International Journal of Advanced Life Sciences (IJALS ... · CMC (1.5% w/v) and gelatin (0.5%, w/v) were recorded as the best carbon and nitrogen sources for the production of enzyme

Int. j. Adv. Lif. Sci., Available online on at www. 27

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Page 27

International Journal of Advanced Life Sciences (IJALS) ISSN

2277 – 758X

Singh. J IJALS, Vol .2. Feb – April : 2012 RESEARCH ARTICLE

Introduction

Microbes used to produce extra-cellular enzymes

that were able to degrade cellulose material into

their smaller components (Bedford and Partridge, 2001).

Natural cellulolysis occurs as combined action of fungi

and bacteria that shift their biomasses depending on the

substrate which is being metabolized (Hu and Van

Bruggen, 1997). Enzyme production is closely controlled

in microorganisms and therefore, to improve its

productivity these controls can be exploited and modified

(Gautam et al., 2011). Compared to fungi the bacterial

cellulase producers are less effective but they produce

enzymes quicker and can be genetically engineered

effectively (Ponnambalam et al., 2011).The production of

cellulase by microbe would bring benefits, such as

promoting rural economy, biomass utilization in an eco-

friendly manner (Himmel et al., 1999) and enhancing

national energy security (Lynd et al., 2008; Zhang

et al., 2008). They have attracted considerable attention

in recent years due to their great biotechnological

and industrial applications, for example, formulation

of washing powder, animal feed production (Han

and He, 2010), textile industry, pulp and paper

industry (Prasetyo et al., 2011), starch processing, grain

alcohol fermentation, malting and brewing, extraction of

fruits and vegetable juices (Bhat, 2000). At present most

Abstract

Cellulase biosynthesis has been established for a variety of bacteria. A novel

bacterium, Bacillus sphaericus JS1 showing production of carboxymethylcellulase

(CMCase) was isolated from the premises of cotton mill using Hirokoshi medium pH

9.5. CMC (1.5% w/v) and gelatin (0.5%, w/v) were recorded as the best carbon and

nitrogen sources for the production of enzyme. The production of CMCase was

maximum when the pH of the media was set at 7.0, inoculated with 12h inoculum of

6% (v/v), aerated at 150 rev/min and incubated at 40oC. Bacillus sphaericus was

found resistant to very few antibiotics (Clindamycin, Bacitracin, Fusidic acid,

Clindamycin, Amikacin, Novabiocin, Oxytetracyclin and Vancomycin) and sensitive

to many on the plates. The Km and Vmax were calculated to be 1.9 mg/ml and 10

µM/min/ml respectively. The outstanding stability of the CMCase from Bacillus

sphaericus JS1 to alkaline pH and high temperature makes this enzyme, a candidate

to be used as an effective additive to laundry detergents.

Keywords: Bacillus sphaericus JS1, carboxymethylcellulase, substrate specificity,

kinetic parameters, carbon source and nitrogen source.

Optimization of nutrient sources and process parameters for the production of carboxymethylcellulase from Bacillus sphaericus JS1

J. Singh Department of Biotechnology, Panjab University, Chandigarh, India

Email : [email protected]

Corresponding Author

J. Singh Department of Biotechnology,

Panjab University, Chandigarh, India

[email protected]



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of the advanced countries like United States and some

parts of Europe almost invariably use the detergents

incorporated with cellulases. Bacillus sp. continues to be

dominant bacterial workhorses due to the capacity of

some selected species to produce and secrete large

quantities of extracellular enzymes (Aa et al., 1994;

Ariffin et al., 2006; Mawadza et al., 1996; Rastogi et al.,

2010; Schallmey et al., 2004 and Singh et al., 2004).

Reports available on strains belonging to species such as

Bacillus sphaericus and Bacillus subtilis express high

cellulose degradation activities (Mawadza et al., 1996;

Rastogi et al., 2010; Schallmey et al., 2004; Singh et al.,

2004). To exploit the potentials of biodiversity of Indian

cellulolytic organisms, the current study was undertaken

to screen the potent strain of Bacillus sphaericus JS1 and

optimization of its process parameters for the production

of alkaline CMCase.

Materials and Methods

Dinitrosalicylic acid (DNS) and CMC (Ulta low

viscosity, SD=0.60-0.95) were purchased from Fluka Rdh

(Sigma-Aldrich, Cat. No. 21901). All media components

were of bacteriological grade and purchased from

HiMedia (Mumbai, India). All other chemicals were of

analytical grade and procured from Qualigens Fine

Chemicals (Mumbai, India) and SD Fine Chemicals

(Mumbai, India).

