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Purification and characterization of extracellular xylanase from Streptomyces cyaneus SN32
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Bioresource Technology 99 (2008) 1252–1258
Purification and characterization of extracellular xylanase from Streptomyces cyaneus SN32 Suchita Ninawe a
a,b
, Mukesh Kapoor a, Ramesh Chander Kuhad
a,*
Lignocellulose Biotechnology Laboratory, Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110 021, India b Department of Biotechnology, Ministry of Science and Technology, Government of India, CGO Complex, Block 2, 7th Floor, Lodi Road, New Delhi 110 003, India Received 4 January 2007; received in revised form 10 February 2007; accepted 11 February 2007 Available online 3 April 2007
Abstract Streptomyces cyaneus SN32 was used in this study to produce extracellular xylanase, an important industrial enzyme used in pulp and paper industry. The enzyme was purified to homogeneity by ammonium sulfate precipitation followed by anion exchange chromatography using DEAE–Sepharose column, with 43.0% yield. The enzyme was found to be a monomer of 20.5 kDa as determined by SDS gel electrophoresis and has a pI of 8.5. The optimum pH and temperature for purified xylanase activity was 6.0 and 60–65 C, respectively. The half-lives of xylanase at 50 and 65 C were approximately 200 and 50 min, respectively. The xylanase exhibited Km and Vmax values of 11.1 mg/ml and 45.45 lmol/min/mg. The 15 residue N-terminal sequence of the enzyme was found to be 87% identical up to that of endoxylanases from Steptomyces sp. Based on the zymogram analysis, sequence similarity and other characteristics, it is proposed that the purified enzyme from S. cyaneus SN32 is an endoxylanase and belongs to Group1 xylanases (low molecular weight – basic proteins). The purified enzyme was stable for more than 20 week at 4 C. Easy purification from the fermentation broth and its high stability will be highly useful for industrial application of this endoxylanase. 2007 Elsevier Ltd. All rights reserved. Keywords: Streptomyces cyaneus; Purification; Xylanase; Endoxylanase; N-terminal sequence
1. Introduction Xylan, a major structural component of plant cell walls and the most abundant renewable hemicellulose, constitutes 20–40% of total plant biomass. Therefore, hydrolysis of xylan becomes an important step towards proper utilization of abundantly available lignocellulosic material in nature (Kuhad and Singh, 1993; Kuhad et al., 1997; Beg et al., 2001; Polizeli et al., 2005). Chemical hydrolysis of xylan applied extensively by the industries, though faster, is accompanied with the formation of toxic compounds and is hazardous to the environment (Beg et al., 2001). Xylan hydrolysis using enzymes such as xylanases provides a via*
Corresponding author. Tel.: +91 11 24112062; fax: +91 11 24115270, +91 11 688 5270. E-mail address: [email protected] (R.C. Kuhad). 0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.02.016
ble alternative to chemical hydrolysis as it is highly specific in nature apart from being an environment friendly process (Bajpai, 1997; Kuhad et al., 1997). The continuous effort in this direction has stimulated basic and applied research on microbial hemicellulases and has not only produced significant scientific knowledge, but also revealed their enormous biotechnological potential as food additives in poultry, in wheat flour for improving dough handling and the quality of baked products, for extraction of coffee, plant oils, and starch, in the improvement of nutritional properties of agricultural silage and grain feed, in combination with pectinase and cellulase for clarification of fruit juices and recovery of fermentable sugars from hemicelluloses and production of xylo-oligosaccharides (Bedford and Classen, 1992; Kuhad et al., 1997; Beg et al., 2001; Kapoor and Kuhad, 2007). Special attention has been given to their use in the pulp and paper industry for bleaching purposes,
