Enhanced production of cellulase-free, thermo-alkali-solvent-stable
xylanase from Bacillus altitudinis DHN8, its characterization and application in sorghum straw saccharication Dharmesh N. Adhyaru a , Nikhil S. Bhatt a,n , H.A. Modi b a P.G. Department of Microbiology, Gujarat Vidyapeeth, Sadra-382 320, Gujarat, India b Department of Life Sciences, School of Sciences, Gujarat University, Ahmadabad, Gujarat, India a r t i c l e i n f o Article history: Received 16 September 2013 Received in revised form 7 October 2013 Accepted 11 October 2013 Available online 23 October 2013 Keywords: Bacillus altitudinis DHN8 Cellulase-free xylanase Submerged fermentation Partial purication Chemical pretreatments Sorghum straw saccharication a b s t r a c t A newly isolated Bacillus altitudinis DHN8 was assessed for xylanase production by utilizing sorghum straw. The highest xylanase production was recorded at sorghum straw 3% w/v, inoculum size 1% v/v, inoculum age 18 h, incubation time 42 h, pH 7.0, temperature 35 1C and agitation speed 250 rpm. Moreover, xylose 0.5%, gelatine 0.5% and KNO 3 0.3% (w/v) further enhanced the production. The detailed optimization study resulted in a 3.74-fold increase in xylanase production as compared to that of the initial conditions. The partially puried xylanase showed 70% pH stability after 18 h at pH range of 610. Thermostability study revealed more than 60% xylanase activity at temperature range 4565 1C after 60 min. The presence of metal ions (10 mM CaCl 2 , MnCl 2 and FeCl 3 ) and solvents (10% v/v isopropanol, methanol, ethanol and acetone) were increased xylanase activities remarkably. During saccharication study, 3% alkaline hydrogen peroxide treatment was found to be benecial for the maximum enzymatic hydrolysis of sorghum straw (34.94 mg/g reducing sugar) after 36 h. As such, this xylanase could be considered as a cellulase-free, thermo-alkali-solvent stable biocatalyst being important tool for many biotechnological industries. To the best of our knowledge, this is the rst report on the production of xylanase by this Bacillus species. & 2013 Elsevier Ltd. All rights reserved. 1. Introduction Xylan is the principal hemicellulose which is the major compo- nent of plant cell wall. It is the second plentiful polysaccharide after cellulose, in both hard woods and annual plants (Qie et al., 2010; Polizeli et al., 2005) and accounts for 2035% of the total dry weight in tropical plant biomass. Endo-xylanases (EC 3.2.1.8) are an impor- tant group of industrial enzymes responsible for complete hydrolysis of xylan in to short xylooligosaccharides and xylose in synergism with other accessory enzymes such as -xylosidase, -L-arabinofur- anosidase and -glucuronidase (Chavez et al., 2006; Collins et al., 2005). Xylanases have been reported from bacteria, fungi, actino- mycetes and yeast (Qie et al., 2010; Sunna and Antranikian, 1997) but bacteria due to their high metabolic diversity are widely used for xylanase production. Several Bacillus sp. are known to secrete high levels of extracellular xylanases which are either poor or free in cellulase activity. In recent years, xylanases have attracted a great deal of attention because of their biotechnological potential in the various industrial processes such as, in food industry in order to enhance the digestibility of animal feed, in textile industry and in the paper and pulp industry for bleaching purpose resulting in a decrease of chlorine utilization and consequently lowering environ- mental impact (Chivero et al., 2001). Xylanases in synergism with several enzymes can be used for the generation of biological fuels, such as ethanol and xylitol from lignocellulosic biomass (Cormana et al., 2005; Beg et al., 2001). The major obstacle for wide range application of the xylanases in industries is high cost of the production. Pure substrates being highly expensive and thus it cannot be afforded at the industrial level bulk production of enzymes. Therefore, it is necessary to explore cheap substrates for cost-effective enzyme production. Agricultural residues represent one such cheap raw material for industrial production of enzymes. Furthermore, each bacterium has its own special conditions for maximum enzyme production. Thus, the hyper production of industrial enzymes by optimizing various physiological and nutritional parameters is of prime importance because an improper optimization of these growth parameters leads to a lower enzyme production. In addition of the above problems, majority of the reported xylanases do not withstand the robust industrial Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/bab Biocatalysis and Agricultural Biotechnology 1878-8181/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bcab.2013.10.003 n Corresponding author. Tel.: 91 98794 83847/079 23274274; fax: 079 232 74 272. E-mail addresses: bhattnikhil2114@gmail.com, bhattnikhil_brc@yahoo.in (N.S. Bhatt). Biocatalysis and Agricultural Biotechnology 3 (2014) 182190 process conditions of high alkaline pH and high temperatures (Sharma and Bajaj, 2005). Therefore, search for still better enzymes which comply well with the industrial processes is in progress (Kumar and Satyanarayana, 2011). In view of the demand for novel xylanases with industrially important properties, an attempt has been made to assess the potentiality of newly isolated Bacillus species for the xylanase production using low cost agricultural residue under submerged fermentation. The partially puried enzyme was characterized for few properties and further examined for its application in the saccharication of sorghum straw. 2. Material and methods 2.1. Isolation, screening and identication of xylanase producing strain 2.1.1. Primary screening Different soil samples were collected from the active compost pit (10 cm depth) during mid of the April 2011. Each sample was mixed thoroughly and 1 g of that was suspended in 50 ml sterile distilled water. Mixtures were allowed to settle down and then serial dilutions were prepared. From each dilution, 0.1 ml was spread on oat spelt xylan (1% w/v) agar plates and incubated at 37 1C for 48 h. Those colonies showed a clear zone of xylan hydrolysis were isolated and retained for further secondary screening. 