Screening, identification and checking the antibiotic

sensitivity of potent bacterial strain

Soil samples were collected from the premises of

different industries i.e. paper, cotton and wool industries

and screened for alkaliphilic bacteria using congo red

staining method (Beguin and Auber, 1994). The isolation

medium (g/l) contained (Carboxymethyl cellulose 10 g;

peptone 5g; yeast extract 5g; NaCl 5g, KH2PO4 1g

and the pH was adjusted to 9.5 with Na2CO3 (10%,

w/v). Cultures were grown on the same solid medium

(containing 2.0% agar (w/v)) at 40oC in Petri plates. The

isolate producing the maximum zone of hydrolysis was

selected, inoculated into the sterilized liquid medium and

incubated in a rotary shaker (40°C, 150 rev/min). The

samples were withdrawn at regular intervals of time,

centrifuged at 10,000 rev/min at 4°C for 10 min and

supernatant was assayed for enzyme activity. The potent

strain was sent to Microbial Type Culture Collection and

Gene Bank (MTCC-An Internationally depository

Authority), Institute of Microbial Technology,

Chandigarh (India) for identification and was used for

further experimentation.

Effect of antibiotics was studied by pouring an

optimized, sterilized media in the Petri plates and inoculating

them with ten microliter of liquid B. sphaericus culture

(grown over nightly) uniformly. Then, antibiotic discs

(OD 001, OD 002, OD 003, OD 0049) were placed on

them with different combinations and the plates were

incubated. After 24-48 hour of incubation the diameter

around the disc were measured and recorded.

Enzyme assay

The total reducing sugar was determined by

dinitrosalicylic acid reagent (Summer and Somers, 1954).

The yellow colored dinitrosalicylic acid curve of glucose

was made using pure glucose. The diluted enzyme (0.25)

was mixed with 0.25 ml CMC (4%, w/v, in Tris-HCl

buffer pH 8.0) and after incubation for 1h, 0.75 ml

solution of dinitrosalicylic acid was added. The mixture

was then heated in a boiling water bath for 10 minutes.

This was followed by the addition of 1.25 ml buffer and

absorbance was measured at 600nm (Beckman

spectrophotometer DU 640B). Glucose (0-2mg/ml) was

used for the preparation of standard curve. Glucose

concentration was measured in terms of color intensity. A

standard is required to catalyze the formation of 1.0

µM/min/ml of reducing sugar (expressed as glucose).



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Optimization of nutrient sources and process parameters for production of CMCase

Different carbon sources (arabinose, CMC,

cellobiose, inositol, lactose, maltose, mannose, methyl

cellulose, salicine, starch, sucrose, xylose, glucose,

aesculin, dulcitol, fructose, galactose, glycerol, glycogen,

raffinose, sorbitol, and trehalose) were used at a

concentration of 0.5% (w/v). The pH of the medium was

adjusted to 8.5. The inoculum was prepared by incubating

the flasks in a rotary shaker at 40oC for overnight at 150

rpm. Different sets (in triplicate) of 250 ml Erlenmeyer

flasks each containing 50 ml medium was inoculated with

2.0 % (v/v) inoculum. Samples were withdrawn at regular

intervals of time and enzyme activity was measured.

Different concentrations (0-2%) of selected substrates

were further optimized by following the same steps.

Similarly different organic nitrogen sources (biopeptone,

beaf extract, casein, gelatin, luria broth, malt extract,

nutrient broth, peptone, soyabean casein, soyabean meal,

tryptone and yeast extract) at a concentration of 0.5 and

1.0% (w/v) were optimized. Different concentrations (0-

2%) of the selected nitrogen sources were further

optimized. The effects of pH and temperature on enzyme

activity were observed in a range of 6-10 and 30-55oC

respectively. Cultures of different ages (3-21 h) and

volume (1-10, % v/v) were used as inoculates at different

shaking speeds (0-200 rpm). Samples were withdrawn at

regular intervals of time and relative activity of CMCase

(%) was measured.

Determination of kinetic parameters with purified CMCase

Extracellular CMCase was precipitated with

ammonium sulphate (30–80% saturation) and dialyzed

against Tris-HCl buffer (50 mM, pH 8.0). The dialysate

was applied to a DEAE Sephadex A-50 column (3.6 x

30 cm) and absorbed material was eluted with a linear

gradient of NaCl in the range of 0.05–0.5M. The active

fractions (20–28) were concentrated using a Centrisart

filter (Sartorius, Gottingen,Germany), before loading

onto a Sephadex G-100 column (2.0 x 35 cm). Fractions

(2 ml; 12–18) corresponding to CMCase activity was

checked for purity on native PAGE and this purified

enzyme was subsequently used for characterization

work (Singh et al., 2004). The kinetic parameters like

reaction velocity (Vmax) and kinetic constant (Km) for

purified CMCase were determined using cellulose

modified substrates: carboxymethyl cellulose. For the

determination of Km and Vmax, the Lineweaver-Burk plots

were used under the conditions of 60oC and pH 8.0.