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resulting in a decrease of chlorine utilization and consequently lowering environmental impact (Viikari et al., 1994; Bajpai, 2004; Ninawe and Kuhad, 2006; Kapoor et al., 2007). A purified xylanase is the pre-requisite for various biochemical studies needed to be done for better understanding of the xylanolytic system (Beg et al., 2001; Polizeli et al., 2005). Streptomyces cyaneus SN32 was reported previously to be a good producer of xylanases when grown on lignocellulosic material and xylan as the substrates (Ninawe and Kuhad, 2005). The most significant effect of a cellulase-free crude enzyme preparation from S. cyaneus SN32 on pulp bleaching was on the delignification of kraft pulp and reduction of the pulp viscosity (Ninawe and Kuhad, 2006). In this present work, we describe the purification and characterization of a new xylanase from S. cyaneus SN32. 2. Methods 2.1. Chemicals Commercial xylans and BSA were purchased from Sigma Chemical Co., USA. Protein marker was obtained from Amersham Pharmacia Biotech, UK. All other chemicals and materials used in the present study were of highest purity grade. 2.2. Microorganism and culture conditions S. cyaneus SN32 was isolated from garden soil using Actinomycete Isolation Agar and maintained on Nutrient Agar Medium (Ninawe and Kuhad, 2006). The organism was identified using molecular methods (Accession no. AY232254 for 16S rRNA sequence) and is deposited with Microbial Type Culture Collection and Gene bank, Institute of Microbial Technology, Chandigarh, India (Accession no. MTCC 7060). For xylanase production, S. cyaneus SN32 was cultivated in 250 ml Erlenmeyer flasks containing 50 ml of optimized production medium containing wheat bran (3% w/v), peptone (1% w/v), KH2PO4 (0.1% w/v), MgSO4 Æ 7H2O (0.01% w/v); pH 9.0 under shaking (200 rpm) conditions at 42 C for 48 h (Ninawe and Kuhad, 2005). The contents of the flasks were filtered through Whatman No. 1 filter paper and the filtrate was centrifuged at 10,000g for 30 min to obtain cell free culture fluid. 2.3. Purification of xylanase The purification of xylanase (cell free supernatant) was carried out in two steps. The first step involved ammonium sulphate precipitation (0–60% saturation) of 500 ml cell free supernatant. The saturated solution was left overnight at 4 C, centrifuged and precipitates were dissolved in 35 ml of 50 mM Tris–HCl buffer (pH 7.5). The dialyzed fraction of concentrated proteins (40 ml) was then loaded on to the anion-exchange DEAE–Sepharose column
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(0.9 cm · 20 cm; Pharmacia LKB Biotechnology, Uppsala, Sweden) with a with a bed volume of 40 ml was pre-equilibrated with Tris–HCl buffer. The column was operated at a flow rate of 30 ml/h. A continuous NaCl gradient (0–1 M) was applied and fractions (1.5 ml each) were collected. 2.4. Electrophoresis of protein samples SDS–PAGE of the samples from above steps was carried out by using BIORAD electrophoresis apparatus. Stacking gel (5%) and resolving gel (12%) with prescribed compositions were used (Sambrook and Russel, 2001). Protein bands in the gel were visualized by Coomassie Brilliant Blue R-250 staining. Molecular weight of test protein(s) was compared with standard LMW protein marker (Amersham Pharmacia, Biotech, UK). Native PAGE of the samples at different steps of purification was carried out with the aim to study the zymographic pattern of purified xylanase. The procedure followed was mostly similar to SDS–PAGE described above with few modifications. 250 ll of 1% (w/v) birchwood xylan was added to 1.75 ml of water during the preparation of gel. SDS and mercaptoethanol were not added in the buffer/tracking dye solution. The step of boiling samples during protein sample preparation was eliminated. The samples were loaded in duplicate sets. After completion of electrophoresis, the gel was cut in two halves, each with one set of samples. One half was stained with Coomassie Brilliant Blue solution to locate the position of the purified protein and the other portion was used for zymogram analysis. The gel for activity staining was incubated in 0.2 M citrate–phosphate buffer, pH 6.0 (stock solutions for 1 M buffer consist of 192 g/l of citric acid and 141.9 g/l Na2HPO4) for 2 min