2.1.2. Secondary screening The isolates from the primary screening were cultured in basal liquid media containing (g/l): Oat spelt xylan, 10.0; beef extract, 3.0; NaCl, 5.0; KNO 3 , 2.0; K 2 HPO 4 , 1.0 and MgSO 4 7H 2 O, 0.5 at pH 7.0 in 100 ml Erlenmeyer asks and incubated at 37 1C under shaking (150 rpm). After 48 h of incubation the fermented broth was centrifuged at 10,000g for 10 min and the supernatant was used for enzyme assay. 2.1.3. Bacterial identication A total of 15 bacterial cultures were selected for the xylanase production based on the secondary screening. The most promising isolate DHN8 was identied on the basis of its morphological and biochemical characterization. Further conrmation was done by 16S rRNA gene sequencing at Gujarat State Biotechnology Mission (GSBTM), Gandhinagar, India. The gene sequence has been sub- mitted to Gene Bank at NCBI (GenBank accession no. KF258832). The sequence was compared with sequences available in NCBI database and dendrogram was generated using Mega 5.2 software. 2.2. Selection of agro-residue for enhanced cellulase free xylanase production For enhanced xylanase production various agro-residues such as caster shell, sugarcane bagasse, saw dust, rice straw, barley straw, sorghum straw, wheat straw, groundnut shell and maize straw were evaluated. Xylanase production was carried out using 2% (w/v) agro-residue in the 100 ml basal liquid medium (pH 7.0) containing same medium components but without oat spelt xylan. The asks were sterilized at 121 1C for 15 min, cooled and inoculated with 1% (v/v) of bacterial culture. The contents were incubated at 37 1C under shaking condition (150 rpm) for 48 h. The fermented broth was centrifuged at 10,000g for 10 min at 4 1C and the clear supernatant was analysed for enzyme assay. 2.3. Analytical methods All the experiments were done in triplicates and the values presented are mean values7SD. Chemicals such as oat spelt xylan, carboxy methyl cellulose (CMC), bovine serum albumin (BSA) and dinitrosalicylic acid (DNSA) were purchased from Hi Media Laboratories Ltd. All the other chemicals, media, salts and reagents used were of analytical grade (Sigma- Aldrich, Hi Media, Qualigens and Merck). The xylanase activity was measured according to Bailey et al. (1992). The reaction mixture consisting 450 ml of 1% oat spelt xylan in 50 mM sodium phosphate buffer (pH 7.0) and 50 ml of enzyme was incubated for 10 min at 50 1C. The amount of reducing sugar released was quantied using 3,5-dinitrosalicylic acid (DNS) method as described by Miller (1959). The CMCase and FPase activities were determined according to IUPAC recommendation (Ghose, 1987). One unit of xylanase or cellulase activity was dened as the amount of enzyme required to liberate 1 mmol of xylose or glucose equivalent per min under the specied conditions. The protein content was measured according to Lowry et al. (1951) with BSA as the standard. 2.4. Assessment of process parameters for cellulase free xylanase production 2.4.1. Optimization of physiological parameters 2.4.1.1. Inoculum size. To study the effect of inoculum size, produc- tion media were inoculated at a level of 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0% (v/v) from the 12 h old bacterial culture broth. 2.4.1.2. Inoculum age. To study the effect of inoculum age on xylanase production, inoculum was prepared by inoculating 50 ml of basal liquid medium with bacterium DHN8 and incubated at 37 1C under shaking condition (150 rpm). At regular interval of 6 h incubation, 1% (v/v) of inoculum was transferred to 100 ml fresh fermentation medium followed by incubation at 37 1C for 48 h under shaking at 150 rpm. The culture ltrate was centrifuged and nally used for enzyme assay. 2.4.1.3. Incubation time. In order to achieve maximum xylanase production, incubation time was varied from 12 to 60 h. The crude enzyme was extracted and assayed at regular interval of 6 h. 2.4.1.4. pH. Initial pH of the fermentation mediumwas varied from 3 to 10. The pH was adjusted by using 1 MHCl or NaOH prior to autoclaving. 2.4.1.5. Temperature. Xylanase production was studied at different temperatures ranging from 25 to 50 1C. 2.4.1.6. Agitation speed. The rate of agitation was studied at 0 to 300 rpm with a difference of 50 rpm. 2.4.2. Optimization of nutritional parameters 2.4.2.1. Inuence of carbon sources. Inuence of various carbon sources on the xylanase production were assessed at a concentration of 0.5% (w/v). 2.4.2.2. Inuence of organic and inorganic nitrogen sources. Various organic and inorganic nitrogen sources were evaluated for enhan- ced xylanase production at concentration of 0.5 and 0.3% (w/v), respectively. D.N. Adhyaru et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 182190 183 2.5. Partial purication and characterization of xylanase Solid ammonium sulphate was slowly added to the culture supernatant in an ice bath under constant stirring to achieve 70% saturation. After centrifugation at 10,000g for 10 min at 4 1C, the supernatant was removed and the precipitate was resus- pended in 10 ml of 50 mM sodium phosphate buffer (pH 7.0). The nal solution was dialysed against the same buffer overnight at 4 1C with three intermittent changes of the buffer. 2.5.1. Effect of temperature on xylanase activity and stability The optimum temperature was determined by incubating the enzyme extract in 1% oat spelt xylan solution at various tempera- tures (3575 1C). For thermostability assay, enzyme was incubated at different temperatures in the absence of substrate for 120 min. Aliquots were withdrawn at regular time intervals to test the residual xylanase activity. 2.5.2. Effect of pH on xylanase activity and stability The pH optima was determined by measuring relative activity at various pH values using sodium citrate (pH 36), sodium phosphate (pH 68) and glycine-NaOH (pH 810) buffers (50 mM). The stability of xylanase was assessed by incubating enzyme samples for 24 h in different buffers. At regular interval of 3 h samples were withdrawn and residual enzyme activity was determined. 2.5.3. Effect of metal ions on xylanase activity The effect of metal ions on enzyme activity was determined by incubating the xylanase preparations in 10 mM metal solution for 30 min. Residual activity was measured by standard enzyme assay. 2.5.4. Effect of various solvents on xylanase activity The enzyme was incubated with various alcoholic and non alcoholic solvents at 10% (v/v) concentration for 30 min and residual activity was measured by the standard assay procedure. 