production of cellulase-free xylanase by bacillus sp

More & Laxman, Int. J.BiotechBiosci., Vol 1 (2) :pp 164-174 (2011)

Original Paper

PRODUCTION OF CELLULASE-FREE XYLANASE BY BACILLUS SP. 90-10-50 More S V & Laxman R S* Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, India Tel: +91-20-25902720, Fax: +91-20-25902648, Email: [email protected]

Abstract A Bacillus strain 90-10-50 isolated from local soil and identified as Bacillus pumilus secretes high levels of cellulase-free xylanase. Effect of various cultural parameters such as concentration of wheat bran, xylan, yeast extract, carbon and nitrogen sources and temperature on xylanase production were studied and optimized. 3% wheat bran and 1% yeast extract were found to be optimal for xylanase production. Comparable activities were also obtained when yeast extract was replaced with inorganic nitrogen sources at equivalent nitrogen concentration. Among the soluble sugars tested, only xylose gave low levels of activities that were less than 10% of the activities obtained with xylan or wheat bran. Xylanase production in xylan containing medium was repressed by addition of xylose, repressive effect increasing with increase in xylose concentration. Tween 80 enhanced extra-cellular enzyme yields by 15-20%. Maximum xylanase activities of 400450 IU/ml were obtained in 72 h. The xylanase activity was found to be optimum at 50°C and pH 7 and stable in the pH range of 6-10 and 40-50°C. End product analysis of xylan hydrolysate indicated the enzyme to be endoxylanase.

Keywords: Cellulase-free xylanase, Bacillus, production, fermentation parameters

INTRODUCTION

animal feed, dietary supplements, textiles (Subramaniyan & Prema 2002; Polizeli et al. 2005).

Hemicelluloses constitute 20-35% by weight of wood and agricultural residues and serve as an abundant and inexpensive source of fermentable carbohydrates (Kuhad & Singh 1993). Xylan is the major component of hemicellulose and is a hetero-polysaccharide containing substituent groups of acetyl, 4-O-methyl-D-glucouronosyl and a-arabinofuranosyl residues linked to the b-1,4, linked xylose units. Due to the heterogeneity, xylans require minimum two enzyme activities for hydrolysis, endoxylanases (EC.3.2.1.8) responsible for the random hydrolysis of xylan backbone and b-xylosidase (EC 3.2.1.37), releasing xylosyl residues by endwise attack on xylooligosaccharides (Bajpai 1997). Xylanases find biotechnological applications in paper and pulp industry, in the bioconversion of lignocelluloses to sugar, ethanol and other useful substances, clarification of juices and wines, extraction of plant oils, coffee and starch, in

Xylan degrading enzymes are reported from bacteria, actinomycetes and fungi (Sunna & Antranikian 1997). Xylanases especially from fungal systems are generally associated with cellulases (Juhasz et al. 2005; Khalil 2002). Interest in cellulase-free xylanases during the last few years is mainly due to the potential use of these enzymes in paper and pulp industry (Nagar et al. 2010; Sanghi et al. 2009; Polizeli et al 2005; Subrama-niyan & Prema 2002; Kulkarni et al. 1999). The present paper reports isolation of a Bacillus strain 90-10-50 secreting high levels of cellulase-free xylanase. The organism is identified as Bacillus pumilus on the basis of biochemical and molecular characterization. Optimization of fermentation parameters for xylanase production and properties of the enzyme are discussed.