at 60 C followed by staining the gel in Congo-Red solution (0.5% w/v Congo-Red and 5% v/v ethanol in distilled water) for 15 min. The gel was de-stained with 1 M NaCl to visualize the clearing zone of hydrolysis. The gel was further exposed to 5% acetic acid to increase the colour contrast between the hydrolysis zone and the remaining portion of the gel (Chadha et al., 1999). Iso-electric focusing of purified xylanase was carried out using a mini 111 IEF apparatus (Amersham Pharmacia, Sweden) with ampholytes in the pH range of 3–11. Polyacrylamide gel was casted on the hydrophobic surface of the gel support film. 2 ll of each protein sample and marker were loaded on to the gel. Focusing was carried out under constant voltage conditions in a step-wise manner (100 V for 15 min, 200 V for 15 min and 450 V for 60 min). The gel was stained with Coomassie Brilliant Blue solution to locate the position of the purified xylanase protein. pI for the purified xylanase was determined with respect to pI marker proteins in the gel. 2.5. Characterization of xylanase The optimal pH for xylanase activity was determined by using different buffers (0.2 M) ranging between 3.0 and 11.0
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(citrate–phosphate, pH 3.0–6.5; phosphate buffer, pH 6.0– 7.5; Tris–HCl, pH 7.5–9.2; glycine–NaOH, pH 8.5–10.5 and carbonate–bicarbonate, pH 9.0–11.0) and the optimum temperature for xylanase activity was determined between 4 and 70 C. The pH stability was determined by incubating equal volume of enzyme solution with different buffers ranging between 4.0 and 11.0 at 60 C for 1 h. For thermostability determination, the enzyme was incubated at 50 and 65 C up to 4 h at pH 6.0, and the residual activities were determined periodically. The rate of substrate hydrolysis, Km and Vmax values for birch wood xylan were determined using standard methods. 2.6. Analysis of hydrolytic products
acids of N-terminal of the enzyme was determined using automatic gas phase Edman degradation on Procise sequencer. 2.9. Xylanase assay The xylanase activity was determined by measuring the release of reducing sugars from birch wood xylan (1% w/ v) by dinitrosalicylic acid method (Miller, 1959). One unit of xylanase was defined as amount of enzyme required to release 1 lmol of xylose from birch wood xylan in 1 min under standard assay conditions (60 C, 0.2 M glycine– NaOH buffer, pH 9.0). 2.10. Protein estimation
Hydrolytic product analysis was carried out by TLC. Purified xylanase (50 ll) was added to 450 ll (1% w/v) birch wood xylan (pH 6.0, 0.2 M citrate–phosphate buffer) and incubated at 60 C. At regular time intervals, aliquots were withdrawn, centrifuged at 10,000g for 10 min at 4 C and the supernatant obtained, were spotted on the silica gel plates. A solvent system comprising of chloroform:acetic acid:water (6:7:1 v/v) was used to separate the end products. The Chromatogram was developed by spraying a solution of concentrated H2SO4:C2H5OH (5:95 v/v) and incubating the plate at 100 C for 15 min (Lopez et al., 1997). 2.7. Shelf life of xylanase The shelf life stability of xylanase was studied at room temperature (25 C) and under refrigeration (4 C) conditions. Enzyme samples were withdrawn at different time intervals up to 52 weeks to monitor residual xylanase activity under the standard assay conditions.
The extracellular protein was precipitated with 10% (w/ v) trichloroacetic acid at 4 C. After centrifugation at 10,000g for 10 min, the pellet was dissolved in 0.1 N NaOH solution. Thereafter, the protein content was estimated by Lowry’s method (Lowry et al., 1951) using bovine serum albumin (BSA) as standard. 3. Results 3.1. Purification of xylanase The xylanase from S. cyaneus SN32 was precipitated using ammonium sulphate (0–50% saturation) to yield an active pellet. The active pellet was dialyzed and used as a starting material for further purification using DEAE– sepharose ion-exchange chromatography. A 2.25-fold purification was achieved with 43.6% recovery of xylanase activity, yielding a specific activity of 893.56 IU/mg protein. The results of xylanase purification are summarised in Table 1.
