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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