2.6. Chemical pretreatment of sorghum straw Prior to the enzymatic hydrolysis, the sorghum straw was given three separate chemical pretreatments. Ten grams of dried and powdered sorghum straw was taken for each pretreatment experiment. The biomass was mixed with 200 ml of 1 M NaOH (for 24 h), 1 M HCl (for 12 h) and 3% alkaline hydrogen peroxide, pH 11.0 (for 12 h) separately to obtain a solid:liquid ratio of 1:20. The contents were kept under shaking (100 rpm) at 50 1C. The pre- treated substrates were washed until neutrality and dried in oven at 50 1C till constant weight was achieved. 2.7. Enzymatic hydrolysis Enzymatic hydrolysis of sorghum straw was carried out in 50 ml Erlenmeyer ask with 2.5% (untreated and pre-treated) substrate and 20 ml of appropriately diluted crude enzyme at 50 1C, 100 rpm for 48 h. Controls were kept in which active enzyme was replaced with heat inactivated enzyme. The reaction system was supplemented with 0.005% sodium azide to prevent microbial growth. Aliquots were taken at regular intervals of 12 h, centrifuged and the supernatant was assayed for total reducing sugar by 3,5-dinitrosalysilic acid method (Miller, 1959). 2.8. Analysis of hydrolysed products by TLC The products of enzymatic hydrolysis of sorghum straw were examined by ascending thin-layer chromatography (TLC) on pre- coated silica gel plates (60 F254, Merck) using acetone/ isopropyl alcohol/water (6:3:1.5, v/v/v) as mobile phase. At dened times, reaction mixtures were sampled, the enzyme activity was stopped by boiling for 10 min and 3.0 ml samples were applied on TLC plates. The separated products were detected by spraying the plate with -naphthol (3.5% in ethanol and 10% sulphuric acid) followed by heating at 100 1C. 3. Results and discussion 3.1. Isolation and identication of strain DHN8 The newly isolated DHN8 strain was Gram positive, strictly aerobic, spore forming, rod shaped and peritrichous. It reacted positively in the catalase and oxidase tests. According to these results, it was clear that the bacterium belonged to the genus Bacillus species. Based on the results of 16S rRNA gene sequencing the bacterium was identied as Bacillus altitudinis (accession no. Fig. 1. The phylogenetic dendrogram of Bacillus altitudinis DHN8 was labelled with an aster and related species by the neighbour-joining approach. Bootstrap values obtained with 1000 resamplings are indicated as percentages at all branches. The scale bars represent 0.05 substitutions per nucleotide position. Numbers following the names of the strains are accession numbers of published sequences. D.N. Adhyaru et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 182190 184 KF258832) and designated as B. altitudinis DHN8. Through the alignment of homologous sequences of known bacteria, phyloge- netic position of the strain is shown in Fig. 1. 3.2. Effect of various agro-residues on xylanase production Among tested agro residues, B. altitudinis DHN8 exhibited clear preference towards sorghum straw for highest xylanase produc- tion (65.4972.18 IU/ml) followed by wheat straw, sugarcane bagasse, barley straw and rice straw (62.0071.91, 47.0071.68, 42.6871.16 and 34.7371.21 IU/ml, respectively). However, lower xylanase activities were observed in presence of maize straw, caster shell, groundnut shell and saw dust (Fig. 2). The higher xylanase production in presence of sorghum straw might be due to the nature of hemicellulose, the presence of some activators in the carbon source, surface, pore size and favourable degrad- ability of carbon source (Gomes et al., 1993). However, many research groups used hemicellulosic substrates such as wheat bran, rice straw, wheat straw, soybean akes, rice bran, sugarcane bagasse and groundnut shells for xylanase production (Battan et al., 2007; Sharma et al., 2007; Taneja et al., 2002; Gawande and Kamat, 1999). These data suggested that the xylanase induc- tion is a complex phenomenon and the level of response to different inducers varies with the strains. This study highlighted that choosing an appropriate agro-residue can improve enzyme production markedly. Further different concentrations of sorghum straw ranging from 1.0 to 6.0% (w/v) were tested under submerged fermentation. The maximum xylanase production (68.4972.37 IU/ml) was achieved at 3% (w/v) sorghum straw (Fig. 3). The gradual decrease in xylanase production was observed above 3% sorghum straw concentration. The possible reason behind such xylanase produc- tion behaviour could be formation of thick suspension in presence of higher substrate concentration resulted in improper mixing of the substrate under agitation condition. 3.3. Effect of inoculum size, inoculum age and incubation period An optimum inoculum level is necessary for maintaining balance between the proliferating biomass and available nutrients to produce maximum enzyme level. The highest xylanase production (72.847 2.64 IU/ml) was achieved when the production medium was inocu- lated with 1% (w/v) of 12 h old inoculum (Fig. 4). The enzyme titre declined drastically with increase in inoculum size beyond the optimum values could be due to faster nutrient consumption. In earlier studies, Battan et al. (2007) and Nagar et al. (2010) also showed the use of 1.05.0% (v/v) inoculum size for hyper xylanase production. Higher concentration of inoculum is not preferable in industrial fermentations (Lincon, 1960). The effect of inoculum age was studied from 6 to 48 h old culture of B. altitudinis DHN8. The maximum xylanase production 91.3872.68 IU/ml was obtained with 18 h old inoculum (Fig. 5) but decreased thereafter. These results obtained might be due to the fact that, the maximum enzyme titre is produced during early to late exponential phase of the organism. It also suggests that the partial association existed between growth of the organism and its enzyme production pattern. Kumar et al. (2012) and Nagar et al. (2010) also showed the use of 18 h old inoculum for xylanase production. The time of fermentation for maximum enzyme production varies among different bacteria and is dependent upon the organism 0 0.5 1 1.5 2 2.5 0 10 20 30 40 50 60 70 80 CS SB SD RS BS SS WS GS MS P r o t e i n
