Ltd. Mumbai, India. Bovine serum
albumin, glutaraldehyde and ethanolamine were procured from Sigma Chemical Co.
(St. Louis, MO) USA. Ethylenediamine tetracetic acid (EDTA) and sodium azide
were purchased from the Merck Chemicals Pvt. Ltd. Worli, Mumbai, India. Bitter
gourd was purchased from the local vegetable market. Other chemicals and reagents
employed were of analytical grade and were used without any further purification.
3.2.2. Ammonium sulphate fractionation of bitter gourd proteins
Bitter gourd (100 g) was homogenized in 200 mL of 100 mM sodium acetate
buffer, pH 5.0. Homogenate was filtered through four layers of cheese-cloth. The
filtrate was then centrifiiged at 10,000 g on a Remi R-24 Cooling Centrifuge for 20
min at 4 °C. The clear supernatant was subjected to salt fractionation by adding 20-
80% (w/v) ammonium sulphate. The solution was stirred overnight at 4 °C and the
obtained precipitate was collected by centrifugation at 10,000 g on a Remi R-24
Cooling Centrifiige for 20 min at 4 °C. The collected precipitate was redissolved in
100 mM sodium acetate buffer, pH 5.0 and dialyzed against the assay buffer (Akhtar
et al., 2005a).
3.2 J . Preparation of Con A-BGP complex
Jack bean extract (10%, w/v) was prepared according to the method described
in Chapter II, Section 2.2.3. The collected supernatant was used as source of Con A.
BGP (1200 U) was incubated with increasing concentrations of jack bean
extract (0.1-1.0 mL) containing Con A and the final volume was adjusted to 4.0 mL
with 100 mM sodium phosphate buffer, pH 6.2. The mixtures were incubated at 37 °C
for 12 h. The insoluble complex was collected by centrifugation at 3000 g for 15 min
at room temperature and the precipitates were washed thrice with sodium phosphate
buffer, pH 6.2 to remove unbound protein. Finally each precipitate was suspended in
assay buffer and peroxidase activity was determined.
The Con A-BGP complex (840 U) was crosslinked prior to entrapment in
calcium alginate-starch beads with 0.5% glutaraldehyde for 2 h at 4 °C with constant
shaking. Ethanolamine was added to a final concentration of 0.1% (v/v) to stop
56
crosslinking. The solution was allowed to stand for 90 min at room temperature and
the complex was collected by centrifugation at 3000 g for 15 min at room
temperature. The precipitate was washed thrice with assay buffer and then suspended
in the same buffer (Jan et al., 2006).
3.2.4. Entrapment of crosslinked Con A-BGP into calcium alginate-starch beads
The crosslinked Con A-BGP complex (792 U) was mixed with sodium
alginate (2.5%, w/v) and starch (2.5%, w/v) prepared in 10.0 mL of assay buffer. The
resulting mixture was slowly extruded as droplets through a 5.0 mL syringe with
attached needle No. 20 into 200 mM calcium chloride solution and further gently
stirred for 2 h. The obtained calcium alginate-starch entrapped crosslinked Con A-
BGP (E-BGP) was washed with 100 mM sodium acetate buffer, pH 5.0 and stored in
the assay buffer at 4 °C for its further use.
3.2.5. Immobilization of BGP on the surface of Con A layered calcium alginate-
starch beads
Sodium alginate (2.5%, w/v) and starch (2.5%, w/v) beads were prepared
without enzyme according to the procedure described in the above section and these
beads were incubated in 10.0 mL jack bean extract, a source of Con A for 12 h at
room temperature with slow stirring. After incubation period. Con A bound calcium
alginate-starch beads were collected and washed with assay buffer. Con A layered
calcium alginate-starch beads were then incubated with BGP (1200 U) overnight at
room temperature with slow stirring on a magnetic stirrer. Unbound enzyme was
removed by repeated washing with 100 mM sodium acetate buffer, pH 5.0. BGP
immobilized on the surface of Con A layered calcium alginate-starch beads was
crosslinked by 0.5% glutaraldehyde for 2 h at 4 °C (Jan et al., 2006). Surface
immobilized and glutaraldehyde crosslinked BGP (SI-BGP) was stored at 4 °C for its
further use.
3.2.6. Measurement of peroxidase activity
Peroxidase activity was estimated in 100 mM sodium acetate buffer, pH 5.0 at
57
37 °C (Chapter II, Section 2.2.6).
3.2.7. Determination of protein concentration
The concentration of protein was determined as described in Chapter II,
Section 2.2.7. Bovine serum albumin was used as a standard.
3.2.8. Effect of pH
Appropriate and equal amounts of S-BGP, SI-BGP and E-BGP were taken to
determine the activity of peroxidase in the buffers of different pH. The buffers used
were glycine-HCl (pH 2.0 and 3.0), sodium acetate (pH 4.0 and 5.0) and Tris HCl (pH
6.0-10.0). The activity at pH-optimum was considered as control (100%) for the
calculation of percent activity at other pH.
3.2.9. Effect of temperature
The activity of soluble and immobilized BGP (1.3 U) was determined at
various temperatures (30-80 °C) in 100 mM sodium acetate buffer, pH 5.0. The
activity at temperature-optimum was considered as control (100%) for the calculation
of percent activity at other temperatures.
In another set of experiment, all three BGP preparations were incubated at 60
°C for varying time intervals in 100 mM sodium acetate buffer, pH 5.0. After each
incubation period the enzyme was quickly chilled in crushed ice for 5 min. The
enzyme was brought to room temperature and the peroxidase activity was measured.
The activity without incubation at 60 °C was taken as control (100%) for the
calculation of remaining percent activity.
3.2.10. Effect of urea
Soluble and immobilized BGP (1.3 U) were incubated with 4.0 M urea for
varying time intervals in 100 mM sodium acetate buffer, pH 5.0 at 37 °C. Peroxidase
activity was determined at the indicated time intervals. The activity of enzyme
58
without incubation witli urea was taken as control (100%) for the calculation of
remaining percent activity.
3.2.11. Effect of water-miscible organic solvents
Soluble and immobilized BGP (1.3 U) were incubated independently with
varying concentrations of water-miscible organic solvents; dioxane and «-propanol
(10-60%, v/v) in 100 mM sodium acetate buffer, pH 5.0 at 37 °C for 1 h. Activity of
enzyme without organic solvent was taken as control (100%) for the calculation of
remaining percent activity.
3.2.12. Effect of detergents
Soluble and immobilized BGP (1.3 U) were incubated with Tween 20 (0.5-
5.0%, v/v) in 100 mM sodium acetate buffer, pH 5.0 at 37 °C for 1 h. The activity of
enzyme without Tween 20 was taken as control (100%) for the calculation of
remaining percent activity.
3.2.13. Effect of sodium azide and EDTA
The inhibitory effect of sodium azide/EDTA (0.01-0.1 mM) was examined on
BGP preparations (1.3 U). Soluble and immobilized BGP were pre-incubated with
inhibitors in 100 mM sodium acetate buffer, pH 5.0 at 37 °C for 1 h. The activity of
enzyme without exposure to sodium azide/EDTA was considered as control (100%)
for the calculation of remaining percent activity.
3.2.14. Effect of HgCh/CdCh
Soluble and immobilized BGP (1.3 U) were incubated independently with
HgCb/CdCb (0.01-0.1 mM) in 100 mM sodium acetate buffer, pH 5.0 at 37 °C for 1
h. The activity of enzyme without exposure to heavy metal was taken as control
(100%) for the calculation of remaining percent activity.
59
3.2.15. Effect of NaCI
Soluble and immobilized BGP preparations (1.3 U) were incubated with
chloride (0.1-1.0 M) in 100 mM sodium acetate buffer, pH 5.0 at 37 °C for 1 h.
Activity of enzyme without sodium chloride was taken as control (100%) for the
calculation of remaining percent activity.
3.2.16. Reusability of immobilized BGP
E-BGP and SI-BGP were taken in triplicates for assaying the peroxidase
activity. After each assay the immobilized enzyme preparations were taken out,
washed and stored in 100 mM sodium acetate buffer, pH 5.0 overnight at 4 °C. The
activity was assayed for seven successive days. The activity determined for the first
time was considered as control (100%) for the calculation of remaining percent
activity after each use.
3.3. RESULTS
3.3.1. Entrapment and surface immobilization of BGP
The entrapment and surface immobilization of BGP into/on calcium alginate-
starch beads is demonstrated in Fig 7. Insoluble Con A-BGP complex retained 70% of
the initial activity. In order to prevent the dissociation of Con A-BGP complex, this
complex was crosslinked by 0.5% glutaraldehyde. The activity of enzyme was
decreased after crosslinking and crosslinked preparation retained 66% of the activity.
However, the entrapment of crosslinked Con A-BGP complex into calcium alginate-
starch beads ftjrther resulted in a loss of 14% activity (Table 7).
BGP immobilized on the surface of Con A layered calcium alginate-starch
beads exhibited 69% of the original activity. Moreover, the surface immobilized
enzyme was also crosslinked by 0.5% glutaraldehyde in order to maintain its integrity.
The crosslinking of surface immobilized BGP also resulted in a loss of 6% of its
initial activity (Table 7).
60
Alginate
Con A
BGP
Starch
[a]
aJSc
* ^
^
^
^ ^ c jgc
l ® c ?Sc
a ? ^ i ^
»?Sc
AlgiiMte
BGP
Con A
Starch
[b]
Fig. 7: Schematic diagram of (a) E-BGP and (b) SI-BGP
61
Table 7: Immobilization of BGP into and on calcium alginate-starch beads
Enzyme immobilized preparations
Con A-BGP complex
Crosslinked Con A-BGP complex
E-BGP
Peroxidase immobilized on Con A
layered calcium alginate-starch beads
SI-BGP
Activity expressed (%)
70
66
52
69
63
Each value shows the mean for three-independent experiments performed in
duplicates, with average standard deviation, < 5%.
62
3.3.2. Effect of pH
Fig. 8 demonstrates the effect of pH on the activity of soluble and immobilized
BGP. Both immobilized BGP preparations showed no change in pH-optima, pH 5.0
but had remarkable broadening in pH-activity profiles as compared to S-BGP.
However, E-BGP retained significantly very high enzyme activity at acidic and
alkaline side of pH-optima as compared to SI-BGP and S-BGP. The E-BGP retained
57% and 60% of the maximum activity at pH 3.0 and pH 8.0, respectively whereas
soluble enzyme exhibited only 36% and 41% activity under similar incubation
conditions.
3.3.3. Effect of temperature
Both immobilized BGP preparations exhibited same temperature-optima as its
soluble counterpart at 40 °C (Fig. 9). E-BGP retained remarkably higher fraction of
catalytic activity at temperatures below and above the temperature-optima as
compared to SI-BGP and S-BGP. E-BGP exhibited 50% activity at 80 °C while SI-
BGP and S-BGP retained 40% and 30% activity at this temperature, respectively.
Soluble and immobilized BGP were incubated at 60 "C for various time
intervals. Incubation of S-BGP at 60 °C for 2 h resulted in a loss of 53% of initial
activity. However, E-BGP and SI-BGP retained 73% and 60% of the original activity
under similar incubation conditions, respectively (Fig. 10).
3.3.4. Effect of urea
Fig. 11 demonstrates the effect of 4.0 M urea on the activity of BGP. E-BGP
and SI-BGP retained 70% and 50% of their activity after 2 h incubation, whereas
soluble enzyme lost nearly 68% of the initial activity under identical urea exposure.
3.3.5. Effect of organic solvents
The effect of increasing concentrations of water-miscible organic solvents;
dioxane and «-propanol (10-60%, v/v), on the activity of soluble and immobilized
BGP is shown in Table 8. E-BGP showed more than 55% of the initial activity when
63
(0 O) _c 'E (5 E
100
80
60
S 40
20
— I
10
PH
Fig. 8: pH-activity profiles of soluble and immobilized BGP
Soluble and immobilized BGP (1.3 U) were incubated in the buffers of
varying pH. The morality of each buffer was 100 mM. The activity at pH 5.0
for all the preparations was taken as control (100%) for the calculation of
remaining percent activity. The symbols show S-BGP (•), SI-BGP (o) and E-
BGP(T).
64
100
80
• > 5 60
u (Q
O) c "c (0
E 0}
en
£ 40
20
20 40
Temperature (°C)
60 80
Fig. 9: Temperature-activity profiles for soluble and immobilized BGP
The activity of soluble and immobilized BGP (1.3 U) was measured in 100
mM sodium acetate buffer, pH 5.0 at various temperatures (30-80 °C). The
activity obtained at 40 °C was taken as control (100%) for the calculation of
remaining percent activity. The symbols show S-BGP (•), SI-BGP (o) and E-
BGP(T).
65
60
Time (min)
120
Fig. 10: Thermal denaturation of soluble and immobilized BGP
Soluble and immobilized BGP (1.3 U) were incubated at 60 "C for various
times in 100 mM sodium acetate buffer, pH 5.0. Aliquots of each preparation
were taken out at indicated time intervals and chilled quickly in crushed ice
for 5 min. Enzyme activity was determined as described in text. Activity
obtained without incubation at 60 °C was taken as control (100%) for the
calculation of remaining percent activity. The symbols show S-BGP (•), SI-
BGP(o)andE-BGP(T).
66
Fig. 11: Effect of 4.0 M urea on soluble and immobilized BGP
Soluble and immobilized BGP (1.3 U) were incubated with 4.0 M urea in
100 mM sodium acetate buffer, pH 5.0 for various times. Activity obtained
without urea exposure was taken as control (100%) for the calculation of
remaining percent activity. The symbols show S-BGP (•), SI-BGP (o) and
E-BGP(T).
67
Table 8: Effect of organic solvents on soluble and immobilized BGP
Organic
solvent
(v/v, %)
10
20
30
40
50
60
Remaining activity (%)
Dioxane
S-BGP
76
64
41
34
26
19
SI-BGP
85
72
58
40
31
24
E-BGP
93
85
67
58
38
29
«-propanol
S-BGP
51
38
29
23
18
14
SI-BGP
68
50
40
35
30
27
E-BGP
89
81
77
56
43
35
Soluble and immobilized BGP (1.3 U) were incubated independently with dioxane/«-
propanol (10-60%, v/v) in 100 mM sodium acetate buffer, pH 5.0 at 37 °C for 1 h.
The activity of BGP without exposure to organic solvent was taken as control (100%)
for the calculation of remaining percent activity. Each value represents the mean for
three independent experiments performed in duplicates, with average standard
deviation, < 5%.
68
exposed to 40% (v/v) dioxane/«-propanol for 1 h at 37 °C. However, S-BGP exhibited
only 34% and 23% of activity after exposure to 40% (v/v) dioxane and «-propanol,
respectively.
3.3.6. Effect of detergent (Tween 20)
The effect of Tween 20 on the activity of soluble and immobilized BGP is
shown in Fig. 12. E-BGP and SI-BGP retained 57% and 40% of the original activity
when exposed to 5.0% (v/v) Tween 20 for 1 h at 37 °C. However, soluble enzyme was
sensitive to Tween 20 and it lost nearly 70% of its activity under similar exposure.
3.3.7. Effect of sodium azide/EDTA
Table 9 demonstrates the effect of sodium azide/EDTA on the activity of
soluble and immobilized BGP. E-BGP and SI-BGP retained 43% and 40% activity
after 1 h exposure to 0.05 mM sodium azide. Moreover, SI-BGP and E-BGP retained
68% and 80% activity after 1 h exposure to 0.05 mM EDTA, respectively while the
soluble BGP lost nearly 50% of its activity under identical exposure (Table 9).
3.3.8. Effect of HgCU/CdCb
The chemical contamination of water from a wide range of toxic derivatives,
in particular heavy metals is a serious environmental problem owing to their potential
human toxicity. In view of their presence in wastewater, it becomes important to
examine the effect of some heavy metals on the activity of BGP. E-BGP and SI-BGP
retained 71% and 58% activity in the presence of 0.1 mM HgCb, respectively
whereas S-BGP exhibited only 53% activity under similar treatment conditions (Table
10).
Immobilized BGP preparations were more resistant to inactivation induced by
CdCl2. E-BGP and SI-BGP retained 69% and 59% activity after 1 h exposure to 0.1
mM CdCl2, respectively. However, S-BGP lost 48% of its original activity when it
was pre-incubated to 0.1 mM CdCb (Table 10).
69
100
(0 O) c 'E (0 E 0)
2.0 2.6 3.0
Tween 20 (%)
5.0
Fig. 12: Effect of Tween 20 on soluble and immobilized BGP
Soluble and immobilized BGP (1.3 U) were incubated with Tween 20 (0.5-
5.0%, v/v) in 100 mM sodium acetate buffer, pH 5.0. The activity of BGP
without Tween 20 was taken as control (100%) for calculation of remaining
percent activity. The symbols show S-BGP (•), SI-BGP (o) and E-BGP (T).
70
Table 9: Effect of sodium azide/EDTA on soluble and immobilized BGP
Inhibitor
(mM)
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
Remaining activity (%)
sodium azide
S-BGP
70
63
55
43
32
28
24
21
16
12
SI-BGP
82
71
65
52
40
35
30
27
24
20
E-BGP
89
75
69
59
43
39
36
31
27
24
EDTA
S-BGP
82
76
71
60
52
49
46
41
34
30
SI-BGP
87
85
78
72
68
64
61
57
54
51
E-BGP
92
90
87
83
80
76
74
70
65
61
Soluble and immobilized BGP (1.3 U) were incubated independently with (0.01-1.0
mM) of sodium azide and EDTA in 100 mM sodium acetate buffer, pH 5.0 at 37 °C
for 1 h. The activity of soluble and immobilized BGP without exposure to inhibitor
was taken as control (100%) for the calculation of remaining percent activity. Each
value represents the mean for three independent experiments performed in duplicates,
with average standard deviation, < 5%.
71
Table 10: Effect of HgCii/CdChon soluble and immobilized BGP
HgCl2/CdCl2
(mM)
0.01
0.02
0.04
0.06
0.08
0.10
Remaining activity (%)
HgCb
S-BGP
72
69
64
60
56
53
SI-BGP
80
72
70
63
61
58
E-BGP
90
86
82
76
74
71
CdCb
S-BGP
77
73
69
62
56
52
SI-BGP
85
80
74
69
63
59
E-BGP
92
88
82
76
74
69
Soluble and immobilized BGP (1.3 U) were incubated with HgCb/CdCh (0.01-1.0
mM) in 100 mM sodium acetate buffer, pH 5.0 at 37 °C for 1 h. The activity of BGP
without exposure to HgC^/CdCb was taken as control (100%) for the calculation of
remaining percent activity. Each value represents the mean for three independent
experiments performed in duplicates, with average standard deviation, < 5%.
72
3.3.9. Effect of sodium chloride
The effect of NaCl on the activity of BGP has been illustrated in Fig. 13. The
activity of immobilized BGP preparations was slightly enhanced in the presence of
0.2 M NaCl. E-BGP and SI-BGP showed 104% and 114% activity after 1 h of
incubation with 0.2 M NaCl. However, S-BGP exhibited a marginal loss of 6% of the
enzyme activity under similar incubation conditions.
3.3.10. Reusability of immobilized BGP
Reusability of two immobilized preparations of BGP has been shown in Fig.
14. After 7* repeated use the E-BGP retained 75% of the original activity, whereas SI-
BGP showed 69% activity.
3.4. DISCUSSION
Sodium alginate has been considered since a long time for the entrapment of
enzymes, cell organelles, microorganisms due to its biocompatibility and
processibility (Zhu et al., 2005; Kasipathy et al., 2006). Calcium alginate entrapped
enzyme preparations have one inherent limitation that the large Mr substrates or
products cannot easily diffuse in and out of gel beads (Norouzian, 2003). In order to
circumvent this problem, the immobilization of enzymes on the surface of such beads
would be a preferred choice. In this work an effort has been made to prepare a hybrid
calcium alginate-starch gel bead which has successfully been employed both for
entrapment of BGP and its immobilization on the surface of such beads layered with
Con A.
The immobilization of BGP on the surface of Con A layered calcium alginate-
starch beads has presented a strategy to overcome the problem of diffusion limitation
of the substrate/product and to increase the surface area of contact between enzyme
and substrate so that such preparation could be exploited for the treatment of large
volume of industrial waste. The enzyme activity expressed by SI-BGP was more than
E-BGP due to the compact structure of E-BGP, which decreased the flexibility of
enzyme as well as considerably limited diffusion of the substrate (Fig. 7, Table 7). It
73
140
120
—' >> •*-» > 4 - *
o CD O) c c
E 0)
cc
100
80
60
^n
20-
1 1 1 1 1 1 1 1 1 1
00 01 0.2 03 0.4 0.5 0.6 0.7 08 0.9 10
Sodium chloride (M)
Fig. 13: Effect of NaCI on soluble and immobilized BGP
Soluble and immobilized BGP (1.3 U) were incubated with NaCl (0.1-1.0 M)
in 100 mM sodium acetate buffer, pH 5.0 at 37 °C for 1 h. The activity of
BGP without incubation with NaCl was taken as control (100%) for the
calculation of remaining percent activity. The symbols show S-BGP (•), SI-
BGP(o)andE-BGP(T).
74
• SI-B6P
DE-BGP
2 3 4 5 Number of uses
Fig. 14: Reusability of immobilized preparations of BGP
E-BGP and SI-BGP (1.3 U) were independently assayed in 100 mM sodium
acetate buffer, pH 5.0 as described in the text. After each assay immobilized
enzyme preparations were taken out and stored in assay buffer overnight for
next use. This procedure was repeated for seven consecutive days.
75
has already been reported that the enzyme immobilized on the surface of gel beads
has minimum mass transfer constrain (Le-Tien et al., 2004; Gomez et al., 2006).
Immobilization of an enzyme to a support often limits its freedom to undergo
drastic conformational changes and thus resulted in increased stability towards
denaturants such as organic solvents, heat, inhibitors and urea (Fig. 9-13, Table 8-10).
However, some earlier workers have demonstrated that the stability of surface
immobilized enzymes was significantly higher against pH, heat and proteolysis than
the free enzyme (Le-Tien et al., 2004). Thus the enhanced resistance to the stability of
immobilized BGP against denaturants offered a potential advantage for the
application of such type of enzyme preparation in treatment of wastewater.
The pH-activity profiles of E-BGP and Sl-BGP have the same pH-optima as
that of S-BGP (Fig. 8). However, entrapped and surface immobilized BGP
preparations showed significant broadening in pH activity profiles indicating a
marked increased stability in the buffers of varying pH. This predicts that the
entrapment of enzyme in the gel beads provides a microenvironment for the enzyme,
which might play an important role in the state of protonation of the protein
molecules. However, the surface immobilized BGP provided stability due to multiple
point attachment between enzyme and Con A. The crosslinking of BGP present on the
Con A layered calcium alginate-starch beads by glutaraldehyde further increased
stability.
Furthermore, immobilized peroxidase preparations retained their structure and
remarkably high activity at elevated temperatures as compared to soluble peroxidase
(Fig. 9, 10). These observations were in agreement with the findings of some earlier
investigators (Al-Adhami et al., 2002; Dodor et al., 2004). It is well established that
thermal exposure initiate unfolding of protein molecules which is followed by
irreversible changes due to aggregation and formation of scrambled structures which
takes place more in soluble form as compared to the immobilized enzyme (Querol et
al., 1996).
SI-BGP and E-BGP were markedly more stable to the denaturation induced by
4.0 M urea (Fig. 11). Although the action mechanism of urea on the protein structure
has not yet been completely understood, some earlier workers have proposed that the
presence of carbohydrate moieties in enzymes increased their resistance to
inactivation caused by urea (Kwon and Yu, 1997).
76
ioi;e > rdsi^ant • toA m ' U The E-BGP followed by SI-BGP was remarkably hio^e
inactivation mediated by water-miscible organic solvents; dibiitane^nd «-propanoly - /^
(Table 8). It has already been reported that the stabilization of inisoluble cwnpjexe^^
against various organic solvents which could possibly be due to low water
requirement or enhanced rigidity of the enzyme structure (Xin et al., 2005).
Immobilized BGP was significantly more resistant to denaturation induced by
Tween 20 as compared to its soluble counterpart (Fig. 12). These observations
suggested that the presence of lower concentrations of detergents was not harmful to
the enzyme's native conformation. Flock et al. (1999) reported an increase in the
activity of soybean peroxidase when the enzyme was treated with low concentrations
of SDS and Tween 20. The enhancement of enzyme activity by 0.5% (v/v) Tween
20/Triton X 100 and stabilization of BGP immobilized on a bioaffmity support (Con
A-Sephadex) against high concentrations of such type of detergents has also been
reported by some earlier workers (Akhtar et al., 2005c).
The immobilized peroxidase preparations showed more than 80% of their
original enzyme activity in the presence of 0.1 mM inhibitory compounds; sodium
azide and EDTA (Table 9). A number of studies have already been performed on the
inhibitory effect of such compounds on HRP (Ortiz de Montellano et al., 1988).
Sodium azide has been shown to be a potent inhibitor of many hemeprotein-catalyzed
reactions (Kvaratskhelia et al., 1997). Peroxidase in the presence of sodium azide and
H2O2 mediates one electron oxidation of azide ions and forming azidyl free radicals,
which bind covalently to the heme moiety thus inhibiting the enzyme activity
(Tatarko and Bumpus, 1997).
