w.sciencedirect.com

J o u rn a l o f R a d i a t i o n R e s e a r c h and A p p l i e d S c i e n c e s 9 ( 2 0 1 6 ) 1 3 9e1 4 7

HOSTED BY Available online at ww

ScienceDirectJournal of Radiation Research and Applied

Sciencesjournal homepage: http : / /www.elsevier .com/locate/ j r ras

Optimization of process parameters for xylanaseproduction by Bacillus sp. in submergedfermentation

Muhammad Irfan a,b,*, Umar Asghar b, Muhammad Nadeem b,Rubina Nelofer b, Quratulain Syed b

a Department of Zoology, University of the Punjab, New Campus Lahore, 54590, Pakistanb Food & Biotechnology Research Center (FBRC), Pakistan Council of Scientific and Industrial Research (PCSIR),

Laboratories Complex, Ferozpure Road Lahore, 54600, Pakistan

a r t i c l e i n f o

Article history:

Received 10 September 2015

Received in revised form

12 October 2015

Accepted 24 October 2015

Available online 6 November 2015

Keywords:

Xylanase

Bacillus sp. agricultural waste

Submerged fermentation

* Corresponding author. Department of Zool

Peer review under responsibility of The Ehttp://dx.doi.org/10.1016/j.jrras.2015.10.0081687-8507/Copyright© 2015, The Egyptian Socopen access article under the CC BY-NC-ND l

a b s t r a c t

In this study an attempt was made to optimize the cultural and nutritional conditions for

xylanase production by Bacillus species in submerge fermentation process. Whole

fermentation process was carried out in 250 ml Erlenmeyer flask with agitation speed of

140 rpm. Bacillus subtilis exhibit maximum xylanase production at initial medium pH of 8,

substrate concentration of 2% with inoculum size of 2% at 35 �C for 48 h of fermentation

period. Further supplementation of sucrose, (NH4)2SO4 and peptone as a carbon and ni-

trogen sources favored enzyme production. The other strain Bacillus megaterium showed its

peak xylanase production at initial medium pH of 8, inoculum size of 1.5% with substrate

concentration of 1.5% at incubation temperature of 40 �C for 72 h of fermentation period.

The best carbon and nitrogen sources are xylose, KNO3 and malt extract. Both strains can

also utilize molasses at 0.5% concentration for xylanase production can grow in medium

containing 0.2% NaCl (B. subtilis BS04) and 0.8% NaCl (B. megaterium BM07) respectively. The

optimum temperature of xylanase was 50 �C and pH was 5 and 5.5 by B. subtilis BS04 and B.

megaterium BM07 respectively.

Copyright © 2015, The Egyptian Society of Radiation Sciences and Applications. Production

and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Xylanase (E.C 3.2.1.8) is the enzyme which degrades b-1, 4

xylan by cleaving b-1, 4 glycosidic linkages thus forming us-

able products such as xylose, xylobiose like xylo-

oligosaccharides (Bernier, Desrochers, Jurasek, & Paice, 1983;

Chakrit, Khin, & Khanok, 2006). In annual plants and hard-

woods, xylan is the most abundant non-cellulosic poly-

saccharide which accounts for 20e35% of the total dry weight

ogy, University of the Pun

gyptian Society of Radiat

iety of Radiation Sciencesicense (http://creativecom

in biomass (Bernier et al., 1983; Elegir, Szakacs, & Jeffries,

1994). Xylanases can be produced by bacteria and fungi in

both liquid culture and solid culture. The production of mi-

crobial xylanases is preferred over plant and animal sources,

because of their availability, structural stability and easy ge-

netic manipulation (Bilgrami & Pandy, 1992). Mostly bacteria

especially from the genus Bacillus are world widely used for

the production of extracellular hemicellulases (Coughlan &

Hazlewood, 1993; Srinivasan & Meenakshi, 1999).

jab, New Campus Lahore, 54590, Pakistan.

ion Sciences and Applications.

andApplications. Production and hosting by Elsevier B.V. This is anmons.org/licenses/by-nc-nd/4.0/).

J o u r n a l o f R a d i a t i o n R e s e a r c h and A p p l i e d S c i e n c e s 9 ( 2 0 1 6 ) 1 3 9e1 4 7140

Mostly the production of xylanases has been studied in

submerged liquid culture but there are few reports concerning

the xylanase production in solid state fermentation using

lignocellulosic wastes (Couri, Terzi, Pinto, Freitas, & Costa,

2000; de Souza, de Souza, & Peralta, 2001; Kalogeris,

Christakopoulos, Kekos, & MacRis, 1998). Submerged

fermentation process is mostly preferable because of more

nutrients availability, sufficient oxygen supply and less time

required for the fermentation than other fermentation tech-

niques (Gomes, Gomes, & Stiener, 1994; Gouda, 2000; Hoq,

Hempel, & Deckwer, 1994; Veluz, Taksuo, Hiroshi, & Yusaku,

1999). Today, xylanases production is highly important due

to its industrial exploitation like, pulp and paper industry,

baking industry, clarification of juices and also in liquification

of fruits and vegetables (Gupta & Kar, 2009). In this study we

reported here the xylanase production from Bacillus subtilis

BS04 and Bacillus megaterium BM07 in submerged fermenta-

tion. To our knowledge this is the first report on xylanase

production by B. megaterium in submerged fermentation.

