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Chapter VI
Part b: Production, purification
and characterization of
alkaliphilic mannanase by
Exiguobacterium sp. VSG-1
219
6.1. Introduction
Mannans are the major component of the hemicelluloses fraction in soft woods. These
polysaccharides are also found in various seeds, where they play an important role in the
mechanical resistance and the swelling that occur during germination. Mannanase (β-1,
4-D-mannan, mannanohydrolase; EC 3.2.1.78) catalyzes the random hydrolysis of β-1, 4
mannosidic linkages in β-1, 4-mannan, glucomannan and galactomannan. Mannans and
heteromannans are widely distributed in nature as part of the hemicellulose fraction in
hardwoods, softwoods (Capoe et al. 2000), seeds of leguminous plants (Handford et al.
2003). They were composed of a backbone of β-1, 4-linked mannose (and glucose) units,
which are often substituted with galactose and acetate residues depending on their origin.
For complete hydrolysis of these mannans, many mannanolytic micro-organisms
synthesize the multiple mannanolytic enzymes for co-operative actions. These enzymes
include endo- β-1, 4-mannanase (EC 3.2.1.78), β-mannosidase (EC 3.2.1.25) and
enzymes that cleave side chain sugars from the mannan backbone, such as, α-
galactosidase (EC 3.2.1.22) and acetyl esterase (EC 3.1.1.6).
Mannanases from a vast array of microorganisms have been purified and evaluated for
various applications (Moreira and Filho 2007). Mannanases have many possible
applications. They were useful in pulp bleaching (Gubitz et al. 1997), reduction of
viscosity of instant coffee, clarification of fruit juices and wines (Coughlan et al. 1993)
bioconversion of biomass wastes to fermentable sugars and upgrading of animal feed
stuff. Some of the alkaline mannanases were used in detergent (Cueves et al. 1996), paper
industries (Gubitz et al. 1997) as well as in food industries (Coughlan et al. 1993). This
220
triggered research interest into the biochemical properties of these enzymes. As a result,
β-mannanases have been purified from both bacterial and fungal sources (Ademark et al.
1998; Ferriera et al. 2004). The β-mannanases reported so far exhibit acidic to neutral pH
optima, molecular mass ranging from 18 to 162 kDa, and mesophillic to moderately
thermophilic temperature optima (Hatada et al. 2005).
Many mannan based carbon sources have been used to cultivate bacteria. These included
locust bean gum (LBG) (Ademark et al. 1998), konjac flour (Oda et al. 1993), guar gum
(McCutchen et al. 1996) and copra meal (Hossain et al. 1996; Ademark et al. 1998).
Although LBG represents the most common carbon source, there are only a few reports
in literature for the best carbon source to cultivate microorganisms (Ademark et al. 1998).
Copra, a well-dried coconut kernel from coconut palm usually regarded as a byproduct of
coconut extraction, which contains a large amount of mannose in the form of mannan,
consisting of repeating β-1, 4 mannose backbone (Hossain et al. 1996). Thus, it seems to
be a best carbon source for the production of mannanase by different microorganisms.
Given the natural abundance and complexity of mannan, many microorganisms, produce
enzyme system to hydrolyze mannan completely that can be used as energy, feed and
food sources.
Relatively, very few alkaline, halotolerent and thermostable mannanase have been
reported which were active between pH 9.0-10.0 (Akino et al. 1989; Hatada et al. 2005).
The most potent use of β-mannanase was enzymatic bleaching of softwood pulps. Since
the pulp used in paper industry for enzymatic bleaching is hot and alkaline, the use of
thermostable alkaline mannanase is highly desirable at the industrial and economic point
221
of view. Pulp treatment under alkaline conditions hydrolyzes hemicelluloses covalently
bound to lignin and helps subsequent removal of lignin. However alkaline treatment of
wood pulps poses environmental pollution. The application of mannanases under such
conditions equally facilitates lignin removal in pulp bleaching and the results were
comparable to alkaline pretreatment (Cuevas et al. 1996). As a result, search for novel
mannanases for paper industries has continued. In the present work, we report the
production, purification and characterization of an extremely alkaline, thermostable β-
mannanse from Exiguobacterium sp. VSG-1.
