role of microbial extracellular enzymes in the ... · amplified and sequenced. sequence analysis...

237

J Pharm Chem Biol Sci, September - November 2018; 6(3):237-249

Journal of Pharmaceutical, Chemical and Biological Sciences

ISSN: 2348-7658

CODEN: JPCBBG

September - November 2018; 6(3):237-249 Online available at https:/ /www.jpcbs.info

Role of Microbial Extracellular Enzymes in the Biodegradation of

Wastes

Monisha Khanna Kapur1*, Payal Das1, Prateek Kumar1, Munendra Kumar1, Renu

Solanki2

1Acharya Narendra Dev College, University of Delhi, Govindpuri, Kalkaji, New Delhi 110019, India 2Deen Dayal Upadhyaya College, University of Delhi, Sector 3, Dwarka, Opp NSIT, New Delhi,110078,

India

*CORRESPONDING AUTHOR

Dr. Monisha Khanna Kapur, Principal Investigator, Microbial

Technology Lab, Acharya Narendra Dev College, University of

Delhi , Govindpuri, Kalkaji, New Delhi 110 019

Email: [email protected],

[email protected]

ARTICLE INFORMATION

Received July 07, 2018

Revised September 02, 2018

Accepted September 12, 2018

Published November 30, 2018

INTRODUCTION

Actinomycetes are group of gram positive

filamentous soil bacteria [1, 2] well known as

producers of various types of extracellular

enzymes. These enzymes are produced within

the cell but are secreted and function outside the

cell [3]. Extracellular enzymes are of great

importance because they have large scale

applications in various industries such as paper,

textile, detergent, cosmetics, biosensors,

pharmaceuticals, agricultural, bioremediation

and biorefineries [5,6]. They can easily degrade

wastes into useful raw materials that can be

directly recycled in industries.

Research Article

The work is licensed under

ABSTRACT

Several soil bacteria produce extracellular enzymes having potential to degrade wastes into useful raw

materials that can be directly recycled in industries. Actinomycetes are well-known producers of

extracellular enzymes. In our study, during primary screening, isolate 196 was found to be efficient in

degrading sugarcane bagasse and corn stover and showed high cellulase activity. Similarly isolate 197

showed high xylanase activity, degrading rice straw and wheat bran; isolate 136 showed high chitinase

activity and degraded crustacean shells and isolate 244 showed maximum phosphatase activity,

degrading fruit pulp. Taxonomic characterization using 16S rDNA study revealed that these bacteria

isolated from soil belong to genus Streptomyces. Based on the results of primary screening, 196 and 197

were selected for secondary analysis. In secondary screening isolate 196 showed cellulase activity of

2.86 IU/ml/min at pH 6.80-7.0 and 2.92 IU/ml/min activity at 300C. Isolate 197 showed xylanase

activity at the rate of 3.86 IU/ml/min at pH 7.2 and 3.72 IU/ml/min at temperature range of 280C-300C.

Statistical analysis of fermentation conditions using one way ANOVA and Post Hoc test analyses

signified a notable pH and temperature effect on enzyme activity of isolates 196 and 197.

KEYWORDS: Extracellular enzymes; Actinomycetes; environmental wastes; fermentation conditions;

statistical analysis.

Kapur et al 238


Traditional chemical and physical treatments on

biodegradable waste materials are not efficient

methods for degradation because these processes

are costly, take longer time and involve use of

chemicals having hazardous properties.

Biodegradation using microbial extracellular

enzymes is an efficient and economical

alternative to the traditional methods [7].

Microorganisms can convert hazardous waste

materials into non-hazardous form by enzymatic

processes [8]. Microbes like bacteria, fungi and

yeast involved in biodegradation process use

organic waste material as the source of energy

resulting in the complete biodegradation of

wastes.

In the present study, biodegradable waste

samples were collected from agro-industrial,

fishery industries and household sites [9].

Samples were pretreated for converting them

into powdered substrates. Actinomycete cultures

from diverse habitats and producing

extracellular enzymes were subjected to primary

screening. Isolates showing high production of

enzymes were selected and subjected to

taxonomic characterization by polyphasic

approach. 16S rRNA genes of the strains were

amplified and sequenced. Sequence analysis was

done using “Sequencing analysis 5.1.1” software

and “EzTaxon” server, and it was found that

isolates 196, 197, 136 and 244 belong to the

genus Streptomyces. After analysis of sequence,

evolutionary trees based on 16S rDNA were

designed with the help of “Phylip-3.69” software

package. These isolates were then subjected to

secondary screening to determine range of

enzyme activity at different pH and temperature.