Metals induce conformational changes in enzymes; however peroxidases
remains active even in the presence of a number of metal ions, as a part of their
detoxifying role. BGP has exhibited more resistant to its stability against heavy metals
induced inhibition. Some recent reports have also indicated that HRP was remarkably
inhibited by heavy metal ions (Keyhani et al., 2003; Einollahi et al., 2006). However,
in this study the strength of inhibition of immobilized BGP by heavy metal ions was
quite low as compared to soluble enzyme (Table 10). The stability of immobilized
BGP against several metal compounds showed that such preparations could be
exploited to treat aromatic pollutants even in the presence of heavy metals.
Enzyme reuse provides a number of cost effective advantages that are often an
essential prerequisite for establishing an economically viable enzyme catalyzed
77
process (Norouzian, 2003). However, E-BGP and SI-BGP retained more than 60% of
their original activity even after its seven successive uses (Fig. 14). The activity loss
during repeated use might be due to the inhibition of enzyme by product or by
leaching of enzyme from the gel bead or damage to the beads (Gaserod et al., 1999;
Musthapa et al., 2004).
On the basis of results obtained in the present work, it can be concluded that
the stability offered by E-BGP followed by SI-BGP against various denaturants
suggested that these preparations could successfully be employed in reactors for the
treatment of effluents containing phenolic and other aromatic pollutants.
78
chapter l¥ (Decoforization of direct dyes 6y
sa ft fractionated turnip proteins
in tfte presence of ^20^2 ^^^
redox Mediators
m '" »"•
m 2.-E
4.1. INTRODUCTION
Recently, enzymatic approach has attracted much attention in the removal of
phenolic pollutants from aqueous solutions as an alternative strategy to the
conventional chemical as well as microbial treatments that pose some serious
problems (Duran and Esposito, 2000; Husain and Jan, 2000; Torres et al., 2003).
Oxidoreductive enzymes such as laccases/peroxidases have been employed for the
degradation/removal of aromatic pollutants from various contaminated sites (Akhtar
et al., 2005a; 2005b; Mohan et al., 2005; Couto, 2007). These enzymes can act on a
broad range of substrates and catalyze the degradation/removal of organic pollutants
present in a very low concentration (Akhtar and Husain, 2006; Husain, 2006; Husain
and Husain, 2008). In view of their potential for treating phenolic compounds, several
microbial and plant peroxidases have been considered for the decolorization and
removal of dyes from polluted water but none of them has been exploited at large
scale due to low enzymatic activity in biological materials and high cost of
purification (Bhunia et al., 2001; Shaffiqu et al., 2002; Verma and Madamvar, 2002b).
Recently the use of enzymes has been further extended by employing several
compounds, which mediate oxidative reactions catalyzed by oxidoreductases (Claus et
al., 2002; Camarero et al., 2005; Akhtar and Husain, 200()).
Here an attempt has been made to investigate tlie mediating role of various
low Mf compounds in the decolorization of direct dyes/mixture of direct dyes by
partially purified peroxidase from turnip roots. The decolorization was examined in
the presence of different concentrations of redox mediators and enzyme, at varying
pH and temperatures.
4.2. MATERIALS AND METHODS
4.2.1. Materials
Direct dyes; Direct Red 23, Direct Red 239 (DR 239), Direct Blue 80 and
Direct Yellow 4 (DY 4) were a gift from Atul Pvt. Ltd. Gujrat, India. Violuric acid
was obtained from Fluka Chemicals, Austria and other redox mediators including 1-
hydroxybenzotriozole, vanillin mentioned in Table 11 were purchased from SRL
79
Chemicals Pvt. Ltd. Mumbai, India. o-dianisidine-HCi was obtained from the Centre
for Biochemical Technology, CSIR, India. Turnip roots were purchased from a local
vegetable market and other chemicals employed were of analytical grade and were
used without any further purification.
4.2.2. Ammonium sulphate fractionation of turnip proteins
Proteins were fractionated from the buffer extract of turnip by using
ammonium sulphate. The detailed procedure is described in Chapter II, Section 2.2.2.
4.2.3. Measurement of peroxidase activity
Peroxidase activity was estimated as described in Chapter II, Section 2.2.6.
4.2.4. Procedure for screening dye decolorization mediating role of various
compounds
The synthetic solutions of direct dyes (50-100 mg L"') were prepared in 100
mM sodium acetate buffer, pH 5.5 to examine their decolorization by TP. The
structures of the used dyes are represented in Fig. 15. Mixtures of direct dyes were
prepared by mixing different dyes in equal proportion in terms of absorbance.
Each dye (5.0 mL) was incubated with TP (0.094 U mL'') in 100 mM sodium
acetate buffer, pH 5.5 in the presence of 0.72 mM H2O2 for 1 h at 30 °C. Compounds
mentioned in Table 11 were used as redox mediators for the selected experiments.
Dye decolorization was monitored at specific wavelength maxima for each dye. The
reaction was stopped by heating in a boiling water bath for 5 min and the insoluble
product was removed by centrifugation at 3000 g for 15 min. Untreated dye solution
was used as control (100%) for the calculation of remaining percent color.
4.2.5. Calculation of percent dye decolorization
To compare various experiments, the decolorization was calculated for each
dye or mixture of dyes. Parameter percent decolorization was defined as:
80
N^ .CHi
Direct Red 23
NaOaS NaOsS NHCOH SOaNa
Direct Red 239
rx r « 3 / ^ ^ ^ ^ ^
Direct Blue 80
HO—f ^> N=N f ^ CH=CH f ^ N=N f \ OH
SOaNa NaO
Direct Yellow 4
Fig. 15: Chemical structures of investigated direct dyes
81
Absorbance of the untreated dye - Absorbance after treatment Percent decolorization = x 100
Absorbance of the untreated dye
4.2.6. Effect of varying concentrations of redox mediators on TP mediated dye
decolorization
The decolorization of direct dyes by TP (0.094 U mL"') was monitored in tlae
presence of varying concentrations of HOBTA^LAAT^ (0.2-0.8 mM) and incubated
under the conditions mentioned in Section 4.2.4.
4.2.7. Treatment of dyes with increasing concentrations of TP
Each dye solution (5.0 mL) was treated by increasing concentrations of TP
(0.094-0.330 U mL"') for 1 h with 0.6 mM HOBTA^LAA^ at 30 °C. The reaction
mixture was incubated under the experimental conditions mentioned in Section 4.2.4.
Untreated dye solution was used as control (100%) for the calculation of remaining
percent color.
4.2.8. Effect of pH on the decolorization of dyes
Each dye solution (5.0 mL) was treated with TP (0.094 U mL"') in the buffers
of various pH (3.0-10.0) imder the experimental conditions mentioned in Section
4.2.4.
4.2.9. Effect of temperature on the decolorization of direct dyes
Each dye solution (5.0 mL) was treated by TP (0.094 U mL'') at various
temperatures (20-80 °C) for 1 h under similar experimental conditions mentioned in
Section 4.2.4.
4.2.10. Decolorization of mixtures of dyes
Four mixtures of direct dye were prepared by mixing various dyes in equal
proportion in terms of absorbance (Table 17). Each mixture of dyes was treated with
82
TP (0.094 U mL"') in 100 mM sodium acetate buffer, pH 5.5 and under the other
experimental conditions mentioned in Section 4.2.4. Untreated dyes mixture was
considered as control (100%) for the calculation of remaining percent color. Decrease
in absorbance in TP treated solutions was monitored at specific wavelength maxima
of the mixture.
4.2.11. Decolorization of mixtures of dyes in stirred batch processes
The mixtures which showed maximimi decolorization in the presence of
HOST and VLA were selected for the stirred batch processes. Dye mixture 3 and 4
(250 mL) each was treated with TP (25 U) in the presence of 0.72 mM H2O2 and 0.6
mM HOBTA' LA for 8 h at room temperature (30-32 °C) under stirring conditions.
After treatment, aliquots of mixtures were taken out at various time intervals and the
reaction was stopped by heating in a boiling water bath for 5 min and the insoluble
product was removed by centrifiigation at 3000 g for 15 min. The absorbance was
recorded in visible region at their specific wavelength maxima. The untreated dye
mixture was considered as control (100%) for the calculation of remaining percent
color.
4.2.12. Data collection and analysis
DB 80 and mixture 4 were treated by TP (0.094 U mL"') in the presence of
0.6 mM HOBTA^LA and 0.72 mM H2O2 for 1 h at 30 °C. The completion of dye
decolorization was followed by centrifiigation and analysis of the clear supernatant
for UV-visible spectra; the spectra of control and TP treated dye samples were taken
on Cintra \0e UV-visible Spectrophotometer.
Each dye and mixture of dyes was independently incubated with TP (0.094 U
mL'') in 100 mM sodium acetate buffer, pH 5.5 in the presence of HOBTA^LA for 1
h at 30 °C. The dye solutions treated with TP were kept in boiling water bath for 5
min in order to stop the decolorization reaction. The treated dye and mixture of dyes
were further incubated with activated silica (1.0 mg mL"') for 2 h at room temperature
with constant stirring. Activated silica was prepared by incubating 1.0 g of silica gel
in an oven (120 °C) for 12 h. After the incubation, silica gel was suspended in 10 mL
of distilled water and stirred for 1 h at room temperature. The suspended particles
83
were decanted by washing thrice in distilled water. The colored product, adsorbed on
the activated silica was separated from the reaction mixture by centrifugation at 3000
g for 15 min. After the removal of the colored product, the TOC content of the clear
supernatant was evaluated by using a TOC analyzer (Multi N/C 2000, Analytic Jena,
Germany). Control and treated samples of each dye and mixture of dyes was diluted
20 fold before measuring their TOC.
4.3. RESULTS
4.3.1. Screening of dye decolorization mediating activity often compounds
Among the ten compovmds selected to investigate the mediating effect on the
TP-catalyzed direct dyes decolorization, six compounds promoted decolorization of
DR 23, three of DR 239, six of DB 80 and five of DY 4. HOBT and VLA enhanced
more than 60% decolorization of all four investigated direct dyes (Table 11).
4.3.2. Effect of increasing concentrations of mediators
Table 12 demonstrates the effect of varying concentrations of mediators (0.2-
0.8 mM) on the TP-catalyzed decolorization of direct dyes. It was observed that 0.6
mM concentration of HOBT/VLA could remove more than 50% of the color from all
four dyes.
However, DY 4 was decolorized marginally in the presence of HOBT.
Although the addition of more than 0.6 mM mediator (HOBTA^LA^VN) had no
significant effect on the decolorization of DR 23 and DR 239, while it was observed
that in case of DB 80 decolorization was continuously decreased with increasing
concentration of VLA.
4.3.3. Effect of varying concentrations of TP
Each direct dye was treated with increasing concentrations of TP (0.094-0.330
U mL"') for 1 h in the presence of 0.6 mM redox mediator (HOBTA^LAA^). An
increase in the concentration of TP resulted in an enhancement in dye decolorization.
However, in all treated dyes there was a marginal decrease in percent decolorization
84
Table 11: Compounds investigated as redox mediators for TP-catalyzed dye
decolorization
Name of
compound
HOBT
VN
L-Histidine
VLA
Bromo-phenol
4-nitrophenol
a-Naphthol
Catechol
Gallic acid
Quinol
DR23
(X 505 mn)
96
19
7
86
6
5
0
0
0
0
Decolorization (%)
DR239
(k 505 nm)
93
21
0
83
0
0
0
0
0
0
DB80
(X 572 nm)
86
55
29
89
37
4
0
0
1
0
DY4
(X 405 nm)
66
37
36
72
0
0
0
0
7
0
Each compound (2.0 mM) was used along peroxidase (0.094 U mL"') for
decolorization mediating property. Each dye (5.0 mL) was incubated in 100 mM
sodium acetate buffer, pH 5.5 and in the presence of 0.72 mM H2O2 for 1 h at 30 °C.
Each value represents the mean for three independent experiments performed in
duplicates, with average standard deviation, < 5%.
85
Table 12: Effect of various concentrations of HOBTA^LAA^ on dye
decolorization by TP
HOBTA^LAA^
(mM)
0.2
0.4
0.6
0.8
Decolorization (%)
HOBT
DR
23
63
90
94
94
DR
239
81
86
86
86
DB
80
58
73
86
86
DY
4
57
57
67
64
VLA
DR
23
77
87
85
84
DR
239
74
76
80
78
DB
80
84
86
89
87
DY
4
64
63
73
73
VN
DR
23
6
10
11
11
DR
239
5
7
7
7
DB
80
39
43
50
51
DY
4
17
21
23
23
Each dye (5.0 mL) was treated by TP (0.094 U mL'') in the presence of increasing
concentrations of redox mediator (0.2-0.8 mM) as described in the text. Each value
represents the mean for three independent experiments performed in duplicates, with
average standard deviation, < 5%.
86
Table 13: Effect of TP concentration on decolorization of direct dyes in the
presence of HOBTA LAAHV
TP
(U mL-')
0.094
0.143
0.186
0.236
0.281
0.330
Decolorization (%) in the presence of HOBTA^LAAnvf
HOBT
DR
23
93
93
93
93
92
92
DR
239
90
90
90
90
89
88
DB
80
84
86
88
88
85
85
DY
4
66
70
72
72
71
71
VLA
DR
23
84
84
85
86
87
87
DR
239
83
83
84
84
85
85
DB
80
89
90
91
92
93
93
DY
4
72
73
73
74
75
76
VN
DR
23
11
20
20
23
23
30
DR
239
8
10
14
14
16
18
DB
80
50
58
60
62
66
66
DY
4
23
29
36
36
36
43
Each dye (5.0 mL) was independently treated with increasing concentrations of TP
(0.094-0.330 U mL'') for 1 h in the presence of 0.6 mM redox mediator
(HOBTA^LAAH ) in 100 mM sodium acetate buffer, pH 5.5 at 30 °C. Each value
represents the mean for three independent experiments performed in duplicates, with
average standard deviation, < 5%.
87
after 0.236 U mL-' of TP in the presence of HOBT. While as in the presence of VLA
and VN there was slight increase in percent decolorization after 0.236 U mL'' of TP
(Table 13).
4.3.4. Effect of pH on the decolorization of direct dyes
All the four direct dyes were treated with TP in the buffers of different pH
(3.0-10.0) in the presence of 0.6 mM redox mediator (HOBTA^LAATN). The dyes
were maximally decolorized at pH 5.5. HOBT and VLA showed significantly higher
rate of dye decolorization as compared to VN (Table 14).
4.3.5. Effect of temperature on the decolorization of direct dyes
The effect of different temperatures (20-80 °C) on the dye decolorization was
monitored and it was observed that all the dyes were decolorized maximally over 60%
at 30 °C (Table 15). The dye decolorization was decreased above and below
temperature-maxima.
4.3.6. Treatment of mixture of dyes with TP
The influence of redox mediators, HOBT and VLA on the TP-catalyzed
decolorization of complex mixtures of dyes was also examined. There was a
significant reduction in the color of the dyes treated with TP even if they were present
in the form of complex mixtures (Table 17). Mixture 3 and 4 were decolorized more
than 50% in the presence of redox mediators, HOBT and VLA whereas mixture 1 and
2 were decolorized less than 50% in the presence of HOBT. Moreover, the
decolorization of mixture 1 and 2 was quite significant in the presence of VLA (Table
17). The decolorization of mixtures of dyes was slower than that of pure dye solutions
(Table 16).
4.3.7. Treatment of dye mixtures in a stirred batch process
Dye mixture 3 and 4 were independently treated by TP in the presence of
88
b .tt
N3
n O s
a u w u a .a
H .A
>» -a u u
o e o
• • M
.a
V
-a e o
M a.
< H-l
^ H CQ O '•M O u o c 1/1
u l-r
(D J3
i=l ,_
o N b«
Q
>-Q
o 00 CQ Q
o\
oi Q
Q
ex
5
<
>
H
O
^
^
<
>
H
O K
^ C!
<
>
H P3 O ffi
^
<
>
H U-1
O
-
m
00
m
(N
r-
m
;::;
00 1-^
«o
ON
O
(
^
00
00
en
*o (N
M3
en
VO
•*
O
90 • *
00
00
VO
00
O OS
as
00
OS
^Tt
o >n
00 00
m 00
r-
oo
•<
r--00
ON
^
00
00
• *
(N
O
m
• ^
o
en
m
wn
VO
O
Os
O
r (N
O
so
^
O
^
ON
o
o
t^
o
o
o
00
o
m
o
o
o
>o
o
o
00
o
o
o
1—1
o
(N
o
o
o
Tt
o
o
OS
o
o
o
o 1-^
o
-^
o
o
o
o
o
o
o
13
0^ oo
,A
H
Table 15: Effect of temperature on decolorization of direct dyes by TP in the
presence of HOBTA^LA
Temperature
rc)
20
30
40
50
60
70
80
Decolorization (%) in the presence of HOBTA/'LA
DR23
HOBT
41
94
90
64
34
26
23
VLA
40
85
79
75
26
11
8
DR239
HOBT
26
93
83
41
36
24
20
VLA
15
81
78
69
30
21
17
DB80
HOBT
18
80
75
50
33
18
13
VLA
24
84
79
66
39
30
21
DY4
HOBT
10
60
55
47
20
16
14
VLA
22
70
67
53
44
31
22
Each dye (5.0 mL) was incubated with TP (0.094 U mL'') in 100 mM of sodium
acetate buffer, pH 5.5 in the presence of 0.72 mM H2O2 and 0.6 mM redox mediator
(HOBTA^LA) at 20-80 "C for 1 h. Each value represents the mean for three
independent experiments performed in duplicates, with average standard deviation, <
5%.
90
Table 16: TOC content of direct dyes after treatment with TP
Name of
direct dye
DR23
DR239
DB80
DY4
(nm)
505
505
572
405
Decolorization
(%)
HOBT
90
93
86
66
VLA
86
83
89
72
% TOC removal
HOBT
91
90
89
70
VLA
88
85
89
76
Each dye (5.0 mL) was treated by TP (0.094 U mL'') in 100 mM sodium acetate
buffer, pH 5.5 in the presence of 0.6 mM HOBTA'LA and 0.72 mM H2O2 for 1 h at
30 °C. The rest of the conditions required for TOC determination of independent dyes
are described in Section 4.2.12. Each value represents the mean for three independent
experiments performed in duplicates, with average standard deviation, < 5%.
91
Table 17: Treatment of mixtures of direct dyes by TP in the presence of
HOBTA^LA
Mixture of direct dyes
Mixture 1: DB 80+DY 4+DR 23
Mixture 2: DB 80+DY 4+DR 239
Mixture 3: DR 23+DY 4+DR 239
Mixture 4: DR 23+DB 80+DR 239
^max
(nm)
520
470
502
515
Decolorization
(%)
HOBT
35
45
55
57
VLA
57
65
70
75
% TOC removal
HOBT
39
48
50
57
VLA
60
66
75
11
The conditions for the treatment of mixtures of dyes were described in Sections
4.2.10 and 4.2.12 of the text. Each value represents the mean for three independent
experiments performed in duplicates, with average standard deviation, < 5%.
92
100 n •mitureSwlthHOBT
•mixtue3withVLA
• mixture 4 with HOBT
• mixture 4 with VLA
o o o o o o o o o o o o o o o o o
Time (nm)
Fig. 16: Decolorization of mixture 3 and 4 by TP in the presence of HOBTA^LA
in stirred batcli processes
Dye mixture 3 (DR 23+DY4+DR 239) and mixture 4 (DR 23+DB 80+DR
239) were treated with TP (25 U) in a stirred batch process for 8 h at room
temperature. The aliquots were removed at the interval of 30 min and
intensity of color was recorded at the specific wavelength maxima for each
mixture.
93
redox mediator (HOBTA^LA) for different times in stirred batch processes. These
observations showed a loss of more than 80% color from both mixtures. The
treatment of dye mixtures by TP in the presence of redox mediator was followed by
the formation of insoluble product within 1 h (Fig. 16).
4.3.8. Analysis of dyes and their mixtures
Fig. 17 demonstrated that the model wastewater containing an individual dye,
DB 80 and mixture of direct dyes treated with TP in the presence of redox mediator
(HOBTA^LA) resulted in the loss of color from the solution in visible region. The
diminution in absorbance peaks in visible region was clear evidence regarding the
removal of dyes from treated wastewaters containing complex mixtures of dyes.
All the tested direct dyes and their mixtures were treated with TP and their
insoluble product was removed by centrifugation. TOC content of individual dye and
mixtures of dyes before and after treatment with TP is given in Table 16 and 17. As
compared to control, the level of TOC was remarkably decreased when it was treated
by TP in the presence of HOBT and VLA.
4.4. DISCUSSION
In this study for the first time peroxidase from an inexpensive source (turnip
roots) was used for the decolorization of direct dyes. The dye solutions were found to
be stable upon exposure to H2O2, redox mediators, enzyme or activated silica alone.
Thus, the dye decolorization and precipitation was a resuh of H202-dependent
enzymatic action, possibly involving free-radical formation followed by
polymerization and precipitation.
It is well known that low Mr compounds, known as redox mediator can
mediate oxidation reaction between a substrate and an enzyme and thus enhance the
reaction rate (Bourbonnais and Paice, 1990). Redox mediators have different mediator
efficiency, and two major factors which determine their efficiency are suggested in
the Hterature: redox potential of a mediator and interaction with enzyme (Baciocchi et
al., 2002). In this study, an experiment has been designed for screening TP-catalyzed
94
3 3 D J 9 IOOJO tSOD SOOO ySOO GOOD 630 JO 70OD
Wavelength (nm)
QDJD
Wavelength (nm)
Fig. 17: Absorption spectra (a) DB 80 and (b) mixture 4 in the presence of
HOBTAO^A.
DB 80 and mixture 4 were treated with TP (0.094 U mL"') in presence of 0.6
mM HOBTA^LA and 0.72 mM H2O2 in 100 mM sodium acetate buffer, pH
5.5 at 30 °C for 1 h, respectively. Spectra for the control and TP treated dye
mixture were taken on Cintra \0e UV-visible spectrophotometer. Spectra
correspond to DB 80 (a) and mixture 4 (b). All the spectra in figure are
labeled.
95
dye decolorization mediating property of ten compounds in the presence of H2O2
(Table 11). The observation suggested that TP in the presence of redox mediators
could remove remarkably very high percentage of color from dye solution
Akhtar et al. (2005a) have reported the presence of HOBT drastically
enhanced decolorization of recalcitrant dyes, same was true in this study as HOBT
was a better redox mediator as compared to other tested compounds.
Several workers have demonstrated that the use of redox mediator-enzyme
system enhanced the rate of dye decolorization by several folds. However, these redox
mediators were required in very high concentrations; 5.7 mM VLA/laccase system,
11.6 mM of HOBT/laccase system, 2.0 mM HOBT/TP, 1.0 mM HOBT/BGP (Soares
et al., 2001a; 2001b; Glaus et al, 2002; Akhtar et al., 2005b; Kulshrestha and Husian,
2007). Such high concentrations may be suitable for some processes, particularly in
closed-loop systems where the mediator is retained. However, high concentrations
may not be appropriate for applications, such as wastewater treatment processes
where important considerations include the potentially prohibitive costs of mediators
and the possibility of creating negative impacts on effluent toxicity (Kim and Nicell,
2006). However, in this study very low concentration (0.6 mM) of mediators
(HOBT/VLAAHV) has been used to enhance the rate of TP-catalyzed dye
decolorization (Table 12).
The amount of enzyme also plays an important role in enzymatic dye
decolorization. However, in this study very low concentration of TP 0.094 U mL"'
was used to enhance the rate of dye decolorization (Table 13). Thus, it indicated high
potential of plant peroxidases in treatment of industrial effluents.
Decolorization of all the direct dyes by TP was optimum at pH 5.5 and 30 °C
(Table 14, 15). This was in agreement with earlier studies that plant peroxidases could
decolorize and degrade dyes maximally at acidic pH and low temperatures (Bhunia et
al, 2001, Akhtar et al., 2005a).
The decolorization rate of mixtures of dyes catalyzed by TP was slower as
compared to pure dye solution (Table 16, 17). This finding supported an eariier
observation that the biodegradation of various phenolic compounds in the form of
mixtures was quite slow as compared to an independent phenol (Kahru et al., 2000).
Mixture 3 and 4 were maximally decolorized in the presence of HOBT, 55% and 57%
respectively. However these mixtures were significantly decolorized in the presence
of VLA, 70% and 75%), respectively (Table 17). These observations were in
96
agreement with several earlier reports which have described an enhancement in
percent decolorization by laccase in the presence of redox mediators (Reyes et al.,
1999; Soares et al., 2001a; 2001b; Claus et al., 2002).
In order to confirm the removal of aromatic compounds from the TP treated
polluted water in the presence of redox mediators spectral analysis was also
performed (Fig. 17). The decrease in absorbance was a clear indication of dye
decolorization. The decrease in the peaks of dye/mixture was a result of either the
removal of dyes product as an insoluble complex or the breakdown of chromophoric
groups present in direct dyes. This observation was in agreement with earlier findings
that the treatment of aromatic compounds resulted in the formation of insoluble
aggregate, which could be easily removed by simple filtration, centriftigation or
sedimentation (Keimedy et al., 2002; Akhtar and Husain, 2006; Husain, 2006). Such
spectral measurements also provide strong evidences for the removal of aromatic
pollutants from wastewater.
The individual dye solutions and the mixtures of dyes exhibited a significant
loss in the TOC content when treated by TP at pH 5.5 (Tables 16, 17), which was an
important signal for the detoxification of wastewater (Kulshrestha and Husain, 2007).
Salt fractionated peroxidase from turnip (Brassica rapa) was able to catalyze
polymerization and precipitation of direct dyes and their mixtures using wide range of
reaction conditions and compounds which acted as redox mediators. The presence of
0.6 mM of HOBTA^LA drastically increased decolorization of direct dyes by TP. The
application of peroxidase that is easily available and inexpensive could be
successfully used for the treatment of wastewater contaminated with colored products.