6.2. Materials and methods
6.2.1. Microorganism
The bacterium used in this study was strain of VSG-1 isolated and identified earlier in
our laboratory as Exiguobacterium sp. as described in chapter 2.
6.2.2. Growth and cultural conditions
The growth and cultural conditions for the production of cellulase by Exiguobacterium
sp. VSG-1 is as explained in the chapter 3.
6.2.3. Enzyme assay
The cellulase activity was determined by measuring the release of reducing sugars using
the DNS method (Miller, 1959) as described in the chapter 3. Protein concentration was
determined by the method of Lowry et al. (1951) using bovine serum albumin as
standard.
222
6.2.4. Defatting of copra
Copra is a well-dried coconut kernel. The copra was finely ground with a grinder for 5
min and sieved (25-30 mesh) and the powder was boiled for 2 h with two volumes of
distilled water. The cooled suspension was then placed at 4°C overnight to allow the oil
to solidify and finally be removed. The dried and sieved residues were then defatted by
the solvent extraction using n-hexane for 24 h. One liter of solvent n-hexane was mixed
with 100 g of ground copra in a beaker and left overnight. The copra suspension was then
filtered through Whatman filter paper no. 1. The residues were oven dried and sieved. All
samples were kept in a desiccator until used.
6.2.5. Effect of temperature, pH and NaCl concentrations on the growth and
mannanase production
The effect of temperature, pH and NaCl concentrations on the growth and enzyme
production was studied as given in the chapter 6a.
6.2.6. Effect of carbon, inexpensive agro waste and nitrogen sources on the growth
and mannanase production
The VSG-1 was grown in different carbon sources such as 1 % (w/v) xylose, arabinose,
galactose, fructose, glucose, mannose, lactose, maltose, sucrose and starch. The
inexpensive 1 % agro waste materials used for the production of mannanase were
defatted copra meal, molasses, wheat bran, corn husk and jowar straw powder. The
nitrogen sources (0.5 %) tested were ammonium sulfate, ammonium nitrate, ammonium
chloride, casein, urea, tryptone, peptone, beef extract, yeast extract and feather
hydrolysate.
223
6.2.7. Enzyme purification
The mannanase enzyme was purified according the standard protocols as described in the
chapter 6a.
6.2.8. Gel electrophoresis and molecular weight determination
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was done
essentially as described by Laemmli (1970) with 12 % acrylamide as described in the
chapter 6a.
6.2.9. Enzyme Kinetics
The kinetic property of the enzyme was determined using LBG and guar gum at a
concentration range of 1-10 mg/ml using Lineweaver-Burk plot; the apparent Km and
Vmax were calculated.
6.2.10. Effect of pH, temperature stability and NaCl concentration on purified
enzyme
The effect of pH and its stability on the activity of purified mannanase was measured in
the as explained in the chapter 6a. The residual activity was determined under standard
assay conditions.
6.2.11. Effect of divalent metal ions on the activity of mannanase
The effect of various cations (1 mM) such as CaCl2, MgCl2, FeCl3, CuSO4, BaCl2,
MnSO4, Pb(NO3)2 and ZnCl2 were tested on purified cellulase and mannanase. The
degree of activation or inhibition of enzyme activity was expressed as a percentage of the
enzyme activity in the control sample.
224
6.2.12. Effect of inhibitors, surfactants, local detergents and bleach on the activity of
mannanase
The purified enzymes were pre-incubated for 30 min before with the following detergents
like SDS (5, 10 and 20 %) and Triton X-100 (1 and 5%), Tween 20 and 80 (1 %), H2O2
(20 %) and local detergents (1 %). Cellulase and mannanase activity was determined as
explained earlier in the chapter 6a.
6.3. Results
6.3.1. Growth and mannanase production
Exiguobacterium sp. VSG-1 strain showed the clear zones on LBG agar plates followed
by staining with 1 % Cong Red solution, indicating that it secretes considerable amounts
of mannanase. The growth and mannanase production of Exiguobacterium sp. VSG-1
(Fig. 6.1b) indicate that there is distinct growth associated with enzyme production.