2. Materials and methods

2.1. Procurement of substrates

Sugarcane bagasse was procured from local market of Lahore,

city and used as substrate for xylanase enzyme production in

submerged fermentation.

2.2. Bacterial strain

Two bacterial strains B. subtilis BS04 and B. megaterium BM07

were locally isolated from soil. The strain was maintained on

nutrient agar (Oxoid) slants and store at 4 �C.

2.3. Cultivation of bacterial cells

Twenty five milliliter of nutrient broth (Oxoid) was sterilized

in each two 250 ml Erlenmeyer flask at 121 �C for 15 min. After

sterilization, the media was inoculated with a loopful of 24 h

old strains of B. subtilis BS04 and B. megaterium BM07 incubated

at 37 �C for 24 h with the agitation speed of 140 rpm. Vegeta-

tive cells were used as a source of inoculum throughout the

study.

2.4. Fermentation technique

Twenty five milliliter of fermentation media (g/l: Sucrose 20,

K2HPO4 0.5, NaCl 0.2, MgSO4.7H2O 0.16, Yeast extract 0.5.) with

2% substrate (sugarcane bagasse) were sterilized in each

250 ml Erlenmeyer flask at 121 �C for 15 min. After steriliza-

tion, the media was inoculated with 2% solution containing

vegetative cells of 24 h old B. subtilis BS04 and B. megaterium

BM07 in each flask and incubated at 37 �C for 48 h of

fermentation period with the agitation speed of 140 rpm.

2.5. Preparation of enzyme

After the termination of fermentation period, the fermented

broth was filtered through muslin cloth and finally by

centrifugation at 4 �C, 8000 � g for 10 min to remove the

bacterial cells and unwanted particles. The clear filtrate ob-

tained after centrifugation was used as a source of crude

enzyme.

2.6. Assay of xylanase enzyme

Xylanase enzyme in the culture filtrate was estimated as re-

ported earlier (Irfan, Nadeem, Syed, & Baig, 2012). Reaction

mixture containing 0.5 ml of appropriately diluted culture

filtrate with 0.5 ml of 1% birchwood xylan (Sigma) solution

prepared in citrate buffer (0.05 M, pH5.0) was incubated for

15 min at 50 �C. After incubation the reaction was stopped by

the addition of 1.75 ml of 3, 5 dinitrosalicylic acid and heated

for 10 min in boiling water bath. After cooling the reducing

sugars liberated were measured by spectrophotometrically at

550 nm and expressed as xylose equivalent. Xylose was taken

as standard. One unit enzyme activity was defined as the

amount of enzyme required to produce 1 mmole reducing

sugar as a xylose equivalent per minute under standard assay

conditions. Units were calculated by using following

formulae.

Xylanase activity ðIUÞ ¼ Reducing sugars ðmg=mlÞ � 1000Incubation time ð15 minÞ � 150

2.7. Optimization of cultural and nutritional conditionsfor xylanase production

Different cultural conditions like time course of fermentation

(24e120 h), initial medium pH (4e10), incubation temperature

(25e50 �C), inoculum size (0.5e3%), substrate concentration

(0.5e3.0%) and various nutritional conditions such as addi-

tional carbon sources (glucose, sucrose, fructose, CMC, arab-

inose & xylose), nitrogen sources (KNO3, NaNO3,

(NH4)2SO4,NH4Cl, ammonium citrate, peptone, yeast extract,

tryptone, Malt extract & urea) molasses supplementation

(0.5e3.0%) and NaCl concentration (0.2e1.0%) were optimized

for enhanced production of xylanase by two tested strains of

B. subtilis BS04 and B. megaterium BM07 in submerged

fermentation process.

2.8. Effect of pH on activity of xylanase

The optimum pH for the enzyme was determined by incu-

bating crude enzyme with substrate (1% xylan) prepared in

appropriate buffers; 0.05 M citrate buffer (pH 3.0e6.0), 0.05 M

sodium phosphate buffer (pH 6.0e8.0), 0.05 M TriseHCl (pH

8.0e9.0) and 0.05 M glycine-NaOH (pH 9.0e11.0). Enzyme and

substrate was incubated for 30 min at 50 �C. After incubationthe reaction was stopped by the addition of DNS reagent and

absorbance was measured at 550 nm.