Statistical analyses of fermentation conditions

including temperature and pH was done using

Post Hoc tests (Turkey HSD) and one way

ANOVA by IBM SPSS Statistics 19 software.

MATERIALS & METHODS

Collection of waste samples from selected

sites and their conversion into substrates

Collection of waste samples

Waste samples were collected from diverse

ecological habitats and were transferred to the

laboratory in zip-lock bags (Table 1).

Table 1: Collection sites for waste materials

Pre-treatment of waste samples to be used

as substrate

A multitude of different pre-treatment methods

have been suggested in literature. These are

placed under (a.) physical (grinding, irradiation,

milling), (b.) chemical methods (alkali/dilute acid

treatment, effects of organic solvents, oxidizing

acids), (c.) physicochemical (steam pre-

treatment/auto-hydrolysis, wet oxidation, hydro-

thermolysis) (d.) biological (combined use of

lignin degrading enzymes like peroxidases and

laccases) [10-12].

Screening of isolates at primary level for

checking production of Xylanase, Cellulase,

Chitinase, Phosphatase

Reviving of actinomycete cultures

Actinomycete cultures representing different

ecological habitats and known to be producing

extracellular enzymes were selected (Table 2).

Waste material used as

substrate

Collection site Waste degraded by

enzyme

Sugarcane bagasse Govindpuri Local Juice Corner

Cellulase

Corn Stover Okhla PhaseI, New Delhi

Wheat Bran Local atta chakki, Govindpuri, New

Delhi

Xylanase

Rice straw Matloda, Hisar, Haryana

Crustacean shells Ghazipur, New Delhi, Fish Market

Chitinase

Fruit pulp Jawahar Colony, Faridabad Local

Juice

Phosphatase

Kapur et al 239


The cultures were revived and restreaked on

Yeast extract – Malt extract (YM agar) [13, 14]

for getting purified culture plates.

Table 2: List of actinomycete isolates selected for Screening

Primary Screening

Media were prepared and supplemented with

specific pre-treated waste samples or with

commercial substrates.

1. Basal agar medium (pH 6.8-7.0) for

cellulase (composition (gL-1) - (NH4)2SO4

S.No. Properties Habitats Number assigned to

isolates/Colonies

1.

Cellulose

degrading

isolates

Agricultural Soil- Dhanaura, UP,

India

84

2. Agricultural Soil- Yamuna River,

Delhi, India

196

3. Dumping Site- Sarai Kale Khan,

Delhi, India

191

4. Purana Quila- Delhi, India 106

5. Diversity Park- Sarai Kale Khan,

Delhi, India

186

6. Dumping Site- Sarai Kale Khan,

Delhi, India

Isolate 194, Positive Control

(Previous study)

1.

Xylan degrading

isolates


India

43


Delhi, India

88

3. Sarai kale Khan, Dumping Site,

Delhi, India

197

4. Sugar Plant- Dhanaura, UP, India 61

5.

Sugar Plant- Dhanaura, UP, India Isolate 169, Positive Control

(Previous study)

1.

Chitin

degrading

isolates


India

4


Delhi, India

222

3. Sarai Kale Khan, Dumping Site

Delhi, India

88

4. Sugar Plant- Dhanaura, UP, India 66

5. Chemical Plant- Faridabad, HR,

India

136

6. Chemical Plant- Faridabad HR,

India


(Previous study)

1. Phosphate

degrading

isolates

Agricultural Soil- Nainital, UK,

India

116

2. Agricultural Soil- Kashipur, UK,

India

161

3. Yamuna Bank- Delhi, India 79

4. Great Himalayan National Park-

Teerthan Valley, HP, India

244

5. Agricultural soil- Kashipur, UK,

India


(Previous study)

Kapur et al 240


2.64, KH2PO4 2.38, K2HPO4.3H2O 5.65,

MgSO4.7H2O, trace salt solution

(composition gL-1) 1ml. CuSO4.5H2O

6.43, FeSO4.7H2O 1.1, MnCl2.4H2O 7.9,

ZnSO4.7H2O 1.5, agar 15 [13]. The

medium was supplemented with

sugarcane bagasse, corn stover and

cellulose (commercial substrate)

respectively.