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PRODUCTION OF CELLULASE-FREE XYLANASE BY BACILLUS SP. 90-10-50

Materials and Methods Oat spelt xylan and 3,5, dinitro salicylic acid (DNSA) were obtained from M/s Sigma Chemical Co, USA. All chemicals used were of analytical grade. Wheat bran, rice bran, bagasse and corncobs were purchased from local market. Isolation, identification and cultural characteristics of the organism - The Bacillus strain 90-10-50 used in the present investigation was isolated from local garden soil. One loopful of soil suspension after appropriate dilution was plated on malt extract, yeast extract, peptone agar medium containing 1% oat spelt xylan (MXYP) and incubated at 28°C for 7 days. For identification of the organism, physiological and biochemical tests were carried out according to the key for identification for Bacillus (Gordon et al. 1973). DNA isolation and sequencing - For molecular identification, DNA was isolated, purity checked, amplified by PCR and nucleotide sequences were analyzed with the NCBI database using BLAST program. For isolation of DNA, the organism was grown in MGYP broth at 28°C for 48 h. Cells were collected by centrifugation and resuspended in 5 ml of 50 mM Tris (pH 8.0) containing 50 mM EDTA and 10 mg/ml lysozyme was added and placed on ice for 45 min. One ml of 0.5% SDS in 50 mM Tris (pH 7.5), containing 0.4 M EDTA, 1 mg/ml proteinase K were added and incubated at 50°C in water bath for 60 min. This was followed by addition of 6 ml of Trisequilibrated phenol. To the supernatant collected after centrifugation (8000rpm, 10 min), 0.1 volume of 3M Na acetate and two volumes 95% ethanol were added. DNA was spooled out and transferred to 5 ml of 50 mM Tris (pH 7.5) contining 1 mM EDTA, 20 mg/ml RNase and dissolved overnight at 4°C. It was extracted with equal volume of chloroform and centrifuged (10,000rpm, 5 min) followed by addition of 0.1 volume of 3M Na acetate and 2 volumes of 95% ethanol to the supernatant to precipitate the DNA. DNA was spooled out and washed with 70% ethanol and dissolved in 5ml of 0.1M Tris EDTA buffer pH 8.0 and stored. The purity of DNA was checked on 0.8% agarose gel electrophoresis. PCR amplification of 16S ribosomal DNA was performed using universal primers. The sequencing reactions of

PCR product were carried out using Taq DNA polymerase dye terminator cycle applying automated DNA sequencing method based on dideoxynucleotide chain termination method using universal primers. The nucleotide sequence was analyzed with the NCBI database using BLAST program. Analysis of nucleotide sequence - The nucleotide sequence was analyzed with the NCBI database using BLAST program. The multiple sequence alignment of homologous sequences was done in Clustal X software and sequences were trimmed in DAMBE software. The phylogenetic tree was inferred using the neighbor-joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test with 1000 replicates is shown next to the branches (Felsenstein 1985). The evolutionary distances were computed using the maximum composite likelihood method and are in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated from the dataset (complete deletion option). Phylogenetic analyses were conducted in MEGA4 (Tamura et al. 2007). Fermentation conditions - Fermentations were carried out at 28°C with shaking at 180-200rpm. Parameters for optimum growth such as pH and temperature were investigated in MGYP liquid medium (g/L): malt extract3; yeast extract-3; peptone-5 and glucose-10 at pH 7. Media were normally sterilized at 121°C for 20 min. Growth was monitored spectrophotometrically by reading the absorbance at 600nm. Enzyme production was studied in 250 ml Erlenmeyer flasks containing 50 ml medium adjusted to pH 7.0. Stocks grown on MGYP slant for 7-10 days were used for developing the inoculum. Five to ten percent (v/v) inoculum grown in MGYP liquid for 24 h was used unless otherwise mentioned. Samples withdrawn at various time intervals during the fermentation were centrifuged at 10000rpm for 10 min and cell free supernatant was used for measurement of enzyme activity. Data presented is mean of two or more independent experiments run in duplicates. Effect of carbon source on growth and xylanase production - Broth cultures were grown in shake flasks with