97
(Decalorization of taj(tife
efffuent 5y Bitter gourd
perojQdase immoBiBzed on
concanavafin A (hyered ca[dum
afginate-starcH Beads
5.1. INTRODUCTION
Recently bioaffinity based procedures for enzyme immobilization have gained
much attention over the other known classical methods, as this method can be
exploited for the direct immobilization of enzymes from partially purified preparation
or even from crude homogenates (Kulshrestha and Husain, 2006a; Khan and Husain,
2007). Surface immobilized enzymes are far more superior to entrapped enzymes as
in the latter case the diffusion of large molecular size products from inside the gel
beads is difficult (Le-Tien et al., 2004). In order to prevent the possibility of
accumulation of products inside polymeric matrices, immobilization of enzymes on
the surface of support will be a preferred choice.
In this study BGP has been immobilized on the surface of Con A layered
calcium alginate-starch beads and this preparation was used for the decolorization of
colored textile effluent. This study describes the optimization of various experimental
conditions for the effective removal of colored compounds from the textile effluent by
immobilized BGP (I-BGP). Immobilized BGP has also been used successfully in
batch process as well as in continuous reactor for the remediation of colored toxic
pollutants from industrial effiuent.
5.2. MATERIALS AND METHODS
5.2.1. Materials
Violuric acid was purchased from Fluka Chemicals, Austria. Sodium alginate
was the product of Koch-Light, England. Glutaraldehyde and ethanolamine were
obtained from Sigma Chemical Co. (St. Louis, MO) USA. Redox mediators; 1-
hydroxybenzotriazole, syingaldehyde, vanillin and veratryl alcohol were all procured
from SRL Chemicals Pvt. Ltd. Mumbai, India. The untreated textile industrial effluent
was obtained from cotton textile industry located in Sector 7, Noida, U.P, India. Jack
bean meal was procured from DIFCO, Detroit, USA. Bitter gourd was purchased
from local vegetable market. Other chemicals and reagents employed were of
analytical grade and were used as supplied.
98
5.2.2. Ammonium sulphate fractionation of bitter gourd proteins
The salt fractionated bitter gourd proteins were obtained from the procedure
described in Chapter III, Section 3.2.2.
5.2.3. Measurement of peroxidase activity
Peroxidase activity was estimated in 100 mM sodium acetate buffer, pH 5.0 at
37 °C (Chapter II, Section 2.2.6).
5.2.4. Immobilization of BGP on the surface of Con A layered calcium alginate-
starch beads
Jack bean extract (10%, w/v) was prepared as described in Chapter II, Section
2.2.3. Immobilization of BGP (4900 U) on Con A layered calcium alginate-starch
beads was done by the procedure described in Chapter 111, Section 3.2.5.
5.2.5. Effluent processing and dilution
The textile effluent was collected from the industrial site situated in Sector 7,
Noida, U.P, India. The effluent was centrifliged; and after centrifugation the clear
supernatant was diluted by 100 mM sodium acetate buffer, pH 5.0 till the effluent
exhibited optical density of 0.550 (approx) at 580 nm (Fig. 18). The Xmax of the
effluent was determined by using Cintra 10 e UV-visible spectrophotometer.
Textile effluent decolorization was calculated by the same procedure as
mentioned in Chapter IV, Section 4.2.5.
5.2.6. Role of redox mediators on the BGP-catalyzed decolorization of textile
industrial effluent
The effect of various redox mediators (1.0 mM) on BGP (0.28 U mL'')
catalyzed effluent decolorization was investigated in 100 mM sodium acetate buffer,
pH 5.0 in the presence of 0.72 mM H2O2 for 1 h at 37 °C. Effluent decolorization by
soluble BGP (S-BGP) was stopped by heating reaction mixture in a boiling water bath
99
for 5 min and the insoluble product was removed by centrifugation at 3000 g for 15
min. However, in the case of I-BGP treated effluent, the reaction was stopped by
removing enzyme by centrifugation. The residual effluent decolorization was
monitored at 580 nm. The percent decolorization was calculated by taking untreated
effluent as control (100%).
5.2.7. EHect of HOBT on BGP catalyzed effluent decolorization
The decolorization of textile effluent by soluble and immobilized BGP (0.28
U mL"') in 100 mM sodium acetate buffer, pH 5.0 was monitored in the presence of
varying concentrations of HOBT (0.1-1.4 mM) and 0.72 mM H2O2 for 1 h at 37 °C.
The percent decolorization was calculated by taking untreated effluent as control
(100%).
5.2.8. Effect of BGP concentration on the decolorization of textile effluent
Textile industrial effluent was treated with increasing concentrations of
soluble and immobilized BGP (0.08-0.32 U mL"') in 100 mM sodium acetate buffer,
pH 5.0 under the other experimental conditions as described in Section 5.2.6.
Untreated effluent was used as control (100%) for the calculation of percent
decolorization.
5.2.9. Effect of pH and temperature on the decolorization of effluent by BGP
The decolorization of textile effluent by soluble and immobilized BGP (0.28
U mL'') was investigated in the buffers of varying pH (2.0-10.0) in the presence of
1.0 mM HOBT and 0.72 mM H2O2 for 1 h at 37 °C. The molarity of each buffer was
100 mM. Untreated effluent in each buffer was considered as control (100%) for the
calculation of percent decolorization.
The effect of temperature on the decolorization of textile industrial effluent by
soluble and immobilized BGP (0.28 U mL"') was investigated at various temperatures
(20-80 °C) in the presence of 1.0 mM HOBT and 0.72 mM H2O2 for 1 h. Treated
effluent exhibiting maximum decolorization at optimal temperature was considered as
control (100%) for the calculation of percent decolorization.
100
5.2.10. Effluent decolorization in batch processes
Industrial effluent (250 mL) was treated with soluble and immobilized BGP
(37.5 U) in batch processes for varying times at 37 °C under the experimental
conditions mentioned in Section 5.2.6.
5.2.11. Effluent decolorization reusability of immobilized BGP
The textile effluent (5.0 mL) was incubated with immobilized BGP (1.4 U) for
1 h at 37 °C under similar experimental conditions described in Section 5.2.6. After
the completion of reaction, enzyme was separated by centrifugation and stored in
assay buffer for over 12 h at 4 °C. The similar experiment was repeated 8 times with
the same preparation of immobilized BGP and each time with a fresh batch of diluted
effluent. Effluent decolorization was monitored at specific wavelength maxima of the
industrial effluent, 580 nm. The percent decolorization was calculated by taking
untreated effluent as control (100%).
5.2.12. Continuous treatment of effluent by using two-reactor system
A two-reactor system was developed for the continuous removal of colored
compounds from the textile effluent. The first column (10.0 x 2.0 cm) was filled with
immobilized BGP (1162 U) connected to another column filled with activated silica
(10.0 X 2.0 cm). Activated silica was prepared by the procedure described in Chapter
IV, Section 4.2.12. The colored effluent (O.D. 0.550) in the presence of 1.0 mM
HOBT and 0.72 mM H2O2 at room temperature was passed through the reactor
containing immobilized enzyme. Second column received the effluent treated by
enzyme in the first reactor; this activated silica column adsorbed most of the oxidized
colored compounds. The flow rate of the reactor was maintained at 16 mL h"'. After a
gap of 10 d, the samples of treated effluent from the second column were collected,
centrifuged and their spectrophotometric analysis was done. The collection and
analysis of textile sample from the continuous two-reactor system was continued for
over 2 months.
101
A parallel two-reactor system was operated in which the first column
contained calcium alginate-starch beads without enzyme and the second column was
filled with activated silica. The effluent was passed through the continuous two-
reactor system and samples were collected after a gap of 10 d for over 1 month and
finally samples were centrifuged and analyzed.
5.2.13. UV-visible spectra
BGP treated textile effluent samples collected from the two-reactor system
were followed by UV-visible spectrophotometric analysis on Cintra lOe UV-visible
spectrophotometer. Spectral profiles fi-om 200-700 nm were recorded to measure the
progress of effluent decolorization.
5.3. RESULTS
The collected industrial effluent was suitably diluted and its absorption
spectrum was recorded. The spectrum of effluent exhibited maximum absorption at
580 nm (Fig. 18). All the effluent decolorization measurements were carried at this
wavelength. The effluent was found to be stable upon exposure to alone H2O2,
enzyme, HOBT, calcium alginate-starch beads and activated silica. It was also
recalcitrant to the combined action of BGP and H2O2. Thus the effluent decolorization
was due to combined action of BGP, H2O2 and a redox mediator, HOBT.
5.3.1. EfHuent decolorization by BGP in the presence of various redox mediators
Fig. 19 demonstrates the effect of various redox mediators on BGP mediated
decolorization of effluent. Among the redox mediators used for the effluent
decolorization, HOBT showed highest decolorization 28% and 70% in the presence of
soluble and immobilized BGP, respectively. However, syringaldehye, VA, VLA and
VN mediated less decolorization as compared to HOBT.
102
1—I—I—1 I I I I I — I — I — I — I — I — I — I — I — r 200.0 29D.0 3000 350.0 «0.0 450.0 900.0 550.0 600.0 6900 7000
Wavelength (nm)
Fig. 18: UV-visible spectra for textile effluent
The textile effluent obtained from the industrial site was centrifuged and
clear supernatant was diluted with 100 mM sodium acetate buffer, pH 5.0.
The optical density and maximum wavelength of the textile effluent was
approxiately 0.550 and 580 nm, respectively.
103
100
80
DS-BGP
l l -BGP
HOBT rninsiildrfi*?* W Redox Mediator
VA MA
Fig. 19: Treatment of textile effluent by BGP in the presence of various redox
mediators
Decolorization of effluent was catalyzed by BGP (0.28 U mL"') In the
presence of various redox mediators (1.0 mM). The diluted effluent (5.0 mL)
was incubated in the presence of 0.72 mM H2O2 for 1 h at 37 °C.
104
5.3.2. Effect of increasing concentration of HOBT
The effluent decolorization by BGP in the presence of varying concentrations
of HOBT (0.1-1.4 mM) has been described in Fig. 20. There was an enhancement in
the effluent decolorization upto 1.0 mM HOBT. After this concentration there was a
marginal increase in effluent decolorization. Soluble and immobilized BGP could
decolorize 28% and 70% color from effluent in the presence of 1.0 mM HOBT,
respectively.
5.3.3. Effect of enzyme concentration
The effect of enzyme concentrations was studied to determine the minimum
amount of enzyme required for maximum decolorization. The rate of effluent
decolorization increased from 0.08-0.28 U mL"' BGP. However, the decolorization of
effluent remained almost constant after 0.28 U mL'' of BGP. Effluent was decolorized
to 28% and 70% by soluble and immobilized BGP (0.28 U mL"'), respectively (Table
18).
5.3.4. Effect of pH
Table 19 shows effluent decolorization by BGP in buffers of various pH (2.0-
10.0). About 28% and 70% of the colored effluent was decolorized by soluble and
immobilized BGP at pH 5.0, respectively. Subsequently, the effluent removal dropped
significantly at pH 6.0 and onwards.
5.3.5. Effect of temperature
The influence of temperature on the decolorization of textile industrial effluent
was evaluated by treating effluent at various temperatures by soluble and immobilized
BGP (Table 20). The textile effluent was maximally decolorized 28% and 70% in the
presence of soluble and immobilized BGP at 40 °C, respectively. However, above
optimum temperature effluent decolorization was further decreased.
105
100
0.1 0.2 0.4 0.6 0.8 1 HOBT(mM)
1.2 1.4
Fig. 20: £ffect of HOBT concentration on BGP-catalyzed effluent decolorization
The effluent (5.0 mL) was incubated with varying concentrations of HOBT
(0.1-1.4 mM) in the presence of BGP (0.28 U mL-'), 0.72 mM of H2O2 in
100 mM sodium acetate buffer pH 5.0 for 1 h at 37 °C.
106
Table 18: Effect of BGP concentration on efHuent decolorization
Enzyme
concentration
(U mU')
0.08
0.12
0.16
0.20
0.24
0.28
0.32
Decolorization (%)
S-BGP
11
18
22
24
26
28
28
I-BGP
31
38
46
53
61
70
70
Textile effluent (5.0 mL) was treated with increasing concentrations of BGP (0.08-
0.32 U mL"') for 1 h in the presence of 1.0 mM HOBT and 0.72 mM H2O2 in 100 mM
sodium acetate buffer, pH 5.0 at 37 °C. Each value represents the mean for three
independent experiments performed in duplicates, with average standard deviation, <
5%.
107
Table 19: Effect of pH on BGP-catalyzed decolorization of textile effluent
pH
2
3
4
5
6
7
8
9
10
Decolorization (%)
S-BGP
15
18
22
28
17
15
12
9
8
I-BGP
46
52
59
70
52
48
41
36
29
Textile effluent (5.0 mL) was treated by BGP (0.28 U mL"') in the buffers of various
pH (2.0-10.0) in the presence of 1.0 mM HOBT and 0.72 mM H 2O2 for 1 h at 37 °C.
Each value represents the mean for three independent experiments performed in
duplicates, with average standard deviation, < 5%.
108
Table 20: Effect of temperature on BGP-catalyzed decolorization of textile
effluent
Temperature
rc)
20
30
40
50
60
70
80
Decolorization (%)
S-BGP
10
16
28
19
11
7
5
I-BGP
49
54
70
59
50
44
32
Textile effluent was incubated with BGP (0.28 U mL"') in 100 mM of sodium acetate
buffer, pH 5.0 in the presence of 0.72 mM H2O2 and 1.0 mM HOBT at 20-80 "C for 1
h. Each value represents the mean for three independent experiments performed in
duplicates, with average standard deviation, < 5%.
109
5.3.6. Decolorization of effluent in batch processes by BGP
Table 21 depicts the'effluent decolorization by BGP in batch processes. It was
observed that after 150 min of incubation, immobilized BGP decolorized more than
90% textile effluent. However, the decolorization of effluent by soluble BGP was
only 48% under similar incubation period. Immobilized BGP showed its superiority
over the soluble enzyme in the removal of color from the industrial effluent on long
incubation.
5.3.7. Effluent decolorization reusability of immobilized BGP
In order to consider an immobilized BGP for its use in a reactor, it is
necessary to investigate the reusability of immobilized enzyme. The effluent
decolorization reusability of immobilized BGP was continuously decreased on its
repeated use (Fig. 21). Immobilized BGP retained 59% effluent decolorization
capacity even after its 8* repeated use.
5.3.8. Continuous treatment of effluent through a two-reactor system
The schematic diagram for the decolorization of textile effluent by
immobilized enzyme in a two-reactor system has been shown in Fig. 22. Immobilized
enzyme could remove more than 90% colored compounds from the effluent during
initial 10 d of operation. However, the effluent decolorization efficiency of the reactor
was gradually decreased. Immobilized BGP present in two-reactor system was
capable of removing 40% colored compounds from the textile effluent even after 60 d
of its operation (Fig. 23).
The textile effluent was passed through a parallel continuous two-reactor
system (without immobilized BGP) in order to check the adsorption of colored
compounds from effluent on calcium alginate-starch beads as well as on activated
silica. There was no change in the optical density of the textile effluent after passing
continuously through the two-reactor system.
110
Table 21: Decolorization of textile effluent by BGP in batch processes
Time
(min)
30
60
90
120
150
180
210
240
Decolorization (%)
S-BGP
22
28
33
41
48
48
48
48
I-BGP
51
62
70
79
86
93
93
93
Textile effluent was incubated with BGP (37.5 U) in 250 mL of 100 mM
sodium acetate buffer, pH 5.0 in the presence of 0.72 mM H2O2 and 1.0 mM
HOBT for varying times. Each value represents the mean for three
independent experiments performed in duplicates, with average standard
deviation, < 5%.
Il l
c
a N 'll 0 0 u V Q
• l-BGP
3 4 5 6 Number of uses
Fig. 21: Textile effluent decolorization reusability of immobilized BGP
Immobilized BGP was incubated with textile effluent for 1 h at 37 °C in
triplicates. Decolorization of textile effluent was determined after incubation
period. After the completion of the reaction, the immobilized enzyme was
collected by centrifugation and stored in assay buffer at 4 °C overnight. Next
day, the similar experiment was repeated. This procedure was repeated for 8
successive days.
112
^
m Effluent
^ s
Immobilized BGP
Activated silica
S Treated effluent
Fig. 22: Schematic representation of a two-reactor system used for decolorization
and removal of colored compounds from textile effluent
A column (10.0 x 2.0 cm) filled with immobilized BGP (1162 U) was
connected to a second column containing activated silica. Textile effluent
having O.D. approximately 0.550 was continuously passed through the
reactor in the presence of 1.0 mM HOBT and 0.72 mM H2O2 for a period of
60 d. Effluent removal in two-reactor system was done under the similar
experimental conditions as described in Section 5.2.12.
113
100 1
^ 80-
E 1 ^° •
0 40-
0
° 20-
0 •
M 1 1 1 IM-10
1 1 X 20
•
111 30 40 50
Number of Days
60
• l-BGP
Fig. 23: Evaluation of decolorized textile effluent collected from two-reactor
system
Diluted effluent was treated in a continuous two-reactor system as described in
the Section 5.2.12. Samples were collected after a gap of 10 d for over 2
month from the second column (containing activated silica) of the two-reactor
system. The collected samples were centrifuged and analyzed
spectrophotometrically for the presence of remaining color.
114
5.3.9. Spectral analysis of treated effluent
UV-visible spectrum of textile effluent before and after enzymatic treatment in
two-reactor system is shown in Fig. 24. The decrease in absorbance peaks in UV-
visible regions with respect to the number of days of operation showed remarkable
variation in the absorbance at various wavelengths and thus removal of the colored
pollutants from textile effluent treated by immobilized BGP.
5.4. DISCUSSION
BGP has earlier been employed for the decolorization of a number of textile
dyes in synthetic solutions (Akhtar et al., 2005a; 2005b). To the best of our
knowledge, BGP has not ever been used for the decolorization/removal of aromatic
compounds present in industrial effluents. For the first time an effort has been made
to treat industrial effluent by BGP immobilized on the surface of Con A layered
calcium alginate-starch beads.
It was observed that the diluted effluent was recalcitrant to the action of alone
H2O2, enzyme, HOBT, calcium alginate-starch beads and activated silica. Thus, the
effluent decolorization was a result of redox mediated H202-dependent enzymatic
action, possibly involving free-radical formation followed by the adsorption of the
activated compounds on silica. Several earlier workers have also reported the removal
of peroxidase treated aromatic pollutants by adsorbing to an adsorbent (Kinsley and
Nicell, 2000; Tonegawa et al., 2003).
Some of the chemicals serving as redox mediators facilitate dye degrading
activity of enzymes and enhance their specificity to a wide range of dyes (Reyes et al.,
1999; Soares et al., 2001b; Husain and Husain, 2008). Among the redox mediators,
those presenting the >NOH moiety (HOBT, N-hydroxyphthalimide, VLA) have been
proved to be very efficient towards benzylic substrates through a radical H-abstraction
route of oxidation involving the aminoxyl radical (>N-0) intermediate (d'Acunzo et
al., 2006).
HOBT, a potential redox mediator plays a critical role in enhancing the rate of
enzyme mediated dye/effluent degradation, bleaching of pulp and other environmental
pollutants (Garcia et al., 2003). Here we have found that, HOBT had an important role
115
1 r 200.0 250.0 300D 350.0
1—I—I—r—I—r 400.0 450.0 500.0 550.0
Wavelength (nm)
1—I—r 600.0 6SDD 700D
Fig. 24: UV-visible spectra of the textile effluent
Absorption spectrum of industrial effluent treated in a two-reactor system
was recorded on UV-visible spectrophotometer Cintra \Qe. Absorption
spectra of effluent were taken before and after treatment by I-BGP. Spectra
in the figure are labeled.
116
in the decolorization of industrial effluent as compared to other redox mediators (Fig.
19), HOBT satisfied all the three properties of a redox mediator for the decolorization
of effluent; (i) it is oxidized by one-electron directly by the action of enzyme to
produce free radical, (ii) it produces radical which is stable enough to diffuse and
react with the target compound and (iii) it has an appropriate redox potential (Tinoco
et al., 2007).
The schematic representation of enzymatic oxidation of recalcitrant substrate
(Sub-H) by means of peroxidase and a >NO-H containing redox mediator (HOBT) is
given below;
H 2 0 2 | | ^ ^ g | Peroxidase ^ ^ ^ ^ >N0'' ^ ^ ^ ^ Sub-H
H2O Peroxidaseox >NO-H Sub
The concentration of HOBT is an important factor for the enzyme catalyzed
decolorization/degradation of aromatic compounds (Murugesan et al., 2007). In most
of the earlier reports, mediators are used at a very high concentration in the range of 6
to 57 mM (Kumiawati and Nicell, 2007). However, some earlier studies indicated a
significant laccase inactivation by 10 mM HOBTA' LA (Li et al., 1999). In the present
study, 1.0 mM HOBT was sufficient to mediate BGP catalyzed maximum effluent
decolorization, 70% (Fig. 20).
The optimization of enzyme concentration was carried to aim at high
efficiency of effluent decolorization by BGP and 0.28 U mL"' enzyme was sufficient
for maximum decolorization (Table 18). A similar observation has been reported in an
earlier study in which HRP (2.985-29.85 U mL"') was evaluated for the decolorization
of Remazol Turquoise G 133%. When the concentration of HRP was 14.985 U mL"',
the decolorization of the dye was 58%. However, when the concentration of HRP was
doubled, the decolorization of dye was increased only by 4%. On the basis of such
results it can be concluded that by using higher concentration of enzyme,
decolorization was not significantly influenced (Ulson de Souza et al., 2007).
Most enzymes have a characteristic pH at which their activity is maximum.
The immobilized BGP showed high effluent decolorization at pH 5.0 (Table 19). This
study was in accordance with an earlier report in which the decolorization of reactive
117
textile dye by soluble and immobilized BGP was maximum in the buffers of acidic
pH (Akhtar et al., 2005b). The decoiorization of textile effluent by BGP was optimum
at 40 °C (Table 20). This is in line with the work demonstrated by earlier investigators
that the decoiorization of acid/reactive dyes by plant peroxidases was also maximum
at 40 °C (Akhtar et al., 2005a; 2005b; Kulshrestha and Husain, 2007).
It has been previously shown that immobilizing bioactive agents (catalase and
a bacteriocin) on the surface of calcium alginate beads offer greater stability against
various experimental parameters as compared to the free enzyme (Le-Tien et al.,
2004). A similar pattern has been observed in this study as the surface immobilized
BGP was remarkably more efficient in decolorizing effluent (90%) as compared to
48% color removal by soluble enzyme within 3 h in a stirred batch process (Table 21).
The advantage of immobilized enzymes does not lie only in increasing the
stability but also in its reusability. I-BGP retained remarkably very high effluent
decoiorization activity (59%) even during its 8* repeated use (Fig. 21). The activity
loss of the surface immobilized BGP preparation during 8* repeated use in effluent
decoiorization was appreciably much higher as compared to 50% acid dye removal by
polyacrylamide gel entrapped HRP only after its 5* repeated reuse (Mohan et al.,
2005). Our observations have suggested that surface immobilized enzyme has more
advantages in removing higher color from industrial effluent.
Considering the success of *surface immobilized BGP in effluent
decoiorization, next step was to evaluate its efficiency at large scale by using a
continuous two-reactor system (Fig. 22). The two-reactor system used in this study
was able to operate continuously and could decolorize textile effluent without any
operational problems. An enhancement in decolorization/degaradation of effluent by
immobilized enzyme was probably due to increased adsorption of oxidized colored
compounds/products on adsorbent, activated silica (Fig. 23). Kim and Nicell (2006)
have shown that bisphenol A oxidation by laccase was significantly enhanced by the
addition of polyethylene glycol because its product got easily adsorbed on
polyethylene glycol.
The decoiorization and remediation of textile effluent by immobilized BGP in
a two-reactor system was further strengthened by UV-visible spectral analysis (Fig.
24). The significant loss of color in UV-visible region with respect to number of days
of operation was attributed to the formation of free radical compounds which got
adsorbed on the activated silica present in the second column. This indicated the
118
suitability of the two-reactor system to treat huge volume of effluents from the textile
industries. In addition, continuous decolorization of dyes has been scarcely
investigated especially dealing with oxidative enzyme (Lopez et al., 2002; Mielgo et
al., 2002; Selvam et al., 2003). Thus it pointed out the relevance as well as the novelty
of the results obtained in the present work.
Here, we have demonstrated that BGP immobilized on the surface of calcium
alginate-starch beads was suitable candidate for wastewater treatment, since it was
used efficiently for the decolorization and removal of colored compounds from textile
effluent in batch process as well as in a continuous two-reactor system. Indeed the
described system was developed with a cheap biocatalyst that was efficient at low
concentration. Thus this work may provide a reasonable basis for development of an
effective biotechnological process for the removal of colored pollutants from the
textile effluents.