2. Mineral Salt agar medium (pH 7.2) for

xylanase (composition (gL-1) NaNO3 2.0,

KCl 0.5, Fe2 (SO4)3 0.01, MgSO4.7H2O

0.5, KH2PO4 0.14, K2HPO4.3H2O 1.2,

Yeast extract 0.02, agar 15 [15]. The

medium was supplemented with rice

straw, wheat bran and birchwood xylan

(commercial substrate) respectively.

3. Chitin medium (pH 8.0) for chitinase

(composition gL-1) KH2PO4 0.3,

ZnSO4.7H2O 0.001, MgSO4.5H2O 0.5,

K2HPO4 0.7, FeSO4.7H2O 0.01, agar 20

[16, 17]. The medium was supplemented

with crustacean shells and colloidal chitin

(commercial substrate) respectively.

4. Pikovskaya’s medium (pH 7.2) for

phosphatase (composition gL-1)

ZnSO4.7H2O 0.001, K2HPO4 0.7,

MgSO4.5H2O 0.5, KH2PO40.5,

FeSO4.7H2O 0.01, Bromophenol blue

(composition- 0.4g in ethanol, pH 6.5

using NaOH, working concentration=

0.08%, 0.16%, 0.24%, 0.32%, 0.40%), agar

20 [18, 19]. The medium was

supplemented with fruit pulp and

tricalcium phosphate (commercial

substrate) respectively.

The strains were subjected to plate assay

(primary screening) by spot inoculating

them on different media plates.

Inoculated plates were incubated for 7-21

days at 280C for secretion of extracellular

enzymes. Clear zone surrounding the

bacterial colonies indicates production of

enzyme. Zones of hydrolysis were

calculated by subtracting the size of

inoculum from the total diameter of zone.

Each experiment was carried out

independently with three replicates.

Study of taxonomic status of the strains

by Polyphasic approach using

Bioinformatics tools:

The highest enzyme producers (isolates

196,197,136 and 244) were selected for

determining their taxonomic status.

Genomic DNA isolation of highest enzyme

producers by Phenol-Chloroform method:

Genomic DNA was isolated by methods

standardized for actinomycetes [13, 20].

Polymerase chain reaction (PCR) of 16s

rRNA gene of isolates 196, 197, 136, 244:

Genomic DNA of the selected isolates was

subjected to 16s rRNA gene amplification

using specific primers and under defined

conditions (Table 3 and 4). The final product

was analyzed on agarose gel [1, 13, 20, 21].

Table 3: 16s rRNA gene specific Primers

Table 4: The PCR conditions used for 16s rRNA gene amplification

S.No. Primer Sequence of Primer

1. 8F 5’-AAGGAGGTGATCCAGCCGCA-3’

2. 1492R 5’-AGAGGTGATCCAGCCGCA-3’

3. 27F 5’-AGAGTTTGATCCTGGCTCA-3’

4. 1542R 5’-AAGGAGGTGATCCAGCCGCA-3’

PCR reaction mix (100µl) PCR conditions

MQ= 74.2µl 35 cycles

gDNA(template)= 3µl Initial denaturation: 5min. at 95°C

10X PCR Buffer= 10µl Denaturation: 30sec. at 95°C

dNTP(s)= 1.5µl Primer annealing: 30sec. at 55°C

Primer 8F „OR‟ 27F= 5µl Primer extension: 1min. 30sec. at 72°C

Primer 1492R „OR‟ 1542R= 5µl Final extension: 10min. at 72°C

Taq DNA polymerase= 1.3µl

Kapur et al 241


Elution and sequencing of amplified PCR

product(s) of isolates 196, 197, 136, 244:

The amplified PCR fragments were processed

using the QIAGEN gel extraction kit and were

submitted for sequencing. The sequences

obtained were analyzed and assembled using

Sequence analysis 5.1.1 software. The resultant

assembled sequence was then searched using EZ

Taxon and NCBI database and the FASTA

format sequences of its homologies (20 in total)

were copied and pasted in a notepad file [13,20

,22].