Results and discussion

Based on the morphological, physiological and

biochemical characteristics, the isolate JS1 was identified

as Bacillus sphaericus by the Microbial Type Culture

Collection and Gene Bank (MTCC-An Internationally

depository Authority), Institute of Microbial Technology,

Chandigarh (India) and designated an accession number

of 3243. The resistance to antibiotics was less with

Clindamycin (2 mcg), Bacitracin (10 mcg), Fusidic acid

(10 mcg), but it was much more with Clindamycin

(10 mcg), Amikacin (10 mcg), Novabiocin (30 mcg),

Oxytetracyclin (30 mcg) and Vancomycin (30 mcg).

The Bacillus sphaericus was sensitive to Amoxyclav

(10 mcg), Cephalexin (10 mcg), Ciprofloxacin 10 mcg),

Cloxacillin (1 mcg), Co-trimoxazole (25 mcg), Erythro-

mycin (15 mcg), Tetracycline (30 mcg), Ampicillin (10 mcg),

Carbenicillin (100 mcg), Cephotaxime (30 mcg),

Chloramphenicol (30 mcg), Co-trimazine (25 mcg),

Gentamicin (10 mcg), Norfloxacin (10 mcg), Oxacillin

(5 mcg), Amoxycillin (10 mcg), Cephalothin (30 mcg),

Erythromycin (15 mcg), Ampicillin (1unit), Erythromycin

(10 mcg), Gentamycin (2 mcg), Tetracycline (10 mcg),

Co-trimoxazole (25 mcg), Penicillin-G (25 mcg). Bacillus

sphaericus was found resistant to very few antibiotics and

sensitive to many on the plates (Table - 1).



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Table 1: Effect of antibiotics on the growth of Bacillus sphaericus JS1.

Name of antibiotic Conc. (mcg)

Growth radii (mm)

Name of antibiotic Conc. (mcg)

Growth radii (mm)

OD 001 OD 002

Amoxyclav (Ac) 10 - Ampicillin (A) 10 - Cephalexin (Cp) 10 - Carbenicillin (Cb) 100 - Ciprofloxacin (Cf) 10 - Chloramphenicol (C) 30 - Cloxacillin (Cx) 01 - Cephotexime (Ce) 30 - Co-Trimoprin (Co) 25 - Co-Trimazine (Cm) 25 - Erythromycin (E) 15 - Gentamycin (G) 10 - Tetracycline (T) 30 - Norfloxacin (Nx) 10 - Clindamycin (Cd) 02 13 Oxacillin (Ox) 05 -

OD 0049 - OD 003 Clindamycin (Cd) 10 6.0 Amikacin (Ak) 10 13 Ampicillin (A) 1 unit - Amoxycillin (Am) 10 - Erythromycin (E) 10 - Bacitracin (B) 10 6.0 Gentamycin (G) 02 - Cephalothin (Ch) 30 - Fusidic Acid (Fc) 10 6.0 Erythromycin (E) 15 - Tetracycline (T) 10 - Novobiocin (Nv) 30 13 Co-Trimoxazole (Co) 25 - Oxytetracycline (O) 30 15 Penicillin-G (P) 25 - Vancomycin (Va) 30 10

(-) No growth of Bacillus sphaericus was seen and measured.

Table - 2: Effect of carbon sources on the production of CMCase by Bacillus sphaericus JS1.

Carbon source (0.5%, w/v)

Growth at 600 nm

Enzyme activity µµµµM/min/ml

Carbon source (0.5%, w/v)

Growth at 600 nm

Enzyme activity

µµµµM/min/ml Control 1.6 1.2 Control 1.6 1.2

Aesculin 0.8 0.5 Lactose 4.5 0.7

Arabinose 0.4 0.8 Maltose 0.8 0.2

CMC 3.4 3.3 4.1 4.1

2.8 2.9 3.8 3.9

Mannitol 2.5 0.5

Cellobiose 2.4 1.1 Mannose 0.6 0.2

Cellulose 2.7 2.5 2.8 2.6

2.5 2.7 2.6 2.5

Methyl cellulose 1.7 1.4



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2277 – 758X


Dulcitol 0.8 1.6 Raffinose 2.7 1.2 Fructose 2.5 1.1 Salicine 2.2 0.2 Galactose 2.6 1.1 Sorbitol 0.7 0.6 Glucose 2.1 0.3 Starch 3.4 1.4 Glycerol 2.2 0.7 Sucrose 2.4 0.1 Glycogen 2.8 2.1

Trehalose 2.9 1.3

Inositol 3.6 0.7 Xylose 4.3 4.2 4.3 3.8

2.7 2.6 2.8 2.0

Table – 3. Effect of organic nitrogen sources on the production of CMCase by Bacillus sphaericus JS1.