119
¥ t
Oecoforization of direct dyes By
immoSifized turnip peroj(idase
in Bated and continuom
processes
6.1. INTRODUCTION
Several methods have been used for the immobilization of peroxidases from
various sources but most of these either use commercially available enzyme or
expensive supports, which increase the cost of the processes (Norouzian, 2003;
Husain, 2006). Such type of immobilized enzymes cannot fulfill the requirements for
the treatment of hazardous compounds. Among, the known techniques used for the
immobilization of enzymes, physical adsorption on the basis of bioaffinity is useful
as this process can immobilize enzymes directly from the crude homogenate and thus
avoids the high cost of purification. The ease of immobilization, lack of chemical
modification usually accompanied with enhanced stability, are some of the
advantages offered by the bioaffinity based procedures (Akhtar et al., 2005a; 2005c;
Kulshrestha and Husain, 2006a). Besides the mentioned advantages offered by the
bioaffinity based procedures, there is an additional benefit such as the enzymes get
properly oriented on the supports (Mislovicova et al., 2000; Khan et al., 2005). Thus
supports provide high yield and stable immobilization of glycoenzymes/enzymes.
In this part of work an effort has been made to find a cheaper and an easily
available alternative to the commercial enzymes, its immobilization/utilization at
large scale. Wood shaving (WS) has been used as support because of it is low cost
and easy availability. Jack bean extract has been employed as a source of Con A for
the preparation of bioaffinity support, Con A-WS. Con A-WS was employed for the
immobilization of TP directly from salt fractionated turnip proteins. Con A-WS
bound TP (I-TP) was compared with its fi-ee form for the decolorization of a direct
dye and mixture of direct dyes in batch processes as well as in continuous reactors
under various experimental conditions.
6.2. MATERIALS AND METHODS
6.2.1. Materials
Direct dyes; Direct Red 23, Direct Red 239 (DR 239) and Direct Blue 80 were
a gift from Atul Pvt. Ltd. Gujarat, India. HOBT was purchased from SRL Chemicals
Pvt. Ltd. Mumbai, India, o-dianisidine HCl was obtained from Centre for
120
Biochemical Technology, CSIR, India. Jack bean meal was piorchased from DIFCO,
Detroit, USA. Turnip roots and wood shavings were collected from the local market.
The chemicals and other reagents employed were of analytical grade and were used
without any further purification.
6.2.2. Ammonium sulphate fractionation of turnip proteins
Proteins were fractionated from the buffer extract of turnip (pH 5.0) by using
ammonium sulphate (Chapter II, Section 2.2.2).
6.2.3. Preparation of Con A-WS complex
Jack bean extract (10%) was prepared in five batches as explained in Chapter
II, Section 2.2.3. Three different types of Con A-WS preparations were made in
several batches. WS was taken as 10 mg, 100 mg and 6.0 g and mixed with 0.5 mL,
4.0 mL and 240 mL jack bean extract respectively, and incubated overnight at 4 °C
imder stirring conditions. Con A-WS complex was collected fi-om all the three
preparations by centrifiigation at 3000 g for 15 min at room temperature and washed
thrice with 100 mM sodium phosphate buffer, pH 6.2.
6.2.4. Immobilization of TP on Con A-WS
Con A bound WS; 10 mg, 100 mg and 6.0 g were incubated with 10.46 U,
104.6 U and 6276 U of TP in the total volume of 0.5 mL, 4.0 mL and 50 mL sodium
phosphate buffer, pH 6.2. The mixtures were incubated at 37 °C for 12 h. Each
immobilized preparation was collected by centrifiigation at 3000 g for 15 min at room
temperature and washed thrice with sodium phosphate buffer, pH 6.2 to remove
unbound proteins.
Con A-WS bound TP preparations; 10 mg, 100 mg and 6.0 g were used for
assaying the activity, decolorization of dyes in batch processes and decolorization of
dyes in continuous reactors, respectively.
191
6.2.5. Preparation of synthetic dye solutions
The synthetic solutions of direct dyes (50-100 mg L' ) and a mixture of direct
dyes consisting of DR 23, DR 239, DB 80 were prepared in sodium acetate buffer, pH
5.0 by the procedure described in Chapter IV, Section 4.2.4.
6.2.6. Calculation of percent dye decolorlzation
The decolorlzation for DR 23 or a mixture of direct dyes was calculated by the
procedure mentioned in Chapter IV, Section 4.2.5.
6.2.7. Decolorlzation of dye solution by soluble and immobilized peroxidase in
batch processes
The dye solution (250 mL) was treated with soluble (S-TP) and immobilized
turnip peroxidase (22.7 U) in 100 mM sodium acetate buffer, pH 5.0 in the presence
of 0.6 mM HOBT and 0.72 mM H2O2 for 1 h at 30 °C. The reaction was stopped by
the same procedure described in Chapter IV, Section 4.2.4. The residual dye solution
was measured spectrophotometrically at specific wavelength maxima of the
dye/mixture of dyes. The absorbance at 505 nm for DR 23, and 515 nm for mixture of
direct dyes was recorded. Untreated dye solution was considered as control (100%)
for the calculation of percent decolorlzation.
6.2.8. Effect of pH and temperature on the decolorlzation of dyes by TP
The decolorlzation of DR 23 and mixtvire of direct dyes was also carried out in
the buffers of varying pH (3.0-10.0) for 1 h under the other assay conditions as
mentioned in Section 6.2.7. The molarity of each buffer was 100 mM.
The effect of temperature on the TP catalyzed decolorlzation of dyes was also
performed at temperatures (20-80 °C). The dye or mixture of dyes (250 mL) was
treated with TP (22.7 U) at various temperatures in the presence of 0.6 mM HOBT
and 0.72 mM H2O2 for 1 h.
199
6.2.9. Decolorization of dyes in the presence of salt and heavy metals
The decolorization of dye solutions was independently conducted in the
presence of 500 mM NaCl, 0.1 mM ZnCb, 0.3 mM CdCh for 1 h under other
experimental conditions as mentioned in Section 6.2.7. The dye solution without any
treatment was considered as control (100%) for the calculation of percent
decolorization.
6.2.10. Effect of water-miscible organic solvent on dye decolorization
The effect of dioxane; 10%, 30% and 60% (v/v) on the TP mediated
decolorization of dye was investigated under the similar experimental conditions as
mentioned in Section 6.2.7. The untreated dye solution was considered as control
(100%) for the calculation of percent decolorization.
6.2.11. Reusability of immobilized TP in the decolorization of direct dyes
DR 23 and mixture of dyes were independently incubated with immobilized
TP for 1 h under the experimental conditions as mentioned in Section 6.2.7. The
enzyme was separated by centrifugation and stored in the assay buffer for over 12 h.
The similar experiment was repeated 8 times with the same I-TP preparation and each
time with a fresh batch of dye solution. Dye decolorization was monitored at specific
wavelength maxima of the dye solutions. The percent decolorization was calculated
by taking imtreated dye or mixture of dyes as control (100%).
6.2.12. Continuous dye decolorization using a two-reactor system
A two-reactor system was developed for the continuous removal of dyes from
solutions as shown in Fig. 22. A column (10 x 2.0 cm) was filled with 6.0 g Con A-
WS bound TP (1362 U) connected to second column (10 x 2.0 cm) containing
activated silica. Activated silica was prepared by the same procedure described in
Chapter IV, Section 4.2.12. The flow rate was maintained at 7.2 mL h'' and the feed
solution contained DR 23 and mixture of dyes in two independent reactor systems,
respectively. Dye solutions were run under the similar experimental conditions as
123
mentioned in Section 6.2.7. Reactors were operated at room temperature for a period
of 4 and 3 months for DR 23 and mixture of dyes, respectively. Dye solution treated
in first column by I-TP passed to the next column where it got adsorbed on the
activated silica. The samples from the second column were collected at an interval of
15 d and the absorbance of each sample was measured at their respective wavelength
maxima.
6.2.13. Data collection and analysis
To examine the ability of immobilized TP to decolorize solution containing
dye/mixture of dyes, spectra were taken for the samples collected from continuous
reactor using Cintra 10 e UV-visible spectrophotometer. Decrease in absorbance, at
Xmax of the dye solutions and spectral profile from 400-700 nm were recorded to
measure the progress of decolorization.
Procedure for the dye decolorization from the continuous reactor was followed
by centrifiigation and clear diluted solution was considered for TOC determination.
TOC was determined by using a TOC analyzer (Multi N/C 2000, Analytic Jena,
Germany). Each control and treated DR 23 or mixture of dyes was diluted 20 fold
before measuring their TOC content.
6.3. RESULTS
6.3.1. Preparation of bioaffinity support and immobilization of TF
WS (1.0 g) adsorbed nearly 22 mg Con A from jack bean extract. Con A-WS,
bioaffmity support has been used for the direct immobilization of glycoenzymes from
ammonium sulphate fractionated and dialyzed turnip proteins. Some isoenzymes of
tumip peroxidases are glycosylated in nature (Duarte-Vazquez et al., 2003b). In view
of the glycoproteinic nature, tumip peroxidases can be adsorbed on Con A-WS from
ammonium sulphate fractionated proteins. Con A-WS adsorbed 227 U of peroxidase
per g of the matrix (Table 22). The effectiveness factor 'ri' of the immobilized
enzyme preparation was 0.67 (Table 22). High effectiveness factor for immobilized
TP suggested that immobilized preparation was quite effective in catalysis.
MA
Table 22: Immobilization of TP on Con A-WS
Enzyme
loaded
(X)
(U)
1046
Enzyme
activity
in
washes
(Y)
(U)
708
Activity bound g' of Con A-WS
(U)
Theoretical
(X-Y=A)
(A)
338
Actual
(B)
227
Effectiveness
factor
(n) (B/A)
0.67
% Activity
yield
(B/AxlOO)
67
The effectiveness factor of an immobilized enzyme is a measure of internal diffusion
and reflects the efficiency of the immobilization procedure. Each value represents the
mean for three independent experiments performed in duplicates, with average
standard deviation, < 5%.
19'^
6.3.2. Effect of pH
The pH optimization was done by measuring the initial rate of enzymatic dye
decolorization in batch processes in the buffers of different pH. The decolorization of
DR 23 and mixture of dyes by TP was maximum at pH 5.0. At pH greater than 5.0,
the rate of dye decolorization by S-TP was very low as compared to I-TP (Fig. 25).
6.3.3. Effect of Temperature
Decolorization of DR 23 and mixture of direct dyes by soluble and
immobilized TP was maximum at 30 °C (Fig. 26). However, immobilized enzyme
preparation decolorized higher percent of color from the dye solutions at temperatures
lower and higher than the temperature-optima, 30 °C.
6.3.4. Effect of salt/heavy metals on TP mediated dye decolorization
The effect of 500 mM NaCl on the decolorization of dyes by TP has been
illustrated in Table 23. I-TP decolorized 88% and 72% DR 23 and mixture of dyes
after 1 h of incubation, respectively. However, immobilized TP was more effective as
compared to its soluble counterpart in the decolorization of both DR 23 and a mixture
of dyes.
Table 24 demonstrates the effect of two different salts of heavy metals on the
decolorization of DR 23 and mixture of direct dyes by soluble and immobilized TP. I-
TP decolorized 86% and 73% of color from DR 23 and mixture of direct dyes even in
the presence of 0.1 mM ZnCb after 1 h of incubation respectively. On the other hand,
soluble enzyme decolorized 77% and 60% of color under above mentioned
experimental conditions.
In the case of CdCb, a peculiar feature was observed; there was no change in
decolorization of DR 23 by I-TP incubated for various times. Decolorization of
mixture of dyes by S-TP/I-TP in the presence of CdCl2 for various time intervals was
marginal (Table 24).
126
100 -1
c .o
o o o
Fig. 25: Effect of pH on the decolorization of direct dyes by soluble and
immobilized TP
The solution of DR 23 or mixture of dyes (250 mL) was incubated with TP
(22.7 U) in the presence of 0.6 mM HOBT, 0.72 mM H2O2 at 30 °C for 1 h
in the buffers of different pH. The molarity of each buffer was 100 mM.
Symbols indicate treatment of DR 23 by S-TP (•), DR 23 by I-TP (o),
mixture of direct dyes by S-TP (T) and mixture of direct dyes by I-TP (V).
197
100
80 -
c 60 .O
(0 N •c o 8 40 0)
O
20
10 — I —
20 — I —
30 40 50 — I —
60 — I —
70 80
Temperature (° C)
Fig. 26: Effect of temperature on the decolorization of direct dyes by soluble and
immobilized TP
The decolorization of DR 23 and mixture of direct dyes (250 mL) by soluble
and immobilized TP was studied out at various temperatures (20-80 °C) in
the presence of 0.6 mM HOBT and 0.72 mM H2O2 for 1 h at 30 °C. Symbols
indicate treatment of DR 23 by S-TP (•), DR 23 by I-TP (o), mixtures of
direct dyes by S-TP (T) and mixture of direct dyes by I-TP (V).
128
Table 23: Effect of NaCl on TP-catalyzed dye decolorization
Time
(min)
30
60
90
120
180
Decolorization (%)
DR23
S-TP
77
77
78
78
78
I-TP
88
88
88
88
88
Mixture of
direct dyes
S-TP
66
66
65
65
65
I-TP
72
72
72
72
72
DR 23 or mixture of direct dyes (250 mL) was treated with soluble and immobilized
TP (22.7 U) in the presence of 500 mM NaCl for varying times in batch processes as
described in Section 6.2.9. Each value represents the mean for three independent
experiments performed in duplicates, with average standard deviation, < 5%.
129
Table 24: Effect of heavy metals on TP-catalyzed dye decolorization
Time
(min)
30
60
90
120
180
Decolorization (%)
O.lmMZnCb
DR23
S-TP
76
77
77
77
77
I-TP
86
86
86
86
86
Mixture of
direct dyes
S-TP
60
60
60
60
60
I-TP
72
72
73
73
73
0.3 mM CdCb
DR23
S-TP
55
60
71
71
71
I-TP
92
92
92
92
92
Mixturfe of
direct dyes
S-TP
27
29
31
31
31
I-TP
69
75
76
76
76
DR 23 or mixture of direct dyes (250 mL) was treated independently with TP (22.7 U)
in the presence of 0.1 mM ZnCl2/0.3 mM CdCb for various times in batch processes
as described in Section 6.2.9. Each value represents the mean for three independent
experiments performed in duplicates, with average standard deviation, < 5%.
no
6.3.5. Effect of organic solvent on TP mediated dye decolorization
The effect of dioxane on decolorization of DR 23 and mixture of dyes by
soluble and immobilized TP has been demonstrated in Table 25. Decolorization of
DR 23/mixture of direct dyes was decreased in the presence of increasing
concentrations of dioxane. DR 23 and mixture of direct dyes were decolorized 85%
and 50% by I-TP in the presence of 10% dioxane whereas S-TP could remove slightly
less dye color 66% and 39% under similar experimental conditions, respectively.
6.3.6. Reusability of immobilized TP with respect to dye/dyes mixture
decolorization
In order to make immobilized TP use more practical in a reactor, it was
necessary to investigate the reusability of I-TP. The reusability of I-TP in the
decolorization of direct dyes has been considered. The dye decolorization reusability
of I-TP was continuously decreased up to its 8* repeated use (Fig. 27). Immobilized
TP showed 47% DR 23 and 30% dye mixture decolorization after its S**" repeated use.
6.3.7. Dye decolorization by I-TP in a two-reactor system
The DR 23 and mixture of direct dyes decolorization performance by the two-
reactor system is shown in Table 26. I-TP decolorized 93%» and 85% of the initial
color from DR 23 and mixture of direct dyes after 15 d of its continuous operation,
respectively. Considerable color removal 64% from DR 23 and 50% from the mixture
of direct dyes was found even after 4 months and 3 months of operation of the two-
reactor system, respectively.
6.3.8. Analysis of dyes and their mixtures
In order to confirm the decolorization of DR 23/mixture of direct dyes by TP
in the presence of HOBT, some spectral analysis was also performed. Fig. 28
demonstrates the absorption spectra of treated and untreated DR 23/mixture of direct
dyes with respect to number of days of operation of the two-reactor system.
The absorbance peaks in visible region of DR 23/mixture of dyes was clear evidence
111
Table 25: Effect of dioxane on TP-catalyzed dye decolorization
Dioxane
(v/v, %)
10
30
60
Decolorization (%)
DR23
S-TP
66
15
0
I-TP
85
30
0
Mixture of
direct dye
S-TP
39
10
0
I-TP
50
25
0
DR 23 or mixture of direct dyes (250 mL) was treated with TP (22.7 U) in the
presence of increasing concentrations of dioxane in batch process as described in
Section 6.2.10. Each value represents the mean for three independent experiments
performed in duplicates, with average standard deviation, < 5%.
n o
•mlxhireofdyes
aDR23
1 2 3 4 5 6 Niimbwofuses
7 8
Fig. 27: Dye/dyes mixture decolorization reusability of immobilized TP
Immobilized TP (22.7 U) was independently incubated with DR 23 and
mixture of direct dyes (250 mL) in the presence of 0.6 mM and 0.72 mM
H2O2 at 30 °C. Dye decolorization was determined after incubation period of
1 h. After completion of reaction the immobilized enzyme was collected by
centriftigation and stored in assay buffer at 4 °C overnight. Next day, the
similar experiment was repeated and this procedure was repeated for eight
days.
1 1 - 2
Table 26: Continuous removal of color and TOC from DR 23 and a mixture of
direct dyes by I-TP in a two-reactor system
Number
of days
15
30
45
60
75
90
105
120
DR23
Decolorization
(%)
93
90
87
84
81
76
70
64
% TOC
removal
94
92
89
86
82
77
71
65
Mixture of direct dye
Decolorization
(%)
85
81
77
70
61
50
39
26
% TOC
removal
87
82
77
71
65
60
53
42
DR 23 or mixture of dyes was treated with I-TP in a two-reactor system as described
in the text. Each value represents the mean for three independent experiments
performed in duplicates, with average standard deviation, < 5%.
11/1
-| 1 1 1 1 1 1 1 1 1 1 1 r 3500 3750 4000 4250 4500 4750 5000 5250 5500 5750 6000 6250 6500 6750 7000
"Wavelength (nm)
Fig. 28: Absorption spectra of DR 23 and a mixture of direct dyes treated in a
continuous two-reactor system
DR 23 and mixture of direct dyes were treated as described in Section 6.2.12
and their absorption spectra were recorded before and after treatment by I-TP
in the presence of HOBT. Spectra were taken from the samples collected for
the treated DR 23 and mixtures of dyes (from the two reactor system) on
different days are labeled in figure. Spectra correspond to DR 23 (a) and
mixture of direct dyes (b).
regarding the removal of dyes from treated wastewaters.
TOC content of DR 23 and mixture of dyes treated by immobilized TP present
in a continuous reactor is given in Table 26. In case of DR 23, 65% of TOC was
removed after 4 months of operation of the continuous reactor. While the TOC
removal from the treated mixture of dyes was 60% after 3 months of reactor
operation.
6.4. DISCUSSION
The aim of this study was to assess the use of an inexpensive immobilized TP
for the decolorization of dyes. Con A-WS bound TP retained 67% of the original
activity (Table 22). Binding of enzyme to support pre-adsorbed with Con A by non-
covalent bonds was a successftil strategy to increas(; the yield of glycoprotein
immobilization (Kulshrestha and Husain, 2006a).
Immobilized TP catalyzed higher dye decolorization at different pH as
compared to its soluble counterpart (Fig. 25). The immobilization of TP on Con A-
WS provides an additional resistance to the enzyme against extreme conditions of pH.
The immobilized TP was quite effective in the decolorization of DR 23 and mixture
of dyes in batch processes at higher temperatures compared to the soluble TP (Fig.
26). The tolerance to elevated temperatiire was attributed to the complexing of
enzymes with lectin, which enhanced its thermal stability (Akhtar et al., 2005c;
Kulshrestha and Husain, 2006b).
Immobilized TP decolorized more than 70% dye in the presence of (500 mM)
sodium chloride for both dye solutions after 1 h of incubation (Table 23). It is
supported by an earlier observation that the magnitude of inhibition of laccases by
halides depends on the accessibility of the copper atoms and can vary between
different laccases and inhibitors (Abadulla et al., 2000).
Effluents from textile industries also contain heavy metals (Hatvani and Mecs,
2003; Einollahi et al., 2006). The decolorization of dii-ect dyes in the presence of
metal ions exhibited that the immobilized TP was significantly more efficient than S-
TP. Thus the immobilized TP could be exploited to treat aromatic pollutants present
in industrial effluents even in the presence of heavy metals (Table 24).
136
Enzymes exploited for the treatment of wastewater contaminated with
aromatic pollutants would be affected by the presence of water-miscible organic
solvents. I-TP has shown remarkable potential in the decolorization of dyes in the
presence of 10% and 30% dioxane (Table 25). It has already been shown that
immobilization of enzymes by multipoint attachment protects them ifrom denaturation
mediated by organic solvents (Kulshrestha and Husain, 2006a). Enzymatic catalysis
in organic solvents is possible if the organic solvent does not substantially disturb the
active site structure (Ryu and Dordick, 1992).
One of the important advantages of immobilized enzyme is its reusability,
which influences the cost of industrial applications (Akhtar et al., 2005a; Mohan et
al., 2005). The immobihzed TP could retain more than 40% DR 23 decolorizing
activity even after its S"* repeated use (Fig. 27).
A two-reactor system was operated for the continuous decolorization/removal
of direct dyes. The reactors were operated without any operational problem and thus
indicating high effluent removal efficiency. The performance of the two-reactor was
quite effective as it fiilfilled the following requirements; (i) limited accessibility to the
immobilized enzyme does not seem to play a role since decolorization by fi-ee enzyme
was never better than with immobilized preparation (Kandelbauer et al., 2004), (ii)
retention of the immobilized enzyme in the reactor in order to maintain an effective
operation (Lopez et al., 2002), (iii) maintenance of a good flow rate in order to ensure
efficient dye decolorization/degradation (Palmieri et al., 2005) and (iv) the
performance of the continuous reactor was improved in terms of treated dye
adsorption on activated silica in the second reactor, which helped in the adsorption of
toxic reactive species (Idris and Saed, 2003).
The treatment of DR 23 and mixture of dyes by passing through a double
reactor system provided almost water fi-ee fi-om dyes. The dyes treated by I-TP
present in the first column, got adsorbed in the second column, which contained
activated silica. Both the reactors worked for more than 90 d approximately, thus
explaining there efficiency towards dye decolorization (Table 26).
A significant loss of color in the visible region of the spectra appeared when
DR 23 or mixture of dyes was treated with Con A-WS bound TP in the presence of
redox mediator, HOBT in a continuous reactor system (Fig. 28). It has earlier been
reported that the disappearance of peak in visible region was either due to the
breakdown of chromophoric groups present in dyes or the removal of pollutants in the
form of insoluble products (Bhunia et al., 2001; Moreira et al,, 2001; Akhtar et al.,
2005b).
However, the difference in the dye decolorization of DR 23 and mixture of
dyes was attributed to the earlier observation that biodegradation of various phenolic
compounds and dyes in the form of complex mixtures was quite slow compared to
individual phenol/dye (Kahru et al., 2000; Akhtar et al., 2005b). It was probably due
to the competition between various phenols/dyes for the active site of the enzyme or
certain aromatic compounds were found to be recalcitrant or slowly transformed
substrates by the action of enzyme. These observations suggested that the industrial
effluents containing mixture of direct dyes coiald be successfully treated with Con A-
WS bound TP.
The level of TOC in case of continuous reactors was significantly decreased in
the presence of immobilized TP treated polluted water. However, I-TP treated dye
solutions exhibited great loss of TOC from the wastewater (Table 26), which
suggested that the major toxic compounds have been removed out of the treated
samples. Immobilized HRP has removed 88% of TOC from model wastewater
containing mixture of chlorophenols (Tatsumi et al., 1996). Recently, Akhtar et al.
(2005a) has reported a significant loss of TOC from polluted water containing
dyes/dye-mixtures and dyeing effluent after treatment by soluble and immobilized
BGP. These evidences strongly suggested that Con A-WS bound TP could be
successfully used for the removal of dye effluents from various textile, printing and
other industries.
We also observed that there was no physical adsorption of direct dye/mixture
of direct dyes on the support, as all the possibilities of adsorption were checked. The
dye solutions were fo\md to be stable upon exposure to Con A-WS support, H2O2,
activated silica or to enzyme alone, respectively. Thus, the dye decolorization was a
result of H202-dependent enzymatic reaction, possibly involving free-radical
formation followed by polymerization and precipitation. In batch processes the loss of
dye color was due to removal of aromatic compounds by precipitation but the
decrease in aromatic pollutants from the dye solutions in a continuous reactor was
because of the adsorption of oxidized product, free radical compound on the activated
silica present in second coliunn.
n 8
Enzymologists are searching for new advances in environmental fields, from
enzymatic bioremediation to the development of renewable methods for the
biochemical cleaning of 'wastewater'. Peroxidases in particular have been underlined
for this purpose due to their low energy requirements, operation over a wide range of
conditions and having minimal environmental impact. A tailoring tool for this
achievement is enzyme immobilization with two main benefits, enhancement of
storage/operational stability and reusability. Meeting the demand for "green
biotechnology", turnip and bitter gourd peroxidase have enormous potential for
replacing conventional wastewater treatment processes.
The present study aimed to work out an inexpensive, simple and high yield
procedure for the immobilization of turnip peroxidase, which would be of extreme
interest in the remediation of several types of aromatic compounds present in polluted
water. Turnip peroxidase was fractionated from the buffer extract by 10-90%
ammonium sulphate and was insolubilized by using jack bean extract, a source of
concanavalin A. Soluble and concanavalin A complex of turnip peroxidase were
entrapped into calcium alginate-pectin beads and these entrapped enzyme
preparations retained 52% and 63% of the original activity, respectively. The stability
against various denaturing agents is an important factor when selecting an appropriate
enzymatic system for any application. Calcium alginate-pectin entrapped
concanavalin A-tumip peroxidase showed impressive gains in resistance to
inactivation induced by pH, heat, urea, detergents and organic solvents. The exposure
of soluble and immobilized turnip peroxidase to low concentrations of detergents;
Sodium dodecyl sulphate and Surf Excel caused activation in enzyme activity.