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( I U / m l ) Agro-residues Xylanase activity Protein Fig. 2. Effect of various agro residues on xylanase production. CS: caster shell, SB: sugarcane bagasse, SD: saw dust, RS: rice straw, BS: barley straw, SS: sorghum straw, WS: wheat straw, GS: groundnut shell, MS: maize straw. The fermentation medium containing 2% w/v agro residue, beef extract, 3.0; NaCl, 5.0; KNO 3 , 2.0; K 2 HPO 4 , 1.0 and MgSO 4 7H 2 O, 0.5 at pH 7.0 was inoculated with 12 h old bacterial culture at 1% v/v. The enzyme production was carried out at 37 1C under shaking (150 rpm) for 48 h. 0 0.5 1 1.5 2 2.5 3 0 10 20 30 40 50 60 70 80 1 2 3 4 5 6 P r o t e i n
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Sorghum straw concentration (%) Xylanase activity Specific activity Protein Fig. 3. Effect of sorghum straw concentration (16% w/v) on xylanase production. The fermentation medium (pH 7.0) with varying concentration of sorghum straw was inoculated with 12 h old bacterial culture at 1% v/v. The enzyme production was carried out at 37 1C under shaking (150 rpm) for 48 h. 0 0.5 1 1.5 2 2.5 0 10 20 30 40 50 60 70 80 0.5 1 2 3 4 5 P r o t e i n
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Inoculum size (% v/v) Xylanase activity Specific activity Protein Fig. 4. Effect of inoculum size on xylanase production. The 3% w/v sorghum straw containing fermentation medium (pH 7.0) was inoculated with 12 h old bacterial culture at 1 to 5% v/v. The enzyme production was carried out at 37 1C under shaking (150 rpm) for 48 h. 0 0.5 1 1.5 2 2.5 3 0 10 20 30 40 50 60 70 80 90 100 6 12 18 24 30 36 42 48 P r o t e i n
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Inoculum age (h) Xylanase activity Specific activity Protein Fig. 5. Effect of inoculum age on xylanase production. The fermentation medium (pH 7.0) was inoculated at 1% v/v level with 12 to 60 h old bacterial culture. The enzyme production was carried out at 37 1C under shaking (150 rpm) for 48 h. D.N. Adhyaru et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 182190 185 type, its enzyme production pattern, cultural conditions and genetic makeup of the organism. During course of incubation period, high- est xylanase production (81.5672.85 IU/ml) was obtained at 42 h of incubation and thereafter it declined gradually (Fig. 6). The reduction in xylanase yield could be due to nutrients depletion or due to proteolysis (Flores et al., 1997). Xylanase produced by Bacillus sp. was found to be growth-associated, reaching a maximum after 24 h and the enzyme production remained more or less constant up to 48 h (Anuradha et al., 2007). On the other hand, Bacillus halodurans PPKS-2 (Prakash et al., 2011) and Bacillus SSP-34 (Subramaniyan and Prema, 2000) produced maximum xylanase after 48 and 96 h, respectively. 3.4. Effect of pH, temperature and agitation speed The B. altitudinis DHN8 was cultivated in the production medium adjusted at different pH (3.010.0). The bacterium did not produce satisfactory xylanase yield at initial medium pH 3.0 to 5.0 but at pH 6.0 xylanase titre was increased and reached maximum at pH 7.0 (85.7372.23 IU/ml). Moreover, afterwards from pH 8.0 to 10.0 substantial xylanase production was observed (Fig. 7). Xylanase production by various bacteria and fungi has been shown to be markedly dependent on pH (Wong et al., 1982). Acidic pH (4.06.0) generally favours fungal xylanases (Bajpai, 1997) while higher pH favours bacterial xylanases (Bisaria and Ghose, 1991). In tuning with this nding, Bacillus pumilus ASH (Battan et al., 2007) and Bacillus subtilis ASH (Sanghi et al., 2008) showed elevated xylanase produc- tion at pH 7.0. However, Bacillus mojavensis AG137 (Sepahy et al., 2011) and Bacillus NT 9 (Han et al., 2004) showed maximum xylanase production at medium pH 8.0 and 10, respectively. The xylanase production was conducted at different temperatures (2550 1C). The maximum xylanase production (91.1472.01 IU/ml) was obtained at 35 1C (Fig. 8) and reduced sharply at higher temperature range of 40 to 50 1C. Although the physiological changes induced by high temperatures during enzyme production are not completely understood, it has been suggested that at higher tem- peratures microorganisms may synthesize only a reduced number of proteins essential for growth and other physiological processes (Gawande and Kamat, 1999). These results might also be corresponds to the growth prole of the microorganism where as no other temperature was suitable to such extant for growth and enzyme secretion. Many researchers reported 37 1C temperature for max- imum xylanase production from Bacillus sp. (Nagar et al., 2010; Sanghi et al., 2008; Battan et al., 2007). During submerged fermentation process, agitation and aeration are two important parameters used for uniform mixing of the nutrients and to meet the oxygen demand. The B. altitudinis DHN8 exhibited signicant amount of xylanase production in the range of 150 to 300 rpm agitation speed with optimum xylanase yield (103.8773.46 IU/ml) at 250 rpm. However, under static and lower agitation (50 rpm) conditions only 10.21 and 45.31% of the maximum xylanase activity was observed (Fig. 9). The lower xylanase produc- tion under static to low agitation conditions may be attributed to the dissolved oxygen (DO) limitation, improper mixing of media compo- nents and cell clumping. Various researchers showed maximum xylanase production at an agitation rate of 200250 rpm (Kumar et al., 2012; Battan et al., 2007; Taneja et al., 2002; Beg et al., 2001; Gawande and Kamat, 1999). 3.5. Inuence of carbon and nitrogen sources Carbon source is one of the most important factors during the growth and metabolic process of microorganisms. The presence 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0 10 20 30 40 50 60 70 80 90 12 18 24 30 36 42 48 54 60 P r o t e i n
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Incubation time (h) Xylanase activity Specific activity Protein Fig. 6. Effect of incubation time on xylanase production. The fermentation medium (pH 7.0) was inoculated at 1% v/v level with 18 h old bacterial culture. The enzyme production was carried out at 37 1C under shaking (150 rpm) and samples were analysed at 6 h intervals. 0 0.5 1 1.5 2 2.5 0 10 20 30 40 50 60 70 80 90 100 3 4 5 6 7 8 9 10 P r o t e i n