Mannanase production was observed in the fermentation broth as soon as the bacterium
entered the exponential phase (18 h) and reached maximum in the stationary phase at 48
h (Fig. 6.2b). The optimum cultural conditions for growth and mannanase production
were up to 48 h of incubation which will remain more or less stable until 52 h and then
decreased with increase in incubation time. This is because of continuing depletion of
nutrients and builds up of metabolic wastes results death of the cells at a rapid and
uniform rate.
225
Fig. 6.1b. Hydrolysis of locust bean galactomannan (LBG) by Exiguobacterium sp.
VSG-1.
226
Fig. 6.2b. The effect of incubation time on growth (●) and alkaline mannanase
production (○) by Exiguobacterium sp. VSG-1. at pH 9.0 and temperature 37°C in
presence of 1% LBG under submerged fermentation conditions. Each value represents
the mean ± SD of the three independent experiments.
0
4
8
12
16
20
0 10 20 30 40 50 60
Incubation time (h)
Act
ivity
U/m
l
0
1
2
3
4
Bio
mas
s at 6
60nm
227
Fig. 6.3b. The effect of pH on growth (●) and alkaline mannanase production (○) by
Exiguobacterium sp. VSG-1 at temperature 37°C in presence of 1% LBG under
submerged fermentation conditions. Each value represents the mean ± SD of the three
independent experiments.
0
0.5
1
1.5
2
2.5
0
4
8
12
16
20
5 6 7 8 9 10 11 12
Bio
mas
s at 6
60 n
m
Act
ivity
(U/m
l)
pH
228
Fig. 6.4b. The effect of temperature on growth (●) and alkaline mannanase production
(○) by Exiguobacterium sp. VSG-1 at pH 9.0 in presence of 1% LBG under submerged
fermentation conditions. Each value represents the mean ± SD of the three independent
experiments.
048
121620
20 25 30 35 40 45 50
Temparature C
Act
ivity
U/m
l
0
1
2
3
4
Bio
mas
s at
660
nm
229
Fig. 6.5b. The effect NaCl concentration on growth (●) and alkaline mannanase
production (○) by Exiguobacterium sp. VSG-1 at pH 9.0 and temparature 37°C in
presence of 1% LBG under submerged fermentation conditions. Each value represents
the mean ± SD of the three independent experiments.
048
121620
0 4 8 12 16
NaCl concentration (%)
Act
ivity
U/m
l
0
1
2
3
4
Bio
mas
s at
660
nm
230
6.3.2. Effect of pH, temperature and NaCl concentrations on the growth and
production of alkaline mannanase
Highest growth and enzyme production were observed in alkaline pH (9-12) with an
optimum at 9.0 (Fig. 6.3b). Maximum growth and enzyme secretion were observed in the
temperature range of 35–45oC with optimum at 37oC (Fig. 6.4b). No growth was
observed at 20ºC whereas low growth and enzyme secretion observed at 50ºC. The
Exiguobacterium sp. VSG-1 was able to grow up to 12 % NaCl and produce the
extracellular mannanase in a broad-range NaCl concentration (0–12 %) (Fig. 6.5b).
However, it required minimum 1 % NaCl for growth and enzyme production. This
clearly indicates the halo-tolerant nature of the strain VSG-1.
6.3.3. Effect of carbon and nitrogen sources on the growth and production of
alkaline mannanase
Different carbon and nitrogen sources were employed in preliminary studies to determine
the growth and production of extracellular alkaline mannanase after incubation for 2
days. The strain VSG-1 grew well in all the media, but the production of the enzyme was
different in different media. Among the organic nitrogen sources used, peptone and
feather hydrolysate had significant effect on the production of extracellular mannanase
and the highest level of production was achieved when the cells were grown in a medium
containing 1.0 % defatted copra meal (24.7 U/ml) as shown in Table 6.1b. The inorganic
nitrogen sources studied here were less favorable for growth and no extracellular
mannanase production was observed ((Fig. 6.6b)
231
Table 6.1b. The effect of various carbon sources on the growth and mannanase
production by Exiguobacterium sp. VSG-1 at pH 9.0 and temperature 37 °C after 48 h.