Phylogenetic tree construction using

“Phylip-3.69” for Taxonomical analysis of

Isolates 196,197,136,244:

The multiple FASTA sequence file was aligned

using ClustalX software. The resultant output

sequences were then used to construct

phylogenetic tree using Phylip 3.69. The tree was

viewed using Treeview software [1, 21, 23].

Secondary screening

Optimization of fermentation conditions

for cellulase and xylanase Based on the

results of primary screening, the colonies

showing maximum zone of clearance in case of

cellulase and xylanase were subjected to

submerged fermentation process (SmF). The

selected isolates were cultured in 25ml 148G

broth (g/L-1)- Beef extract 4g, Glucose 2.2g,

Bacto-peptone 5g, Tryptone 3g, NaCl 1.5g, Yeast

extract 0.5g (pH 7.5) incubated at 28 ֯C (5 to 6

days) at 200rpm on incubator shaker [6]. A

standard inoculum having average viable count

105 to 107 CFUs/ml was inoculated in respective

medium for enzyme production. Culture flasks

were kept for incubation at range of

temperature, pH values for determination of

optimum conditions for enzyme action.

Enzyme activity estimation for cellulase

and xylanase

Well grown, week old cultures were centrifuged

to obtain cell free supernatant which was taken

as crude enzyme source. Dinitrosalicyclic (DNS)

method was used to estimate enzyme activity for

both enzymes in crude extract. 0.5ml of crude

cell free extract from the culture flasks of

both processed wastes and commerc ial

substrates were added to 1.5ml volume of 2%

cellulose, prepared in 0.05M sodium citrate

buffer having pH value of 4.8 and incubated at

40°C for 30 minutes. To each tube, 3ml DNS

reagent was added, tubes were then incubated

for 5 minutes at 100°C [3, 13, 24, 25].

Absorbance was recorded at 575 nm using

spectrophotometer (UV Vis Elico SI-159).

Glucose standard curve was made and by

extrapolating the absorbance values on the

graph, amount of glucose in each sample was

determined. One unit of enzyme activity is the

amount of enzyme that releases 1μM of

glucose/ml/min. Each experiment was carried

out independently with three replicates.

Analyses of enzymatic activity by

univariate and multivariate methods using

statistical software

Experimental data was statistically evaluated

using Post-Hoc and ANOVA analyses at p < 0.05

(significance level) by using software IBM SPSS

19.

RESULTS

Collection of waste samples from selected

sites and their conversion into substrates.

In the present study, the waste samples collected

from various sites (Figure 1) were processed and

converted into powdered substrates by different

pre-treatment methods.

Utilization of enzymatic potential of bacterial

cultures for degradation of waste

Kapur et al 242


Figure 1: Collection sites of waste samples from varied habitats and their processing into

substrates

Reviving of actinomycete cultures

Actinomycete cultures representing different

ecological habitats and known to be producing

extracellular enzymes were selected and revived.

Primary Screening

During primary screening, isolate no. 196 was

found to be most efficient in degrading sugarcane

bagasse and corn stover and showed maximum

cellulase activity (32mm on bagasse and 24 mm

on corn stover substrates) followed by isolate no.

194 (positive control, 26mm and18mmm), isolate

no. 106 (12mm), isolate no. 84 (7mm and 5mm),

isolate 186 (3mm) and isolate 191 (no zone on

bagasse and corn stover substrates) (Figure 2).

Figure 2: Colonies with Cellulase enzyme activity showing degradation of processed waste

supplemented in growth medium plates in form of zones of clearance (denoted by arrows)

Colony 196 on BM +

bagasse plate (congo red

staining)

Colony 186 on BM +

bagasse

plate (congo red staining)

Colony194 (Positive

control) on BM +

bagasse plate (congo red

staining)

Kapur et al 243


Similarly isolate 197 showed maximum xylanase

activity (21mm on rice straw 15mm and wheat

bran substrates) followed by isolate no. 169

(positive control, 13mm and 18mm), isolate no.

61 (10mm and 7mm), isolate no. 85 (6mm and

4mm) and isolate no. 43 (3mm) (Figure 3).