Nitrogen source Conc. (%, w/v) Growth at 600 nm Enzyme activity (µµµµM/min/ml)

Control - Control -

Biopeptone 0.5 1.0

1.3 2.3

0.9 1.0

Beaf extract 0.5 1.0

1.2 2.6

1.3 1.8

Casein 0.5 1.0

4.1 4.2

1.2 1.1

Gelatin 0.5 1.0 1.5 2.0

2.0 2.7 2.3 2.8

2.4 2.2 2.3 2.3

Luria broth 0.5 1.0

1.5 1.4

1.4 0.9

Malt extract 0.5 1.0

1.3 2.8

1.3 1.1

Nutrient broth 0.5 1.0

1.3 1.3

1.6 1.2

Peptone 0.5 1.0

1.6 2.6

1.4 1.1

Soyabean casein 0.5 1.0

1.6 2.0

1.3 1.3

Soyabean meal 0.5 1.0

2.9 5.6

1.3 2.3

Trypton 0.5 1.0

1.6 2.9

1.5 1.2

Yeast extract 0.5 1.0

1.2 2.9

0.9 0.6



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Optimization of carbon and organic nitrogen

sources for the production of CMCase

Effect of carbon source on the production of

CMCase by Bacillus sphaericus was shown in Table - 2.

The rate of production of enzyme was maximum with

CMC, cellulose and xylose than other carbon sources.

Less than 50% of CMCase was observed in media

containing arabinose, cellobiose, inositol, lactose, starch,

aesculin, dulcitol, fructose, galactose, glycerol, raffinose,

sorbitol, trehalose and glycogen. Very low level (less

than 12%) of enzyme activity was seen with maltose,

mannose, glucose, sucrose and salicine. The different

concentrations (0.5, 1.0, 1.5 and 2.0%, w/v) of CMC,

cellulose or xylose was optimized. At both the

concentrations (0.5, 1.0% w/v), production of enzyme

was comparable among three substrates, but beyond this,

the CMC had shown the upper edge. It displayed 150%

of the enzyme activity as compared to xylose and

cellulose at 1.5% (w/v) concentration and reported to be

the best for the production of enzyme by JS1 isolate. All

other concentrations (1.0-2.0%, w/v) displayed the same

yield. Figure - 1 shows the course of cultivation of B.

sphaericus for the production of CMCase in shake flasks

in the optimized media. In the early hours of fermentation

(12-36h), the active cell growth took place and then

started decreasing. After 12 hours, CMCase activity

started increasing and continued to do so up to 48 h. After

the exponential phase, cell mass concentration started

deceasing and it continued up to 60h of fermentation.

Total residual sugar (as equivalent to glucose)

concentration started deceasing from the very beginning.

Rate of CMC utilization was highest during log phase of

growth. At the end of fermentation, total reducing sugar

utilization was 56% and 12% of initial sugar remained

unutilized, while the enzyme activity showed an

increasing trend. Many authors reported inducible

CMCases while optimizing carbon source. Cellobiose or

CMC (1% w/v) induced CMCase production in

alkalophilic Bacillus sp. No 1139 (Fukumori et al., 1985).

Production of alkaline cellulases required the presence of

CMC (1%) in the strains of alkalophilic Bacillus sp.

KSM-64 and KSM-520 (Shikata et al., 1990). CMCases

from Bacillus sp. KSM-S237 and KSM-N252 were

produced when the media was supplemented with CMC

(Hakamada et al., 1997 and Endo et al., 2001). Alkalophilic

Bacillus circulans synthesized neutral endoglucanases

when grown on N3 medium supplemented with glucose,

fluffy cellulose or wheat bran celluloses (Landaud et al.,

(1996). The highest yield was obtained on wheat bran,

cellobiose, lactose or polysaccharides such as crystalline

cellulose (Avicel, MN 300). Endoglucanase synthesis

was not completely repressed by glucose or cellobiose,

which was generally the case in cellulase producing

microorganisms. The effect of carbon sources on

cellulase production by the candidate species after 72

hours of incubation period at 37°C was observed. The

maximum cellulose production by Bacillus pumilus

EWBCM1 was recorded in galactose (0.5851 ± 0.006

IU/ml) supplemented medium (Shankar and Isaiarasu,

2011).