2.8. N-terminal sequence of the pure xylanase 3.2. SDS–PAGE, native PAGE and IEF analysis Electro-blotting of the xylanase protein from unstained SDS–PAGE gel on to poly-vinyl difluoride (PVDF) membrane was carried out at constant voltage of 37 V (100 mA) for 60 min at room temperature (Matsudaira, 1987). The membrane was removed from the trans-blotting sandwich and saturated with 100% methanol followed by staining with Coomassie Brilliant Blue R-250. The blotted membrane was de-stained with 50% methanol and the band of xylanase protein was excised. The sequence of amino
The purified xylanase preparation was represented in the form of a single band on SDS–PAGE (Fig. 1a). The molecular weight of purified xylanase was estimated to be 20.5 kDa by SDS–PAGE. Zymogram revealed the presence of a zone of hydrolysis that corresponded with Commassie stained band of purified xylanase on native PAGE, confirming the purified protein as xylanase (Fig. 1b). The xylanase exhibited a pI value of 8.5.
Table 1 Summary of the purification steps of an extracellular xylanase produced by S. cyaneus SN32 Purification step
Volume (ml)
Total enzyme activity (IU)
Total soluble protein (mg)
Specific enzyme activity (IU/mg)
Yield (%)
Purification fold
Culture fluid Concentrated with ammonium sulfate (60% saturation) DEAE–sepharose chromatography
500 40
358,000 210,934
903.33 359.51
396.31 586.73
100 58.92
1.000 1.480
240
156,159
174.76
893.56
43.62
2.254
S. Ninawe et al. / Bioresource Technology 99 (2008) 1252–1258
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Fig. 1. (a) SDS–PAGE of extracellular proteins from S. cyaneus SN32 (Lane 1: LMW protein marker; Lane 2: Culture fluid; Lane 3: Concentrated enzyme (precipitated by ammonium sulfate); Lane 4: Purified xylanase after chromatography on DEAE–Sepharose). (b) Confirmation of the purified protein as xylanase from S. cyaneus SN32 ((I) Native PAGE of purified xylanase; (II) Zymogram stained with Congo-Red followed by treatment with 5% acetic acid). A – ammonium sulphate precipitated xylanase, C – crude xylanase, P – purified xylanase.
3.3. Characterization of N-terminal sequence
3.4. Characterization of xylanase
BLAST homology search of 15 amino acid N-terminal sequence of the purified xylanase from S. cyaneus SN32 determined by automatic gas phase Edman degradation method revealed its similarity with peptide sequences of endoxylanases from Streptomyces sp. (Table 2).
The purified xylanase was active between pH 5.0 and 8.0 and retained more than 60% of its activity. It exhibited pH optima at 6.0 (Fig. 2). The xylanase was stable between pH 4.0 and 9.5, at 60 C for 1 h, retaining 80% of its activity (Fig. 2). S. cyaneus SN32 xylanase exhibited temperature
Table 2 Sequences indicating significant alignment with N-terminal of xylanase of S. cyaneus SN32
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optima of 60–65 C at pH 6.0 (Fig. 3). The half-lives of xylanase at 50 and 65 C were approximately 200 and 120 min, respectively (Fig. 4). The xylanase exhibited Km and Vmax values of 11.1 mg/ml and 45.45 lmol/min/mg. TLC analysis of hydrolysates formed by the action of purified xylanase on birch wood xylan was characterized by an initial release of xylo-oligosaccharides (Xoligo > X2) that was followed by further hydrolysis to release xylobiose and xylose as the end products, respectively. The mode of action of xylanase from S. cyaneus SN32 was found to be endo-type as it produced xylobiose (X2) as the predominant end product form birch wood xylan along with higher xylo-oligosaccharides as intermediates. The xylanase retained 100% of its activity for up to 20 weeks at 4 C. Fig. 4. Thermostability profile of xylanase from S. cyaneus SN32 Thermostability was determined at temperatures of 50 C (d) and 65 C (m) as a function of time. 100% xylanase activity was equivalent to 500.0 IU/mL.
4. Discussion
Fig. 2. Effect of pH (d) and pH stability (j) on xylanase activity assayed at 65 C for 10 min. The xylanase activity was assayed in the pH range 4.0–10.0 using different buffers (Citrate–phosphate, pH 4.0–6.0; Tris–HCl, pH 7.0–9.0 and Glycine–NaOH, 9.0–10.0). 100% xylanase activity was equivalent to 500.0 IU/mL.