Si No. Carbon Source (1%)
Activity U/ml
Biomass at 660nm
1 LBG 15.2 3.564 2 Xylose 10.8 2.724 3 Arabinose 11.4 2.523 4 Glucose 9.3 2.702 5 Galactose 8.5 2.192 6 Fructose 10.7 2.408 7 Mannose 12.7 2.268 8 Lactose 13.6 2.256 9 Maltose 13.2 2.352 10 Sucrose 9.5 2.374 11 Starch 10.5 2.232 12 Molasses 12.1 2.428 13 Wheat bran 13.8 2.224 14 Copra mannan 21.7 3.264 15 Corn husk 16.3 3.158
232
Fig. 6.6b. The effect of various nitrogen sources on the growth and mannanase
production by Exiguobacterium sp. VSG-1. at pH 9.0 and temperature 37°C after 48 h.
Each value represents the mean ± SD of the three independent experiments.
48
121620
Pepton
e
Beef ex
tract
Yeast
extra
ct
Ammonium
nitrate
Ammonium
sulphate
Ammonium
chlor
ide
Potassi
um nitrate
Casein
Urea
Feather
hydro
lysate
Nitrogen Source (1%)
Act
ivity
U/m
l
01234
Bio
mas
s at 6
60nm
Biomass Activity
233
Fig. 6.7b. SDS-PAGE analysis of crude and purified mannanase with zymogram activity.
Lane M, molecular mass markers; phosphorylase (97 kDa), bovine serum albumin (66
kDa), ovalbumin (43 kDa), cacrbonic anhydrase (29 kDa), and lysozyme (14.3); Lane 1,
crude extract; Lane 2, purified mannanase, Lane 3, Zymogram of the mannanase purified
from Exiguobacterium sp. VSG-1.
234
6.3.4. Purification of mannanase
Strain VSG-1 was found to secrete mannanase into the extracellular medium as
confirmed by activity staining in the gel. β-mannanase secreted extracellularly by strain
VSG-1 was purified to homogeneity by ammonium sulfate precipitation and ion
exchange chromatography. The dialyzate from ion-exchange chromatography was
concentrated to a small volume and subjected to gel filtration G-200. After simple
purification steps, PAGE and SDS-PAGE of the final enzyme preparation showed a
single band. Molecular mass of mannanase was estimated as 38 kDa, by comparison with
molecular mass standards (Fig. 6.7b). The summary of the purification of mannanase
from alkaliphilic, thermostable mannanase from Exiguobacterium sp. VSG-1 is given in
Table 6.2b. The apparent Km and Vmax for LBG was found to be 3.65±0.5 and 402±25
respectively (Fig. 6.8b).
6.3.5. Effect of pH, temperature and NaCl concentration on the purified enzyme
The mannanase was active in a broad range of pH 8–12 at an optimum of 9 (Fig. 6.9b).
The maximum mannanase activity was recorded between 45-70oC (Fig. 6.10b), while it
decreased rapidly above 70oC. The enzyme is stable and active for more than 5 days at
room temperature to 45oC, and retained 100 % activity at 70oC for 3 h. The enzyme was
active over a broad range of NaCl (0–16 %) by retaining 80 % of activity at 14 % (Fig.
6.11b).
235
Table 6.2b. Summary of the purification of mannanase from alkaliphilic, thermostable
mannanase from Exiguobacterium sp. VSG-1.
Steps Total activity (U/ml)
protein (mg/ml)
Specific Activity (U/mg)
Fold Yield (%)
Crude 61000 800 76.25 1 100
(NH4)2SO4 Precipitation
55980 286 195.73 2.56 91.77
Ion-exchange (DEAE- Sepharose)
36600 113 323.89 4.24 60.05
Gel permeation (G-200)
20150 25 806 10.57 33.03
236
Fig. 6.8b. Lineweaver-Burk plot for mannanase purified (○) from the Exiguobacterium
sp. VSG-1. Mannanase activity was measured under standard assay conditions.
00.050.1
0.150.2
-1 -0.5 0 0.5 1 1.5 2
1/V
U/m
l
1/S (mg)
237
Fig. 6.9b. The pH and stability on the activity of purified mannanase by Exiguobacterium
sp. VSG-1 at 9.0 (●), 10.0 (♦), 11.0 (▲) and 12.0 (■). The activity of enzyme stored at
4°C was calculated as 100 %. Each value represents the mean ± SD of the three
independent experiments.