The production of alkaline CMCase by utilizing

gelatin as the nitrogen source has been reported for the

first time. It displayed 230% of enzyme activity and was

optimized as the best nitrogen source. Enzyme activity

was induced (150% approximately) by glycogen, beaf

extract, casein, luria broth, malt extract, nutrient broth,

peptone, soyabean meal and trypton, while the yeast

extract and biopeptone showed no effect (Table - 3).The

enzyme activity was same (2.3 µM/min/ml) at both the

concentrations (0.5, 1.0% w/v) of gelatin. By studying

enzyme synthesis on different concentrations (0.5, 1.0,

1.5, 2.0%, w/v), 0.5% (w/v) was reported to be the best

for the production of enzyme. Different workers had

reported different nitrogen sources for the production of



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enzyme. The utilization of polypeptone and yeast extract

as the complex nitrogen source for the maximum

production of alkaline CMCase (Fukumori et al., 1985).

For higher production of alkaline cellulase K, Bacillus

subtilis KSM-635 required the presence of complex

nitrogen such as meat and yeast extract and three strains

of alkalophilic Bacillus KSM-19, KSM-520, and KSM-

64 required the presence of complex nitrogen such as

polypeptone, corn steep liquor and yeast extract

(Ito et al., 1989 and Shikata et al., 1990). These

observations were confirmed by many workers later on

(Lusterio et al., 1992; Landaud et al., 1996; Hakamada

et al., 1997 and Endo et al., 2001). Maximum amount of

enzyme production (0.5666 ± 0.002 IU/ml) by Bacillus

pumilus EWBCM1 in malt extract supplemented medium

(Shankar and Isaiarasu, 2011). The production of

CMCase in shake flasks from Bacillus subtilis at early

log phase of growth was very quick in the logarithmic

phase and amount of sugars started decreasing sharply in

the log phase (Au and Chan, 1987). Screening of most

significant medium components by Plackett-Burman

Design for Bacillus subtilis AS3 which produce

maximum cellulase activity of 0.49 U/mL appearing at

CMC (18 g/L), peptone (8 g/L), and yeast extract (4.798

g/L) (Deka et al., 2011).

Optimization of process parameters (pH, temperature,

agitation and inoculum) for the production of

alkaline CMCase

The effect of initial pH and temperature on the

growth and CMCase production by Bacillus sphaericus

was observed that after 84 h of fermentation, maximum

CMCase activity was obtained at pH 7.0 (100% relative

activity, while it was reported to be 89, 85 and 57%

relative activity at alkaline pH of 8.0. 9.0 and 10.0

respectively. The cell mass concentration was approximately

same at three pHs (7, 8, 9), less at pH 10 and no growth

at pH 6. The total reducing sugar concentration in the

T im e (h)

0 12 24 36 48 60 72 84 96

Gro

wth

at 6

00 n

m

Enz

yme

activ

tiy ( µµ µµ

M/m

in/m

l)

o

0.0

0.5

1.0

1.5

2.0

2.5

3.0R

esid

ual r

educ

ing

suga

rs (

g/l)

0

2

4

6

8

10

Figure - 1. The course of production of CMCase by B. sphaericus JS1.



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2277 – 758X


culture broth reduced to same level at all pHs (Data not

shown). The maximum enzyme production took place at

pH 7.0-9.0, by Bacillus sphaericus (Fig – 2A). While

optimizing pH, the highest yields of cellulase activity at

pH 7.0 were 1.652 and 1.978U/ml of CMCase extracted

by isolates Bacillus sp. (8) and Bacillus sp. (17), respectively

was reported by Sohair et al. (2011).

As is evident from figure 2B, Bacillus sphaericus

grew over a wide range (30-55oC) of temperature.

However, the maximum enzyme production took place at

40oC. After 48h of fermentation, the substrate uptake rate

was same between 35-45oC, approximately 65% of the

total reducing sugars were consumed (Data not shown).

At 35 and 45oC the relative enzyme activity was 70 and

78 respectively as compared to 100% at 40oC. All the

subsequent production experiments were carried out at

40oC. Bacillus brevis VS-1 was reported to produce cellulase

optimally at 37oC (Singh and Kumar, 1998). Bacillus

strain CTP-09 yielded maximum productivity (1120

IU/L) of extracellular endoglucanase (CMCase) on 0.5%

cellobiose after 10 h fermentation at 55oC (Saleem et al.,

2008). Bacillus subtilis and Bacillus circulans shows

optimal cellulolytic activities at 35oC (Otajevwo et al.,

2010).