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pH Xylanase activity Specific activity Protein Fig. 7. Effect of pH on xylanase production. The fermentation medium containing different pH from 3 to 10 was inoculated at 1% v/v level with 18 h old bacterial culture. The enzyme production was carried out at 37 1C under shaking (150 rpm) for 42 h. 0 0.5 1 1.5 2 2.5 3 0 10 20 30 40 50 60 70 80 90 100 25 30 35 40 45 50 P r o t e i n
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Temperature ( C) Xylanase activity Specific activity Protein Fig. 8. Effect of temperature on xylanase production. The fermentation medium (pH 7.0) was inoculated at 1% v/v level with 18 h old bacterial culture. The enzyme production was carried out at different temperature range from 25 to 50 1C under shaking (150 rpm) for 42 h. 0 0.5 1 1.5 2 2.5 3 0 20 40 60 80 100 120 0 50 100 150 200 250 300 P r o t e i n
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Agitation speed (rpm) Xylanase activity Specific activity Protein Fig. 9. Effect of agitation speed on xylanase production. The fermentation medium (pH 7.0) was inoculated at 1% v/v level with 18 h old bacterial culture. The enzyme production was carried out at 35 1C under static and shaking (0 to 300 rpm) conditions for 42 h. D.N. Adhyaru et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 182190 186 of carbon sources in the fermentation medium exerted profound effect on the enzyme production behaviour of the bacterium. Among various monosaccharide and disaccharides tested only xylose, sucrose and starch (145.1373.85, 115.4173.66 and 111.073.91 IU/ ml, respectively) stimulated xylanase production. However, other carbon sources failed to support high yield of xylanase production as compared to control (Table 1). The most plausible explanation for decrease in xylanase yield with other carbon sources is that these sources exerted catabolite repression. Such carbon source dependent enzyme production regulation has been noticed in different xylanase producing microbial strains (Lakshmi et al., 2009; Oliveira et al., 2006; Gassesse and MaMo, 1999). To check the effect of nitrogen sources on xylanase production different organic and inorganic nitrogen sources were added individually to the fermentation medium (Table 2). Amongst various organic nitrogen sources, maximum xylanase production was obtained with gelatine (197.9476.00 IU/ml) followed by urea, yeast extract, tryptone and beef extract. However, casein, meat extract, malt extract and peptone did not showed stimulatory effect on xylanase production. The enhanced production of xylanase in presence of gelatine as organic nitrogen sources may be attributed to organic nitrogen source mediated regulation of microbial growth and metabolism (Gupta et al., 2000). Moreover, it has been also observed that nitrogen can signicantly affect the pH of the medium during the course of fermentation (Haapala et al., 1994) thereby inuences the microbial metabolism. Bajaj and Manhas (2012) reported 24.0% xylanase improvement in the presence of gelatine. The most suitable organic nitrogen source for the xylanase production by Geobacillus thermoleovorans (Sharma et al., 2007) and Bacillus circulans AB16 (Dhillon and Khanna, 2000) was tryptone. B. moja- vensis AG147 yielded maximum xylanase when tryptone and yeast extract was used in combination (Sepahy et al., 2011). During the evaluation of inorganic nitrogen sources KNO 3 showed maximum xylanase production (245.0473.15 IU/ml). All other tested inorganic nitrogen sources also showed increase in xylanase produc- tion except (NH 4 ) 2 H 2 PO 4 (103.7974.76 IU/ml) as compared to the control set devoid of any nitrogen source. In accordance with these results, Nagar et al. (2012) observed stimulatory effect of KNO 3 in combination with peptone and yeast extract on xylanase production. 3.6. Partial characterization of xylanase produced by B. altitudinis DHN8 3.6.1. Effect of temperature on activity and stability The partially puried xylanase was active in the broad range of temperatures 35 to 75 1C, with temperature optima at 50 1C. The enzyme exhibited more than 60% of its activity in the range from 45 to 65 1C. The xylanase activity was dropped at temperature values above 70 1C and only 20% of relative xylanase activity was detected at 75 1C (Fig. 10). The enzyme thermostability depends upon molecular interac- tions such as hydrogen bonds, electrostatic and hydrophobic inter- actions, disulde bonds, and metal binding which can promote a superior conformational structure for the enzyme with a higher packing efciency, reduced entropy of unfolding, conformational strain release and stability of -helices (Li et al., 2005). Thermo- stability studies of B. altitudinis DHN8 xylanase showed residual activity between 70 and 90% at 45 to 60 1C temperature after 60 min of incubation. Even at 70 1C after 30 min of incubation 47% of the residual activity was noticed. The obtained results clearly indicate the nature of xylanase as thermostable. The suitable temperature range for industrial application of present xylanase lies in 35 to 65 1C (Fig. 11). Most of the bacterial xylanases showoptimumactivity at 50 to 60 1C (Bajaj and Singh, 2010). Identical temperature optima at 50 1C were reported for the xylanases from Bacillus sp. (Nagar et al., 2010; Polizeli et al., 2005). However, xylanase produced by B. halodurans exhibited activity over 30100 1C, with an optimum at Table 1 Inuence of carbon sources on cellulase-free xylanase production by Bacillus altitudinis DHN8. Carbon sources Xylanase activity (IU/ml) Protein (mg/ml) Specic activity (IU/mg) Control a 103.1872.80 2.5470.09 40.5371.11 Glucose 62.0773.04 2.1070.06 29.6072.29 Maltose 83.9372.29 2.2870.07 36.7170.99 Starch 111.0073.91 2.2870.07 36.7172.30 Cellulose 68.3672.86 2.4470.09 27.9370.16 Sucrose 115.4173.66 2.9370.15 39.4170.80 Fructose 88.2972.66 2.2670.11 39.1171.69 Xylose 145.1373.85 3.1370.13 46.3172.02 Galactose 68.8773.70 2.3370.16 29.6672.72 Lactose 62.1271.71 2.3370.14 26.7272.36 Mannitol 85.3374.01 2.9370.06 29.1371.38 a The control medium used was devoid of carbon source. Table 2 Inuence of nitrogen sources on cellulase-free xylanase production by B. altitudinis DHN8. Nitrogen source Xylanase activity (IU/ml) Protein (mg/ml) Specic activity (IU/mg) Organic Peptone 133.9673.13 3.0470.13 44.1071.40 Beef extract 144.2974.61 3.3370.07 43.3572.04 Yeast extract 173.6476.38 3.5470.08 48.9970.75 Casein 83.0372.66 2.5070.11 33.1470.54 Malt extract 101.0672.52 2.9070.10 34.8572.00 Trypton 154.7672.40 3.4970.14 44.3471.67 Gelatine 197.9476.00 3.8770.10 51.1772.12 Skim milk powder 144.8273.47 3.4270.09 42.2771.13 Meat extract 94.7873.94 2.3870.11 39.7971.21 Urea 183.8477.93 3.1870.08 57.6971.87 Inorganic (NH 4 ) 2 HPO 4 195.6578.69 3.8670.11 50.6770.79 (NH 4 ) 2 H 2 PO 4 103.7974.76 3.2670.14 31.8470.93 KNO 3 245.0473.15 4.0070.17 61.3171.95 (NH 4 ) 2 S 2 O 8 163.7372.49 3.6470.14 45.0071.10 NH 4 NO 3 213.8874.90 3.9170.16 54.6471.25 NH 4 Cl 177.3176.09 3.1070.09 57.1172.93 NaNO 3 198.1372.18 3.6670.15 54.0871.70 (NH 4 ) 2 SO 4 207.0673.27 4.0870.19 50.8372.64 (NH 4 ) 2 HPO 4 195.6578.69 3.8670.11 50.6770.79 Control a 135.3973.87 3.1370.14 43.2371.08 a The control medium used was devoid of nitrogen source. 0 20 40 60 80 100 120 35 40 45 50 55 60 65 70 75 R e l a t i v e