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sugars/glycerol and 1% yeast extract as carbon and nitrogen sources respectively. Xylose, sucrose, glucose, lactose and glycerol were used at 1% concentration. Effect of xylan and wheat bran concentrations - Since the organism showed large clearance zone on xylan containing MXYP plate, effect of xylan concentration on enzyme production was studied in medium containing 1% yeast extract and xylan concentrations varying from 0.25 to 2%. Effect of wheat bran concentration ranging from 0.5 to 5% was studied with 1% yeast extract as nitrogen source. Effect of yeast extract concentration - Effect of yeast extract on xylanase production was studied with 3% wheat bran as carbon source and inducer and concentration of yeast extract varying from 0.5 to 2%. Effect of nitrogen sources - Effect of different nitrogen sources on xylanase production was investigated in medium containing 3% wheat bran and 1% yeast extract. Yeast extract was replaced with either organic nitrogen sources such as peptone, tryptone and casein hydrolysate or inorganic nitrogen sources viz. ammonium sulphate, di-ammonium hydrogen phosphate, sodium nitrate and urea at nitrogen concentration equivalent to 1% yeast extract. Effect of catabolite repression by xylose on xylanase production - Effect of xylose repression on xylanase production was studied in medium containing 0.5% xylan, 1% yeast extract. Xylose was separately autoclaved (10lb, 10 min) and added aseptically to a final concentration of 0.05 to 0.5% before inoculation. One ml bacterial spore suspension in sterile water was used to inoculate the fermentation medium.

(FPA), carboxymethyl cellulase (CMCase) and amylase activities in the crude culture filtrate were determined at pH 4.8 as well as at pH 7. CMCase and amylase assays were carried out by incubating the reaction mixture at 50°C for 30 min and using 1% carboxymethyl cellulose and 1% soluble starch as substrates respectively. Filter paper activity (PFA) was determined by incubating Whatman No 1 filter paper (25mg) in a total reaction mixture of 1 ml containing 0.5 ml of suitably diluted enzyme and 0.5 ml buffer and incubated at 50°C for 60 min. The reducing sugar liberated was measured by dinitro salicylic acid method (Bernfeld 1955). One unit of activity is defined as the amount of enzyme required to release 1µM of reducing sugar as xylose equivalents per min under the assay conditions. One unt PFA, CMCase and amylase activity is defined as the amount of enzyme required to release 1µmole of reducing sugar as glucose equivalents per min under the assay conditions. pH optima and stability - Optimum pH for xylanase activity was determined at 50ºC by incubating the reaction mixture at pH values ranging from 3 to 9. Citrate buffer was used for pH 3 and 4, acetate buffer for 5, phosphate buffer for pH 6 to 8 and carbonate bicarbonate buffer for 9 and 10. Stability of xylanase was examined at different pH values by incubating the enzyme at room temperature in buffers at pH values ranging from 6 to 10 up to 2 h. Samples were removed after every 15 min. Residual activity was estimated and expressed as percentage of the initial xylanase activity taken as 100%.

Effect of surfactants - Various surface active agents viz. Tween-40; Tween-60; Tween-80 and Triton-X-100 were tested at 0.1% concentration for their influence on xylanase production.

Temperature optima and stability - Optimum temperature for xylanase was determined at pH 7 by incubating the reaction mixture at temperatures ranging from 30 to 70ºC. Thermal stability of xylanase was examined by incubating the enzyme at different temperatures up to 2 h. Samples were withdrawn at an interval of 15 min and residual activity was estimated at 50°C and pH 7 and expressed as percentage of initial activity taken as 100%.

Enzymatic assays - Xylanase activity was measured using 1% soluble xylan (w/v) in 50mM potassium phosphate buffer pH 7 in a total reaction mixture of 1 ml containing 0.5 ml of suitably diluted enzyme and 0.5 ml substrate and incubated at 50°C for 30 min. Filter paper

End product analysis - Fifty milligrams of oat spelt xylan was incubated with crude xylanase (50 IU) at 50°C up to 2 h. Hydrolysed products were analyzed by descending paper chromatography run for 18 h with butanol: acetic acid: water solvent system (3:1:1). The paper was dried

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and sprayed with mixture of pthalic acid and panisidine to visualize the spots.