However, the immobilized enzyme preparations exhibited further activation in turnip
peroxidase activity at higher concentrations of detergent as compared to soluble
counterpart.
Another promising plant peroxidase involved in environmental application is
bitter gourd peroxidase. In order to increase its potential use, calcium alginate-starch
hybrid gel was employed as an enzyme carrier both for surface immobilization and
entrapment of bitter gourd peroxidase. The insoluble concanavalin A-bitter gourd
peroxidase complex retained 70% of the initial activity. In order to prevent the
dissociation of concanavalin A-bitter gourd peroxidase complex, this preparation was
crosslinked by 0.5% glutaraldehyde. Entrapped crosslinked concanavalin A-bitter
gourd peroxidase retained 52% of the initial activity while surface immobilized and
139
glutaraldehyde crosslinked enzyme showed 63% activity. Crosslinking of bitter gourd
peroxidase resulted in small loss of enzyme activity but glutaraldehyde based
chemistry is an effective method for increasing mechanical and operational support of
enzymes.
A comparative stability of both forms of immobilized bitter gourd peroxidase
was investigated against pH, temperature, chaotropic agent; like urea, heavy metals,
water-miscible organic solvents, detergent and inhibitors. The pH and temperature-
optima for both immobilized preparations was the same as for soluble counterpart at
pH 5.0 and 40 °C. Entrapped bitter gourd peroxidase was remarkably more stable than
surface immobilized and soluble peroxidase when incubated for different times at 60
°C. Entrapped peroxidase was significantly more stable as compared to surface
immobilized enzyme followed by soluble form of enzyme under various physical and
chemical denaturing conditions. Enzyme reuse provides a number of cost effective
advantages that are often an essential pre-requisite for establishing an economically
viable enzyme catalyzed process. Entrapped crosslinked concanavalin A-bitter gourd
peroxidase showed 75% of the initial activity while the surface immobilized and
crosslinked bitter gourd peroxidase retained 69% activity after their seventh repeated
uses.
The ability of partially purified peroxidases to treat direct dyes used in textile
industries was also reviewed. Dye solutions (50-100 mg L"') were treated with 0.094
U mL'' of turnip peroxidase under various experimental parameters. Direct dyes were
recalcitrant to the action of turnip peroxidase, however the rate and extent of
decolorization of direct dyes by turnip peroxidase was significantly enhanced in the
presence of different kinds of redox mediators. Six out often investigated compounds
showed their potential in enhancing the decolorization of direct dyes. Various
parameters such as pH, temperature, enzyme and redox mediator concentrations were
standardized in order to obtain maximum rate of decolorization of direct dyes.
Maximum decolorization of dyes over 60% occurred in the presence of 0.6 mM 1-
hydroxybenzotriazole/violuric acid in sodium acetate buffer, pH 5.5 at 30 °C. In order
to prove the capability of plant peroxidase in the treatment of industrial effluents, the
treatment of mixtures of direct dyes was also investigated. Complex mixtures of dyes
were also maximally decolorized in the presence of 0.6 mM redox mediator (1-
hydroxybenzotriazole/violuric acid). In order to examine the operational stability of
the enzyme, the enzyme was exploited for the decolorization of mixtures of dyes for
140
different times in a stirred batch process. More than 80% of the dye mixtures were
decolorized within first hour of incubation with turnip peroxidase. However, the
treatment of direct dyes/mixtures in the presence of redox mediators by turnip
peroxidase caused the formation of insoluble precipitate, which could be removed by
the process of centrifligation, filtration or adsorption. Thus indicating the removal of
end products of the enzyme mediated dye decolorization. Total organic carbon
analysis of treated dyes or their mixtures showed that these resuhs were quite
comparable to the loss pf color fi-om solutions. The results suggested that catalyzed
oxidative coupling reactions might be important for natural transformation pathways
for dyes and indicated their potential use as an efficient means for removal of dyes
color from waters and wastewaters.
Surface immobilized bitter gourd peroxidase has been used for the effective
decolorization of textile industrial effluent. Effluent was recalcitrant to the action of
bitter gourd peroxidase, however in the presence of some redox mediators, it was
successfully decolorized. Effluent decolorization was maximum upto 70% in the
presence of 1.0 mM 1-hydroxybenzotriazole within 1 h of incubation. However,
immobilized bitter gourd peroxidase had optimum decolorization at pH 5.0 and 40 °C.
Immobilized bitter gourd peroxidase decolorized more than 90% effluent color after 3
h of incubation in a batch process. In order to evaluate the efficiency of salt
fractionated plant peroxidase for the decolorization of textile effluent, it was
necessary to decolorize textile effluent in a continuous mode. A two-reactor system,
one reactor containing immobilized peroxidase and the other had adsorbent; activated
silica was used for the effective decolorization of textile effluent. The system was
capable of decolorizing 40% effluent even after 2 months of continuous operation
with a flow rate of 16 mL h"'. The absorption spectra of the untreated and treated
effluent exhibited a marked difference in absorbance at various wavelengths. Thus
immobilized peroxidase/1-hydroxybenzotriazole system has been successfully
employed for the treatment of a large volume of effluent in a continuous reactor.
Further an attempt has been made to use a simple, inexpensive concanavalin
A-wood shaving bound turnip peroxidase for the decolorization of a direct dye and
mixture of direct dyes in batch processes and continuous reactors. The study has
shown that wood shaving could be exploited as an inexpensive material for the
preparation of bioafflnity support due to its high affinity for concanavalin A; being
the natural source of cellulose. Wood shaving (1.0 g) adsorbed nearly 22 mg of
141
concanavalin A from jack bean extract. Wood shaving adsorbed concanavalin A was
used for the immobilization of peroxidase directly from ammonium sulphate
fractionated proteins of turnip. Concanavalin A-wood shaving bound turnip
peroxidase showed very high effectiveness factor 'T|' of 0.67. The comparative results
between soluble and immobilized turnip peroxidase obtained from the batch process
revealed the ability of immobilized turnip peroxidase, to decolorized Direct Red 23
and mixture of dyes up to 92% and 83% after 1 h of incubation in the presence of 0.6
mM 1-hydroxybenzotriazole, respectively. Enzymes used for the treatment of
wastewater contaminated with aromatic pollutants could also be affected by the
presence of water-miscible organic solvents/salts/heavy metals. Therefore, the
decolorization of direct dye and mixture of direct dyes by turnip peroxidase was
carried in the presence of water-miscible organic solvent, salt and heavy metals. The
immobilized turnip peroxidase could effectively remove more than 70% of color in
the presence of metals/salt. The dye decolorizing reusability was gradually decreased
upto 8*'' repeated uses. Immobilized peroxidase could decolorize 47% and 30% of the
initial color from Direct Red 23 and mixture of direct dyes even after 8* repeated use,
respectively. Decolorization of Direct Red 23/mixture of direct dyes carried in a
continuous reactor containing a cheap biocatalyst, immobilized enzyme with a flow
rate of 7.2 mL h'' was highly efficient even till 4 and 3 months of operation, removing
64% and 50% of color from the solution of Direct Red 23 and mixture of direct dyes,
respectively. Total organic carbon analysis of treated dye or mixture of dyes exhibited
that these results were quite comparable to the loss of color from solutions. Thus, this
work may provide a reasonable basis for development of biotechnological processes
for continuous color and aromatic compounds removal from various industrial
effluents at large scale.
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171
LIST OF PUBLICATIONS AND PRESENTATIONS
List of Publications:
1. Mahreen Matto and Qayyum Husain. (2006) Entrapment of porous and
stable Con A-peroxidase complex into hybrid calcium alginate-pectin gel.
Journal of Chemical Technology and Biotechnology 81, 1316-1323.
2. Mahreen Matto and Qayyum Husain. (2007) Decolorization of direct dyes by
salt fractionated turnip proteins enhanced in the presence of hydrogen
peroxide and redox mediators. Chemosphere 69,338-345.
3. Mahreen Matto and Qayyum Husain. (2008) Decolorization of direct dyes by
immobilized turnip peroxidase in batch and continuous processes.
Ecotoxicology and Environmental Safety (in press).
4. Mahreen Matto and Qayyum Husain. (2008) Redox mediated decolorization
and removal of Direct Red 23 and Direct Blue 80 catalyzed by bioaffmity
immobilized tomato peroxidase {Lycopersicon esculentum). Biotechnology
Journal (in press).
5. Mahreen Matto, Sara Naqash and Qayyum Husain. (2008) An economical
and simple bioaffinity support for the immobilization and stabilization of
tomato (Lycopersicon esculentum) peroxidase. Acta Chimica Slovenica (in
press).
6. Rukshana Satar, Mahreen Matto and Qayyum Husain. (2008) Studies on
calcium entrapped alginate-pectin gel entrapped concanavalin A-bitter gourd
{Momordica charantia) peroxidase complex. Journal of Scientific and
Industrial Research (in press).
7. Mahreen Matto and Qayyum Husain. (2008) A novel calcium alginate-starch
hybrid support for both surface immobilization and entrapment of bitter gourd
(Momordica charantia) peroxidase. Journal of Molecular Catalysis B:
Enzymatic (in revision).
8. Mahreen Matto and Qayyum Husain. (2008) Decolorization of textile
effluent by bitter gourd peroxidase immobilized on concanavalin A layered
calcium alginate-starch beads. Journal of Hazardous Materials (in revision).
9. Mahreen Matto and Qayyum Husain. (2008) Removal of colour and aromatic
compounds from textile effluent in batch as well as in continuous reactors
containing calcium alginate-starch entrapped bitter gourd {Momordica
charantid) peroxidase. Applied Biochemistry Biotechnology (communicated).
Seminars and Symposia attended:
1. Mahreen Matto, Rukhsana Satar and Qayyxun Husain. Studies on
concanavalin A-bitter bourd {Momordica charantid) peroxidase complex
entrapped into alginate-pectin gel. Presented in the Ilnd Jammu & Kashmir
Science Congress held at University of Kashmir, Srinagar, India. July 25-27,
2006. Abstract no: Mol-19.
2. Mahreen Matto, Huidrom Rully and Qayyum Husain. Studies on
immobilized bitter gourd {Momordica charantia) peroxidase. Presented in the
75* Annual Meeting of Society of Biological Chemists (India) on
"Metabolism to Metabolome" held at Jawaharlal Nehru University, New
Delhi, India. Dec 8-11,2006. Abstract no: PIII-97.
3. DBT sponsored workshop on "Genome Analysis: a Bioinformatic approach"
sponsored by the Department of Biotechnology. Ministry of Science and
Technology, Govt of India, New Delhi held at Interdisciplinary Biotechnology
Unit, Aligarh Muslim University, Aligarh, India. February 24-25,2007.
4. Rukhsana Satar, Mahreen Matto and Qayyum Husain. Studies on calcium
entrapped alginate-pectin gel entrapped concanavalin A-bitter gourd
{Momordica charantia) peroxidase complex. Presented in 95*'' Indian Science
Congress, Visakhapatnum, India. January 3-7,2008. Abstract no: 37.
5. Mahreen Matto and Qayyimi Husain. Decolorization of dyes and their
mixtures by using immobilized turnip peroxidase in batch as well as in
continuous processes. Presented in the International Symposium on the
Predictive, Preventive and Mechanistic Mutagenesis and XXXIII EMSI
Annual Meeting. Department of Agriculture Microbiology, Aligarh Muslim
University, Aligarh, India. January 1-3,2008. Abstract no: P-65.
6. Mahreen Matto and Qayyum Husain. 1-hydroxybenzotriazole mediated
decolorization and removal of direct dyes from polluted water mediated by
concanavalin A cellulose immobilized tomato peroxidase {Lycopersicon
173
esculentum). Presented in the Society for Free Radical Research-India (SFRR)
Satellite Meeting. Department of Biochemistry, AIIMS, New Delhi, India.
February 11-12,2008. Abstract no: PI-61.
7. Mahreen Matto, Saba Naseem Ansari and Qayyum Husain. Studies on celite
adsorbed mushroom {Agaricus Bisporus) tyrosinase. Presented in the National
Symposium on Emergence of Modem Techniques and Development in
Biobusiness, held at Department of Zoology, Modem College of Arts,
Sciences and Commerce, Pune, India. Febuary 18-20,2008. Abstract no: 40.
8. Mahreen Matto and Qayyum Husain. Remediation of textile colored effluent
by bitter gourd peroxidase immobilized on concanavalin A layered calcium
alginate-starch beads. Presented in National Symposium on Recent Advances
in Biochemistry and Allied Sciences, held at Department of Biochemistry,
Aligarh Muslim University, Aligarh, India. March 25,2008. Abstract no: A-7.
9. Mahreen Matto and Qayyum Husain. A novel calcium alginate-starch hybrid
support for the surface immobilization of bitter gourd {Momordica Charantia)
peroxidase. Presented in National Symposiimi on Recent Advances in
Biochemistry and Allied Sciences, held at Department of Biochemistry,
Aligarh Muslim University, Aligarh, India. March 25,2008. Abstract no: A-9.
174
Journal of Chemical Technology and Biotechnology JChem TechnolBtotechnom:13l6-l323 (2006)
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Entrapment of porous and stable concanavalin A-peroxidase complex into hybrid calcium alginate-pectin gel Mahreen Matto and Qayyum Husain* Department of Biochemistry, Faculty of Ufe Science, Aligarh Muslim University, Aligarh-202 002, India
Abstract: Ammonium sulfate-fractionated proteins of turnip (Brassica rapa) were used for tlie simultaneous purification and immobilization of peroxidase by using crude jack bean extract. The concanavalin A-tumip peroxidase complex retained nearly 70% of the original activity. Calcium alginate-pectin-entrapped soluble turnip peroxidase and the concanavalin A complex of peroxidase retained 63% and 52% of the original activity, respectively. The concanavalin A-peroxidase complex, the alginate-pectin-entrapped soluble peroxidase and the alginate-pectin-entrapped concanavalin A-peroxidase complex showed very high stability against denaturation mediated by heat, pH, urea, organic solvents and detergents. The exposure of soluble and immobilized turnip peroxidase to trypsin resulted in an enhancement of the peroxidase activity. The concanavalin A-peroxidase complex entrapped preparation was markedly more stable as compared with the directly entrapped soluble enzyme preparation. The results suggested that such preparations have great potential in the construction of bioreactors to be used for the remediation of aromatic compounds present in polluted wastewater/industrial effluents. © 2006 Society of Chemical Industry
Keywords: alginate; concanavalin A; entrapment; inunobilization; pectin; peroxidase; stability; stabilization; turnip
NOTATION Con A Concanavalin A DMF Dimethyl formamide SDS Sodium dodecyl sulfate STP Soluble turnip peroxidase TP Turnip peroxidase
INTRODUCTION Peroxidases (EC 1.11.1.7) are ubiquitous proteins having applications in a wide range of fields such as medicine, chemical synthesis and in the analysis of food, chemical, clinical and environmental samples.'-^ More recently, peroxidases have been employed for several novel applications such as detoxification and removal of various organic pollutants, e.g. phenols, aromatic amines, dyes, etc., from polluted wastewater.^"^ They have also been used as catalysts in phenolic resin synthesis, in fuel and chemical production from wood pulp, in the production of dimeric alkaloids, in bio-bleaching processes and in the oxi-dation/biotransformation of organic compounds.^"'° This great diversity of applications is due to the wide substrate specificity of peroxidase catalysis." The use of soluble enzyme is not practical due to the huge amount of enzyme required and extreme denaturing conditions encountered during the process.
Immobilized enzymes have received a lot of attention for their use as biocatalysts in the removal/biotransformation of toxic aromatic organic compounds present in wastewater.'^'^ There are different methods which can be used for the immobilization of enzymes. However the high cost and low yield of immobilized enzyme preparations are two important limitations in their applications in the treatment of industrial efifiuents.'*'' The immobilized enzymes must be obtained by a cost eflfective and technologically convenient method. Among the techniques used for immobilization, entrapment in natural biopoly-mers is favored for various reasons; e.g. non-toxicity of the matrix, variation in the bead size of the gel and high yields of immobilization.'*'^ Calcium alginate-mediated entrapment has attracted much attention in the detoxification of phenolic compounds present in industrial effluents.'* Recentiy some workers have reported that pectin beads were significantly more stable than alginate beads." The cost of pectin is further making the immobilization procedure more expensive.
There are several reports about the leakage of enzymes from porous polymeric matrices, calcium alginate gdj'^^o^i poly(vinyl) hydrogel^^ and sol-gel.^^ In order to prevent the leaching of enzymes from physically entrapped gels, a number of attempts have been made to increase the molecular dimension
• Correspondence to: Qayyum Husain, Department of Biochemistry, Faculty of Life Science, AJigarti Muslim University, Aligaiti-202 002, India E-mail qayyum husain©redlffmail .com Contract/grant sponsor University Grants Commission Contract/grant sponsor DST, New Delhi, India Received 22 October 2005; revised version received 5 December 2005; accepted 5 December 2005) Publishedonline26May2006;DOI: I0.1002/)ctb.l540 { j f e i f ^ f ^ C ' •
© 2006 Society of Chemical Industry. J Chem Technol Bwtechnol 0268-2575/2006/J30.00 ^ ^ o.ico.i . io>iti,i.a . . i « .
Entrapment of concanavalin A-peroxidase complex
of the enzymes pnor to their entrapment The leakage could be avoided by entrapping crosslinked turnip peroxidase into calcium alginate gel,^° crosslinked penicillin G acylase into lentikats^* and pre-immobilized glucose oxidase on Sepabeads denvatized with glu-taraldehyde into sol-gel.^^
In this study, an effort has been made to entrap insoluble Con A-tumip peroxidase (TP) complex in calcium alginate-pectm hybnd beads. In order to investigate the better performance of the Con A-TP complex, the soluble TP (STP) and concanavalin A (Con A)-peroxidase complex, preparations were independently entrapped in algmate-pectin beads. A comparative study of the stability of STP, Con A-TP complex, entrapped STP and entrapped Con A-TP complex has been made. The ptirpose of this study was to reduce the cost of commercially available enzyme for large-scale immobilization and utilization. Alginate-pectin-entrapped preparations exhibited very high immobilization yields and good stability agamst various forms of denaturants.
MATERIALS AND METHODS Materials Sodium alginate was the product of Koch-Light Lab (Colnbrook, UK). Bovme serum albumin was obtained firom Sigma Chemical Co. (St Lotiis, MO) USA. Jack bean meal was procured from DIFCO, Detroit, USA. o-Dianisidine HCl was purchased from the Centre for Biochemical Technology, CSIR, India. Dioxane, dimethyl formamide, sodium dodecyl sulfate and pectm were obtamed from SRL Chemicals, Mumbai, India. Surf Excel and turnip were purchased from the local market. Other chemicals and reagents employed were of analytical grade and were used without any further purification
Ammonium sulfate fractionation of turnip proteins Turnip (250.0 g) was homogenized in 250 mL of O.lmolL"^ sodium acetate buffer, pH 5.6. Homogenate was filtered through four layers of cheesecloth. The filtrate was then centrifuged at a speed of 10 000 X ^ on a Remi C-24 Cooling Centrifuge. The clear solution obtained was subjected to salt fractionation by adding 0-90% (w/v) (NH4)2S04. It was continuously stirred overnight at 4 °C for complete precipitation of protein. The precipitate was collected by centnfuganon at 10 000 x ^ on a Remi C-24 Cooling Centrifuge. The precipitate obtained was redissolved in 0.1 molL~' sodium acetate buffer, pH 5.6 and dialyzed agamst the assay buffer.^"
Preparation of insoluble Con A-TP complex Jack bean extraa (10%, w/v) was prepared by adding 5.0 g of crude jack bean meal to 50 mL of O.lmolL"' sodium phosphate buflfer, pH 6.2. The jack bean extract was used for the insolubihzation of peroxidases from the ammoniujn sulfate-fracnonated
J Chem Technol Biotechnol 81:1316-1323 (2006) DOI: 10.1002/jctb
turnip proteins. To a senes of tubes, 200 enzyme units of peroxidase were mixed with increasing concentration of 10% jack bean extract.^* The final volume was adjusted to 2.0 mL with assay buffer The mixtures were incubated at 37 °C for 12 h. Each precipitate was collected after centnfiigation at 3000 X ^ for 5 mm at room temperature and was washed once with the same buffer. Finally the precipitate was suspended in 2.0 mL assay buffer. Each precipitate was analyzed for the acDvity of enzyme. The precipitate with maximum activity' was taken for further stability studies exhibited maximum activity was taken for further stability studies.
Entrapment of soluble and Con A-TP complex in calcium alginate-pectin beads The soluble peroxidase (250 units) and Con A-peroxidase complex (260 umts) were mixed mde-pendently with an aqueous mixtvire of sodium alginate (2.5%) and pectin (2.5%) made in assay buffer. The resulting mixture was slowly extruded as droplets through a 5.0 mL syringe with attached needle No. 20 mto 0 2molL~' calcium chloride solution. The formation of calcium algmate-pectin beads was instantaneous and the solutions were further gently stirred for 2 h . ' ' ^ The beads were then washed and stored in 0 I rnolL"' acetate buffer, pH 5.6 at 4 °C, unol use.
Effect of pH on the activity of soluble and immobilized TP The aaivities of STP, Con A-TP complex and alginate-pectin-entrapped TP preparations (1.25 units) were measured in buffers of various pH values. The molarity of each buffer was 0.1 mol L~'.
Effect of temperature on the activity of soluble and alginate-entrapped TP STP, Con A-TP complex and algmate-pectm-entrapped TP preparations (1.25 umts) were incubated at 60 °C in 0.1 mol L~' sodium acetate buffer, pH 5.6. Aliquots of each preparation were removed at each indicated time interval and activity was measured. The activity obtained without mcubaoon at 60 °C was taken as control (100%i) for the calculation of percent activity.
The enzyme activities of STP, Con A-TP complex and algmate-pectin-entrapped TP preparations were measured at vanous temperatures under standard assay conditions. The activity obtamed at 40 °C was taken as 100% for the calculation of percent activity.
Effect of urea on soluble and immobilized TP Soluble and immobilized TP preparations (1.25 units) were mcubated in 4 0molL~' urea dissolved m 0 ImolL" ' sodium acetate buffer, pH 5.6. Aliquots were removed at vanous ome intervals and activity was determmed. The activity obtamed without incubauon with urea was taken as 100% for the calculation of percent activity.
1317
M Matto, Q Husain
Effect of water-soluble organic solvents on soluble and immobilized TP Soluble and immobilized TP preparations (1.25 units) were incubated with 10-60% (v/v) of water-miscible organic solvents; dioxane/DMF prepared in 0.1 molL"' sodium acetate buffer, pH 5.6, at 37°C, for 1 h. Peroxidase activity was determined at all the organic solvent concentrations indicated. The activity obtained without exposure with organic solvent was taken as 100% for the calculation of percent activity.
Effect of detergents on soluble and immobilized TP SDS and Surf Excel (0.1-1.0%, w/v) were used to observe the effect of detergents on the activity of TP.^* Soluble and immobilized enzyme preparations (1.25 units) of peroxidase were incubated with increasing concentration of detergents in 0.1 molL"' sodium acetate buffer, pH 5.6, at 37 °C, for 1 h. Peroxidase activity was determined at all the detergent concentrations indicated. The activity obtained without exposure with detergent was taken as control (100%) for the calculation of percent activity.
Effect of trypsin-mediated proteolysis on the activity of soluble and immobilized TP Soluble and immobilized TP preparations (1.25 units) were incubated with increasing concentrations of (0.025-0.125 mg) trypsin per mL of incubation mixture at 37 °C for 1 h.^*
Measurement of peroxidase activity Peroxidase activity was determined from a change in the optical density (/l460nra) by measuring the initial rate of oxidation of 6.0mmolL~' o-dianisidine HCl in the presence of 18nmiolL~' hydrogen peroxide in 0.1 molL"' sodium acetate bioffer, pH 5.6, for 15min at37°C.
The immobilized preparations were continuously agitated for the entire duration of the assay. The assay was highly reproducible with immobilized preparations. The significant absorption of the color complexes formed on the gel matrix could be observed.^'
One unit of peroxidase activity was defined as the amount of enzyme protein that catalyzes the oxidation of 1 jxmol of o-dianisidine HCl per min at 37 °C.
Determination of protein concentration The protein concentration was determined by the procedure of Lowry et al^° Bovine serum albtmiin was used as standard.