Fig. 3. Effect of assay temperature on activity of xylanase. 100% xylanase activity was equivalent to 500.0 IU mL 1.
Purification and characterization of the enzyme are important pre-requisites for its successful biotechnological application. Purification of xylanases from microorganisms generally requires precipitation of the active protein followed by two to three chromatographic steps (Sunna and Antranikian, 1997). A single step gel filtration for purification of xylanase from Aspergillus ochraceus with a 50% recovery of the active protein has been reported earlier (Biswas et al., 1990). Li et al. (2000) have obtained 40% xylanase yield using ethanol precipitation and a single anion-exchange chromatography. One step affinity purification of xylanase from Aspergillus sp. (Gawande and Kamat, 1999) and Bacillus sp. strain K-1 (Ratanakhanokchai et al., 1999) using Eudragit S100 and affinity adsorption–desorption on insoluble xylan, respectively, has also been reported. In the present study, ammonium sulfate precipitated xylanase from S. cyaneus SN32 was purified to homogeneity following the single step anion exchange chromatography (DEAE–Sepharose). As ion exchange chromatography can be easily scaled up in comparison to gel filtration or alcohol precipitation based procedures, the current procedure aim at high through put purification of the xylanases for commercial application. Molecular weight of the purified xylanase of S. cyaneus SN32 was found to be 20.5 kDa and is well within the average range of molecular weight for xylanases i.e. 11–85 kDa (Wong et al., 1988; Beg et al., 2001). Comparative analysis of molecular weight (20.5 kDa) and pI (8.5) of the purified enzyme from S. cyaneus SN32 indicated that it follows dichotomous pattern and falls under Group 1 xylanases (low molecular weight – basic proteins) based on the classification suggested by Wong et al. (1988). Presence of only one band on zymogram of xylanase on native gel indicated the presence of a single active xylanase. However, native-
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PAGE of partially purified xylanases from Aspergillus foetidus strain and subsequent activity staining revealed three clear and distinct bands, indicating the presence of multiple forms of xylanases (Shah and Madamwar, 2005). The purified xylanase was optimally active at pH 6.0 and retained more than 80% of its activity in a wide range of pH (4.0–9.5). There are very few reports of xylanases optimally active under alkaline conditions (Gessesse and Mamo, 1998; Gupta et al., 2000; Khandeparkar and Bhosle, 2006). The uses of alkaline xylanases, which are operationally stable at higher temperature, are more beneficial because of savings in cooling cost and time. In this regard, the present xylanase is expected to operate under conditions close to those of most mills, i.e. high pH and temperature. It may also find application in waste management programs, where xylanases can be used to hydrolyze xylan in industrial and municipal waste. The pH stability of xylanases between 6.5 and 10.0 (40 C), 5.0–11.0 (50 C), 3.0–8.0 (30 C), and 7.5 and 9.2 (50 C) have been reported from Micrococcus sp. AR-135 (Gessesse and Mamo, 1998), Acidobacterium capsulatum (Inagaki et al., 1998) and Streptomyces chattanogenesis CECT 3336 (Fernandez et al., 1998) and Staphylococcus sp. SG-13 (Gupta et al., 2000), respectively. S. cyaneus SN32 xylanase exhibited temperature optima of 60–65 C at pH 9.0 (Fig. 3). A similar temperature optima of 60–65 C has been reported earlier (Beg et al., 2000; Basaran and Hang, 2000; Sa-Pereira et al., 2002; Anthony et al., 2003; Heck et al., 2006). The half-lives of S. cyaneus SN32 xylanase at 50 and 65 C were approximately 200 and 50 min, respectively. Anthony et al. (2003) reported that Aspergillus fumigatus xylanase was stable up to 40 C over long period of incubation and at 50 C it lost activity gradually after 30 min. At high temperatures a rapid loss of activity was observed with half-life for deactivation of 17, 12, and 7 min at 55, 60, and 70 C, respectively. Purified xylanase from Bacillus sp. was capable of retaining full activity after incubation at 50 C for more than 23 h (Sapre et al., 2005). However, xylanase from alkalophilic Micrococcus sp. AR-135 was very stable up to 40 C