0
20
40
60
80
100
120
0 20 40 60 80 100
Res
idua
l act
ivity
(%)
Time (h)
238
Fig. 6.10b. The thermal stability on the activity of purified mannanase by
Exiguobacterium sp. VSG-1 at 40°C (●), 50°C (■), 60°C (▲), 70°C (♦) and 80°C (○).
The activity of enzyme stored at 4°C was calculated as 100 %. Each value represents the
mean ± SD of the three independent experiments.
0
20
40
60
80
100
120
0 20 40 60 80 100
Res
idua
l act
ivity
(%)
Time (h)
239
Fig. 6.11b. The NaCl stability on the activity of purified mannanase by Exiguobacterium
sp. VSG-1 at 2 % (●), 4 % (■), 6 % (▲), 8 % (♦) and 12 % (○). The activity of enzyme
stored at 4°C was calculated as 100 %. Each value represents the mean ± SD of the three
independent experimentss.
240
Table 6.3b. The effect of various divalent cations on the mannanase activity from
Exiguobacterium sp. VSG-1.
Metal ions
(1mM) Residual activity
(%) Control 100
CaCl2 104
MgCl2 102
FeCl3 86
CuSO4 64
BaCl2 75
MnSO4 78
Pb(NO3)2 53
AgNO3 26
ZnCl2 80
241
Table 6.4b. The effect of various inhibitors, various surfactants and local detergents on
the mannanase activity by Exiguobacterium sp. VSG-1.
Chemicals Relative activity (%)
Control 100
1, 10 Phenanthroline (10mM) 106
PMSF (10mM) 102
EDTA (10 mM) 95
Dithiothretol (10 mM) 100
2-Mercaptoethanol (1 mM) 100 Tween 20, 1% (v/v) 95
Tween 80, 1% (v/v) 98
SDS up to 20% (w/v) 100
Triton X-100 (1%) 98
H2O2 (20 %) 95
Surf 1% (w/v) 68
Wheel (1%) 120
Rin (1%) 100
Tide (1%) 95
Aerial (1%) 98
Nirma (1%) 105
242
6.3.6. Effect of metal ions on the activity of purified mannanase
The effect of various divalent cations on the activity of purified mannanase from
Exiguobacterium sp. VSG-1 was studied. Ag+2 have markedly inhibited the enzyme
activity up to 80 % while Cu+2, Fe+2, Ba+2 and Mn+2 could inhibit 35-40 % of the enzyme
activity which is believed to be the oxidation of aminoacid residues essential for the
enzyme activity. Among the investigated metal ions, no metal ion had significant effects
on the activity of mannanase. The enzyme activity was increased by 6 % and 2 % in
presence of Ca+2 and Mg+2 ions (Table 6.3b).
6.3.7. Effect of inhibitors, surfactants and detergents on the activity of purified
mannanase
Partial inhibition was observed in the presence of EDTA. Dithiothretol did not inhibit the
mannanase activity. Mannanase enzyme was found to be very stable towards laboratory
surfactants such as Tween 20, Tween 80, Triton X-100 as enzyme retained above 95 % of
its activity; when incubated in presence of 1.0 % (w/v) for 48 h. Enzyme possessed a
good stability in presence of commercial detergents such as Rin, Surf, Tide and Aerial as
it retained 95 % of its activity whereas commercial detergents except Nirma and Tide
mannanase, enhanced the mannanase activity (Table 6.4b).
6.4. Discussion
The benefit of employing novel enzymes for specific industrial processes is well
recognized with the discovery of β-mannanases. There are currently about 50
β-mannanase gene sequences in GH families 5 and 26. The increasing number of new
microbial genomes is revealing new mannolytic systems. Major challenges in this field
243
include the design of efficient enzyme system for commercial applications. Mannanases
occur ubiquitously in animals, plants, and microbes. However, microbes are most potent
producers of mannanases and represent the preferred source of enzymes in view of their
rapid growth, limited space and time required for cultivation, and ready accessibility to
genetic manipulation. Advances in genetic manipulation of microorganisms have opened
new possibilities for the introduction of predesigned changes, resulting in the production
of tailor-made mannanases with novel and desirable properties. The development of
recombinant mannanases and their commercialization by P&G, ChemGen and Genencor
is an excellent example of the successful application of modern biology to biotechnology.