RESULTS AND DISCUSSION Isolation and cultural characteristics of the organism Colony exhibiting the largest clearance zone with the code name 90-10-50 was picked up from the isolation plate and purified by repeated sub-culturing on MGYP agar plates. The organism was maintained on MGYP and MXYP slants and transferred to cold after 7-10 days. Large clearance zone was observed after 48 h of bacterial growth on MXYP agar plate, after congo red staining (Fig.1). Growing colonies were off-white in colour, appear flat with dry reticulate surface, which on further incubation tend to become mucoid due to development of endospores. Table 1. Comparison of morphological and biochemical characteristics of Bacillus sp. 90-10-50 with B. pumilus Characters Form Motility Gram stain Sporangia Spores Growth in nutrient broth Catalase Vogus Proskaur Test Growth Temperature PH for growth Acid production from Glucose Mannitol Xylose Sucrose Lactose Use of citrate Decomposition of casein Hydrolysis of gelatin Hydrolysis of starch Reduction of nitrate to nitrite Growth in 7% NaCl

Bacillus sp. 90-10-50 Rods Motile Positive Not swollen Central Positive Positive Positive 15-40°C 5.0-8.0

B. pumilus Rods Motile Positive Not swollen Central Positive Positive Positive 15-40°C 5.5-8.5

Positive Positive Positive Positive Positive Utilized Decomposed Hydrolyzed Not hydrolyzed Not reduced

Positive Positive Positive Positive Positive Utilized Decomposed Hydrolyzed Not hydrolyzed Not reduced

No growth

Positive

Fig. 1. Clearance zone on MXYP plate

The organism was identified as Bacillus pumilus on the basis of their similarities in morphological and biochemical characteristics (Table 1). This was also confirmed by molecular studies. The 16S rDNA sequence showed maximum homology with Bacillus pumilis (Table 2, Fig. 2). 16S rDNA sequence of this organism has been deposited in NCBI GenBank database under accession number HM176596. Effect of pH and temperature on growth - The organism was able to grow in MGYP broth over a pH range of 5-8 with an optimum at pH 7.0. Significant growth was also observed at pH 5 (75%) and pH 8 (85%). The organism showed good growth when incubated over a temperature range of 15-50°C with an optimum at 28°C. More than 70% growth was observed even at 15°C compared to 28°C. Effect of carbon source on growth - Good growth was observed in all the sugars, xylose being the best followed by sucrose, glucose and lactose while glycerol was poorly utilized (Table 3). Growth increased with incubation time up to 48 h and declined thereafter (data not shown). Xylanase production by Bacillus sp. 90-10-50 was studied in a simple medium containing 1% yeast extract and wheat bran/xylan as carbon source and inducer. As fermentation profile of an organism is generally affected by nutritional and physiological factors such as carbon source, nitrogen source, surfactants, inoculum size, pH of the media, incubation temperature, optimization of these parameters is important for maximum production of the enzyme. Hence effects of various parameters such as concentration of wheat bran, xylan, yeast extract, effect of carbon and nitrogen sources and temperature etc. on xylanase production were investigated.



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Table 2. First 10 BLAST hit of 16S rRNA sequence

Accession HM176596.1 HQ317174.1 HQ236083.1 HQ236064.1 HQ161778.1 HQ161775.1 HQ122449.1 HM371418.1 HM027879.1 AB360809.1

Description Bacillus pumilus strain RSL-90-10-50 16S ribosomal RNA gene, partial sequence Bacillus pumilus strain DYJL-A 16S ribosomal RNA gene, partial sequence Bacillus pumilus strain TBT1-3 16S ribosomal RNA gene, partial sequence Bacillus pumilus strain TAG2-5 16S ribosomal RNA gene, partial sequence Bacillus pumilus strain Sol-1 16S ribosomal RNA gene, partial sequence Bacillus pumilus strain Ama-6 16S ribosomal RNA gene, partial sequence Bacillus pumilus strain AU MB 16S ribosomal RNA gene, partial sequence Bacillus pumilus strain UW-02 16S ribosomal RNA gene, partial sequence Bacillus pumilus strain MW-1 16S ribosomal RNA gene, partial sequence Bacillus pumilus gene for 16S rRNA, partial sequence, strain: BHK28

Fig. 2. Phylogenetic tree shows the relationship between Bacillus sp. 90-10-50 with other bacteria on the basis of 16S rDNA sequence.