2.9. Effect of temperature on activity of xylanase

The effect of temperature on activity of Xylanase was deter-

mined by incubating crude enzyme mixture in 1% xylan in

0.05 M citrate buffer, pH 4.8. at temperatures between 40 and

90 �C with regular interval of 5 �C. Enzyme activity was

J o u rn a l o f R a d i a t i o n R e s e a r c h and A p p l i e d S c i e n c e s 9 ( 2 0 1 6 ) 1 3 9e1 4 7 141

assayed by DNS method at different temperatures as

described above.

2.10. Statistical analysis

The data obtained after experimentation was statistically

evaluated using ANOVA at significance level of p < 0.05 by

using computer based program SPSS.

3. Results and discussion

3.1. Time course study for xylanase production

Different experiments were conducted to study the optimum

period for maximum xylanase production in submerged

fermentation process. Results (Fig. 1) described that fermen-

tation period of 48 h was optimum for xylanase production by

B. subtilis BS04 while B. megaterium BM07 exhibited maximum

production after 72 h of fermentation period. Further increase

of fermentation period beyond this resulted decline in enzyme

production which might be due to the production of toxic

metabolites during microbial growth which inhibits the

enzyme synthesis. Gupta and Kar (2009) studied on xylanase

production by Bacillus sp. and reported that maximum xyla-

nase production was observed in 48 h and 72 h using wheat

bran and corn cob as a substrate respectively. Mrudula and

Shyam (2012) reported 48 h of fermentation time for

maximum production of protease from B. megaterium MTTC

2444. Murugan, Arnold, Pongiya, and Narayanan (2011) re-

ported 96 h of fermentation period was optimum for xylanase

production by Arthrobacter sp. MTCC 6915 in SSF using saw

dust as a substrate. In an other study some strains of Bacillus

showed maximum xylanase production after 24 h using

digested bran and 48 h of fermentation using saw dust as a

substrate respectively (Simphiwe, Ademola, Olaniran, &

Pillay, 2011). Heck, Plinho, Marco, and Ayub (2002) isolated B.

subtilis from soil which exhibit highest xylanase activity after

72 h of cultivation in SSF. Bacillus licheniformis MTCC 9415 can

producemaximum xylanase after 72 h of fermentation period

in solid state fermentation (Gupta& Kar, 2008). Sepahy, Ghazi,

Fig. 1 e Time course study of xylanase production by Bacillus sp.

the means of triplicates and different letters differ significantly

and Sepahy (2011) reported fermentation period of 48 h by

Bacillus mojavensis AG137 in submerge fermentation using oat

bran as substrate.

3.2. Effect of initial medium pH for xylanase production

Most of the bacteria are dependent on pH and produce xyla-

nase under high pH (Battan, Sharma, Dhiman, & Kuhad, 2007;

Poorna & Prema, 2006). Many enzymatic processes and

transport of various components across the cell membrane

are strongly affected by the pH of medium (Kapoor, Nair, &

Kuhad, 2008). Different pH ranges (4e10) were tested for

maximum xylanase production by B. subtilis BS04 and B.

megaterium BM07 in submerged fermentation. Results (Fig. 2)

revealed that both strains of Bacillus sp. exhibited its peak

activity at pH 8.0. Lower pH i.e. 4 and higher pH i.e. 10 of the

medium retards xylanase secretion. These results indicated

that both bacterial strains can tolerate in alkaline conditions.

Similar findings were also reported by Sepahy et al. (2011)

showing optimum pH of 8.0 for xylanase production by B.

mojavensis AG137 in submerge fermentation. Simphiwe et al.

(2011) reported that eight different strains of Bacillus sp.

showed maximum xylanase production at pH 8.0. Bacillus

pumilus showed maximum xylanase production at pH 7.0

(Monisha, Uma, & Murthy, 2009).

3.3. Effect of incubation temperature for xylanaseproduction

Various incubation temperatures (25e50 �C) were tested for

maximum production of xylanase enzyme. Fig. 3 illustrates

that, at 25 �C bacterial strains did not grow well thus resulting

in decreased enzyme production. B. subtilis BS04 showed

highest enzyme production (51.60 ± 0.53 IU/ml) at 35 �C while

B. megaterium BM07 producedmaximum titer of xylanase yield

(41.66 ± 0.58 IU/ml) at 40 �C. Increased in incubation temper-

ature up to 50 �C significantly reduced in enzyme production.