followed by a rapid loss of activity above 45 C (Gessesse and Mamo, 1998). The purified xylanase of S. cyaneus SN32 retained almost 100% of its activity up to 20 weeks, when stored at 4 C. Xylanase from A. foetidus retained full activity when stored in deep freeze up to 6 months. At refrigeration temperature no loss of activity was found up to 2 weeks but after 4 weeks a marginal decrease (5–10%) was found (Shah and Madamwar, 2005). Further characterization of a purified xylanase through analysis of N-terminal sequence confirmed it as an endoxylanase. Comparison of the N-terminal sequence of xylanase from S. cyaneus SN32 with other known sequences through BLAST search indicated its similarity with xylanases from Genus – Streptomyces. The homologous proteins from Streptomyces sp. EC3, S. roseiscleroticus and S. coelicolor A3 (2) (Accession numbers – S47512, A57001 and T37005) are extracellular having higher molecular weight
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as compared to the purified xylanase from S. cyaneus SN32. Low molecular weight xylanases as bleaching agents in pulp and paper industry are desirable, since they can easily penetrate into the re-precipitated xylan on the surface of kraft pulp. This alleviates the problem of xylan barrier on the surface of lignin containing pulp during subsequent chemical bleaching steps (Kuhad et al., 1997). In the present study, purification of xylanase from S. cyaneus SN32 was a single step process. Moreover, S. cyaneus SN32 needs economically viable nutritional requirements for xylanase production to the magnitude of 720 IU/ml, the highest production reported so far using actinomycete strain (Ninawe and Kuhad, 2005). These characteristics of xylanase from S. cyaneus SN32 enhance its industrial potentials. Acknowledgements Dr. Suchita Ninawe thankfully acknowledges Department of Biotechnology, Ministry of Science and Technology, Government of India, New Delhi for granting study leave to undertake this work and Department of Microbiology University of Delhi for providing research facilities. References Anthony, T., Chandra Raj, K., Rajendran, A., Gunasekaran, P., 2003. High molecular weight cellulase-free xylanase from alkali-tolerant Aspergillus fumigatus AR1. Enzyme Microb. Technol. 32, 647–654. Bajpai, P., 1997. Microbial xylanolytic enzyme system: properties and applications. Adv. Appl. Microbiol. 43, 141–194. Bajpai, P., 2004. Biological bleaching of pulps. Crit. Rev. Biotechnol. 24, 1–58. Basaran, P., Hang, Y.D., 2000. Purification and characterisation of acetyl esterase from Candida guilliermondii. Lett. Appl. Microbiol. 30, 167– 171. Bedford, M.R., Classen, H.L., 1992. The influence of dietary xylanase on intestinal viscosity and molecular weight distribution of carbohydrates in rye-fed broiler chick. In: Visser, J., Beldman, G., Vansomenen, A.K., Voragen, A.G.J. (Eds.), Xylans and Xylanases. Elsevier, Amsterdam, pp. 361–370. Beg, Q.K., Bhushan, B., Kapoor, M., Hoondal, G.S., 2000. Production and characterisation of thermostable xylanase and pectinase from Streptomyces sp. QG-11-3. J. Ind. Microbiol. Biotechnol. 24, 396–402. Beg, Q.K., Kapoor, M., Mahajan, L., Hoondal, G.S., 2001. Microbial xylanases and their industrial applications: a review. Appl. Microbiol. Biotechnol. 56, 326–338. Biswas, S.R., Jana, S.C., Mishra, A.K., Nanda, G., 1990. Production, purification and characterization of xylanase from a hyperxylanolytic mutant of Aspergillus ochraceus. Biotechnol. Bioeng. 35, 244–251. Chadha, B.S., Jaswinder, K., Rubinder, K., Saini, H.S., Singh, S., 1999. Xylanase production by Thermomyces lanuginosus wild and mutant strains. World J. Microbiol. Biotechnol. 15, 195–198. Fernandez, C.L., Rodreiguez, J., Ball, A.S., Patino, J.L.C., Leblic, M.I.P., Arias, M.E., 1998. Application of affinity binding of xylanase to oat spelt xylan in the purification of endoxylanase CM-2 from Streptomyces chattanogenesis CECT 3336. Appl. Microbiol. Biotechnol. 50, 284–287. Gawande, P.V., Kamat, M.Y., 1999. Purification of Aspergillus sp. xylanase by precipitation with an anionic polymer Eudragit S100. Process Biochem. 34, 577–580. Gessesse, A., Mamo, G., 1998. Purification and characterization of an alkaline xylanase from alkalophilic Micrococcus sp. AR-135. J. Ind. Microbiol. Biotechnol. 20, 210–214.