RESULTS AND DISCUSSION The crude peroxidase preparation was obtained from 250 g of turnip roots and the initial specific activity was 16.6 units mg"' of protein. The ammonium sulfate precipitation followed by dialysis increased
the specific activity 2.5-fold over crude enzyme and this preparation was used for entrapment in calcium alginate-pectin beads. Immobilization by means of entrapment is a very rapid and simple technique. It has been described earlier that soluble enzyme could be leached out of beads on long standing or yjg 18,20-23 jjj order to overcome the leaching effect of enzymes out of porous gel beads, the insoluble complex of TP was prepared by using Con A, jack bean extract and subsequently entrapped into alginate-pectin gel. Several earlier reports indicated that crosslinked enzymes or pre-inamobilized enzymes could remain inside the polymeric matrices for longer periods of time than soluble enzymes. *'' '*' ' ^
Figure 1 demonstrates the precipitation of glycosylated turnip proteins by increasing concentration of jack bean meal extract. The maximum precipitation of peroxidase activity was 70%). It indicated that the major isoenzymes of turnip peroxidases are glycosylated. Further soluble and Con A-peroxidase complex preparations were entrapped into calcivun alginate-pectin beads and retained 63%) and 52% of the original activity, respectively (Table 1). The entrapped Con A-peroxidase complex preparations thus obtained were quite active and exhibited an effectiveness factor'/;' of 0.52 (Table 1). The effectiveness factor of an immobilized enzyme is a measure of internal diffiision and reflects the efficiency of the immobilization procedure.^' Earlier reports also described that the enzymes immobilized into the alginate gels had a moderate effectiveness factor.^"'^*
The effect of pH on the activity of soluble and immobilized enzyme is shown in Fig. 2. The pH-activity profile of the immobilized enzyme has the same pH optima as the soluble peroxidase at pH 5.0, while the entrapped Con A-TP complex was more resistant to pH changes. This predicts that entrapment
100
0.0 0,1 0,2 0,3 0.4 0,5 0.6 0,7 0.8 0.9 1.0 1.1 1.2
Jack bean extract mL
Rgure 1. Insolubilization of TP by using jack bean extract. STP 200 enzyme unit was Incubated with increasing concentrations of 10% jack bean extract (0.1 to 1 mL) in a total volume of 2.0 mL of 0,1 mol L~' sodium phosphate buffer, pH 6,2, tor 12 h at 37 °C. Precipitate of each preparation was collected, washed and activity was determined as described in the text.
1318 yChem Technol Biotechnol S1:1316-1323 (2006) DOI: 10.1002/jctb
Entrapment of concanavalin A-peroxidase complex
Table 1. Immobilization of TP by using jack bean extract and calcium alginate-pectin matnx
120 1
Method
Activity Effectiveness expressed factor
(%) 'rj'
Con A-peroxidase complex Calcium alginate-pectin-entrapped STP Calcium alginate-pectin-entrapped Con A-TP complex
70 63
52
0 7 0 63
0 52
< c S E
ss
Ttie effectrveness factor (i;) value of the immobilized preparation represents ttie ratio of actual and theoretical activity of the immobilized enzyme Each value represents the mesin for three-independent experiments performed in duplicate, with average standard deviation <5%.
120 1
100
80 •5
< I 60
I 40
20
10 12 PH
., STP », Con A -TP complex o, Alginate - pectin-entrapped STP ' , Alginate - pectin-entrapped Con A-TP complex
Figure 2. pH-activity profiles of soluble and immobilized TP preparations. The enzyme activities of soluble and immobilized TP preparations were measured in buffers of various pH values. The molanty of each buffer was 0 1 mol L"'. The activity was optimum at pH 5 6 for all the preparations and was taken for the calculatton of percent activity.
of enzymes in gel beads provides a microenvironment
for the enzyme, which may play an important role in
the state of protonation of the protein molecules.'^-^^
The formation of the Con A - T P complex further
confers additional resistance to the enzyme against
extreme conditions of pH.^"'^^
Native peroxidase is quite stable to heat inactivation.
Immobilization, however, gives additional stability
against heat inactivation. As shown in Fig. 3, STP,
Con A - T P complex, alginate-pectin-entrapped S T P
and Con A - T P complex preparations were incubated
at 60 °C for various times. Incubation of soluble
enzyme at this temperature resulted m the loss
of nearly 92% of activity in 120 min whereas
the entrapped STP retained 22% of the initial
activity. Moreover, the entrapped Con A - T P complex
preparation markedly exhibited very high stability
against thermal denaturation and it retained more
than 40% of the initial activity. It indicates that such
preparations can be used at high temperatures for
60 80 Time (mm)
140
• , STP », Con A-TP complex o, Alginate - pectn-entrapped STP V, Alginate - pectn-entrapped Con A-TP complex
Figure 3. Thermal denaturation of soluble and immobilized TP preparations. STP, Con A-TP complex, entrapped STP and entrapped Con A-TP complex preparations were incutiated at 60 "C for various time penods in 0 1 mol L"' sodium acetate buffer, pH 5 6. Aliquots of each preparation were removed and activity was measured according to tfie procedure descntied in the text.
the treatment of polluted water. Improvement in the
thermal stability of alginate-pectin-entrapped Con
A complex preparations may come from multipoint
complexing of peroxidase with Con A. Earlier findings
described that the complexmg of enzymes with lectins
enhanced its thermal stability. This enhancement in
thermal stability is due to several interactions between
enzyme and Con A. * ^''
The activities of soluble and immobilized peroxidase
preparations were compared at various temperatures.
The resistance of enzymes to high temperatures was
greatiy increased by immobilization (Fig. 4). A large
120
100
S 80
60
40
20
10 20 30 40 50 60
Temperature (°C)
70 80 90
..STP », Con A-TP complex o. Alginate - pectin-entrapped STP ". Alginate - pectin-entrapped Con A-TP complex
Figure 4. Temperature-activity profiles of soluble and immobilized TP preparations. The enzyme activities of soluble and immobilized TP preparations were measured at vanous temperatures under standard assay conditions The activity obtained at 40 °C was taken as control (100%) for the calculation of percent activity
JChem TeckrtolBiotechnol Sl:13l6-\323 (2006) DOI: 10.1002/jctb
1319
M Matto, Q Husain
difiference in the activity of soluble enzyme was observed when the enzyme activity was measured at various temperatures. Soluble enzyme retained marginal enzyme activity between 60 and 80 ""C, whereas the alginate-pectin-entrapped Con A-TP complex retained more than 40% of its original activity at 60 "C. However, the temperature optima of the entrapped STP preparation remained unaltered but the Con A-TP-entrapped preparation exhibited higher stability at high temperatures. This was achieved due to strong bond formation between the enzyme molecules, thus it increased the rigidity of the structure of the native enzyme. ' ' ^-^^ The alginate-pectin-entrapped Con A-TP complex retained its structure and remarkably high activity at elevated temperatures. Therefore, such an enzyme preparation could be very useful at relatively high temperatures, which is an important factor for industrial applications.
Urea at a concentration of 4.0molL"' is a strong denaturant of protein and this irreversibly denatures the soluble enzyme. The Con A-enzyme-entrapped preparation was found to be more stable compared with the other preparations, as shown in Fig. 5. Soluble enzyme lost 58% of its initial activity after 2h incubation in 4.0molL~' urea while Con A-TP complex, alginate-pectin-entrapped STP and alginate-pectin-entrapped Con A-TP complex retained 60%, 56% and 65% of the initial activity, respectively, under similar incubation conditions. It is, therefore, obvious that the enzymes in beads were completely accessible to urea and it is already known that this can significantly affect the ionic interaction of soluble entrapped enzyme.^* Although the action mechanism of urea on the protein structures
is not yet completely understood, several earlier studies have proposed that protein is unfolded by the direct interaction of urea molecules with a peptide backbone via hydrogen bonding/hydrophobic interaction, which contributes to the maintenance of protein conformation.^^ However, the complexing of several enzymes with Con A has been shown to result in an enhancement of their resistance to denaturation.^^-^^ These observations clearly indicate that the complex of TP with Con A protects it from urea-induced inactivation.
Figure 6 shows the activity of soluble and immobilized TP in the presence of increasing concentrations of trypsin (0.025-0.125 mg trypsin per mL of incubation mixture) for 1 h at 37 °C. The activities of all the preparations of TP were activated at each incubated trypsin concentration (Fig. 6). The activity of soluble enzyme was enhanced by up to 111% after 60 min incubation at 37 °C while the activities of Con A-TP complex, entrapped STP and entrapped Con A-TP complex were increased up to 120%, 114% and 125%, respectively. In an earlier study some investigators have shown that bitter govird peroxidase immobilized on a Con A-Sephadex support also exhibited an enhancement in enzyme activity when exposed to low concentrations of trypsin.^*
Wastewater from various elimination sites contains several types of denaturants, including detergents, which can strongly denature the enzymes used for the treatment of polluted wastewater. In order to use such enzymes for the removal of aromatic pollutants from wastewater it becomes necessary to monitor the stability of enzymes in the presence of denaturants. In this study two different detergents have been selected for comparative stability' of soluble and
60 80 100 120 140
Time (min)
•, STP », Con A - T P complex o, Alginate - pectin-entrapped STP ' , Alginate - pectin-entrapped Con A - T P complex
Figure 5. Effect of urea on the activity of soluble and immobilized TP preparations. Soluble and immobilized TP preparations were incubated In 4 0 mol L"' urea dissolved in 01 mol L"' sodium acetate buffer, pH 5.6. Aliquots were removed at various time intervals and activity was determined.
130 120 110 100 90 80 70 60 50 40 30 20 10 0 0. 00 0.02 0.04 0.06 0,08 0.10 0 12 0.14
Trypsin Cone (0.025-0.125 mg L" )
•, STP », Con A - T P complex °, Alginate - pectin-entrapped STP •', Alginate - pectin-entrapped Con A - T P complex
Figure 6. Effect of trypan concentration on activity of soluble and immobilized TP preparations Soluble and immobilized TP (1.25 units) preparations were independently incubated with increasing concentrations of trypsin (0.025-0.125 mg) in a total volume of 1.0 mL of 100 mol L"' sodium acetate buffer, pH 5.6, at 37 °C for 1h. Activity of enzyme was assayed as descnbed in the text.
1320 JChem TecktiolBiotechnolSl-.nie-1323 (2006) DOI: 10.1002/jctb
Entrapment of concanavalin A-peroxidase complex
Table 2. Effect of detergents on the activity of soluble and immobilized TP
Detergent concentration (%, w/v)
0.1 0.2 0.4 0.6 0.8 1.0
STP
80 65 50 41 38 34
Entrapped STP
153 70 55 50 45 40
SDS
Con A-TP
170 78 67 56 50 44
Percent TP activity remaining
Entrapped Con A-TP
190 94 74 62 58 47
STP
210 50 40 30 26 15
Entrapped STP
230 70 52 42 30 24
Surf Excel
Con A-TP
250 90 70 57 44 29
Entrapped Con A-TP
260 160 80 70 60 38
Soluble and immobilized TP preparations (1.25 EU) were incubated with SDS/Surf Excel (0.1-1.0%, w/v) prepared in 0.1 molL"' sodium acetate buffer, pH 5.6, at 37 °C for 1 h. Peroxidase activity was assayed at all the indicated detergents concentrations and other assay conditions were the same as mentioned in the text. The activity of soluble and Immobilized TP in assay buffer without any detergent was taken as control (100%) for the calculation of percent activity. Each value represents the mean for three independent experiments performed in duplicate, with average standard deviation <5%.
immobilized TP. In order to investigate the effect of higher concentrations of detergents on the activity of TP, soluble and immobilized TP preparations were incubated witii 0.1-1.0% (w/v) SDS for 1 h at 37 °C. Pre-incubation of soluble and immobilized enzyme preparations with 1% (w/v) SDS for Ih resulted in the loss of up to 66%, 60%, 56% and 53% of die original enzyme activity, respectively (Table 2). The exposure of entrapped STP and Con A-TP complex preparations with 0.1% SDS showed an enhancement in enzyme activity and these preparations exhibited 153% and 190% of original activity respectively (Table 2). Both soluble and immobilized enzyme preparations were activated by low concentrations of SDS; however, the immobilized TP achieved further activation at higher levels of detergent (Table 2). This experiment indicated that the presence of lower concentrations of SDS is not harmful to the enzyme structure and that enzymes can work more efficientiy on the industrial effluents containing compounds like soap and detergents.^^ Lower concentrations of Triton X 100 and Tween 20 also activated soluble and immobilized TP and in this case the extent of activation was much higher (data not shown).
Surf Excel is a very common detergent used in households and laundries. Unused detergent is normally present in municipal wastewater. This wastewater is somewhere mixed with the effluents released by industry. In order to make immobilized enzyme preparations more efficient for wastewater treatment, we have investigated the effect of Surf Excel on the activity of immobilized TP. STP was more sensitive to exposure to Surf Excel and lost nearly 85% of its enzyme activity after 1 h incubation with 1% (w/v) detergent. However, the entrapped STP, Con A-TP complex and entrapped Con A-TP complex were more resistant to inactivation induced by 1.0% Surf Excel and retained 24%, 29% and 38% of the initial activity, respectively (Table 2).
Industrial effluents and municipal wastewater contain organic solvents together with other aromatic
JChem Techrwl Biotechnol 81:1316-1323 (2006) DOI: 10.1002/jctb
pollutants. Therefore, it is of utmost importance to investigate the role of some water-miscible organic solvents on the activity of immobilized enzymes. The exposure of soluble enzyme to var3dng concentrations of DMF (10-60%, v/v) resulted in the loss of a greater fraction of enzyme activity while the immobilized enzyme was quite resistant to inactivation induced by DMF. Con A-TP complex and Con A-TP complex entrapped preparations exposed to 30% (v/v) DMF for 1 h retained half of the enzyme activity while soluble enzyme lost nearly 70% of its original activity with similar treatment (Table 3). The incubation of soluble and immobilized TP with increasing concentration of dioxane resulted in a continuous loss of enzyme activity. However, the inmiobilized enzyme was more resistant to inactivation mediated by dioxane. The treatment of STP with 60% (v/v) dioxane for 1 h resulted in the loss of 86% of the initial activity while the Con A-TP complex and entrapped Con A-TP complex retained 27% and 33% of the original activity (Table 3). In an earUer study it has been shown that bitter gourd peroxidase immobilized on Con A and other suppons was significantiy more stable against the denaturation induced by water-miscible organic solvents.^^-^^
CONCLUSIONS Several procedures have been described for the immobilization of enzymes but very few can control the amount of immobilization and provide good stability to the enzymes. The present study aimed to work out an inexpensive, simple and high yield procedure for the immobilization of TP, which would be of extreme interest in the remediation of several types of aromatic compounds present in polluted wastewater/industrial effluents. Turnip root peroxidases were partially purified by using ammonium sulfate fractionation and were easily insolubilized by using jack bean extract. Soluble as well Con-A complexes of TP were ftirther entrapped
1321
M Matto, Q Husam
Table 3 Effect of organic solvents on the activity of soluble and immobilized TP
Organic solvent concentration (% v/v)
10 20 30 40 50 60
STP
54 48 39 23 17 14
Entrapped STP
77 67 42 26 19 17
Dioxane
Con A-TP
82 77 63 54 43 27
Percent TP activity remaining
Entrapped Con A-TP
93 86 69 63 54 33
STP
70 44 31 18 13 5
Entrapped STP
82 62 38 21 16 12
DMF
Con A-TP
90 77 48 28 22 18
Entrapped Con A-TP
93 84 50 34 28 22
Soluble and immobilized TP preparations (1 25 EU) were incubated witti increasing concentration of DI F/dioxane (0-60% v/v) in 0 1 molL ^ sodium acetate buffer pH 5 6, at 37 "C for 1 h Peroxidase activity was assayed at all ttie indicated organic solvent concentrations and other assay conditions were same as mentioned in the text The activity of soluble and immobilized TP in assay buffer without any detergent was taken as control (100%) for ttie calculation of percent activity Each value represents the mean for three-independent expenments performed in duplicate, wrtfi average standard deviation <5%
into calcium alginate-pecnn beads. The stability against vanous denatunng agents is an important factor when selectmg an appropnate enzymatic system for any appbcanon. This study mdicates that the alginate-pectm-entrapped Con A - T P preparation has signiiicandy greater stabihty against physical and chemical denaturants compared with soluble enzyme-entrapped preparaoons. Immobilized TP preparaaons with high stabihty could be exploited for developing bioreactors for the treatment of phenohc and other aromatic pollutants present m wastewater, which are derived from vanous agricultural and mdustnal activities.
ACKNOWLEDGEMENTS The authors are thankful to the Umversity Grants Commission, New Delhi and DST, New Delhi, India for providmg special grants to the Department m the form of DRS and FIST, respectively for developing infrastructure facilities
REFERENCES 1 Laborzewsky J and Ginalska G, Industnal use of soluble or
immobilized plant peroxidases Plant Perox Newslett 6 3-7 (1995)
2 Macek T, Dobranskv R, Pespisilova R, Vanek T and Kralova B, Peroxidases from i« vitro cultures of different plant species, in Plant Peroxidases Biochemistry and Physiology III, International Symposium 1993 Proceedings, ed by Welinder KG, Ras-mussen SK, Penel C and Greppin H University of Copenhagen and University of Geneva, pp 451-435 (1993)
3 Husam Q and Jan U, Detoxificaoon of phenols and aromanc amines from polluted wastewater by using phenol oxidases J Sci IndRes 59 286-293 (1999)
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5 Shaffiqu TS, Roy JJ, Nair RA and Abraham TE, Degradanon of textile dyes mediated by plant peroxidases Appl Btochem Bwtechnol 102-103 315-326 (2002)
6 Bhunia A, Durani S and Wangikar PP, Horseradish peroxidase catalyzed degradanon of industnally important dyes Btouch-nolBioengn 562-567 (2001)
7 Akhtar S, Khan AA and Husam Q, Pamally punfied bitter gourd (Momordica charantia) peroxidase catalyzed decolonza-non of textile and other mdustnally important dyes Biores Technol 96 1804-1811 (2005)
8 Dordick JS, Marietta MA and Klibanov AM, Polymenzanon of phenols catalyzed by peroxidase m non-aqueous media Biotechnol Bweng 30 31-36 (1987)
9 Rvu K, McEldon JM, Pokora AR, Cyrus W and Dordick JS, Numencal and Monte-Carlo simulation of phenohc polymerization catalyzed by peroxidase Biotechnol Bweng 42 807-814 (1993)
10 XuY-P, Huang G-L and Yu Y-T, Kinetics of phenolic polymenzanon catalyzed by peroxidase m orgamc media Bwtechnol Bioeng 47 117-119 (1995)
11 McEldon JP and Dordick JS, Unusual thermo-stabihty of soybean peroxidase Biotechnol Prog 12 555-558 (1996)
12 Tatsumi K, Wada S and Ichikawa H, Removal of chlorophenols from wastewater by immobilized horseradish peroxidase Biotechnol Bioeng SI 126-130 (1996)
13 Akhtar S, KhanAA and Husam Q, Potennal of immobilized bitter gourd {Momordica charantia) peroxidases m the decolonzanon and removal of texnle dyes fixjm polluted wastewater and dyeing effluent Chemosphere 60 291-301 (2005)
14 TischerW and KascheV, Immobilized enzymes crystals or earners TIBTECH 17 326-335 (1999)
15 Gupta MN and Matnasson B, Umque apphcanons of immobilized protems in bioanalyncal systems Meth Biochem Anal 36 1-14(1992)
16 Kierstan M and Bucke C, The immobilization of microbial cells, sub-cellular organelles and enzymes m calcium aigmate gels Biotechnol Bioeng 61 726-736 (2000)
17 Smidsr^d O and Gudmund SB, Aigmate as immobilization mamx for ceUs TIBTECH 8 71-78 (1990)
18 Davis S and Bums RG, Decolonzanon of phenolic effluents by soluble and immobilized phenol oxidases Appl Microbiol Biotechnol 32 721-726 (1990)
19 Kunllova L, Gememer P, VikanovskaA, Mikova H, Rosenberg M and Ilavskv M, Calcium pectate gel beads for cell entrapment 6 Morphology of stabilized and hardened calcium pectate gel beads with cells for unmobUized biotechnology J Microencap 17 279-296 (2000)
20 Musthapa MS, Akhtar S, Khan AA and Husam Q, An economical, simple and high vield procedure for the immobilization, stabilization of peroxidases from turnip roots J Sci Ind Res 63 540-547 (2004)
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Entrapment of concanavalin A-peroxidase complex
21 Blandmo A, Maccas M and Cantero D, Glucose oxidase release from calcium alginate gel capsules Enzyme Mtcrob Technol 27 319-324 (2000)
22 Veronese FM, Manomucan C, Schiavon, FO, Lore S, Secum-ndo F, Chilin A, ei al, Pegylated enzvme entrapped in pol> (viny alcohol) hvdrogel for biocatalytic application II Farmaco 56 541-547(2001)
23 Przyb\t iM and Bialkowska B, Enzyme electrode constructed on the basis of oxygen electrode with oxidases immobilized by sol gel techmque Mater Sa 20 63-79 (2002)
24 Wilson L, Dlanes A, Pessela BCC, Abian O, Femadez-Lafuente R and Guisan JM, Encapsulation of crosslmked penicillin G acylase aggregates in lentikats ev aluauon of a novel biocatah st m organic media Bwiechnol Bioeng 86 558-562 (2004)
25 Betancor L, Lopez-Gallego F, Hidalgo A, Fuentes M, Podrasky O, Kuncova G, et al. Advantages of the preimmobilizaoon of enzymes on porous supports for their entrapment m sol-gels BiomacromolecuUs 6 1021-1030 (2005)
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28 Akhcar S, Khan AA and Husam Q, Simultaneous punfication and umnobilization of bitter gourd (Monwrdtca charanttd) peroxidases on bioaffinity support J Ckem Technol Bwtechnol 80 198-205 (2005).
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29 Husain S, Husain Q and Saleemuddm M, Inactivation and reactivation of horseradish peroxidase immobilized by vanety of procedures Indian J Btochem Bwphys 29 482-486 (1992)
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JfChem Technol Btouchnol 81 .1316-1323 (2006) DOI . 10.1002/)Ctb
1323
ELSEVIER
Available online at www.sdencedirect.coni
*%' ScienceDirect Chemosphere 69 (2007) 338-345
CHEMOSPHERE
www.elsevier.coin/locate/chemosphere
Technical Note
Decolorization of direct dyes by salt fractionated turnip proteins enhanced in the presence of hydrogen
peroxide and redox mediators
Mahreen Matto, Qayyum Husain *
Department of Biochemistry, Faculty of Life Sciences. Aligarh Muslim University, Aligarh-202002, UP, India
Received 9 January 2007; received in revised form 29 March 2007; accepted 30 March 2007 Available online 23 May 2007
Abstract
The present paper demonstrates the effect of salt fractionated turnip (Brassica rapa) proteins on the decolorization of direct dyes, used in textile industry, in the presence of various redox mediators. The rate and extent of decolorization of dyes was significantly enhanced by the presence of different types of redox mediators. Six out of 10 investigated compounds have shown their potential in enhancing the decolorization of direct dyes. The performance was evaluated at different concentrations of mediator and enzyme. The efficiency of each natural mediator depends on the type of dye treated. The decolorization of all tested direct dyes was maximum m the presence of 0.6 mM redox mediator at pH 5.5 and 30 °C. Complex mixtures of dyes were also maximally decolorized in the presence of 0.6 mM redox mediator (l-hydroxybenzotriazole/violuric acid). In order to examine the operational stability of the enzyme preparation, the enzyme was exploited for the decolorization of mixtures of dyes for different times in a stirred batch process. There was no further change in decolorization of an individual dye or their mixtures after 60 min; the enzyme caused more than 80% decolorization of all dyes in the presence of 1-hydroxybenzotriazole/violuric acid. However, there was no desirable increase in dye decolorization of the mixtures on overnight stay. Total organic carbon analysis of treated dyes or their mixtures showed that these results were quite comparable to the loss of color from solutions. However, the treatment of such polluted water in the presence of redox mediators caused the formation of insoluble precipitate, which could be removed by the process of centrifugation. The results suggested that catalyzed oxidative coupling reactions might be important for natural transformation pathways for dyes and indicate their potential use as an efficient means for removal of dyes color from waters and wastewaters. © 2007 Elsevier Ltd. All rights reserved.
Keywords: Peroxidase; I-Hydroxybenzotriazole; Dye decolorization; Redox mediator; Turnip
1. Introduction
About 10000 different synthetic dyes and pigments are produced annually world wide and used extensively in textile, dyeing and printing industries. It is estimated that about 10% of the used dyes are lost in industrial effluents (Young and Yu, 1997; Husain, 2006). The discharge of wastewater that contains high concentration of aromatic dyes is a well-known problem associated with dyestulT
Corresponding author. Tel.: +91 571 2720135 (R); +91 571 2700741 (O); fax:+91 571 2721776.
E-mail address: qayyumhusain@redifi'mail.com {Q. Husain).
activities. Their highly variable and complex chemical structures also make them difficult to remove using conventional wastewater treatment systems (Husain, 2006). There are about twelve classes of chromogenic groups, the most common being the azo type, which makes up to 60-70% of all the textile dye stuff produced, followed by the anthra-quinone type. The simple manufacturing process, and the enormous variety of dyes, ranging from the simple insoluble disperse class to simple anionic mono azo dyes, have made azo dyes ubiquitous chemicals which are used in different colorant applications ranging from synthetic to natural dyes (de Moraes et al., 2000). Environmental pressure has impelled the search for new technological development.
0045-6535/S - see front matter © 2007 Elsevier Ltd. All rights reserved. doi:I0.1016/j.chemosphere.2007.03.069
M. Malta, Q. Husain I Chemosphere 69 (2007) 338-345 339
There is a need to find alternative treatments that are effective in removing dyes from large volumes of effluents and are low in cost (Abadulla et al., 2000).