Sepahy et al. (2011) reported optimum temperature of 37 �C for

xylanase production by B. mojavensis AG137 in submerge

fermentation. Different strains of Bacillus sp. gave maximum

yield of xylanase production at incubation temperature of

in submerged fermentation at 37 �C. Values presented were

at p < 0.05.

Fig. 2 e Effect of initial medium pH for xylanase production in submerged fermentation of 48 h. Values presented were the

means of triplicates and different letters differ significantly at p < 0.05.

J o u r n a l o f R a d i a t i o n R e s e a r c h and A p p l i e d S c i e n c e s 9 ( 2 0 1 6 ) 1 3 9e1 4 7142

45 �C and 55 �C (Simphiwe et al., 2011). Monisha et al. (2009)

reported that incubation at 37 �C gave maximum xylanase

production by B. pumilus.

3.4. Effect of substrate concentration on xylanaseproduction

Fig. 4 describes the effect of different substrate (corn cobs)

concentrations (0.5e3.0%) on xylanase production by Bacillus

sp. in submerged fermentation. Results indicated that B. sub-

tilis BS04 showed maximum enzyme (47.33 ± 1.17 IU/ml) pro-

duction with 2% substrate concentration while B. megaterium

BM07 showed its peak production (40.00 ± 1.00 IU/ml) at 1.5%

substrate concentration. Further increase in substrate con-

centration did not significantly affect the enzyme production.

Saleem, Akhtar, and Jamil (2002) isolated a new strain of B.

subtiliswhich can produce best xylanase using 0.5% bagasse as

a substrate in submerge fermentation. Li, Lin, Meng, Lu, and

Gu (2006) worked on endoxylanase production by Aspergillus

awamori ZH-26 under submerged fermentation reporting op-

timumsubstrate (wheat bran) concentration of 4.93%was best

for endoxylanase production. Immanuel, Dhanusha, Prema,

Fig. 3 e Effect of incubation temperature for xylanase productio

fermentation period. Values presented were the means of tripli

and Palavesam (2006) also reported that maximum endoglu-

canase production was maximum with 1.5% substrate (coir

powder) concentration using Cellulomonas sp., Bacillus sp. and

Micrococcus sp.

3.5. Effect of inoculum size on xylanase production

Different inoculum levels (0.5e3.0%) were tested for enhanced

production of xylanase enzyme by Bacillus sp. in submerged

fermentation. When low inoculum level i.e. 0.5% was used,

enzyme production was minimum but as the inoculum level

increased the enzyme production was also increased.

Maximum yield of xylanase enzyme was observed at 2% with

Bacillus subtiltis BS04 and 1.5% inoculum level with B. mega-

terium BM07 (Fig. 5). Increased level of inoculum mostly

reduced xylanase production in industrial fermentation pro-

cess (Battan et al., 2007). This may be due to the depletion of

nutrients from the fermentation medium which resulted

decline in enzyme synthesis. Sepahy et al. (2011) reported

inoculum size of 2% was best for xylanase production by B.

mojavensis AG137 in submerged fermentation.

n by Bacillus sp. with initial medium pH of 8 for 48 h of

cates and different letters differ significantly at p < 0.05.

Fig. 4 e Effect of substrate concentration on xylanase production by Bacillus sp. in submerged fermentation (initial pH 8, 48 h

period). Values presented were the means of triplicates and different letters differ significantly at p < 0.05.

Fig. 5 e Effect of inoculum size on xylanase production by Bacillus sp. in submerged fermentation (initial pH 8, 48 h period).

Values presented were the means of triplicates and different letters differ significantly at p < 0.05.

J o u rn a l o f R a d i a t i o n R e s e a r c h and A p p l i e d S c i e n c e s 9 ( 2 0 1 6 ) 1 3 9e1 4 7 143

3.6. Effect of additional carbon sources on xylanaseproduction

Xylanase production was further enhanced by supplementing

the fermentation medium with suitable additional carbon

source. Table 1 depicts that sucrose and xylose at concentra-

tion of 0.5% were the best xylanase inducer by B. subtilis BS04

and B. megaterium BM07 in submerged fermentation respec-

tively. Any other carbon sources like fructose, glucose, CMC

Table 1 e Effect of supplementation of additional carbonsources on xylanase production by Bacillus sp. insubmerged fermentation (initial pH 8, 48 h period).

Sr. No. Carbon source Xylanase activity (IU/ml)

B. subtilis B. megaterium

1 Control 19.96 ± 1.55a 16.63 ± 1.18a

2 Xylose 48.30 ± 0.61e 43.06 ± 1.10e

3 Fructose 38.80 ± 1.25d 26.60 ± 1.21b

4 Sucrose 54.80 ± 0.52f 39.27 ± 0.94d

5 Glucose 36.93 ± 1.10d 34.90 ± 1.15c

6 CMC 26.23 ± 1.08c 18.16 ± 1.04a

7 Arabinose 23.20 ± 0.72b 25.67 ± 1.53b

Values presentedwere themeans of triplicates and different letters

differ significantly at p < 0.05.