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Temperature ( C) Fig. 10. Effect of temperature on the activity of partially puried xylanase. The enzyme extract was incubated in 1% oat spelt xylan solution at different tempera- tures (35 to 75 1C). The relative activity was measured using standard assay after 10 min. D.N. Adhyaru et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 182190 187 80 1C (Kumar and Satyanarayana, 2011). B. halodurans PPKS-2 xylanase showed optimum temperature of 70 1C (Prakash et al., 2011). Thermostability of different Bacillus sp. xylanases varies between 55 and 80 1C (Kumar and Satyanarayana, 2011). 3.6.2. Effect of pH on activity and stability Enzyme activity is markedly affected by pH because substrate binding and catalysis are often dependent on charge distribution of both substrate and particularly enzyme molecules. The xylanase of B. altitudinis DHN8 exhibited the highest xylanase activity at pH 7.0 and retained more than 60% relative activity over broad range of pH 5 to 10 (Fig. 12). pH stability study revealed that the present xylanase was stable in pH range of 6 to 10 and showed 70% residual activity after 18 h of incubation (Fig. 13). The results indicated that the partially puried xylanase was alkali stable in nature. In similar study, xylanase from Bacillus sp. GRE7 showed pH stability over the pH range of 511 for 30 min (Kiddinamoorthy et al., 2008). Bacillus stearothermophilus T-6 xylanase had stability at pH 6.510.0 for 5 min (Khasin et al., 1993). The differences in pH and temperature stability for extracellular xylanases might be due to the post transcriptional modications in xylanase excretion process, such as glycosylation, that improved stability in more extreme pH and temperature conditions (Savitha et al., 2007). Thus, B. altitudinis DHN8 xylanase possessed cellulase-free nature, broad pH stability spectrum and stability at elevated temperature, could be important tool for application in many industrial processes. 3.6.3. Effect of metal ions and additives on xylanase activity Metal ions can be involved in enzyme catalysis in a variety of ways: they may accept or donate electrons; they may them- selves act as electrophiles; they may mask nucleophiles to prevent unwanted side reactions; they may bring together enzyme and substrate by coordinate bonds; they may hold the reacting groups in the required 3D orientation and they may simply stabilize a catalytically active conrmation of the enzyme (Palmer, 2001). As shown in Table 3, xylanase activity was assayed in the presence of several metal solutions (10 mM). The xylanase activity was markedly stimulated in the presence of metal ions such as CaCl 2 (168.00%), MnCl 2 (126.95%) and FeCl 3 (106.01%). However, signi- cant inhibition in xylanase activity was found in the presence of other metal compounds. HgCl 2 was acted as strong inhibitory compound (64% inhibition), suggesting that there is an impor- tant cystein residue in or close to the active site of the enzyme (Khandeparkar and Bhosle, 2006). Kumar and Satyanarayana (2011) reported that the activity of B. halodurans xylanase was strongly inhibited by Sn 2 , Hg 2 , Cu 2 and Cd 2 . More than a quarter of all known enzymes require the presence of metal atoms for fully catalytic activity (Palmer, 2001). These effects reveal which kind of ions should be precluded or included in industrial processes. The reports on the solvent stable xylanases are very rare. The inuence of various alcoholic and non alcoholic solvent additives on the activity of xylanase is shown in Table 3. The xylanase activity was stimulated in the presence of isopropanol (126.07%), methanol (117.81%), ethanol (108.69%) and acetone (102.96%). However, pre- sence of 1-butanol, 2-butanol, benzene and toluene resulted in reduced xylanase activity. Similar observations for xylanase stimula- tion and suppression in presence of straight chain alcohols were reported by Li et al. (2010). Biocatalysis in organic media offers several 0 20 40 60 80 100 120 0 30 60 90 120 R e s i d u a l
a c t i v i t y
( % )
Time (min) 35 C 40 C 45 C 50 C 55 C 60 C 65 C 70 C 75 C Fig. 11. Effect of temperature on the stability of partially puried xylanase. The enzyme extract was incubated at different temperatures (35 to 75 1C) for 120 min. The residual activity was measured after each 30 min intervals. 0 20 40 60 80 100 120 3 4 5 6 7 8 9 10 R e l a t i v e
a c t i v i t y
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pH pH 3-6 pH 6-8 pH 8-10 Fig. 12. Effect of pH on the activity of partially puried xylanase. The enzyme extract was incubated in different buffers: sodium citrate (pH 36), sodium phosphate (pH 68) and glycine-NaOH (pH 810) buffers (50 mM). 0 20 40 60 80 100 120 0 3 6 9 12 15 18 21 24 R e s i d u a l