Industrial production of β-mannanase is favored by micro-organisms. This is due to its
low cost, high production rate and controlled conditions. Molecular biology and protein
engineering are playing an important role to understand and to improve enzyme catalytic
properties. Sequencing and cloning of β-mannanase genes for homologous and
heterologous expressions in bacterial and fungal strains is common tool to increase
enzyme yields. Nowadays, the use of β-mannanase has become a fact mainly in the
detergent industry and in animal feed. Furthermore, β-mannanase have a great potential
in pulp and paper industry, food processing and in the near future as a diet supplement for
human beings with digestive problems.
The benefit of employing novel enzymes for specific industrial processes is well
recognized with the discovery of β-mannanases. β-mannanases (3.2.1.78) hydrolyze
mannan based hemicelluloses and liberate short β-1, 4 manno-oligomers, which can be
further hydrolyzed to mannose by β-mannosidases (EC 3.2.1.25). There are currently
244
about 50 β-mannanase gene sequences in GH families 5 and 26. The increasing number
of new microbial genomes is revealing new mannolytic systems. Major challenges in this
field include the design of efficient enzyme system for commercial applications.
Mannanases occur ubiquitously in animals, plants, and microbes. However, microbes are
most potent producers of mannanases and represent the preferred source of enzymes in
view of their rapid growth, limited space required for cultivation, and ready accessibility
to genetic manipulation. Microbial mannanases have been used recently in the food, feed
and detergent industries. Advances in genetic manipulation of microorganisms have
opened new possibilities for the introduction of predesigned changes, resulting in the
production of tailor-made mannanases with novel and desirable properties. The
development of recombinant mannanases and their commercialization by P&G,
ChemGen and Genencor is an excellent example of the successful application of modern
biology to biotechnology.
The growth and mannanase production of Exiguobacterium sp. VSG-1 indicate that there
is distinct growth associated with enzyme production. The bacterium can grow from 30 to
40oC suggesting mesophilic properties. In the current work, biomass and enzyme activity
were found to be higher at 37oC. Although mannanolytic bacteria often display optimal
growth and activity at higher temperatures, this is consistent with optimum values
described for mannanolytic Bacillus sp. AM001 (Akino et al. 1989), Bacillus sp. JAMB-
750 (Hatada et al. 2005), Thermoanerobacterium polysaccharolyticum (Cann et al. 1999)
and Trichoderma harzanium strain T4 (Franco et al. 2004) which showed optimum
temperature for growth and mannanolytic enzyme production ranging from 20 to 37oC.
245
Maximum biomass and mannanase activity were observed at pH 9–12, which agrees well
with those described earlier.
As we know, NaCl plays a very important role in the alkaliphiles’ physiology (Ma,
1999). It can function as the driving force of some endergonic processes in the cell
(Detkova and Pusheva, 2006), so it can balance the extracellular and intracellular pH of
the cell, help the cell produce energy, and is also very important in the substrate
transports (Ma, 1999). The enzymes of such organisms display maximum activity in the
presence of salts and, moreover, are inactivated in their absence. Our previous
experiments indicated that alkaliphilic Bacillus sp. N16-5 was also a salt-tolerant
bacterium, but not so much halophilic. When under high pH condition, around 9.5- 10.0,
the cell growth and the production of alkaline β-mannanase were comparatively
desirable.