Recently, enzymatic approach has attracted much interest in the removal of phenolic pollutants from aqueous solutions as an alternative strategy to the conventional chemical as well as microbial treatments that pose some serious limitations (Duran and Esposito, 2000; Husain and Jan, 2000; Torres et al., 2003). Oxidoreductive enzymes such as peroxidases are participating in the degradation/ removal of aromatic pollutants from various contaminated sites (Bhunia et al., 2001; Akhtar et al., 2005a,b; Mohan et al., 2005). These enzymes can act on a broad range of substrates and can also catalyze the degradation/removal of organic pollutants present in a very low concentration at the contaminated site (Akhtar and Husain, 2006; Husain, 2006). In view of the potential of these enzymes in treating the phenolic compounds several microbial and plant peroxidases have been considered for the treatment of dyes but none of them has been exploited on large scale due to low enzymatic activity in biological materials and high cost of purification (Bhunia et al., 2001; Shaffiqu et al., 2002; Verma and Madamwar, 2002). Recent studies have been further extended by the use of several compounds taken in minimal amounts to mediate the oxidative reactions catalyzed by partially purified oxidoreductases (Claus et al., 2002; Camarero et al., 2005; Akhtar and Husain, 2006).
In the present study an attempt has been made to investigate the role of various low molecular weight redox mediators in the decolorization of direct dyes mediated by the action of peroxidase. The decolorization was examined in the presence of different concentration of redox mediators and enzyme, at varying pH and temperatures. Complex matures of direct dyes were also targeted using turnip peroxidase and redox mediators.
2. Materials and methods
2.1. Materials
Direct dyes; Direct Red 23 (DR 23), Direct Red 239 (DR 239), Direct Blue 80 (DB 80) and Direct Yellow 4 (DY 4) were a gift from Atul Pvt. Ltd. India. Violuric acid (VA) was obtained from Sigma Chemical Co. (St. Louis, MO) USA and other redox mediators mentioned in Table 1 were purchased from SRL Chemicals, Mumbai, India. o-dianisidine-HCl was obtained from the IGIB, New Delhi, India. Turnip roots were purchased from a local vegetable market. The chemicals and other reagents employed were of analytical grade and were used without any further purification.
2.2. Ammonium sulphate fractionation of turnip proteins
Turnip (250 g) was homogenized in 250 ml of 100 mM sodium acetate buffer, pH 5.5. Homogenate was filtered
Table 1 Compounds investigated as redox mediators for mediating dye decolorization by TP
Name of compounds
HOBT IX Naphthol Vanillin L-Histidine VA Catechol Gallic acid Quinol Bromopbenol 4-Nitrophenol
% Dye
DR23 (A 5051
96 0 9 7
86 0 0 0 6 5
decolorization
im) DR239 (X 505 nm)
93 0
27 0
83 0 0 0 0 0
DB80 (A572nm)
86 0
37 29 89 0 1 0
37 4
DY4 (X 405 nm)
66 0
37 36 72 0 7 0 0 0
Each compound (2.0mM) was used-with peroxidase (0.094 U ml"') for decolorization mediating property. The mixture was incubated in 100 mM sodium acetate buffer, pH 5.5 and in the presence of 0.72 mM H2O2 for I h at 37 °C. The dyes are written as Direct Red 23 (DR 23), Direct Red 239 (DR 239), Direct Blue 80 (DB 80) and Direct YeUow 4 (DY 4). Each value represents the mean for three independent experiments performed in duplicate, with average standard deviation <5%.
through four layers of cheesecloth. The filtrate was then centrifuged at a speed of 10000^ for 15 min at 37 "C on a Remi Cooling C-24 Centrifiige. The obtained solution was subjected to salt fractionation by adding 10-90% (w/v) ammonium sulphate. This solution was continuously stirred overnight at 4 °C for complete precipitation of protein. Precipitate was collected by centrifiigation at lOOOOg on a Remi C-24 Cooling Centrifuge. The obtained precipitate was re-dissolved in lOOmM sodium acetate buffer, pH 5.5 and dialyzed against the assay buffer (Kulshrestha and Husain, in press).
2.3. Analysis of peroxidase activity and protein estimation
Peroxidase activity was determined from a change in the optical density {A^f^am) by measuring the initial rate of oxidation of 6.0 mM o-dianisidine-HCl in the presence of 18mM H2O2 in 100 mM sodium acetate buffer, pH 5.5 for 15 min at 37 "C (Matto and Husain, 2006).
One unit of peroxidase activity was defined as the amount of enzyme protein that catalyzes the oxidation of 1.0 nmol of o-dianisidine-HCl min"' at 37 °C.
The protein concentration was determined by using the procedure of Lowry et al. (1951). Bovine serum albumin was used as standard.
2.4. Procedure for screening dye decolorization mediating role of various compounds
Direct dyes (50-100 m g p ' ) were prepared in distilled water. The structure of all four used direct dyes is given in Fig. 1. Each dye was incubated with TP (turnip peroxidase) (0.094 U m r ' ) in 100 mM sodium acetate buffer, pH 5.5 in the presence of 0.72 mM H2O2 for 1 h at 37 °C.
340 \f. Malta, Q. Husain I Chemosphere 69 (2007} 338-345
Direct Red 23
NaOjS'
Direct Red 239
NaOjl
SOjNa
NaOjS
SO3N
SOjNa SOjNa
Direct Blue 80
H 0 - ^ Q ^ N . N _ < Q ^ C H . C H - / ^ ) ^ N . N - . Q ^ 0 H
\sO3Na NaO^^ Direct Yellow 4
Fig. 1. Chemical structures of investigated direct dyes.
Compounds (mentioned in Table 1) were used as redox mediators for the selected experiments. Dye decolorization was monitored at specific wavelength maxima of the dye. The percent decolorization was calculated by taking untreated dye solution as control (100%).
2.5. Treatment of dyes with increasing concentration ofTP
Decolorization of direct dyes was examined by treating dyes with increasing concentration of TP "0.094-0.330 U m r ' " for 1 h at 37 °C. The reaction mixture was incubated under the same conditions as mentioned in Section 2.4. The reaction was stopped by heating in a boiling water bath for 15 min and the insoluble product was removed by centrifugation. Untreated dye solution was used as control (100%).
2.6. Effect of varying concentration of redox mediators and temperature on TP mediated dye decolorization
The decolorization of direct dyes by TP (0.094 U ml"') in the presence of varying concentrations of redox mediator "0.2-0.8 mM" and incubated under the same conditions as mentioned in Sections 2.4 and 2.5.
In another set of experiment the dye solutions were treated with TP (0.094 Uml~') at various temperatures "20-80 °C" for 1 h incubated under similar experimental conditions as mentioned in Sections 2.4 and 2.5.
2.7. Effect ofpH on the decolorization of dyes
Each dye was treated with TP (0.094 U mP') in the buffers of various pH values ranging from 3.0 to lO.O in the presence of same conditions used for the treatment of dyes as mentioned in Sections 2.4 and 2.5.
2.8. Decolorization of mixtures of dyes
Four dye mixtures were prepared by mixing various dyes in equal proportions in terms of absorbance. These mixtures of dyes were treated with TP (0.094 U ml"') in 100 mM sodium acetate buffer and other conditions were the same as mentioned in Sections 2.4 and 2.5. Decrease in absorbance in each TP treated solution was monitored at specific wavelength maxima of the mixture.
2.9. Data collection and analysis
Procedure for the dye decolorization was followed by centrifugation and clear dilute solution was considered for TOC determination. TOC was evaluated by using a total organic carbon analyzer (Multi N/C 2000, Analytic Jena, Germany). Independent dye and mixture of dyes were incubated with TP (0.094 U ml"') for over Ih in the presence of HOBT/VA at 37 °C and after centrifugation the TOC content was determined. Each control and
M. Malio. Q. Husain I Cliemosphere 69 (2007) 338-345 341
treated independent dye or mixture of dyes was diluted to 20-fold before measuring their TOC.
DB 80 and mixture 4 were incubated with TP (0.094 U ml"') for 1 h in the presence of HOBT/VA at 37 °C and after centrifugation the clear supernatant was used for UV-vis spectral analysis; the spectra of control and TP treated dye samples were taken on Cintra lOe Spectrophotometer.
2.10. Decolorization of mixtures of dyes in stirred batch processes
The mixtures, which showed maximum decolorization in the presence of HOBT and VA were selected for the experiment. Dye mixtures (250 ml) were treated with TP (25 U) in the presence of 0.72 mM H2O2 and 0.6 mM HOBT/VA for 8 h at room temperature (30 °C) under stirring conditions. After treatment, aliquot of mixtures were taken for the measurement of absorbance in the visible region.
3. Results and discussion
The experiments were designed for screening redox-mediating property of 10 compoxmds as peroxidase mediators in the presence of H2O2. The dye solutions were found to be stable upon exposxire to H2O2 or to the enzyme alone. Thus, the dye precipitation was a result of H202-dependent enzymatic reaction, possibly involving free-radical formation followed by polymerization and precipitation.
structure of the dyes and not to enzyme inactivation (Reyes et al., 1999; Claus et al., 2002). However, it has been earlier reported that redox mediators could enhance the oxidation activity of enzymes; laccases and peroxidases (Bourbonnais and Paice, 1990; Reyes et al., 1999; Akhtar et al., 2005a; Kulshrestha and Husain, in press).
3.2. Effect of varying concentrations of TP
All four direct dyes were treated with increasing concentration of TP "0.094-0.330 U ml"'" for 1 h in the presence of 0.6 mM redox mediators. An increase in concentration of TP has resulted in enhanced decolorization. However, in all the four treated dyes there was marginal decrease in percent decolorization after 0.236 U ml"' of TP in the presence of HOBT, while as in the presence of VA there was slight increase in percent decolorization after 0.236 U ml"' of TP (Table 2). It is attributed to the difference in azo dye oxidation explained by the different electron-donating properties of the substituents and their location on the phenolic ring (Claus et al., 2002). Several workers have demonstrated that the use of redox mediator-enzyme system enhanced the rate of dye decolorization by several folds but these mediators were required in very high concentrations, 5.7 mM VA/ laccase system and 11.6mM of HOBT/laccase system (Soares et al., 2001a,b; Claus et al., 2002). However, in this study we used very low concentration (0.6 mM) of mediators and only 0.0961 U ml"' to enhance the rate of dye decolorization.
3.1. Screening of dye decolorization mediating activity of various compounds
Among the 10 compounds selected to investigate the mediating effect on peroxidase activity, six compounds promoted decolorization of DR 23, three of DR 239, six of DB 80 and five of DY 4. HOBT and VA enhanced more than 60% decolorization of all the four direct dyes (Table 1). It goes in agreement with the fact that some dyes were recalcitrant to enzyme action even in the presence of redox mediators. This was mainly attributed to the molecular
3.3. Effect of increasing concentrations of mediators
Table 3 demonstrates the effect of varying concentrations of mediators "0.2-0.8 mM" on the decolorization of direct dyes by TP. It was observed that in the presence of 0.6 mM concentration of HOBT/VA could decolorize more than 50% all the four dyes. However, DY 4 was decolorized marginally in the presence of HOBT. After addition of more than 0.6 mM mediator concentration there was no significant effect on the decolorization of DR 23 and DR 239, while it was noted that in DB 80
Table 2 Effect of TP concentration on the decolorization of direct dyes in the presence of HOBT/VAA'N
Concentration ofTPUml '
0.094 0.143 0.1 S6 0.236 0.2«1 0.330
% Decolorization in
HOBT
DR23
93 93 93 93 92 92
DR239
90 90 90 90 89 88
the presence
DB80
84 86 88 gg 85 85
of HOBT/VAA'N
DY4
66 70 72 72 71 71
V
DR23
84 84 85 86 87 87
DR239
83 83 84 84 85 85
DB80
89 90 91 92 93 93
DY4
72 73 73 74 75 76
VN
DR23
U 20 20 23 23 30
DR 239
8 10 14 14 16 18
DB80
50 58 60 62 66 66
DY4
23 29 36 36 36 43
All four direct dyes were independently treated with increasing concentrations of TP "0.094-0.330 U ml"'" for 1 h in the presence of 0.6mM redox mediators (HOBT/VA/VN) in 100 mM sodium acetate buffer, pH 5.5 at 37 °C. Each value represents the mean for three independent experiments performed in duplicate, with average standard deviation <5%.
342 M Matto, Q Husain I Chemosphere 69 (2007) 338-345
Table 3 Effect of vanous concentrations of HOBT/VA on dye decolonzation by TP
Concentration of HOBT/VA (mM) % Decolorization in the presence of HOBT/VA
DR23
HOBT
63 90 94 94
VA
77 87 85 84
DR239
HOBT
81 86 86 85
VA
74 76 80 78
DB80
HOBT
58 73 86 86
VA
84 86 89 87
DY4
HOBT
57 57 67 64
VA
64 63 73 73
02 04 06 08
Each dye was treated with TP (0 094 U ml ) in the presence of increasing concentration of redox mediator "0 2-0 8" mM as described m the text Each value represents the mean for three independent expenments performed in duplicate, with average standard deviation <5%
decolorization was continuously decrease in the presence of increasing concentration of VA. It could be due to dual roles played by such compounds; as mediator because they mcreased the rate of recalcitrant dye decolorization and as competitive inhibitor of substrate, these molecules inhibited the activity of enzyme at their higher concentrations.
3.4. Effect ofpH on the decolorization of direct dyes
All the four direct dyes were treated with TP in the buffers of different pH values in the presence of 0.6 mM redox
mediators, HOBT A'A. The dyes were maximally decolorized within the pH range from 5.0 to 5.5. HOBT and VA showed significantly higher rate of dye decolorization as compared to VN (Table 4).
3.5 Effect of temperature on the decolorization of direct dyes
The effect of different temperatures "30-80 °C" on the dye decolorization was monitored and it was observed that all the dyes were decolorized (about 60% or more) maxi-
Table 4 Effect of pH on the decolorization of direct dyes by TP in the presence of HOBT/VA/VN
pH
3 4 5 55 6 7 8 9 10
% Decolorization in the
DR23
HOBT
30 67 93 91 33 0 0 0 0
VA
9 56 87 87 54 0 0 0 0
presence
VN
5 6 9
14 13 9 5 4 0
of HOBT/VA/VN
DR239
HOBT
18 63 93 90 43
1 0 0 0
VA
II 65 83 83 60 0 0 0 0
VN
5 6 7 6 4 1 0 0 0
DB80
HOBT
17 74 83 82 31 26 3 2 1
VA
21 75 88 85 70 0 0 0 0
VN
3 26 50 48 24 27 8
11 10
DY4
HOBT
28 38 66 60 28 0 0 0 0
VA
31 48 72 72 48 . 9 0 0 0
VN
1 4
24 26 16 0 0 0 0
Each direct dye was treated with 0 094 U ml"' of TP in buffers of vanous pH "3 O-IO 0" in the presence of 0 6 mM redox mediator (HOBT/VA/VN) and 0 72 mM H2O2 for I h at 37 °C Each value represents the mean for three independent expenments performed in duplicate, with average standard deviation <5%
Table 5 Effect of temperature on the decolorization of direct dyes by TP m the presence of HOBT/VA
Temperature (°C)
20 30 40 50 60 70 80
% Decolorization
DR23
HOBT
41 94 90 64 34 26 23
1 m the
VA
40 85 79 75 26 11 8
presence of HOBT/VA
DR239
HOBT
26 93 83 41 36 24 20
VA
15 81 78 69 30 21 17
DB80
HOBT
18 80 75 50 33 18 13
VA
24 84 79 66 39 30 21
DY4
HOBT
10 60 55 47 20 16 14
VA
22 70 67 53 44 31 22
Each dye was incubated with 0 094 U ml ' mediator (HOBT/VA) at 20-80 °C for I h standard deviation <Syo
of TP in 100 mM of sodium acetate buffer, pH S 5 in the presence of 0 72 mM H2O2 and 0 6 mM redox Each value represents the mean for three mdependent expenments performed m duphcate, with average
M MaUo. Q Husam I Oiemosphere 69 (2007) 338-345 343
mally at 30 °C (Table 5). The rate of dye decolorization was decreased above and below the temperature-maxima.
120
-B—B—a—B—B—B—B- - B — B — B — S — • t a i ! l l i l l ! l t l ! l l > ^
\ 1 1 1 1 \ 1- H 1 1 1-0 30 60 90 120150180210240270300330360390420450
Time (min)
Fig 2. DecolonzaUon of mixture 3 and 4 by TP in the presence of HOBT/ VA in stirred batch process. Dye mixture 3 (DR 23 + DY4 + DR 239) and mixture 4 (DR 23 + DB 80 + DR 239) were treated with 25 U of TP in a stirred batch process for 8 h at room temperature. The ahquots were removed at 30 mm time intervals and mtensity of color was recorded at the specific wavelength The symbols show (A) mixture 3 with HOBT, (•) mixture 3 with VA, (O) mixture 4 with HOBT and (A) mixture 4 with VA
3.6 Analysis of dyes and their mixtures
In order to confirm the removal of aromatic compounds from the TP treated wastewater in the presence of redox mediators some spectral analysis were also performed. Fig. 3 demonstrates that the model wastewater containing an individual dye, DB 80 and mixture 4 of direct dyes treated with TP in the presence of redox mediator resulted in a loss of dyes from solution in visible as well as from UV regions. The diminution in absorbance peaks in UV and visible regions was clear evidence regarding the removal of dyes from treated wastewaters containing complex muctures of dyes (Fig. 3). Removal of reaction product as insoluble complex formed maximally after 12 h of incubation was an important signal for the detoxification of aromatic compounds from wastewater. It has already been demonstrated that horseradish peroxidase can catalyze free-radical formation followed by spontaneous polymerization of a variety of aromatic compounds including phenols (Cooper and Nicell, 1996; Tatsumi et al., 1996), chlorophenols (Tatsumi et al., 1996) other substituted phenols (Nicell et al., 1992; Akhtar and Husain, 2006) and dyes (Bhunia et al., 2001; Shaffiqu et al., 2002; Akhtar et al., 2005a,b). These observations were in agreement with earlier findings that the treatment of aromatic compoimds
a '»
Wave Length (nm)
Fig 3 Adsorption spectra of DB 80 and mixture 4 DB SO and mixture 4 were treated as described in text and their absorption spectra were taken before and after treatment with TP in the presence of HOBTA'A respectively. Spectra correspond to DB 80 (a) and mixture 4 (b).
344 M Mallo Q Husain I Oiemosphere 69 (2007) 338-345
Table 6 Treatment of mixtures of direct dyes with TP in the presence of HOBT/ VA
Mixture of direct dyes (nm)
% Dye decolonzation
% TOC removal
Mixture 1 DB80 + DY 4 + DR23
Mixture 2 DB80 + DY 4 + DR239
Mixtures DR 23 + DY 4 + DR 239
Mixture 4- DR 23 + DB 80 + DR239
520
470
502
515
HOBT
35
45
55
57
VA
57
65
70
75
HOBT
39
48
50
57
VA
60
66
75
77
The conditions for the treatment of mixtures of dyes were descnbed in Sections 2 8 and 2.9 of the text, respectively Each value represents the mean for three independent expenments performed in duplicate, with average standard deviation <5%
resulted in the formation of insoluble aggregate, which could be easily removed by simple filtration, centrifugation or sedimentation (Akhtar et al., 2005b; Husain, 2006). Such spectral measurements also provide strong evidences for the removal of aromatic pollutants from wastewater.
The influence of redox mediators, HOBTA^A on the decolorization of complex mixtures of dyes was also examined. There was a significant reduction in the color of the dyes treated with TP even if they were present in the form of complex mixtures (Table 6). Mixture 3 and 4 were decolorized more than 50% in the presence of both redox mediators, HOBT/VA whereas mixture 1 and 2 were decolorized less than 50% in the presence of HOBT. Moreover, the decolonzation of mixture 1 and 2 was quite significant in the presence of VA. The decolorization rate of mixtures of dyes was slower than that of pure dye solution. This finding supported an earlier observation that the bio-degradation of various phenolic compounds in the form of mixtures was quite slow compared to the independent phenol (Kahru et al., 2000). The mixture 3 and 4 were maximally decolorized in the presence of HOBT, 55% and 57% respectively whereas these mixtures were significantly decolorized in the presence of VA, 70% and 75%, respectively. These obser\'ations were in agreement with several earlier reports which have described the enhancement of laccase activity by redox mediators (Reyes et al., 1999; Soares et al., 2001 a,b; Claus et al., 2002), and increase in percent decolonzation by peroxidase in the presence of HOBT (Akhtar et al., 2005a; Kulshrestha and Husain, in press).
All the tested direct dyes and their mixtures were treated with TP and their insoluble product was removed by centrifugation. TOC content of individual dyes and mixtures of dyes before and after treatment with TP is given in Table 7 and 6 As compared to control, the level of TOC was markedly decreased when it was treated with TP in the presence of HOBT/VA. These observations suggested that major toxic compounds get easily removed out of the TP treated samples Several earlier workers have reported the removal of dye from wastewater in the form of TOC loss
Table 7 TOC content of direct dyes after treatment with TP
Name of direct dye
DR23 DR239 DB80 DY4
^max
505 505 572 405
(nm) % Dye decolonzation
HOBT
90 93 86 66
VA
86 83 89 72
% TOC removal
HOBT
91 90 89 70
VA
88 85 89 76
Each dye was incubated with 0094Umr' of TP m 100mM sodium acetate buffer, pH 5 5 with 0 6 mM redox mediator (HOBT/VA) for 1 h at 37 °C Each value represents the mean for three independent expenments performed in duplicate, with average standard deviation <5%
when treating with certain chemical methods such as ozonation (Koch et al., 2002).
3.7. Treatment of dye mixtures m a stirred batch process
Dye mixtures treated with TP in the presence of redox mediators (HOBTA^A) at different time intervals in stirred batch processes resulted in significant loss of color. The disappearance of more than 80% of color was followed by insoluble precipitate formation maximally after 12 h of incubation (Fig. 2). The observations suggested that TP in the presence of redox mediators could remove remarkably very high percentage of color. However, HOBT was a better redox mediator as compared to VA.
4. Conclusions
Sak fractionated peroxidase from Brassica rapa was able to catalyze polymerization and precipitation of direct dyes and their mixtures using wide range of reaction conditions and compounds which acted as redox mediators. The presence of 0.6 mM of HOBT/VA drastically increased the decolorization rate of direct dyes by TP. The application of peroxidase that is easily available and inexpensive could be successfully used for the treatment of wastewater.
Acknowledgements
The authors are thankful to the University Grants Commission and Department of Science and Technology, New Delhi, Government of India for providing special grants to the Department in the form of DRS and FIST, respectively for developing mfrastructure facilities.
References
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Ecotoxicology and Environmental Safety I (IIH) I
I sr/^r-R
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/looate/ecoenv
Decolorization of direct dyes by immobilized turnip peroxidase in batch and continuous processes Mahreen Matto, Qayyum Husain * Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University. Aligarh 202002, India
A R T I C L E I N F O
Article hbtory: Received 17 August 2007 Received in revised Form 2 February 2008 Accepted 23 February 2008 Available online XXXX
Keyvmrds: Peroxidase Dye decolorization Turnip Concanavalin A Wood shaving
A B S T R A C T
An inexpensive immobilized turnip peroxidase has been employed for the decolorization of some direct dyes in batch and continuous reactors. Wood shaving was investigated as an inexpensive material for the preparation of bioafiinity support. Concanavalin A-wood shaving bound turnip peroxidase exhibited 67% of the original enzyme activity. Both soluble and immobilized turnip peroxidase could effectively remove more than 50% color from dyes in the presence of metals/salt and 0.6 mM 1-hydroxybenzo-triazole, after 1 h of incubation. The columns containing immobilized peroxidase could decolorize 64% direct red 23% and 50% mixture of direct dyes at 4 and 3 months of operatioa respectively. Total organic carbon analysis of treated dye or mixture of dyes revealed that these results were quite comparable to the loss of color from solutions. Thus, this study showed that the immobilized enzyme could be efficiently used for the removal of synthetic dyes from industrial effluents.
© 2008 Published by Elsevier Inc.
1. Introduction
Treatment of synthetic dyes in wastewater is a matter of great concern. Several physical and chemical methods have been employed for the removal of dyes (Robinson et al., 2001). However, these procedures have not been widely used due to high cost, formation of hazardous by products and intensive energy requirement (Hai et al., 2007).
Extensive research has been directed towards developing processes in which enzymes are employed to remove dyes from polluted water (Bhunia et al., 2001; Shaffiqu et al., 2002; Torres et al.. 2003; Lopez et al., 2004; Husain. 2006). The potential advantages of enzymatic treatment as compared to microbial treatment are mainly associated to several factors; shorter treatment period; operation of high and low concentrations of substrates; absence of delays associated with the lag phase of biomass, reduction in sludge volume and ease of controlling the process (Lopez et al., 2002; Akhtar and Husain, 2006). However, the use of soluble enzymes has some inherent limitations as compared to immobilized form of enzymes, which has several advantages over the soluble enzymes such as enhanced stability.
Abhreviations: TP, turnip peroxidase; HOBT, l-hydroxybenzotriazole; DR 23, Direct Red 23; TOC, total organic carbon; Con K concanavalin A; WS, wood shaving; fTP. immobilized turnip peroxidase; STP, soluble turnip peroxidase.
• Corresponding author. Fax; +915712721776 E-mai'l addresses: qayyumbio®redifrmail.com, qayyumhusaineiyalioo.co.in (Q,
Husain).
easier product recovery and purification, protection of enzymes against denaturants, proteolysis and reduced susceptibility to contamination (Husain and Jan, 2000; Zille et al., 2003; Matto and Husain, 2006).