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Time (h) pH 3 pH 4 pH 5 pH 6 pH 7 pH 8 pH 9 pH 10 Fig. 13. Effect of pH on the stability of partially puried xylanase. The enzyme extract was incubated for 24 h in different buffers (pH 3 to 10) for 24 h. At regular interval of 3 h samples were withdrawn and residual enzyme activity was determined. Table 3 Effect of metal compounds and solvents on cellulase-free xylanase activity. Metal compound (10 mM) Relative xylanase activity (%) Solvents (10% v/v) Relative xylanase activity (%) Control a 100.0071.42 Control 100.0071.42 CoCl 2 72.7072.45 Methanol 117.817 5.04 FeCl 3 106.017 4.98 Ethanol 108.6972.56 MnCl 2 126.9573.21 Isopropanol 126.0774.90 CaCl 2 168.7278.66 1-Butanol 89.3472.47 HgCl 2 46.8772.03 2- Butanol 92.5673.83 ZnCl 2 62.3673.00 Acetone 102.9672.81 NiCl 2 32.3571.21 Toluene 67.2172.99 MgCl 2 62.9571.27 Benzene 76.1071.60 a The control medium used was devoid of any modulator or solvent. D.N. Adhyaru et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 182190 188 advantages viz. higher solubility of hydrophobic substrate enabaling their effective reactions, reduced microbial contamination and reusa- bility (Sardessai and Bhosle, 2004). These results provide essential data for the application of xylanase in an organic phase industrial reactions. 3.7. Enzymatic hydrolysis of sorghum straw The utilization of enzymatic hydrolysis to obtain sugars from agricultural residues is of great interest in modern biotechnology particularly for bio-solvent production. Fig. 13 depicts the effect of enzymatic hydrolysis on reducing sugar production from differ- ently pretreated sorghum straw. The 3% alkaline hydrogen per- oxide pretreated sorghum straw yielded maximum reducing sugar (34.94 mg/g) after 36 h of enzymatic hydrolysis. Whereas, alkali pretreated, acid pretreated and untreated biomass yielded 29.56, 23.81 and 2.58 mg/g reducing sugar after 48 h. In 3% alkaline hydrogen peroxide pretreated sorghum straw after an initial phase of rapid sugar formation during 36 h the rate of hydrolysis decreased which could be due to enzyme inactivation or depletion of an easily hydrolysable fraction of hemicellulose. As compared to alkali and acid pretreatments, alkaline hydrogen peroxide treat- ment is more effective in lignin solubilisation (Chen et al., 2008) and improving of crop residue digestibility (Talebnia et al., 2010) (Fig. 14). 4. Conclusion Due to the increasing economic relevance of xylanases present study was designed and attempt was made to optimize different process parameters. The detailed optimization study resulted in a 3.74-fold enhancement in xylanase production as compared to that of the initial conditions. The cellulase-free and thermo-alkali- solvent stable xylanase produced by the newly isolated B. altitu- dinis DHN8 using least reported sorghum straw as substrate is one of the rare xylanases because of its stability at extreme process conditions prevailing in the paper industry. The xylanase was able to release reducing sugars mainly xylose from alkaline hydrogen peroxide treated sorghum straw biomass which could be further fermented to biofuel. Acknowledgement Authors are greatly acknowledges the nancial assistance from Department of Science and Technology (DST), Ministry of Science and Technology, Govt. of India, for giving INSPIRE Fellowship to Mr. Dharmesh N. Adhyaru, under INSPIRE Program. References Anuradha, P., Vijayalaxmi, K., Prasanna, N.D., Sridevi, K., 2007. Production and properties of alkaline xylanases from Bacillus sp. isolated from sugarcane eld. Curr. Sci. 90, 12831286. Bailey, M.J., Biely, P., Poutanen, K., 1992. Laboratory testing of methods for assay of xylanase activity. J. Biotechnol. 23, 257270. Bajaj, B.K., Manhas, K., 2012. Production and characterization of xylanase from Bacillus licheniformis P11(C) with potential for fruit juice and bakery industry. Biocatal. Agric. Biotechnol. 1, 330337. Bajaj, B.K., Singh, N.P., 2010. Production of xylanase from an alkalitolerant Streptomyces sp. 7b under solid state fermentation, its purication and characterization. Appl. Biochem. Biotechnol. 162, 18041818. Bajpai, P., 1997. Microbial xylanolytic enzyme system properties and applications. Appl. Environ. Microbiol. 43, 141189. Battan, B., Sharma, J., Dhiman, S.S., Kuhad, R.C., 2007. Enhanced production of cellulase-free thermostable xylanase by Bacillus pumilus ASH and its potential application in paper industry. Enzyme Microb. Technol. 41, 733739. Beg, Q.K., Kappor, M., Mahajan, L., Hondal, G.S., 2001. Microbial xylanases and their industrial applications: a review. Appl. Microbiol. Biotechnol. 20, 325333. Bisaria, V.S., Ghose, T.K., 1991. Biodegradation of cellulosic materials: substrates, microorganisms, enzymes and products. Enzyme Microb. Technol. 3, 90104. Chavez, R., Bull, P., Eyzaguirre, J., 2006. The xylanolytic enzyme system from the genus Penicillium. J. Biotechnol. 123, 413433. Chen, H., Han, Y., Xu, J., 2008. Simultaneous saccharication and fermentation of steam exploded wheat straw pretreated with alkaline peroxide. Process Biochem. 43, 14621466. Chivero, E.T., Mutukumira, A.N., Zvauya, R., 2001. Partial purication and char- acterization of a xylanase enzyme produced by a microorganism isolated from selected indigenous fruits of Zimbabwe. Food Chem. 72, 179185. Collins, T., Gerday, C., Feller, G., 2005. Xylanases, xylanase families and extremo- philic xylanases. FEMS Microbiol. Rev. 29, 323. Cormana, E.C., Fialho, M.B., Buchgnani, E.B., Coelho, G.D., Brocheto-Braga, M.R., Jorge, J.A., 2005. Production, purication and characterization of a minor form of xylanase from Aspergillus versicolor. Process Biochem., 359364. Dhillon, A., Khanna, S., 2000. Production of a thermostable alkali-tolerant xylanase from Bacillus Circulans AB 16 grown on wheat straw. World J. Microbiol. Biotechnol. 16, 325327. Flores, M.E., Perez, R., Huitron, C., 1997. -xylosidase and xylanase characterization and production by Streptomyces sp. CH-M-1035. Lett. Appl. Microbiol. 24, 410416. Gassesse, A., MaMo, G., 1999. High- level xylanase production by an alkaliphilic Bacillus sp. by using solid state fermentation. Enzyme Microb. Technol.. 25, 6872. Gawande, P.V., Kamat, M.Y.J., 1999. Production of Aspergillus xylanase by lignocel- lulosic waste fermentation and its application. J. Appl. Microbiol. 87, 511519. Ghose, T.K., 1987. Measurements of cellulase activities. Pure Appl. Chem. 59, 257268. Gomes, J., Purkarthofer, H., Hyan, M., Kapplmuler, M., Sinner, N., Steiner, W., 1993. Production of high levels of cellulase-free xylanase by Thermomyces lanuginosus in laboratory scale and pilot scale using lignocellulosic materials. Appl. Micro- biol. Biotechnol. 39, 700707. Gupta, S., Bhushan, B., Hoondal, G.S., 2000. Isolation, purication and characteriza- tion of xylanase from Staphylococcus sp. SG-13 and its application in biobleach- ing of kraft pulp. J. Appl. Microbiol. 