At low agitation (i.e., 50, 100 rev/min), the enzyme

took longer time to reach its maxima, while at higher speed

(i.e., 200 rev/min) the enzyme production was maximum

after 48 hours (similar as at 150 rev/min). After this it started

degrading, but the enzyme produced at 150 rev/min was

quite stable for long time (Fig – 2C). The optimum

production of cellulose at 175 rev/min was reported in

Bacillus brevis VS-1 (Singh and Kumar, 1998). The optimal

agitation speed for the production of CMCase by B.

subtilus subsp. subtilis A-53 were 300 rev/min. The

highest productions of CMCase by B. subtilus subsp.

subtilis A-53 in 7 and 100 L bioreactors were 150.3 and

196.8 U mL−1, respectively (Bo-Hwa Lee et al., 2010).

Time (h)

0 12 24 36 48

% R

elat

ive

Act

ivit

y o

f C

MC

ase

Pro

du

ctio

n

0

20

40

60

80

100 30oC

35oC

40oC

45oC

50oC

55oC

2B

Time (h)

0 12 24 36 48 60 72 84

% R

elat

ive

Act

ivit

y o

f C

MC

ase

Pro

du

ctio

n

0

20

40

60

80

100pH 7

pH 8

pH 9pH 10

2A



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2277 – 758X


Bacillus sphaericus cells in the log phase were the best

source of CMCase production. After 48 hours of

fermentation, the production of enzyme was 1.3, 1.8, 2.0,

2.8, 1.9, 2.0 and 1.6 µM/min/ml at inoculum ages of 3, 6,

9, 12, 15, 18 and 21h respectively. The age of the

inoculum was optimized to be 12 hour and inoculums

size 6% (v/v). The relative activity obtained at 2.0,

3.0, 4.0, 5.0% (v/v) inoculum sizes was 85%, whereas, it

was 78 % at 1.0% (v/v). At higher inoculum sizes (i.e.,

8.0 and 10 %, v/v) the relative activity was similar to that

obtained with 6.0% (Fig - 2d). Inoculum size 1.25% (v/v)

and inoculum age 2 h were found to be optimal for

maximum xylanase production from free and immobilized

Bacillus pumilus strain MK001 (Kapoor et al., 2008).

Determination of kinetic parameters with purified CMCas

For the determination of Km and Vmax, the

Lineweaver-Burk plots were used. The Km and Vmax were

calculated to be 1.9 mg/ml and 10 µM/min/ml respectively

(Fig - 3). Michaelis- Menten kinetics of monomeric enzyme

exhibiting Km 0.48, 4.0 and 1.7 mg/ml was respectively

obtained for Bacillus sp. No 1139 (Fukumori et al.,

1985), Bacillus subtils AU-1 (Au and Chan 1987),

Bacillus brevis VS-1 (Singh and Kumar, 1998). B. coagulans

shows the Km 28.1 mg/ml while the Vmax 0.79 U/sec

(Odeniyi et al., 2009). The enzyme from Bacillus subtilis

ASH obeyed Michaelis-Menton kinetics towards birch

wood xylan with apparent Km 3.33 mg/ml and Vmax 100

IU/ml (Sanghi et al., 2010).

Time (h)

0 12 24 36 48 60 72

% R

elat

ive

Act

ivit

y o

f C

MC

ase

Pro

du

ced

0

20

40

60

80

100

Stationary50 rev/min 100 rev/min 150 rev/min 200 rev/min

Time (h)

0 20 40 60 80

% R

elat

ive

Act

ivit

y o

f C

MC

ase

Pro

du

ctio

n

0

20

40

60

80

100

2% (v/v)4% (v/v)6% (v/v)8% (v/v)10% (v/v)

2C 2D

Figure – 2. Optimization of process parameters (pH, temperature, inoculum size and aeration) for the production CMCase by from Bacillus sphaericus JS1.



Page 36


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Conclusions

Extremophiles are the need of today’s industrial

world. CMCase strongly hydrolyzed CMC and had a

broad pH activity (7-10). CMC and gelatin were

optimized as the best carbon and nitrogen sources

respectively for the production of enzyme. When

the culture medium was aerated at 150 rev/min with 12h

inoculum of 6.0% (v/v), the production of enzyme was

at peak. This study gives us a hint as well as the

microbial wealth of cellulase producing bacteria which

can be harnessed for biotechnological processes.

Acknowledgment

JS is highly grateful to CSIR, New Delhi for providing

the research grant.

Reference

Aa, K., Flengsrud, R., Lindahl, V. and Tronsmo, A.

1994. Characterization of production and enzyme

properties of an endo-β-1, 4-glucanase from

Bacillus subtilis CK-2 isolated from compost soil.

Anton Leeuw. Int. J. G., 66: 319 – 326.

Ariffin, H., Abdullah, N., UmiKalsom, M.S., Shirai, Y.

and Hassan, M.A. 2006. Production and

characterization of cellulase by Bacillus pumilus

EB3. Int. J. EngTech., 3 : 47 – 53.