(carboxymethyl cellulose) and arabinose gave less enzyme

production. Azeri, Tamer, and Oskay (2010) isolated different

strains of Bacillus sp. and all exhibit maximum xylanase pro-

duction by using birchwood xylan as a carbon source. Saleem

et al. (2002) reported that supplementation of sucrose to the

fermentation medium significantly enhance the xylanase

production by B. subtilis. In 96 h of fermentation, wheat bran

was best carbon source for xylanase production by Strepto-

myces sp. (Sharma & Bajaj, 2005).

3.7. Supplementation of nitrogen sources

Among all the tested inorganic and organic nitrogen sources,

tryptone and (NH4)2SO4 are best for B. subtilis BS04 while KNO3

and malt extract for B. megaterium BM07 for xylanase syn-

thesis in submerged fermentation as shown in Table 2.

Therefore, these nitrogen sources were selected as optimum

for respective bacterial strains. Earlier studies reported that

organic nitrogen sources have been found to stimulate xyla-

nase production in Bacillus species (Battan et al., 2007). Yeast

extract þ tryptone and yeast extract þ NH4NO3 have stimu-

latory effect on xylanase production from B. mojavensis AG137

in submerge fermentation (Sepahy et al., 2011). Sharma and

Bajaj (2005) isolated different species of Streptomyces sp. and

reported that best xylanase production was observed with

Table 2e Effect of different nitrogen sources on xylanase production by Bacillus sp. in submerged fermentation (initial pH 8,48 h period).

Sr.# Inorganic nitrogen sources Xylanase activity (IU/ml) Organic nitrogen sources Xylanase activity (IU/ml)

B. subtilis B. megaterium B. subtilis B. megaterium

1 Control 3.07 ± 0.058a 3.56 ± 0.15a Control 11.70 ± 0.30a 10.10 ± 0.10b

2 KNO3 21.33 ± 0.15d 34.5 ± 0.25f Peptone 48.93 ± 0.90e 16.50 ± 0.20d

3 NaNO3 17.63 ± 0.15c 18.66 ± 0.25c Yeast extract 39.90 ± 0.85d 12.43 ± 0.23c

4 (NH4)2SO4 38.03 ± 0.06e 21.33 ± 0.15d Tryptone 55.66 ± 0.41f 21.30 ± 0.30e

5 NH4Cl 20.27 ± 0.15d 24.03 ± 0.06e Malt extract 23.40 ± 0.72c 43.66 ± 0.06f

6 Ammonium citrate 14.11 ± 1.72b 12.56 ± 0.15b Urea 20.13 ± 1.02b 8.50 ± 0.30a

Values presented were the means of triplicates and different letters differ significantly at p < 0.05.

J o u r n a l o f R a d i a t i o n R e s e a r c h and A p p l i e d S c i e n c e s 9 ( 2 0 1 6 ) 1 3 9e1 4 7144

soybean meal and yeast extract as nitrogen sources in the

medium.

3.8. Effect of different concentrations of molasses onxylanase production

Effect of different concentrations of molasses supplementa-

tion was also evaluated for xylanase production by tested

bacterial strains. Results (Fig. 6) revealed that supplementa-

tion of 0.5% molasses concentration favored enzyme pro-

duction by both bacterial strains i.e. B. subtilis BS04 and B.

megaterium BM07. Increased supplementation of molasses

caused decreased enzyme production which might be due to

increased sugars level and many other nutrients present in

molasses which resulted in catabolite repression. Shabeb,

Younis, Hezayen, and Nour-Eldien (2010) achieved best

cellulase production with 10% molasses containing medium.

Rafi, Asghar, Yaqube, and Ghour (1998) obtained highest yield

of xylanase production in a medium containing 2% cane

molasses. Supplementation of 0.125%molasses concentration

enhances the CMCase yield (Gouri & Malana, 2010).

3.9. Effect of different concentration of NaCl on xylanaseproduction

Different salt concentrations ranging from 0.2 to 1.0% were

tested for xylanase production. B. subtilis BS04 showed

maximum xylanase production (48.1 ± 1.00 IU/ml) at 0.2% NaCl

concentration and further increased concentration resulted

decline in enzyme production up to 12.20 ± 0.35 IU/ml at 1%

Fig. 6 e Effect of different concentrations of molasses on xylana

fermentation (initial pH 8, 48 h period). Values presented were

significantly at p < 0.05.