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Gupta, S., Bhushan, B., Hoondal, G.S., 2000. Isolation, purification and characterization of xylanase from Staphylococcus sp. SG-13 and its application in biobleaching of kraft pulp. J. Appl. Microbiol. 88, 325– 334. Heck, J.X., Soares, L.H.B., Hertz, P.F., Ayub, M.A.Z., 2006. Purification and properties of a xylanase produced by Bacillus circulans BL53 on solid-state cultivation. Biochem. Eng. J. 32, 179–184. Inagaki, K., Nakahia, K., Mukai, K., Tamura, T., Tanaka, H., 1998. Gene cloning and characterization of an acidic xylanase from Acidobacterium capsulatum. Process Biochem. 62, 1061–1067. Kapoor, M., Kuhad, R.C., 2007. Immobilization of xylanase from Bacillus pumilus strain MK001 and its application in production of xylo-oligosaccharides. Appl. Biochem. Biotechnol., in press. Kapoor, M., Kapoor, R.K., Kuhad, R.C., 2007. Differential and synergistic effects of xylanase and laccase mediator system (LMS) in bleaching of soda and waste pulps. J. Appl. Microbiol., in press, doi:10.1111/j.1365-2672.2006.03251.x. Khandeparkar, R.D.S., Bhosle, N.B., 2006. Purification and characterization of thermoalkalophilic xylanase isolated from the Enterobacter sp. MTCC 5112. Res. Microbiol. 157, 315–325. Kuhad, R.C., Singh, A., 1993. Lignocellulose biotechnology: current and future prospects. Crit. Rev. Biotechnol. 13, 151–172. Kuhad, R.C., Singh, A., Eriksson, K.-E.L., 1997. Microorganisms and enzymes involved in the degradation of plant fiber cell wall. Adv. Biochem. Eng. Biotechnol. 57, 47–125. Li, Y.K., Yao, H.J., Cho, Y., 2000. Effective induction, purification and characterization of Trichoderma koningii G-39 b-xylosidase with high transferase activity. Biotechnol. Appl. Biochem. 31, 119–125. Lopez, C., Blanco, A., Pastor, F.I.J., 1997. Xylanase production by a new alkali-tolerant isolate of Bacillus. Biotechnol. Lett. 20, 243–246. Lowry, O.H., Rosebrough, N.J., Farr, A.C., Randall, R.J., 1951. Protein measurement with folin phenol reagent. J. Biol. Chem. 193, 265–275. Matsudaira, P., 1987. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 261, 10035–10038.