Au, K.S. and Chan, K.Y. 1987. Purification and properties

of the endo-1,4 - β-glucanase from Bacillus subtilis.

J. Gen. Microbiol., 133: 2155-2162.

Bedford, M.R. and Partridge, G.G. 2001. Enzyme in

Farm Animal Nutrition. Marlborough Wiltshire:

Finn feeds Cab Publ.

Beguin, P. and Aubert, J.P. 1994. The biological degradation

of cellulose. FEMS Microbiol Rev., 13: 25 – 5.

Bhat, M.K. 2000. Cellulase and related enzymes in

biotechnology. J. Biotechnol Adv.,18: 355–383.

1 /S (m g /m l) -1

-1 .0 -0 .5 0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0

1/V

x 1

0-3 ( µµ µµ

M/m

in/m

l)-1

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

6 0 0

7 0 0

8 0 0

S (m g /m l)0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0

V (

µµ µµM/m

l/min

)0

1

2

3

4

5

6

7

Figure – 3. Line weaver-Burk plot for hydrolysis of CMC. [Inset: Reaction velocity (V) versus CMC concentration (S)]



Page 37


2277 – 758X


Bo-Hwa, L., Bo-Kyung, K., You- Jung, L., Chung-Han,

C. and Jin-Woo, L. 2010. Industrial scale of

optimization for the production of carboxymethyl

cellulase from rice bran by a marine bacterium,

Bacillus subtilis subsp. subtilis A-53. Enzyme

Microbial Technol., 46 : 38-42.

Deka, D., Bhargavi, P., Sharma, A., Goyal, D., Jawed, M.

and Goyal, A. 2011. Enhancement of Cellulase

with Various Cellulosic Substrates Activity from a

New Strain of Bacillus subtilis by Medium Optimization

and Analysis. Enzyme Res., 8.

Endo, K., Hakamada, Y., Takizawa, S., Kubota, H.,

Sumitomo, N. and Kobayashi, T. 2001. A novel

alkaline endoglucanase from an alkalophilic Bacillus

isolate: Enzymatic properties, and nucleotide and

deduced amino acid sequences. App. Microb.

Biotech., 57: 109-116.

Fukumori, F., Kudo, T. and Horikoshi, K. 1985. Purification

and properties of a cellulose formal kalophilic

Bacillus sp. No. 1139. J Gen. Microbiol.,131 : 2239

- 3345.

Gautam, S.P., Bundela, P.S., Pandey, A.K., Khan, J.,

Awasthi, M.K. and Sarsaiya, S. 2011. Optimization

for the production of cellulaseenzyme from municipal

solid waste residue by two novel cellulolytic fungi.

Biotech. Res Int., 2011 : 8.

Hakamada, Y., Koike, K., Yoshimatsu, T., Mori, H.,

Kobayashi, T. and Ito, S. 1997. Thermostable alkaline

cellulases from an alkaliphilic isolate, Bacillus sp.

KSM - S237. Extremophil. ,1: 151-156.

Han, W. and He, M. 2010. The application of exogenous

cellulase to improve soil fertility and plant growth

due to acceleration of straw composition. Bioresour

Technol.,101: 3724–3731.

Himmel, M.E., Ruth, M.F. and Wyman, C.E. 1999.

Cellulase for commodity products from cellulosic

biomass. Curr Opin Biotechnol., 10: 358 – 364.

Hu, S. and Van Bruggen, A.H.C. 1997. Microbial

dynamics associated with multiphasic decomposition

of 14C-labelled cellulose in soil. Microb. Ecol., 33:

134 - 143.

Ito, S. 1997. Alkaline cellulase from alkaliphilic Bacillus:

Enzymatic properties, genetics, and application to

detergents. Extremophil.,1: 61-66.

Kapoor, M., Nair, L.M. and Kuhad, R.C. 2008. Cost-

effective xylanase production from free and

immobilized Bacillus pumilus strain MK001 and its

application in saccharification of Prosopisjuliflora.

J. Biochem. Eng., 38: 88–97.

Landaud, S., Davila, A.M. and Pourquie, J. 1996. Neutral

endoglucanases production by a newly isolated

alkalophilic Bacillus circulans. Biotech Let., 18:

741 - 746.

Lusterio, D.D., Suizo, F.G., Labunos, N.M., Valledor,

M.N., Ureda, S. and Kawai, S. 1992. Alkali-

resistant, alkaline endo-β-glucanase produced by

Bacillus sp. PKM-5430. Biosci Biotech Biochem.,

56: 1671-1672.