Figure 3: Colonies with Xylanase enzyme activity showing degradation of processed waste


In case of chitinase, isolate no. 136 showed

maximum chitinase activity (30mm on

Crustacean shells substrate) followed by isolate

no. 130 (positive control, 22mm), isolate no. 222

(12mm), isolate no. 88 (9mm) and isolate no. 04

(5mm) ( (Figure 4). In phosphatase, isolate no.

244 showed maximum phosphatase activity

(20mm on Fruit pulp substrate) followed by

isolate no. 79 (15mm), isolate no. 165 (positive

control, 12mm), isolate no. 161 (9mm) and

isolate no. 116 (1mm) (Figure 5).

Figure 4: Colonies with Chitinase enzyme activity showing degradation of processed waste


Colony 197 on MSA + wheat

bran plate (congo red staining)

Colony 88 on MSA + wheat

bran plate (congo red

staining)

Colony 169 (Positive control)

on MSA + wheat bran plate

Colony 136 on CM+

crustacean shells plate Colony 61 on CM+

crustacean shells plate

Colony 136 on CM+

crustacean shells plate

Kapur et al 244


Figure 5: Colonies with phosphatase enzyme activity showing degradation of processed

waste supplemented in growth medium plates in form of zones of clearance (denoted by

arrows).

Study of taxonomic status of strains by

Polyphasic approach using Bioinformatics

tools

Genomic DNA isolation of highest enzyme

producers by Phenol-Chloroform method

Taxonomic characterization of highest enzyme

producers (isolates 196,197,136 & 244) was done

by polyphasic approach. Genomic DNA profile on

the gel showed a sharp single band for all the

strains.

Polymerase chain reaction (PCR) of 16s

rRNA gene of isolates 196, 197, 136, 244

Genomic DNA of the selected isolates were

subjected to 16s rRNA gene amplification using

specific primers. The final product was analyzed

on 0.8% agarose gel. Using Lambda DNA

EcoRI/HindIII double digested ladder as

reference it was found that the size of eluted

products of isolates 96, 197, 136, 244 was

≈1500bp which is equal to the size of 16s rRNA

gene.

Elution and sequencing of amplified PCR

product(s) of isolates 196, 197, 136, 244

The amplified product were then eluted and size

of the band obtained in each case was found to

be≈1500bp. The eluted product samples were

submitted for sequencing. The sequences

obtained were analyzed and assembled using

Sequence analysis 5.1.1 software. The resultant

assembled sequence was then searched using EZ

Taxon and the FASTA sequences of it homologs

(20 in total) were copied and pasted in a notepad

file which was used for construction of

phylogenetic trees.

Phylogenetic tree construction using

“Phylip-3.69” for taxonomical analysis of

isolates 196,197,136,244

After sequencing, the resultant assembled

sequences were compared with the sequences of

close Streptomyces species deposited in database.

The results showed that all the strains belong to

the genus Streptomyces. Actinomycete isolates

196, 197, 136, 244 were represented as distinct

clades in their respective rooted evolutionary

tress designed by neighbor-joining method.

Isolate 196: 16s rRNA gene sequence of 1409

nucleotides was generated for isolate 196.

Phylogenetic studies revealed that isolate 196

showed 100% similarity with Streptomyces

albolongus followed by 99.93%, 99.50%, 99.43%

with Streptomyces cavourensis, Streptomyces

puniceus, Streptomyces bacillaris respectively.




has highest 16s rRNA similarity of 100% with

Streptomyces griseorubens followed by 99.79%,

99.79%, 99.79% with Streptomyces althioticus.




has highest 16s rRNA similarity of 100% with

Streptomyces parvulus followed by 99.37%,

99.30%, 99.30% with Streptomyces

malachitospinus, Streptomyces olivaceus,

Colony 161 on PKV+ fruit

pulp plate (bromophenol

blue staining)



blue staining)



blue staining)

Kapur et al 245


Streptomyces pactum.



Phylogenetic studies revealed the isolate 244 has

highest 16s rRNA similarity of 100% with

Streptomyces parvulus followed by 99.37%,

99.30%, 99.30% with

Streptomycesmalachitospinus, Streptomyces

olivaceus, Streptomyces pactum.