NaCl as shown in Fig. 7. The other strain B. megaterium BM07

exhibitedmaximumactivity (45.93± 0.11 IU/ml)with 0.8%NaCl

concentration showing its halophilic character. Sajitha,

Vasanthabharathi, Lakshminarayanan, and Jayalakshmi

(2011) isolated a strain of B. megaterium which have good

growth in medium containing 0.3% NaCl and gave maximum

yield of amylase. Shaheen, Shah, Hameed, and Hasan (2008)

worked on protease production by B. subtilis reporting that

1.5% NaCl concentration was best for maximum production of

protease in submerged fermentation.

3.10. Effect of pH on activity

Different pH ranges from 4 to 9 was tested to check the opti-

mum activity of xylanase by using various buffers. Results

revealed (Fig. 8) that pH 5 and 5.5 was found optimum for

maximum xylanase activity B. subtilis BS04 and B. megaterium

BM07 by using 0.05 M citrate buffer respectively. Further in-

crease in pH resulted decline in enzyme activity. The opti-

mum pH of xylanase enzyme from different Bacillus sp. was

reported 5 (Pratumteep, Sansernsuk, Nitisinprasert, &

Apiraksakorn, 2010) 7 (Sanghi, Garg, Gupta, Mittal, & Kuhad,

2010), 8 (Kamble & Jadhav, 2012) and 9 (Annamalai, Thavasi,

Jayalakashmi, & Blasubramanian, 2009).

3.11. Effect of temperature on activity

The activity of the xylanase enzyme was assayed at various

temperatures ranging from 35 �C to 70 �C to check the opti-

mum temperature. Results (Fig. 9) indicated that enzyme

se production by Bacillus sp. in submerged

the means of triplicates and different letters differ

Fig. 7 e Effect of different concentration of NaCl on xylanase production by Bacillus sp. in submerged fermentation (initial pH

8, 48 h period). Values presented were the means of triplicates and different letters differ significantly at p < 0.05.

Fig. 8 e Effect of pH on activity of xylanase produced by Bacillus sp. in submerged fermentation.

J o u rn a l o f R a d i a t i o n R e s e a r c h and A p p l i e d S c i e n c e s 9 ( 2 0 1 6 ) 1 3 9e1 4 7 145

activity was increased with increase in temperature and

highest activity was observed at 50 �C. After that, as the

temperature was increased from 60 �C to 70 �C, there was a

sharp decline in enzyme activity was observed. Xylanase

enzyme from various Bacillus species has optimum tempera-

ture of 40e60 �C (Khandeparker, Verma, & Deobagkar, 2011;

Kumar, Panday, & Naik, 2011; Pratumteep et al., 2010; Roy &

Rowshanul, 2009; Sanghi et al., 2010; Sa'-Pereira, Costa-

Ferreira, & Aires-Barros, 2002).

Fig. 9 e Effect of temperature on activity of xylanase pr

4. Conclusion

The results of the present study revealed that B. megaterium

BM07 can produce xylanase enzyme using sugarcane bagasse

as a substrate in submerged fermentation which was not re-

ported earlier. In this study bacterial strains were used for

xylanase enzyme production which has advantage of short

period of growth as compared to the fungi. Results of this

oduced by Bacillus sp. in submerged fermentation.

J o u r n a l o f R a d i a t i o n R e s e a r c h and A p p l i e d S c i e n c e s 9 ( 2 0 1 6 ) 1 3 9e1 4 7146

study indicated that nutrients and cultural properties played a

pivotal role in enzyme production. The optimization of all the

process parameters are being considered as pre-requisites to

make the process of enzyme production cost effective at large

scale.

r e f e r e n c e s

Annamalai, N., Thavasi, R., Jayalakshmi, S., &Balasubramanian, T. (2009). Thermostable and alkalinetolerant xylanase production by Bacillus subtilis isolated frommarine environment. Indian Journal of Biotechnology, 8, 291e297.

Azeri, C., Tamer, A. U., & Oskay, M. (2010). Thermoactive cellulase-free xylanase production from alkaliphilic Bacillus strains usingvarious agro-residues and their potential in biobleaching ofkraft pulp. African Journal of Biotechnology, 9(1), 63e72.

Battan, B., Sharma, J., Dhiman, S. S., & Kuhad, R. C. (2007).Enhanced production of cellulase-free thermostable xylanaseby Bacillus pumilus ASH and its potential application in paperindustry. Enzyme and Microbial Technology, 41(6e7), 733e739.

Bernier, R., Desrochers, M., Jurasek, L., & Paice, M. G. (1983).Isolation and characterization of a xylanase from Bacillussubtilis. Applied and Environmental Microbiology, 46(2), 511e514.

Bilgrami, K. S., & Pandy, A. K. (1992). In E. S. K. Jain (Ed.), Industryand fermentation in introduction to biotechnology (pp. 149e165).