Query coverage 100%

Max ident

99%

100%

99%

100%

99%

100%

99%

100%

99%

100%

99%

100%

99%

100%

99%

100%

99%

100%

100%

Table 3. Effect of carbon source on growth and xylanase production

Carbon source (1%)

Growth (OD at 600nm)

Xylanase activity (IU/ml)

Glucose Xylose Sucrose Lactose

24 h 0.550 0.347 0.737 0.352

48 h 0.760 1.400 0.906 0.665

24 h NA* 11.0 0.71 0.41

48 h 0.73 20.3 0.57 0.65

Glycerol

0.185

0.217

NA*

NA*

* No activity HM176596.1 (Bacillus pumilus strain RSL-90-10-50); HQ317174.1 (Bacillus pumilus strain DYJL-A); HQ236083.1 (Bacillus pumilus strain TBT1-3); HQ236064.1 (Bacillus pumilus strain TAG2-5); HQ161778.1 (Bacillus pumilus strain Sol-1); HQ161775.1 (Bacillus pumilus strain Ama-6); HQ122449.1 (Bacillus pumilus strain AU MB); HM371418.1 (Bacillus pumilus strain UW-02); HM027879.1 (Bacillus pumilus strain MW-1); AB360809.1 (Bacillus pumilus strain: BHK28)

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Effect of xylan concentration - Xylanase activities increased with increase in xylan concentration up to 1% (Fig. 3). Maximum activities of around 330 IU/ml and 380 IU/ml were obtained with 0.5% and 1% xylan. Further increase in xylan concentration had no additional benefit. Balakrishnan et al. (1992) reported highest xylanase activities of 100 IU/ml by alkalophilic Bacillus sp in media containing pure xylan. Bacillus polymyxa




produced 12-13 IU/ml xylanase when grown in medium containing yeast extract and oat spelt xylan as nitrogen and carbon sources respectively (Pinaga et al. 1993). The optimum concentration of oat spelt xylan for xylanase production by Bacillus sp was 3.16 g/l (Pham et al. 1998). Bocchini et al. (2005) reported xylanase activities of 7 IU/ml by B. circulans D1 on 0.5% birch wood xylan. Bacillus strains isolated from sugarcane fields produced 50 IU/ml xylanase activities (Anuradha et al. 2007). Recently Nagar et al. (2010) reported xylanase activities of 690 IU/ml and 650 IU/ml on oat spelt and birch-wood xylan respectively by B. pumilus SV-85S. Since xylan is an expensive substrate, alternative xylan rich sources such as wheat bran, rice bran and corncobs were tested for xylanase production. As highest xylanase activity was produced in wheat bran medium it was selected for further study (data not shown). Effect of wheat bran concentration - Xylanase activity increased with increase in wheat bran concentration only up to 3% and further increase inhibited the production (Fig. 3). Maximum activites of around 400-450 IU/ml were were obtained on inexpensive media containing 3% wheat bran and 1% yeast extract which were nearly same or slightly higher to those produced on 1% xylan. 3% wheat bran was also found to be optimum for xylanase production by an alkalophilic Bacillus sp. (Balakrishnan et al. 1992). Rajaram & Varma (1990) reported higher xylanase activities with baggase compared to xylan as substrate. There are few reports where higher activities were obtained in xylan medium compared to agricultural residues like wheat bran or bagasse. Archana & Satyanarayana (1998) reported xylanase activities to be nearly double with xylan compared to wheat bran while Aureobasidium pullulans gave activities nearly 4 times with xylan compared to wheat bran (Karni et al. 1993). Maximum activities of around 400450 IU/ml were obtained on inexpensive media containing 3% wheat bran and 1% yeast extract which were nearly same or slightly higher to those produced on 1% xylan. Poorna & Prema P (2006) reported 1.3 times higher activities in xylan compared to wheat bran medium. Bacillus circulans AB 16 (Dhillon & Khanna 2000) and Bacillus subtilis ASH (Sanghi et al. 2009) were reported to produce 50 and 410 IU/ml, respectively on wheat bran medium. Nagar et al. (2010) found xylanase production B. pumilus SV-85S to be highest with 2% (w/v)