The strain VSG-1 showed a high growth and mannanase production over a wide pH
range of pH 8.0 to 12.0 with maximum at pH 9.0. Growth and enzyme production was
nearly 50 % even at pH 11.0, indicated that strain VSG-1 is an extremely alkaliphilic
bacterium. Many mannan based carbon sources have been used to cultivate micro-
organisms. These include LBG, guar gum and konjac flour. The bacterium is able to
grow and produce appreciable levels of alkaline mannanase using LBG as substrate and
could offer tremendous potential for the development of biotechnological methods for the
hydrolysis of LBG and other substrates. Especially, the high level of mannanase
production by strain VSG-1 even in the absence of any supplement makes it extremely
interesting. Therefore, application of mannanase for the catalyzing are the random
246
hydrolysis of 1, 4-β-D-mannopyranoside linkages in β-1, 4-mannans is as important as
applications of xylanases. Mannanases of microbial origin have been reported to be the
both induce as well as constitutive enzymes and are being secreted extracellularly into the
medium in which the micro-organism is cultured. The bacterial mannanase produced by
Sporocytophaga coccoids and Aerobacter mannanolyticus were found to be intracellular
(Dekker and Richards. 1976). Extracellular mannanases are of considerable commercial
importance, as their bulk production is much easier. Mannanases have been produced
under submerged shaking condition, except for a few thermophilic bacteria where static
submerged fermentation has been reported. Different strains of Bacillus sp. have been
used in submerged fermentation (between pH 7-9) rather than SSF for the production of
mannanases.
The mannanase activity from strain VSG-1 was dependent on various carbon and
nitrogen sources. The mannanase was very active over a wide pH range from 8 to 12.
This is an extremely range compared with other known alkaliphilic mannanases. In
general, detergent compatible enzymes are alkaline thermostable in nature with a high pH
optima because the pH of the laundry detergent is generally in the range of 9-11 and
varying thermostability at laundry temperatures (50oC/ 60oC) (Gupta et al. 1999). Besides
pH and temperature stability, bleach stability is an important because bleach stable
enzymes are not generally available except for a few reports (Bakhtiar et al. 2002). Thus
the reported mannanase of Exiguobacterium sp. VSG-1 outstands with respect to pH,
temperature, stability, detergent compatibility and above all bleach stability for its future
application in detergent formulation. Mannanases with high activity and stability in
247
alkaline range and high temperature are interesting for biotechnological applications.
Since the mannanase secreted by Exiguobacterium sp. VSG-1 was stable up to 45oC for 4
days, inactivation of the enzyme during storage and transportation does not arise. Further,
this enzyme does not require much sophistication for its storage and transportation, as
they are the limiting factors in the industrial applications.
Another potential application of the mannanase constituent with its potential use in the
enzymatic bleaching of softwood pulps (Gubitz et al. 1996). The alternate use of
mannanase equally facilitates lignin removal in pulp bleaching and yields results
comparable to alkaline treatment (Cueves et al. 1996). Mannanases are useful in chlorine-
free bleaching processes for paper pulp (chemical pulps, semi-chemical pulps,
mechanical pulps or Kraft pulps) in order to increase brightness thus decreasing the need
for hydrogen peroxide in the bleaching process (Tenkanen et al. 1997). The enzyme is
also found useful in hydrolysis of coffee mannan, thus reducing viscocity of coffee
extracts (Sachslehner et al. 2000) and to improve nutritional value of poultry feeds.
Alkaline mannanases are also useful to enhance the flow rate of oil or gas in drilling
operations, in oil extraction of coconut meats, in textile industries as well as in food
industries.
248
Conclusion
The alkaliphilic Exiguobacterium sp. strain VSG-1 was shown to produce extracellular
extreme alkaliphilic, halotolerent and thermostable mannanase activity. The cultural
conditions for the maximum enzyme production were optimized with respect to pH,
temperature, NaCl and inexpensive agro wastes as substrates. Mannanase production was
enhanced more than 4 folds in the presence of 1 % defatted copra meal and 0.5 %
peptone or feather hydrolysate at pH 9.0 and 37°C. Mannanase was purified to 10.57 fold
with 33.3 % yield by ion exchange and gel filtration chromatography methods. Its
molecular mass was estimated to be 38 kDa by SDS-PAGE. The mannanase had maximal
activity at pH 9.0 and 60°C and was active over broad range, 0-16 % sodium chloride.
The enzyme was thermostable retaining 100 % of the original activity at 60°C for 3 h.
The apparent Km and Vmax for LBG was found to be 3.85±0.5, 412±25 and guar gum
3.40±0.2, 376±20 respectively. Since the strain grows on cheaper agro wastes such as
defatted copra meal, corn husk, and wheat bran can be exploited for mannanase
production on an industrial scale.
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