Chakrit, T., Khin, L. K., & Khanok, R. (2006). Purification ofxylanase from alkaliphilic Bacillus sp. K-8 by using corn huskcolumn. Process Biochemistry, 41(12), 2441e2445.

Coughlan,M. P., &Hazlewood, G. P. (1993). b-1,4-D-Xylan degradingenzyme systems: biochemistry, molecular biology andapplications. Biotechnology andApplied Biochemistry, 17, 259e289.

Couri, S., Terzi, S. D. C., Pinto, G. A. S., Freitas, S. P., &Costa, A. C. A. D. (2000). Hydrolytic enzyme production insolid-state fermentation by Aspergillus niger 3T5B8. ProcessBiochemistry, 36(3), 255e261.

de Souza, D. F., de Souza, C. G. M., & Peralta, R. M. (2001). Effect ofeasily metabolizable sugars in the production of xylanase byAspergillus tamarii in solid-state fermentation. ProcessBiochemistry, 36(8e9), 835e838.

Elegir, G., Szakacs, M., & Jeffries, T. W. (1994). Purification,characterization and substrate specificities of multiplexylanases from Streptomyces sp. strain B-12-2. Applied andEnvironmental Microbiology, 60(7), 2609e2615.

Gomes, D. J., Gomes, J., & Stiener, W. (1994). Production of highlythermostable xylanase by a wild strain of thermophilic fungusThermoascus aurantiacus and partial characterization of theenzyme. Journal of Biotechnology, 37, 11e22.

Gouri, M. I., & Malana, M. A. (2010). Production of carboxymethylcellulase from local isolate of Aspergillus species. PakistanJournal of Life and Social Sciences, 8(1), 1e6.

Gouda, M. K. (2000). Purification and partial characterization ofcellulose free xylanase produced in solid state and submergedfermentation by Aspergillus tamarii. Advances in Food Science, 22,31e37.

Gupta, U., & Kar, R. (2008). Optimization and scale up of cellulasefree endo xylanase production by solid state fermentation oncorn cob and by immobilized cells of a thermotolerantbacterial isolate. Jordan Journal of Biological Science, 1(3),129e134.

Gupta, U., & Kar, R. (2009). Xylanase production by a thermo-tolerant Bacillus species under solid-state and submergedfermentation. Brazilian Archives of Biology and Technology, 52(6),1363e1371.

Heck, J. X., Plinho, F., Marco, H., & Ayub, A. Z. (2002). Cellulase andxylanase production by isolated amazon Bacillus strains using

soybean industrial residue based solid-state cultivation.Brazilian Journal of Microbiology, 33, 213e218.

Hoq, M. M., Hempel, C., & Deckwer, W. D. (1994). Cellulase freexylanase by Thermomyces lanuginosus RT9: effects of aeration,agitation and medium components on production. Journal ofBiotechnology, 37, 49e58.

Immanuel, G., Dhanusha, R., Prema, P., & Palavesam, A. (2006).Effect of different growth parameters on endoglucanaseenzyme activity by bacteria isolated from coir retting effluentsof estuarine environment. International Journal of EnvironmentScience and Technology, 3(1), 25e34.

Irfan, M., Nadeem, M., Syed, Q., & Baig, S. (2012). Effect of mediumcomposition on xylanase production by Bacillus subtilis usingvarious agricultural wastes. American-Eurasian Journal ofAgriculture & Environmental Science, 12(5), 561e565.

Kalogeris, E., Christakopoulos, P., Kekos, D., & MacRis, B. J. (1998).Studies on the solid-state production of thermostableendoxylanases from Thermoascus aurantiacus, characterizationof two isozymes. Journal of Biotechnology, 60(3), 155e163.

Kamble, R. D., & Jadhav, A. R. (2012). Isolation, purification, andcharacterization of xylanase produced by a new species ofBacillus in solid state fermentation. International Journal ofMicrobiology, 2012, 8. http://dx.doi.org/10.1155/2012/683193(Article ID 683193).

Kapoor, M., Nair, L. M., & Kuhad, R. C. (2008). Cost-effectivexylanase production from free and immobilized Bacilluspumilus strain MK001 and its application in saccharification ofProsopis juliflora. Biochemical Engineering Journal, 38(1), 88e97.

Khandeparker, R., Verma, P., & Deobagkar, D. (2011). A novelhalotolerant xylanase from marine isolate Bacillus subtilischo40, gene cloning and sequencing. New Biotechnology, 28,814e821.

Kumar, S. S., Panday, D. D., & Naik, G. R. (2011). Purification andmolecular characterization of low molecular weight cellulase-free xylanase from thermoalkalophilic Bacillus spp. JB 99.World Journal of Science & Technology, 1, 09e16.