wheat bran and activities declined above 2%. Activities of 400-500 IU/ml secreted by this strain are one of the highest reported for a wild strain of Bacillus sp. High levels of xylanase production are also reported in literature by other strains of Bacillus pumilus. Among bacteria, strains belonging to B. pumilus seem to be good secretors of xylanase (Nagar et al. 2010; Poorna & Prema 2006; Battan et al. 2006). Effect of yeast extract concentration - Effect of yeast extract concentration on xylanase production was studied with 3% wheat bran as carbon source and inducer. Optimal yeast extract concentration for xylanase production was found to be 1% and further increase had no significant effect (data not shown). Yeast extract could be effectively substituted by whole bakers yeast with comparable activities. 1% yeast extract was also optimal for xylanase production by alkalophilic Bacillus sp. (Balakrishnan et al. 1992). Battan et al. (2006) observed that addition of yeast extract stimulated xylanase production by alkalophilic B. pumilus by solid-state fermentation. Effect of nitrogen sources - Xylanase activity was highest with yeast extract and activities were nearly similar in media containing either ammonium salts or nitrates while slightly lower activities were obtained in peptone, tryptone and casein hydrolysate (Table 4). But, activities were very low in urea. Archana & Satyanarayana (1998) reported high xylanase activities with corn steep liquor and di-ammonium hydrogen phosphate. They however found activities to be almost half with urea/ ammonium sulphate while activities were not detectTable 4. Effect of nitrogen sources

Nitrogen source Yeast extract Ammonium sulphate Diammonium hydrogen phosphate Sodium nitrate Urea Casein hydrolysate Peptone Tryptone

Relative Activity (%) 100.0 91.0 89.0 86.0 31.0 69.0 73.0 70.0

able in KNO3. In the present study, significant activities were obtained in presence of NaNO3.



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Hossain et al. (2008) reported yeast extract and ammonium sulphate to be the best nitrogen sources for xylanase production by intenstinal bacteria in termite. Casein hydrolysate and ammonium chloride supported growth and xylanase production in Bacillus sp. (Pham et al. 1998). Virupakshi et al. (2005) reported yeast extract, beef extract and peptone to be good nitrogen sources for xylanase production by thermoalkalophilic Bacillus sp JB-99 in solid state fermentation. Xylanase production by B. pumilus SV-85S was higher in organic nitrogen sources such as peptone, yeast extract and beef extract than with inorganic nitrogen sources (Nagar et al. 2010). Effect of carbon sources - Xylanase is an inducible enzyme and produced in presence of xylan or xylan rich substrates and relatively low levels of xylanase are produced in media devoid of xylan or xylan rich lignocellulosic substrate. The present Bacillus strain 90-10-50 produces low levels of xylanase in 1% xylose medium (510% as compared to 1% xylan or 3% wheat bran), while activity was absent or negligible in presence of glycerol, glucose, fructose and lactose (Table 3). This is in agreement with the results obtained by Archana & Satyanarayana (1998) where glucose completely repressed enzyme synthesis. Virupakshi et al. (2005) observed that fructose/glucose/lactose/rhamnose and sucrose repressed xylanase production by thermoalkaophilic Bacillus sp. Anuradha et al. (2007) reported xylanase activities of 8, 13 and 18 IU/ml on xylose, arabinose and sucrose respectively while the activities on xylan were 52 IU/ml which were several folds higher. In contrast, Bacillus sp. was reported to produce higher xylanase activity on 0.5% xylose than even on equivalent concentration of xylan (Perttula et al. 1993). Alkalotolerant Bacillus circulans gave maximum activities in 2% beech wood xylan while activities were nearly negligible in 2% xylose (Ratto et al. 1992). Xylanase activities by B. pumilus SV-85S in sugars were lower than wheat bran and xylan (Nagar et al. 2010). They reported around 150 IU/ml in xylose as carbon source. Effect of catabolite repression by xylose on xylanase production - Effect of xylose repression on xylanase production was studied in medium containing 0.5% xylan, 1% yeast extract. As shown in Fig. 4a, addition of xylose resulted in repression of xylanase production, the re-