Secondary screening

Based on the results of primary screening,

isolates 196 (for cellulase) and 197 (for xylanase)

were selected for quantitative analyses using

fermentation parameters of pH and temperature

so as to identify the conditions at which enzymes

show highest levels of activity. In case of isolate

196, maximum cellulase activity was 2.86

IU/ml/min at pH 6.8-7.0 and 2.92 IU/ml/min

activity at temperature 300C (Table 5, Figure 6

and 7). Isolate 197 showed maximum xylanase

activity, 3.86IU/ml/min at pH 7.2 and 3.72

IU/ml/min activity at temperature range of

280C-300C (Table 6, Figure 8 & 9).

Table 5: Optimization of fermentation conditions for Isolate 196 (Cellulase producer)

Table 6: Optimization of fermentation conditions for isolate 197 (Xylanase producer)

Analyses of enzymatic activity by

univariate and multivariate methods

using statistical software One way ANOVA was used for statistical

analyses of fermentation conditions including

temperature and pH, which showed a notable

temperature and pH effect on enzyme activity

of isolates 196 and 197. For isolate 196 it was

F (5, 12) = 111288.172, p = .000 for pH and F

(5, 12) = 111288.172, p = .000 for temperature.

For isolate 197 it was F (5, 12) = 66581.475, p

= .000 for pH and F (5, 12) = 137621.778, p =

.000 for temperature.

Results were re-checked using Post Hoc

analysis where a statistically significant

variation in activity was observed at various

values of temperature and pH. In conclusion,

with increasing temperature and pH, enzyme

activity increases initially, then attains a

maximum level and finally decreases gradually

Cultures pH Enzyme activity (IU/ml/min) Temp.

(°C)

Enzyme activity

(IU/ml/min)

Isolate 196

(Agricultural

soil,

Yamuna

river, Delhi)

Substrate-

Sugarcane

baggase

6.4 0.67 26 0.90

6.6 0.89 28 0.92

6.8 2.86 30 2.92

7.0 2.80 32 0.65

7.2 0.63 34 0.12

7.4 0.056 36 0.09

CULTURES pH Enzyme activity

(IU/ml/min)

Temp. Enzyme

activity

(IU/ml/min)

Isolate 197

(Dumping Site, Sarai kale

Khan, Delhi)

(Substrate-Rice straw)

6.6 0.72 240C 0.68

6.8 0.92 260C 0.78

7.0 1.12 280C 3.72

7.2 3.86 300C 3.70

7.4 0.96 320C 1.12

7.6 0.091 340C 0.61

Kapur et al 246


hence producing a bell shaped curve. For

isolate 196, maximum cellulase activity was at

pH range 6.80-7.0, temperature 300C (Figure

10(a, b)) and for isolate 197, maximum

xylanase activity was at pH 7.2, temperature

range 280C -300C (Figure 11 (a, b)).

DISCUSSION

Actinomycetes are well known producers of

extracellular enzymes [26, 27, 28, 29]. In the

current study actinomycetes were isolated from

diverse habitats including Agricultural Soil-

Yamuna River, Delhi; waste dumping site (Sarai

Kale Khan) Delhi; chemical plant (NTPC)-

Faridabad and Great Himalayan National

Park- Teerthan Valley and were screened for

their ability to produce extracellular enzymes.

Isolation of actinomycete strains from varied

habitats have been reported by researchers

earlier too for screening of extracellular enzyme

producers [30-33]. They have isolated bacterial

strains from diverse habitats and screened the

cultures for presence of cellulase, xylanase,

chitinase and phosphatase activity respectively.

During primary screening, isolate no. 196 was

found to be most efficient in degrading

sugarcane bagasse and corn stover and showed

maximum cellulase activity (32mm on bagasse

and 24mm on corn stover substrates) as

compared to other colonies and positive control.

Similarly isolate 197 showed maximum xylanase

activity (21mm on rice straw and 15mm on

wheat bran substrate), isolate 136 showed

maximum chitinase activity (30mm on

crustacean shells substrate) and isolate 244

showed maximum phosphatase activity (20mm

on fruit pulp substrate).

Nayaka and Vidyasagar [34], screened isolates

from different habitats and found them to yield

zones of clearance for degradation of sugarcane

bagasse in the range of 19-52mm. Porsuk et al.,

[35] obtained zones of clearance of 12-25mm for

rice straw degrading bacterial isolates. Similar

experiments with wheat bran, crustacean shell

and fruit pulp degrading bacteria have been

reported in literature [33, 36-38].