Lynd, L.R., Laser, M.S., Bransby, D., Dale, B.E.,

Davison, B., Hamilton, R., Himmel, M., Keller, M.,

McMillan, J.D., Sheehan, J. and Wyman, C.E.

2008. How biotech can transform biofuels. Nat

Biotechnol., 26 : 169 – 172.

Mawadza, C., Boogerd, F.C., Zvauya, R. and Van

Verseveld, H.W. 1996. Influence of environmental

factors on endo-β- 1,4-glucanase production by

Bacillus HR 68, isolated from a Zimbabwean hot

spring. Anton Leeuw. Int J. G., 69 : 363–369.



Page 38


2277 – 758X


Odeniyi, O.A., Onilude, A.A. and Ayodele, M.A. 2009.

Production characteristics and properties of

cellulose polygalacturonase by a Bacillus coagulans

strain from a fermenting palm-fruit industrial

residue. Afr. J. Microbiol. Res., 3: 407 - 417.

Otajevwo, F.D. and Aluyi, H.S. 2010. Cultural conditions

necessary for optimal cellulose yield by cellulolytic

bacterial organisms as they relate to residual sugars

released in broth medium. Nigerian J. Microbiol.,

24: 2168 – 2182.

Ponnambalam, A.S., Deepthi, R.S. and Ghosh, A.R.

2011. Qualitative display and measurement of

enzyme activity of isolated cellulolytic bacteria.

J. Biotech. Bioinf. Bioeng.,1:33-37.

Prasetyo, J., Zhu, J., Kato, T. and Park, E.Y. 2011.

Efficient production of cellulase in the culture of

Acremonium cellulolyticus using untreated waste

paper sludge. Biotechnol. Prog., 7: 104 – 110.

Rastogi, G., Bhalla, A., Adhikari, A., Bischoff, K.M.,

Hughes, S.R., Christopher, L.P., Sani, R.K.2010.

Characterization of thermo stable cellulases produced

by Bacillus and Geobacillus strains. Bioresource

Technol., 101: 8798 – 8806.

Saleem, M., Akhtar, M.S., Yasmin, R., Zahid, M., Malik,

N.N., Afzal, M. 2008. Production, purification and

characterization of beta-1,4 - endoglucanase from a

novel bacterial strain CTP-09 of a Bacillus sp.

Protein Pept. Lett., 15: 402 - 10.

Sanghi, A., Garg, N., Gupta, V.K., Mittalk, A., Kuhad,

R.C. 2010. One-Step Purification and Characterization

of Cellulase Free Xylanase Produced by Alkalophilic

Bacillus subtilis ASH. Braz J. Microbiol., 4: 467-476.

Schallmey, M., Singh, A. and Ward, O.P. 2004. Develop-

ments in the use of Bacillus species for industrial

production. Can. J. Microbiol., 50: 1 – 17.

Shikata, S., Saeki, K., Okoshi, H., Yoshimatsu, T., Ozaki,

K. and Kawai, S. 1990. Alkaline cellulase laundry

detergents: Production by alkalophilic strains of

Bacillus and some properties of the crude enzymes.

Agri. Biol. Chem., 54: 91 - 96.

Shankar, T. and Isaiarasu, L. 2011.Cellulase Production

by Bacillus pumilus EWBCM1 under Varying

Cultural Conditions. Middle East J. Sci. Res., 8:

40 - 45.

Singh, V.K. and Kumar, A. 1998. Production and

purification of an extracellular cellulose From

Bacillus brevis VS-1. Biochem. Mol. Biol. Int., 45:

443 - 452.

Singh, J., Batra, N. and Sobti, R.C. 2004. Purification and

characterization of alkaline cellulose produced by a

novel isolate Bacillus sphaericus JS1. J. Ind.

Microb. Biotech., 31: 51 - 56.

Sohair, A.N., Abozaid, A.A., Hussein, N.A. and Al-

salem, F.A. 2011. Cellulase production by local

bacteria isolated from Taif in Saudi Arabia.

J. Agric. Sci., 19: 163-170.

Summer, J.B. and Somers, G.F. 1954. Dinitrosalicylic

acid for glucose. In: Laboratory experiments in

Biological Chemistry. Academic Press, New York,

PP. 34–39.

Zhang, Y.H.P. 2008. Reviving the carbohydrate economy

via multiproduct biorefineries. Ind. J. Microb.

Biotech., 35 : 367–37.

Corresponding Author : J. Singh, Department of Biotechnology, Panjab University, Chandigarh, India, Email - [email protected].

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International Journal of Advanced Life Sciences (IJALS ... · CMC (1.5% w/v) and gelatin (0.5%, w/v) were recorded as the best carbon and nitrogen sources for the production of enzyme