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pressive effect increasing with increase in xylose concentration. Xylanase secretion started after 24 h where xylose concentration was below 0.1%. However when xylose concentration was above 0.1%, secretion increased only after 48 h, possibly the organism utilized xylose by this time (Fig. 4b). Gessesse & Mamo (1999) reported xylanase production on wheat bran medium by alkaliphilic Bacillus sp. to be repressed upon addition of 5% xylose and lactose while the effect by glucose and sucrose to be mild. Rajaram & Varma (1990) reported xylanase production in media containing bagasse supplemented with 0.25% xylose to be delayed and appeared only after 36 h which was thought to be due to the consumption of xylose.

Fig. 3. Effect of xylan & wheat bran concentrations

Fig. 4. Repression of xylanase production by xylose

Effect of surfactants - Surfactants can stimulate secretion of enzymes (Reese & Maguire 1969). Addition of Tween-80 enhanced production by 15-20% while Triton-X-100 markedly inhibited growth as well as




xylanase production (data not shown). Tween-80 at 0.5% concentration was found to stimulate xylanase production in A. pullulans and the yields increased by 13-20% (Karni et al. 1993). Balakrishnan et al. (1992) reported enhancement in activity by Tween-80 which was concentration dependent while Triton-X-100 resulted in inhibition. Xylanase production by Geobacillus thermoleovorans and B. pumilus SV-85S was also enhanced by Tween 80 (Sharma et al. 2007; Nagar et al. 2010). Effect of inoculum size -Twenty four hours old MGYP grown vegetative inoculum was used for studying effect of inoculum size (5-20%) on xylanase production. Five to ten percent inoculum was found to be optimum and higher incoulum was not beneficial for xylanase production (data not shown). Xylanase production by thermo-alkalophilic Bacillus sp. is highest with 10% inoculum (Sindhu et al. 2006; Virupakshi et al. 2005) while B. pumilus gave highest xylanase activities with 15% inoculum (Battan et al. 2006). However in case of B. pumilus SV-85S and thermophilic B. licheniformis A99, 1 to 2% inoculum was reported to be optimal for xylanase production (Archana & Satyanarayana 1998; Nagar et al. 2010). Effect of temperature on xylanase production -Temperature optima for growth of Bacillus sp. 90-10-50 coincided with temperature optima for xylanase production. Xylanase production was optimum at 28°C with nearly half of these activities at 37°C while less than 5% relative activities were obtained at 50°C. Growth of the organism was comparable over a wide temperature range (15 to 50°C) with an optimum at 28°C (Fig. 5). These results are consistent with the results of Subramaniyan & Prema (1998) where temperature optima for growth and xylanase production for Bacillus SSP-34 were same. Fermentation profile - Fermentation profile for xylanase production in the optimized medium containing 3% wheat bran, 1% yeast extract and 0.1% Tween 80 is showed highest xylanase activity of around 430 IU/ml in 72 h and decreased on further incubation. This is consistent with the report by Karni et al. (1993) where xylanase activity reached a peak at 70 h and decreased thereafter. Xylanase production by Lysinibacillus sp., Bacillus pumilus and thermophilic B. licheniformis A99 reached maximum at 72 h (Alves-Prado et al. 2010;