88, 325334. Haapala, R., Linko, S., Parkkinen, E., Sumimonen, P., 1994. Production of endo-1,4 glucanase and xylanase by Trichoderma reesei immobilized on polyurethane foam. Biotechnol. Tech. 8, 401406. Han, X., Zheng, Lian, S., Xie, Y., 2004. Study on screening and cultivation conditions xylanase producing alkaliphilic bacteria. Wuhan Univ. J. Nat. Sci. 9, 125128. Khandeparkar, R., Bhosle, N.B., 2006. Purication and characterization of thermo- alkalophilic xylanase isolated from the Enterobacter sp. MTCC 5112. Res. Microbiol. 157, 315325. Khasin, A., Alchanati, I., Shoham, Y., 1993. Purication and characterization of a thermostable xylanase from Bacillus stearothermophilus T-6. Appl. Environ. Microbiol. 59, 17251730. Kiddinamoorthy, J., Anceno, A.J., Haki, G.D., Rakshit, S.K., 2008. Production, purication and characterization of Bacillus sp. GRE7 xylanase and its applica- tion in eucalyptus kraft pulp biobleaching. World J. Microbiol. Biotechnol. 24, 605612. Kumar, A., Gupta, R., Shrivastava, B., Khasa, Y.P., Kuhad, R.C., 2012. Xylanase production from an alkaliphilic actinomycete isolate Streptomyces sp. RCK- 2010, its characterization and application in saccharication of second genera- tion biomass. J. Mol. Catal. B: Enzym. 74, 170177. Kumar, V., Satyanarayana, T., 2011. Applicability of thermo-alkali-stable and cellulase-free xylanase from a novel thermo-halo-alkaliphilic Bacillus halodur- ans in producing xylooligosaccharides. Biotechnol. Lett. 33, 22792285. Lakshmi, G.S., Rao, C.S., Rao, R.S., Hobbs, P.J., Prakasham, R.S., 2009. Enhanced production of xylanase by a newly isolated Aspergillus terreus under solid state fermentation using palm industrial waste: a statistical optimization. Biochem. Eng. J. 48, 5157. 0 5 10 15 20 25 30 35 40 0 12 24 36 48 R e d u c i n g
s u g a r
( m g / g )
Time (h) Untreated Acid treated Alkali treated Alkaline peroxide treated Fig. 14. Enzymatic hydrolysis of chemically pretreated sorghum straw using crude xylanase from B. altitudinis DHN8. Saccharication was carried out at 50 1C, 100 rpm for 48 h. Aliquots were taken at regular interval of 12 h and analysed for reducing sugar production. D.N. Adhyaru et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 182190 189 Li, W.F., Zhou, X.X., Lu, P., 2005. Structural features of thermozymes. Biotechnol. Adv. 23, 271281. Li, X., She, Y., Sun, B., Song, H., Zhu, Y., Lv, Y., Song, H., 2010. Purication and characterization of cellulase-free, thermostable xylanase from Streptomyces rameus L2001 and its biobleaching effect on wheat straw pulp. Biochem. Eng. J. 52, 7178. Lincon, R.D., 1960. Control of stock culture preservation and inoculum build up in bacterial fermentation. Microbiol. Technol. Eng. 2, 481500. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 31, 426428. Miller, L.G., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426428. Nagar, S., Gupta, V.K., Kumar, L., Kuhad, R.C., 2010. Production and optimization of cellulase-free, alkali-stable xylanase by Bacillus pumilus SV-85S in submerged fermentation. J. Ind. Microbiol. Biotechnol. 37, 7183. Nagar, S., Mittal, A., Kumar, D., Gupta, V.K., 2012. Production of alkalitolerant cellulase free xylanase in high levels by Bacillus pumilus SV-205. Int. J. Biol. Macromol. 50, 414420. Oliveira, L.A., Porto, A.L.F., Tambourgi, E.B., 2006. Production of xylanase and protease by Penicillium janthinellum CRC 87-M-115 from different agricultural wastes. Bioresour. Technol. 97, 862867. Palmer, T., 2001. The Chemical Nature of Enzyme Catalysis, Enzymes: Biochemistry, Biotechnology and Clinical chemistry. Hardwood Publication Ltd, UK, pp. 191222. (Chapter 11). Polizeli, M.L.T.M., Rizzatti, A.C.S., Monti, R., Terenzi, H.F., Jorge, J.A., Amori, D.S., 2005. Xylanases from fungi: properties and applications. Appl. Microbiol. Biotechnol. 67, 577591. Prakash, P., Jayalakshmi, S.K., Prakash, B., Rubul, M., Sreeramulu, K., 2011. Produc- tion of alkaliphilic, halotolerant, thermostable cellulase free xylanase by Bacillus halodurans PPKS-2 using agrowaste: single step purication and characteriza- tion. World J. Microbiol. Biotechnol. 82, 183192. Qie, Z., Shi, P., Luo, H., Bai, Y., Yuan, T., Yang, P., Liu, S., Yao, B., 2010. A xylanase with broad pH and temperature adaptability from Streptomyces megaspores DSM 41476, and its potential application in brewing industry. Enzyme Microb. Technol. 46, 506512. Sanghi, A., Garg, N., Sharma, J., Kumar, K., Kumar, R.C., Gupta, V.K., 2008. Optimization of xylanase production using inexpensive agro-residues by alkalophilic Bacillus subtilis ASH in solid state fermentation. World J. Microbiol. Biotechnol. 24, 633640. Sardessai, Y., Bhosle, S., 2004. Industrial potential of organic solvent tolerant bacteria. Biotechnol. Progr. 20, 655660. Savitha, S., Sadhasivam, S., Swaminathan, K., 2007. Application of Aspergillus fumigatus xylanase for quality improvement of waste paper pulp. Bull. Environ. Contam. Toxicol. 78, 217221. Sepahy, A.A., Ghazi, S., Sepahy, M.A., 2011. Cost-effective production and optimiza- tion of alkaline xylanase by indigenous Bacillus mojavensis AG 137 fermented on agricultural waste. Enzyme Res. , http://dx.doi.org/10.4061/2011/593624. Sharma, A., Adhikari, S., Satyanarayana, T., 2007. Alkali-thermostable and cellulase- free xylanase production by an extreme thermophile Geobacillus thermoleovor- ans. World J. Microbiol. Biotechnol. 23, 483490. Sharma, P., Bajaj, B.K., 2005. Production and partial characterization of alkalitoler- ant xylanase from an alkaliphilic Streptomyces sp. CD3. J. Sci. Ind. Res. 64, 688698. Subramaniyan, S., Prema, P., 2000. Cellulase free xylanase from Bacillus and other microorganisms. FEMS Microbiol. Lett. 183, 17. Sunna, A., Antranikian, G., 1997. Xylanolytic enzymes from fungi and bacteria. Crit. Rev. Biotechnol. 17, 3967. Talebnia, F., Karakashev, D., Angelidaki, I., 2010. Production of bioethanol from wheat straw: an overview on pretreatment, hydrolysis and fermentation. Bioresour. Technol. 101, 47444753. Taneja, K., Gupta, S., Kuhad, R.C., 2002. Properties and application of a partially puried alkaline xylanase from an alkaliphilic fungus Aspergillus nidulans KK99. Bioresour. Technol. 85, 3942. Wong, K.K.Y., Tan, L.U.L., Saddler, J.N., 1982. Multiplicity of -1,4-xylanase in microorganisms: functions and applications. Microbiol. Rev. 52, 305317. D.N. Adhyaru et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 182190 190