Li, Y., Lin, J., Meng, D., Lu, J., & Gu, G. (2006). Effect of pH,cultivation time and substrate concentration on theendoxylanase production by Aspergillus awamori ZH-26 undersubmerged fermentation using central composite rotarydesign. Food Technology and Biotechnology, 44(4), 473e477.

Monisha, R., Uma, M. V., & Murthy, V. K. (2009). Partialpurification and characterization of Bacillus pumilus xylanasefrom soil source. Kathmandu University Journal of ScienceEngineering and Technology, 5(II), 137e148.

Mrudula, S., & Shyam, N. (2012). Immobilization of Bacillusmegaterium MTCC 2444 by Ca-alginate entrapment method forenhanced alkaline protease production. Brazilian Archives ofBiology and Technology, 55(1), 135e144.

Murugan, S., Arnold, D., Pongiya, U. D., & Narayanan, P. M. (2011).Production of xylanase from Arthrobacter sp. MTCC 6915 usingsaw dust as substrate under solid state fermentation. EnzymeResearch, 2011, 7. http://dx.doi.org/10.4061/2011/696942 (ArticleID 696942).

Poorna, C. A., & Prema, P. (2006). Production and partialcharacterization of endoxylanase by Bacillus pumilus usingagro industrial residues. Biochemical Engineering Journal, 33(2),106e112.

Pratumteep, A., Sansernsuk, J., Nitisinprasert, S., &Apiraksakorn, J. (2010). Production, characterization andhydrolysation products of xylanase from Bacillus subtilisGN156. KKU Research Journal, 15, 343e350.

Rafi, S., Asghar, M., Yaqube, M., & Ghour, M. A. (1998). Productionof xylanase from corn stover by Arachniotus sp. Pakistan Journalof Biological Sciences, 1(4), 380e382.

Roy, N., & Rowshanul, H. M. (2009). Isolation and characterizationof xylanase producing strain of Bacillus cereus from soil. IranianJournal of Microbiology, 1, 49e53.

J o u rn a l o f R a d i a t i o n R e s e a r c h and A p p l i e d S c i e n c e s 9 ( 2 0 1 6 ) 1 3 9e1 4 7 147

Sa'-Pereira, P., Costa-Ferreira, M., & Aires-Barros, M. R. (2002).Enzymatic properties of a neutral endo-1,3(4)-b-xylanase Xyl IIfrom Bacillus subtilis. Journal of Biotechnology, 94, 265e275.

Sajitha, N., Vasanthabharathi, V., Lakshminarayanan, R., &Jayalakshmi, S. (2011). Amylase from an estuarine Bacillusmegaterium. Current Research Journal of Biological Sciences, 3(2),110e115.

Saleem, M., Akhtar, M. S., & Jamil, S. (2002). Production ofxylanase on natural substrates by Bacillus subtilis. InternationalJournal of Agricultural Biology, 4(2), 211e213.

Sanghi, A., Garg, N., Gupta, V. K., Mittal, A., & Kuhad, R. C. (2010).One-step purification and characterization of cellulase-freexylanase produced by alkalophilic Bacillus subtilis ASH.Brazilian Journal of Microbiology, 41, 467e476.

Sepahy, A. A., Ghazi, S., & Sepahy, M. A. (2011). Cost-effectiveproduction and optimization of alkaline xylanase byindigenous Bacillus mojavensis AG137 fermented onagricultural waste. Enzyme Research, 9. http://dx.doi.org/10.4061/2011/593624 (Article ID 593624).

Shabeb, M. S., Younis, A. M., Hezayen, F. F., & Nour-Eldien, M. A.(2010). Production of cellulase in low-cost medium by Bacillussubtilis KO strain. World Applied Science Journal, 8(1), 35e42.

Shaheen, M., Shah, A. A., Hameed, A., & Hasan, F. (2008).Influence of culture conditions on production and activity ofprotease from Bacillus subtilis bs1. Pakistan Journal of Botany,40(5), 2161e2169.

Sharma, B., & Bajaj, B. K. (2005). Production and partialcharacterization of alkali-tolerant xylanase from analkalophilic Streptomyces sp. CD3. Journal of Scientific andIndustrial Research, 64, 688e697.

Simphiwe, P., Ademola, B., Olaniran, O., & Pillay, B. (2011).Sawdust and digestive bran as cheap alternate substrates forxylanase production. African Journal of Microbiology Research,5(7), 742e752.

Srinivasan, M. C., & Meenakshi, V. R. (1999). Microbial xylanasefor paper industry. Current Science, 77(1), 137e142.

Veluz, G., Taksuo, K., Hiroshi, M., & Yusaku, F. (1999). ScreeningRhizopus sp. Journal of Faculty of Agriculture, 43, 419e423.