Taxonomic characterization of selected isolates

was done by polyphasic approach. 16SrRNA gene

sequence analyses showed that isolates 196, 197,

136 and 244 belong to the genus Streptomyces.

Sequence similarity between isolate 196 with

Streptomyces albolongus was 100%, isolate 197

with Streptomyces griseorubens was 100%,

isolate 244 with Streptomyces parvulus was

100% and isolate 136 with Streptomyces

parvulus was 100%. Yassein et al., [39]

performed taxonomic study of the selected

cellulase producing Streptomyces isolate C188 by

16S rRNA gene analysis. The results of 16S

rRNA genes showed high similarity (98 to 100%)

with Streptomyces. Ninawe et al., [40] analyzed

16S rRNA gene sequences of three xylanase

producing strains and found that the strains

belong to the genus Strepomyces. Similar work

has been reported which includes the use of

16SrRNA gene sequence for taxonomic

characterization of actinomycete isolates and

pH

Figure 10 (a): Bell shaped curve of

activity at different pH shown by isolate

196

Temperature

Figure 10 (b): Bell shaped curve of

activity at different temperature shown

by isolate

Kapur et al 247


found that the strains belong to the genus

Streptomyces [1, 21-23, 33].

Based on the results of primary screening,

isolates 196 (for cellulase) and 197 (for xylanase)

were selected for quantitative analyses using

fermentation parameters of pH and temperature

so as to identify the conditions at which enzymes

show highest levels of activity. In case of isolate

196, maximum cellulase activity was 2.86

IU/ml/min at pH 6.8 and 2.92IU/ml/min activity

at 300C. Isolate 197 showed maximum xylanase

activity, 3.86IU/ml/min at pH 7.2 and

3.72IU/ml/min activity at 280C.

Golinska and Dahm, [41] estimated cellulase

activity in different Streptomyces sp. at different

pH and temperature and found enzyme activity

in the range of 8-13IU/ml using sugarcane

bagasse as substrate. Rawashdeh et al., [42]

estimated maximum xylanase activity of

5.12IU/ml/min at pH 6.5 and 5.52IU/ml/min at

temperature 600C using rice straw as substrate.

Statistical study of fermentation conditions

including temperature and pH was done using

Post-Hoc test (Turkey HSD) and ANOVA (one

way) test signified a notable pH and temperature

effect on enzyme activity of isolates 196 and 197.

Researchers used the same approach for

statistical analysis of fermentation conditions

and observed similar results [22, 43-45].

CONCLUSION

Actinomycete isolates from diverse habitats were

studied for their potential of waste degradation

during primary screening. It was observed that

isolates 196, 197, 136 and 244 are high

producers of cellulase, xylanase, chitinase and

phosphatase respectively and were found

efficient in degradation of sugarcane bagasse

and corn stover; rice straw and wheat bran;

crustacean shells; fruit pulp.Secondary

screening was performed for 196 (cellulase

producer) and 197 (xylanase producers) and with

the help of statistical analyses it was concluded

that there is a notable effect of pH and

temperature on enzyme activity. On increasing

pH and temperature, enzyme activity increases

initially, then attains a maximum level and

finally decrease gradually hence producing a bell

shaped curve. As a conclusion of our study,

actinomycete isolates were found efficient in

degrading various agro- industrial, fishery and

household wastes. Future studies involve

lyophilization of the bacterial cultures, using

their liquid formulations for degradation of

wastes kept either in compost bags under lab

conditions or alternatively degradation of wastes

directly in the field.

ACKNOWLEDGEMENT

The authors acknowledge University of Delhi for

granting the financial assistance for research

work under the Innovation Project scheme 2015-

2016. Acharya Narendra Dev College (ANDC),

University of Delhi is gratefully acknowledged

for providing infrastructural facilities.

CONFLICT OF INTEREST

The authors declare no conflict of interest

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Cite this article as:

Monisha Khanna Kapur, Payal Das, Prateek Kumar, Munendra Kumar, Renu Solanki. Role of

Microbial Extracellular Enzymes in the Biodegradation of Wastes. J Pharm Chem Biol Sci 2018;

6(3):237-249

role of microbial extracellular enzymes in the ... · amplified and sequenced. sequence analysis...

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