Battan et al. 2006; Archana & Satyanarayana 1998) while xylanase production by Bacillus SSP-34 reached maximum after 96 h (Subramaniyan & Prema 1998). Activities of crude culture filtrate towards different substrates - The culture broth was free of cellulase activity. No FPA, CMCase or amylase activities were detectable in the culture filtrate. Gessesse & Mamo (1999) reported formation of cellulase and protease activities along with xylanase by alkalophilic Bacillus licheniformis A99 sp. Small amounts of cellulase were produced by B. megaterium when grown in submerged culture and suppressed when grown in SSF under optimized conditions (Sindhu et al. 2006). Production of cellulase-free xylanase by thermophilic B .licheniformis A99 was reported by Archana & Satyanarayana (1998). pH optima and stability - Effect of pH on xylanase activity showed that activity was highest at pH 7.0. The activities were 70% and 55% at pH 6 and 8 respectively. The optimum pH for bacterial xylanases especially from those belonging to Bacillus is in the range of 5.5 to 7 (Subramaniyan & Prema 2002). Optimum pH for xylanase from Bacillus pumilus SV-85S and Lysinibacillus sp.was found to be 6.0 (Nagar et al. 2010; Alves-Prado et al. 2010). Xylanase activity was stable over a pH range of 6 to 10 up to 2 h (Fig. 6). This is in agreement with Gessesse & Mamo (1998) who reported xylanase from Micrococcus sp. AR135 to be stable in the range of pH 6.5 to 10. Crude xylanases from Bacillus strains were stable between pH 7 to 11 (Anuradha et al. (2007). Temperature optima and stability - Maximum xylanase activity was observed at 50°C with 75% and 50% relative activities at 40°C and 60°C respectively. Geesesse & Mamo (1998) reported 55°C to be the optimum temperature for xylanase from Micrococcus sp. AR135. Xylanases from the three Bacillus isolates showed sharp peak at 55°C (Anuradha et al 2007). Xylanase activity was stable at 40°C up to 2 h while it was stable up to 1 h at 50°C (Fig. 7). Maximum stability was reported at 50°C for xylanase from alkalophilic Bacillus sp. (Balakrishnan et al. 1992). Rajaram & Verma (1990) reported xylanase from B. thermoalkalophilus en-



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zyme to be stable at 65°C for 1 h. Xylanases from the three Bacillus strains were stable at 55°C (Anuradha et al. 2007). End product analysis - End product analysis of the xylan hydrolysates showed the presence of xylobiose, xylotriose and xylotetraose and there was no xylose formation up to 2 h indicating it to be endoxylanase (Fig. 8). Ratto et al. (1992) reported formation of xylose, xylobiose and xylotriose as main hydrolysis products of xylan by xylanase from alkalophilic B. circulans. Alkalophilic Bacillus licheniformis A99. produced xylobiose and xylose even at early stages (5 min) and after longer incubations (24 h), they were the predominant products with relatively small amounts of higher oligo-saccharides (Balakrishnan et al 1992).

Fig. 7. Temperature stability

Fig. 5. Effect of temperature on growth and activity

Fig. 8. End product analysis

CONCLUSIONS

Fig. 6. pH stability

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The present isolate Bacillus sp. 90-10-50 was identified as Bacillus pumilus based on biochemical and physiological tests and was also confirmed by molecular studies. The organism produced xylanase activities of around 330-380 and 400-450 IU of xylanase in 72 h when grown on 1% xylan and 3% wheat bran respectively. 1% yeast extract was the best nitrogen source and comparable activities were also obtained with other organic or inorganic nitrogen sources at equivalent nitrogen concentration. Among the soluble sugars, only 1% xylose gave activities around 40 IU/ml which was less than 10% of the activities obtained with 1% xylan or 3% wheat bran. International Journal of Biotechnology and Biosciences



Xylanase production was repressed by supplementing 0.05-0.5% xylose to xylan containing medium. Tween 80 at 0.1% concentration enhanced extra cellular enzyme yields by 15-20%. The enzyme is an endoxylanase having pH and temperature optima of 7.00 and 50°C. The activity was stable over a pH range of 6-10 and up to 2 h at 40°C. Cellulase-free nature of the xylanase renders it a suitable candidate for application in eco-friendly bio-bleaching in paper and pulp industries. Its potential application for enzyme assisted pulp bleaching is yet to be evaluated.

ACKNOWLEDGEMENT Authors wish to thank Dr. M.C. Srinivasan for discussions and useful suggestions during the course of this investigation.

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