Numerous methods have been employed for the immobilization of peroxidases from various sources but most of the immobilized enzyme preparations either use commercially available enzyme or expensive supports, which increase the cost of the processes (Norouzian, 2003; Husain, 2006). Such immobilized enzyme systems cannot fulfill the requirements for the treatment of hazardous compounds coming out of the industrial sites. Besides, the known techniques used for the immobilization of enzyme, physical adsorption on the basis of bioaffinity is useful as this process can immobilize enzyme directly from crude homo-genate and thus avoid the high cost of purification. The ease of immobilization, lack of chemical modification and usually accompanying an enhancement in stability are some of the advantages offered by the adsorption procedures (Akhtar et al., 2005a, b; Kulshrestha and Husain, 2006). Besides the mentioned advantages offered by the bioaffinity-based procedures, there is an additional benefit, such as proper orientation of enzyme on the support (Mislovicova et al., 2000; Khan et al., 2005). These supports provide high yield and stable immobilization of giycoenzymes/enzymes.
In this study, an effort has been made to find a cheaper and easily available alternative for the commercially available enzymes, its immobilization/utilization at large scale. Wood shaving (WS) has been employed in this work as an easily available and
0147-6513/$-see front matter e 2008 Published by Elsevier Inc. doi: 10.1016/j.ecoenvJOOS.02.019
M Mono Q Husam / Ecotoxicology and Environmental Safety I 1111} •ll-in
inexpensive support Jack bean extract has been used as a source of concanavalin A (Con A) for the preparation of bioaffinity support, Con A-WS Con A-WS was employed for the immobthza-tion of turnip peroxidase (TP) directly from salt fractionated turnip proteins Con A-WS bound peroxidase was compared with Its free form for the decolonzation of direct dye and mixture of direct dyes m batch as well as in continuous reactors under various experimental conditions
2. Materials and methods
2 ; Materials
Direct dyes. Direct Red 23 (DR 23) Direct Red 239 (DR 239) and Direct Blue 80 (DB 80) were i gift from Atul Pvt Ltd Gujarat, India 1-Hydroxybenzotnazole (HOBT) was purchased rromSRLGiemicals Mumbai India o-Dianisidine HCI was obtained from the ICIB New Delhi, India. Jack bean meal was purchased from Loba Chemical Co Mumbai India Turnip roots and WSs were collected from local market The chemicab and other reagents employed were of analytical grade and were used without any further punfication
22 Ammonium sulfate fractionation of turnip proteins
Turnip (250 g) was homogenized in 250 ml of 01 M sodium acetate buffer, pH SJO Homogenate was filtered through four layets of cheesecloth The filtrate was then centnfiiged at a speed of 10 DOOg for 20 mm at 37 'C on a Remi C-24 Cooling Centnfuge The obtained solubon was subjected to salt fractionation by adding 10-90% (w/v) ammonium sulfate The solution was continuously stirred overnight at 4 C Precipitate was collected by centnfugation at lOOOQg on a Remi C-24 cooling centrifuge The obtained precipitate was redissolved in 01M sodium acetate bufTer pH 5 0 and dialyzed against the same buffer (Matto and Husain 2006)
2 3 Preparation of Con A-WS complex
Jack bean extract flO*) was prepared in five batches by stirring lOg of jack bean meal in lOOmL of 01 M sodium phosphate buffer pH 62 The jack bean
extract was collected by centnfugation at 3000g for 15 mm at room temperature (Matto and Husam 2006)
Three different types of Con A-WS preparations were made in several batches WS was taken as 10 lOOmg and 6Og and were mixed with 0 5 40 and240mL jack bean extraa respectively and incubated overnight at 4°C under stimng conditions Con A-WS complex was colletted from all the three preparations by centnfugation at 3000g for 15 mm at room temperature and washed at least thnce with the above mentioned buffer
2 4 Immobilization of TP on Con A-WS
Enzyme was immobilized on the three above-mentioned Con A WS preparations Con A-WS, 10, lOOmg, and 6 Og preparations were incubated with 10 46 1046 and 6276U of TP in the total volume of OJ, 40 and SOmL of sodium phosphate buffer pH 6,2 The mixtures were incubated at 37 °C for 12 h Each immobilized preparation was collected by centnfugation at 3000g for IS mm at room temperature and washed thnce with phosphate buffer pH 6 2 to remove unbound protein
Con A-WS bound TP preparations as 10 lOOmg and 6 Og were used for assay of activity for the decolonzation of dyes in batch processes and for the decolonzation of dyes m continuous reactors, respectively
2 5 Preparation of synthetic dye solutions
The synthetic solutions of direct dyes (50-100nigL ') were prepared in distilled water to examine their decolonzation by TP The structures of the used dyes are represented \n Fig. 1 A mixture of direct dyes consisting of DR 23 DR239 DB 80 was prepared by mixing each dye m equal proportion of color intensity (Matto and Husain 2007).
2 6 Calculation of percent dye decolonzation
To compare various expenments the decolonzation was calculated for each dye or mixture of dyes Parameter percent decolonzation was defined as
Percent decolonzation _ fabsorbance of the untreated dye - absorbance after treatment] ~ [ absorbance of the untreated dye j 100
c / ^ Direct Red 23
Du-ect Red 239
N = N
NaOiS NHCOHN
N = N
SO,Na SOjNa
NaOjS
Direct Blue 80
OH NaOjS
SO,Na
fig. 1 Chemical structures of investigated direct dyes
w / SO,Na
M. Marco, Q, Husa'm / Ecocoxicology and Environmental Safety • (••llj i n - n i
2.7. Decotorization of dye solution by soluble and immobilized peroxidase in batch processes
The dye solution (250 mL) was treated with STP/ITP (22.7 U) in 0.1 M sodium acetate buffer, pH 5.0 in the presence of 0.6 mM HOBT and 0.72 mlM HjOj for 1 h at 37 =C. The treated samples were centrifuged at 300Og for lOmin. The residual dye concentration was measured spectrophotometrically at specific wavelength maxima of the dye. The decrease in the absorbance at 505 nm for DR 23, and 515 nm for mixture of direct dyes was recorded. Untreated dye solution was considered as control (100%) for the calculation of percent decolorization.
2.8. Effect of temperature and pH on the decolorization of dyes by TP
The effect of temperature on the TP catalyzed decolorization of dyes was carried out at selected temperatures ranging from 20-80 °C. The dye or mixture of dyes (250 mL) was treated with TP (22.7 U) at various temperatures for 1 h in the presence of 0.6rtiM HOBT and 0.72 mM H2O2.
In another set of experiment the decolorization of DR 23 and mixture of direct dyes was carried out at various pH (3.0-10.0) for 1 h under the other assay conditions mentioned in Section 2.7. The molarity of each buffer was 0.1 M.
2.9. Decolorization of dyes in the presence of salt and heavy metals
The decolorization of dye solutions was independently conducted in the presence of 0.5 M NaCI/0.1 mM ZnCl2/03mM CdClj for 1 h under other identical experimental conditions mentioned in 2.7. The dye solution without any treatment was considered as control (100%) for the calculation of percent decolorization.
collected at an interval of 15 days and the absorbance of each sample was measured.
2.13. Data collection and analysis
To examine the ability of immobilized TP to decolorize solution-containing dye/mixture of dyes, spectra were obtained from samples collected from continuous reactor using Cintra lOe. Decrease in absorbance, at X^tx of the dye solutions and spectral profile from 300 to 700 nm were recorded to measure the progress of decolorization.
Procedure for the dye decolorization was followed by centrifugation and clear diluted solution was considered for TOC determination. TOC was evaluated by using a TOC analyzer (Multi N/C 2000, Analytic Jena, Germany). Each control and treated DR 23 or mixture of dyes was diluted 20-fold before measuring their TOC content.
2.14. Assay of peroxidase activity and protein estimation
Peroxidase activity was determined from a change in the optical density (At«onm) by measuring the initial rate of oxidation of 6.0mM o-dianisidine HCI in the presence of 18 mM H2O2 in 0.1 M sodium acetate buffer, pH 5.0 for ISmin at 37 °C (Matto and Husain, 2006).
One unit (1.0 U) of peroxidase activity was defined as the amount of enzyme protein that catalyzes the oxidation of 1.0 iimol of o-dianisidine HCI min~' at 37 'C.
The protein concentration was determined by using the procedure of Lowry et al. (1951). Bovine serum albumin was used as standard.
2.70. Effea of water-miscible organic solvent on dye decolorization
The effect of dioxane: 10%, 30%, and 60% (v/v) on the TP mediated decolorization of dye was invesrigated under the similar experimental conditions as mentioned in Section 2.7. The untreated dye solution was considered as control (100%) for the calculation of percent decolorization.
2.11. Reusability of immobilized TP in the decolorization of direct dyes
DR 23 and mixture of dyes were incubated with immobilized TP for 1 h under Che experimental conditions as mentioned in Section 2.7. The enzyme was separated by centriAjgation and stored in the assay buffer for over 12 h. The similar experiment was repeated 8 rimes with the same preparation of ITP and each time with a fresh batch of dye solution. Dye decolorization was monitored at specific wavelength maxima of the dye solutions. The percent decolorization was calculated by taking uncreated dye or mixture of dyes as control (100%).
2.J2. Continuous dye decolorization using a tiAfo-reactor system
3. Results
3.1. Preparation of bioaffinity support and immobilization ofTP
WS (1.0 g) adsorbed nearly 22 mg Con A from jack bean extract. Con A-WS, bioaffinity support has been used for the direct immobilization of glycoenzymes from ammonium sulfate fractionated turnip proteins. Some isoenzymes of turnip peroxidases are glycosylated in nature (Duarte-Vazquez et a!., 2003). In view of the glycoproteinic nature, turnip peroxidases can be adsorbed via non-covalent binding on bioaffinity support. Con A-WS from ammonium sulfate fractionated proteins. Con A-WS adsorbed 227 U of peroxidase per g of the matrix (Table 1). The effectiveness factor 'ij' of the immobilized enzyme preparation was 0.67 (Table 1). High effectiveness factor of immobilized TP preparation suggested that immobilized preparation was quite effective in catalysis.
Two-reactor system was developed for the continuous removal of dyes from solutions. A column (10 x 2.0cm) was filled with 6.0g Con A-WS bound TP (136211) connected to second column (10x2.0cm) containing activated silica. Activated silica was prepared by heating 6.0 g of silica in an oven (120°C) for overnight and then washed thrice with 20.0 ml distilled water. The flow rate was maintained at 7.2mLh~' and the feed solution contained either DR 23 or mixture of dyes in two independent reactor systems. Dye solutions were run under the same experimental conditions as mentioned in Section 2.7. Reactors were operated at room temperature (30 C) for a period of 4 and 3 months for DR 23 and mixture of dyes, respectively. fFP treated and activated silica gel adsorbed samples were
3.2. Effect Of pH
The pH optimization was done by measuring the initial rate of enzymatic dye decolorization in batch processes at different pH. The decolorization of DR 23 and mixture of dyes by TP was maximum at pH 5.0. At pH greater than 6.0, the rate of dye degradation by STP was very low as compared to ITP (Fig. 2).
Table 1 Immobilization of TP on Con A-WS
Enzyme loaded (XU) Enzyme activity in washes (V, U) Activity bound/g of Con A-WS (U) % Activity yield CB/Ax 100)
Theoretical (X-y = A) (A)
Actual (B)
Effectiveness factor (1) IB(A)
1046 708 338 227 0.67 67
The effectiveness factor of an immobilized enzyme is a measure of internal diffusion and reflects the efficiency of the immobilization procedure. Each value represents the mean for three independent experiments performed in duplicates, with average standard deviation < 5%.
PJea* pfe Ipf: ^rtidC as:«(fap|\fc MuaiA-.|j,|geOT^ direct 1 ^ ^ bylmroobinz^ ,^MiapiP9i#e|^Mc*^^
M Matto, Q Husam / Ecotoxicology and Environmental Safely I ClHlj n i - i u
100 n
Fig. 2. EfFect of pH on Che decolonzanon ofdirect dyes by soluble and immobilized TP. The soluuon of DR 23 or mixture of dyes (250 mL) was Incubated with TP (22.7 U) in the presence of 0.6 mM HOBT. 0.72 mM H2O2 at 37 X for 1 h in the buffers of different pH. The molarity of each buffer was 0.1 M. Symbols indicate treatment of DR 23 by STP ( • X DR 23 by ITP (o), direct dye mixtures by SIP (•), mixture of direct dyes by fTP (V)
100
80
^ 60 2 o u o O a >>
Q
40
20-
10 20 30 40 50 60 70 Temperature (°C)
80 90 100
Fig. 3. Effect of temperature on the decolonzation of direct dyes by soluble and immobilized TP. The decolorizaaon of DR 23 and mixture ofdirect dyes (250 ml) by soluble and immobilized TP was earned out at various temperatures (20-80 'C) For symbols please refer to legend of Fig. 2
3.3. Effect of temperature
Table 2 TP mediated dye decolonzation in the presence of 0.5 M NaCI
Time (mm)
30 60 90
120 150 180
Dye decolorization {%)
DR23
STP
77 77 78 78 78 78
rrp
88 88 88 88 88 88
Mixture
STP
66 66 65 65 65 65
of direct dyes
ITP
72 72 72 72 72 72
DR 23 or mixture of direct dyes (250 ml) was Created with soluble and immobilized TP (22 7 U) in the presence of 0 3 M NaQ for varying times in batch processes Each value represents the mean for three independent expenments performed in duplicate, with average standard deviation <5%.
Tables TP mediated decolonzation of dyes in the presence of vanous heavy metals
Tiine(min) Dye decolorization (X)
0.1 mM ZnQz 03mMCdCl2
DR23 Mixture ofdirect dyes DR23 Mucture of direct eyes
30 60 90
120 180
STP rrp STP
76 86 60 77 86 60 77 86 60 77 86 60 77 86 60
ITP
72 72 73 73 73
STP ITP STP
55 92 27 60 92 29 71 92 31 71 92 31 71 92 31
ITP
69 75 76 76 76
DR 23 or mixture of direct dyes (250 mL) was treated independently with TP (22.7 U) in the presence of 0.1 mM ZnClj/OJ mM CdClj for vanous times m batch processes. Each value represents the mean for three independent expenments performed in duplicate, with average standard deviation <5%
above mentioned experimental conditions. In the case of CdCl2, a peculiar feature was observed that there was no change in decolorization of DR 23 by ITP incubated for various times intervals. Decolorization of mixture of dyes by STP/ITP was marginal (Table 3).
Decolorization of DR 23 and mixture ofdirect dyes by soluble and immobilized TP was maximum at 30'C (Fig. 3). However, immobilized enzyme preparation decolonzed higher percent of color from the dye solutions at temperatures lower and higher than the temperature-optima, 30 °C.
3.4. Effect of salt/heavy metals on TP mediated dye decolonzation
The effect of 0.5 M NaCl on the decolorization of dyes by TP has been illustrated in Table 2. ITP decolorized 88 and 72% DR 23 and mixture of dyes after I h of incubation, respectively. However, immobilized TP was more effective as compared to its soluble counterpart in the decolonzation of both DR 23 and a mixture of dyes.
Table 3 demonstrates the effect of three different salts of heavy metals on the decolorization of DR 23 and mixture of direct dyes by soluble and immobilized TP. ITP decolorized 86% and 73% of color from DR 23 and mixture ofdirect dyes even m the presence of 0.1 mM ZnCb after 1 h of incubation, respectively. On the other hand, soluble enzyme decolorized 77% and 60% of color under
3 5. Effect of organic solvent on TP mediated dye decoloruation
The effect of dioxane on decolorization of DR 23 and mixture of dyes by soluble and immobilized TP has been demonstrated in Table 4. Decolorization of dye/mixture was decreased in the presence of increasing concentrations •of dioxane DR 23 and mixture ofdirect dyes were decolorized 85% and 50% by ITP in the presence of 10% dioxane whereas STP could remove slightly less dye color (66% and 39%) under similar experimental conditions, respectively.
3 6. Dye/dyes mixture decolonzation reusability of immobilized TP
In order to make immobilized TP use more practical in a reactor, it was necessary to investigate the reusability of ITP. The reusability of ITP in the decolorization of direct dyes has been considered. The dye decolorizing reusability of ITP was continuously decreased up to its eighth repeated use (Fig. 4). Immobilized Tl' retained 47% DR 23 and 30% dye mixture decolorization eificiency after eighth repeated use.
cie »>iri;b^MM:
M Mono. Q Husam / Ecotoxicology and Environmental Safety I ^mij n i - l l l
Table 4 Effect of varying concentration of dioxane on dye decolorization by TP
Dioxane (v/v. %) Dye decolonzanon (%)
DR23 Mixture of direct dyes
10 30 60
STP
66 15 0
ITP
85 30 0
STP
39 10 0
ITP
50 25
0
Each dye solution (250 mL) was treated with TP (22.7 U) in the presence of increasing concentrations of dioxane in batch process. Each value represents the mean for three independent experiments performed in duplicate, with average standard deviation <5.
4 5 No of Uses
Fig. 4. Dye/dyes mixture decolonzation reusability of immobilized TP. Immobi-l.zed TP (22 7U} was independently incubated with DR 23 and mixture of direct dyes (250 mL) for 1 h at 37 "C. Dye decolonzation was determined after incubation period of 1 h. After completion of reaction the immobilized enzyme was collected by centnfugation and stored in assay buffer at 4 'C overnight. Next day, the similar expenment was repeated This procedure was repeated 8 times. Symbols indicate DR 23 by ITP ( • ) . mixture of direct dyes by [TP (o)
3.7. Dye decolonzation by ITP m a two-reactor system
The performance of the two-reactor system in terms of dye decolonzation is shown in Table 5. ITP decolorized 93% and 85% of the initial color from DR 23 and mixture of direct dyes after 15 days, respectively. Considerable color removal from DR 23 (64%) and mixture of direct dyes (50%) was found even after 4 and 3 months, of operation of the two-reactor system, respectively.
3.8. Analysis of dyes and their mixtures
Table 5 Continuous removal of color and TOC from DR 23 and a mixture of direct dyes by ITP in a two-reactor system
No of days
15 30 45 60 75 90
105 120
DR23
Dye decolonzation (%)
93 90 87 84 81 76 70 64
TOC removal («)
94 92 89 86 82 77 71 65
Mixture of direct dyes
Dye decolonzation (%)
85 81 77 70 61 50 39 26
TOC removal
87 82 77 71 65 60 53 42
DR 23 or mixture of dyes was treated with ITP in a two-reactor system as descnbed in text Each value represents the mean for three independent expenments performed in duplicate, with average standard deviation < 5%.
Wave Length (nm) nm
Fig. 5. Absorption spectra of DR 23 and mixture ofdirect dyes DR 23 and mixture of direct dyes were treated as descnbed in text and their absorption spectra were taken before and after treatment by TP in the presence of HOBT. Spectra correspond to DR 23 (a) and mixture of direa dye (b)
treated mixture of dyes was 60% and it was less ss compared to DR 23.
In order to confirm the decolonzation of DR 23/mixture of direct dyes by TP in the presence of HOBT some spectral analysis were also performed. Fig. 5 demonstrates the absorption spectra of treated and untreated DR 23/mixture of direct dyes with respect to number of days of operation of the two-reactor system. The absorbance peaks in visible region of DR 23/mixture of dyes was clear evidence regarding the removal of dyes from treated wastewaters.
TOC content of DR 23 and mixture of dyes treated by immobilized TP present in a continuous reactor is given in Table 5. In case of DR 23, 65% of TOC was removed after 4 months of operation of the continuous reactor. The removal TOC from the
4. Discussion
The aim of this study was to assess the use of an inexpensive immobilized TP for the decolorization of the dyes. Con A-WS bound TP retained 67% of the original activity (Table 1). Binding of enzyme to matrices pre-adsorbed with Con A by non-covalent bonds was a successful strategy to increase the yield of glycoprotein immobilization (Kulshrestha and Husain, 2006). The effectiveness factor of an immobilized enzyme is a measure of internal diffusion and reflects the efficiency of the immobilization procedure (Matto and Husain, 2006).
M. Mano, Q, Husam / Ecotoxicology and Environmental Safety • fun) IH-III
Immobilized TP exhibited a higher dye decolonzation at different pH as compared to its soluble counterpart (Fig. 2). The immobilization of TP on Con A-WS confers additional resistance to the enzyme against extreme conditions of pH. The immobilized TP was quite effective in decolorizing DR 23 and mixture of dyes in batch processes at higher temperatures compared to the soluble TP (Fig. 3). The tolerance to elevated temperature was attributed to the complexing of enzymes with lectin, which enhanced its thermal stability (Akhtar et al., 2005a; Matto and Husain, 2006).
The immobilized TP caused more than 70X dye decolorization in the presence of (0.5 M) sodium chloride for both dye solutions after 1 h of incubation (Table 2). This supports an earlier observation that the magnitude of inhibition of laccases by halides depends on the accessibility of the copper atoms and
Q2 can vary between different laccases and inhibitors (Abadullah et al., 2000).
Effluents from textile industries also contain heavy metals (Hatvani and Mecs. 2003; Einoliahi et al., 2006). The decolorization of direct dyes in the presence of metal ions exhibited that the immobilized TP was significantly more efficient than STP. Thus the immobilized TP preparation could be exploited to treat aromatic pollutants present in industrial effluents even in the presence of heavy metals (Table 3).
Enzymes exploited for the treatment of wastewater containing aromatic pollutants would be affected by the presence of water-miscible organic solvents. ITP has shown remarkable potential in the decolorization of dyes in the presence of 10% and 30% dioxane (Table 4). It has already been reported that immobilization of enzymes by multipoint attachment protects them from denatura-tion mediated by organic solvents (Kulshrestha and Husain, 2006). Enzymatic catalysis in organic solvents is possible if the organic solvent does not substantially disturb the active site structure
0,3 (Ryu and Dorick, 1992). One of the important advantages of immobilized enzyme is its
reusability, which influences the cost of industrial applications (Akhtar et al., 2005c). The immobilized TP could retain more than 40% DR 23 decolorizing activity even after eighth repeated uses (Fig. 4).
Considering the success of immobilized TP in dye decolorization, next step was to evaluate its efficiency at large scale by using two-reactor system. The performance of the reactors was quite efficient as they fulfilled the following requirements: (i) limited accessibility to the immobilized enzyme does not seem to play a role since decolorization by free enzyme was never better than
0,4 with immobilized preparation (Andreas et al., 2004), (ii) retention of the immobilized enzyme in the reactor in order to maintain an effective operation (Lopez et al., 2002), (iii) maintenance of a good flow rate in order to ensure efficient dye decolorization/degrada-tion (Palmien et al., 2005), and (iv) the performance of the continuous reactor was improved in terms of dye degradation by using activated silica in the second reactor, which helped in the adsorption of toxic reactive species (Azni and Katayon, 2003).
The treatment of DR 23 and mixture of dyes by passing through a double reactor system provided almost the water free from dyes. The dyes treated by ITP present in the first column, got adsorbed in the second column, which contained activated silica. Both the reactors worked for more than 90 days approximately, thus explaining there efficiency towards dye decolorization.
A significant loss of color appeared when DR 23 or mixture of dyes was treated with Con A-WS bound TP in the presence of redox mediator, HOBT in a continuous reactor system (Fig. 5). It has eariier been reported that the disappearance of peak in visible region was either due to the breakdown of chromophoric groups present in dyes or the removal of pollutants m the form of insoluble products (Moreira et al., 2001; Bhunia et al., 2001; Akhtar et al., 2005c).
However, the difference in the dye decolorization of DR 23 and mixture of dyes was attributed to the earlier observation that biodegradation of various phenolic compounds and dyes in the form of complex mixtures was quite slow compared to individual phenol/dye (Kahru et al., 2000; Akhtar et al., 2005c). This was probably due to the competition between various phenols/dyes for the active site of the enzyme or certain aromatic compounds were found to be recalcitrant or slowly transformed substrates by the action of enzyme. These observations suggested that the industrial effluents containing mixture of direct dyes could be successfully treated with Con A-WS bound TP.
The level of TOC in case of continuous reactors was significantly decreased in the presence of immobilized TP treated polluted water. However, the ITP treated dye solutions exhibited great loss of TOC from the wastewater (Table 5), which suggested that the major toxic compounds could have been removed out of the treated samples. Immobilized HRP has removed 88% of TOC from model wastewater containing mixture of chlorophenols (Tatsumi et al., 1996). Recently in this laboratory it was reported that a significant amount of TOC from polluted water containing dyes/dye-mixtures and dyeing effluent was removed when treated with soluble and immobilized bitter gourd peroxidase (Akhtar et al., 2005c). These evidences strongly proved that Con A-WS bound TP could be successfully used for the removal of dye effluents from various textile, printing and other industries.
We also observed that there was no physical adsorption of direct dye/mixture of direct dyes on the support, as all the possibilities of adsorption were checked. The dye solutions were found to be stable upon exposure to Con A-WS support, H2O2. silica gel or to enzyme alone, respectively. Thus, the dye decolorization was a result of H202-dependent enzymatic reaction, (Kjssibly involving free-radical formation followed by polymerization and precipitarion. In batch processes the loss of dye color was due to removal of aromatic compounds by precipitation but the decreased in aromatic pollutants from the dye solutions was because of adsorption of initial product free radical compound on the activated silica present in second column.
5. Conclusion
Here an attempt has been made to obtain a simple, inexpensive and stable Con A-WS bound TP preparation. A simply operated two-reactor system for decolorization/degradation of direct dyes has focused on the potential future use of immobilized peroxidases. Indeed, the described system is developed with a cheaper biocatalyst that is quite effective in treating dyes continuously in a small laboratory reactor. Thus, this work may provide a reasonable basis for development of biotechnological processes for continuous color and aromatic compounds removal from various industrial effluents at large scale.
Acknowledgments
The authors are thankful to the University Grants Commission and Department of Science and Technology. New Delhi, Government of India for providing special grants to the Department in the form of DRS and FIST, respectively for developing infrastructure facilities.
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