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Bioresource Technology 99 (2008) 1252–1258
Purification and characterization of extracellular xylanase from Streptomyces cyaneus SN32 Suchita Ninawe a
a,b
, Mukesh Kapoor a, Ramesh Chander Kuhad
a,*
Lignocellulose Biotechnology Laboratory, Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110 021, India b Department of Biotechnology, Ministry of Science and Technology, Government of India, CGO Complex, Block 2, 7th Floor, Lodi Road, New Delhi 110 003, India Received 4 January 2007; received in revised form 10 February 2007; accepted 11 February 2007 Available online 3 April 2007
Abstract Streptomyces cyaneus SN32 was used in this study to produce extracellular xylanase, an important industrial enzyme used in pulp and paper industry. The enzyme was purified to homogeneity by ammonium sulfate precipitation followed by anion exchange chromatography using DEAE–Sepharose column, with 43.0% yield. The enzyme was found to be a monomer of 20.5 kDa as determined by SDS gel electrophoresis and has a pI of 8.5. The optimum pH and temperature for purified xylanase activity was 6.0 and 60–65 C, respectively. The half-lives of xylanase at 50 and 65 C were approximately 200 and 50 min, respectively. The xylanase exhibited Km and Vmax values of 11.1 mg/ml and 45.45 lmol/min/mg. The 15 residue N-terminal sequence of the enzyme was found to be 87% identical up to that of endoxylanases from Steptomyces sp. Based on the zymogram analysis, sequence similarity and other characteristics, it is proposed that the purified enzyme from S. cyaneus SN32 is an endoxylanase and belongs to Group1 xylanases (low molecular weight – basic proteins). The purified enzyme was stable for more than 20 week at 4 C. Easy purification from the fermentation broth and its high stability will be highly useful for industrial application of this endoxylanase. 2007 Elsevier Ltd. All rights reserved. Keywords: Streptomyces cyaneus; Purification; Xylanase; Endoxylanase; N-terminal sequence
1. Introduction Xylan, a major structural component of plant cell walls and the most abundant renewable hemicellulose, constitutes 20–40% of total plant biomass. Therefore, hydrolysis of xylan becomes an important step towards proper utilization of abundantly available lignocellulosic material in nature (Kuhad and Singh, 1993; Kuhad et al., 1997; Beg et al., 2001; Polizeli et al., 2005). Chemical hydrolysis of xylan applied extensively by the industries, though faster, is accompanied with the formation of toxic compounds and is hazardous to the environment (Beg et al., 2001). Xylan hydrolysis using enzymes such as xylanases provides a via*
Corresponding author. Tel.: +91 11 24112062; fax: +91 11 24115270, +91 11 688 5270. E-mail address: [email protected] (R.C. Kuhad). 0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.02.016
ble alternative to chemical hydrolysis as it is highly specific in nature apart from being an environment friendly process (Bajpai, 1997; Kuhad et al., 1997). The continuous effort in this direction has stimulated basic and applied research on microbial hemicellulases and has not only produced significant scientific knowledge, but also revealed their enormous biotechnological potential as food additives in poultry, in wheat flour for improving dough handling and the quality of baked products, for extraction of coffee, plant oils, and starch, in the improvement of nutritional properties of agricultural silage and grain feed, in combination with pectinase and cellulase for clarification of fruit juices and recovery of fermentable sugars from hemicelluloses and production of xylo-oligosaccharides (Bedford and Classen, 1992; Kuhad et al., 1997; Beg et al., 2001; Kapoor and Kuhad, 2007). Special attention has been given to their use in the pulp and paper industry for bleaching purposes,
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resulting in a decrease of chlorine utilization and consequently lowering environmental impact (Viikari et al., 1994; Bajpai, 2004; Ninawe and Kuhad, 2006; Kapoor et al., 2007). A purified xylanase is the pre-requisite for various biochemical studies needed to be done for better understanding of the xylanolytic system (Beg et al., 2001; Polizeli et al., 2005). Streptomyces cyaneus SN32 was reported previously to be a good producer of xylanases when grown on lignocellulosic material and xylan as the substrates (Ninawe and Kuhad, 2005). The most significant effect of a cellulase-free crude enzyme preparation from S. cyaneus SN32 on pulp bleaching was on the delignification of kraft pulp and reduction of the pulp viscosity (Ninawe and Kuhad, 2006). In this present work, we describe the purification and characterization of a new xylanase from S. cyaneus SN32. 2. Methods 2.1. Chemicals Commercial xylans and BSA were purchased from Sigma Chemical Co., USA. Protein marker was obtained from Amersham Pharmacia Biotech, UK. All other chemicals and materials used in the present study were of highest purity grade. 2.2. Microorganism and culture conditions S. cyaneus SN32 was isolated from garden soil using Actinomycete Isolation Agar and maintained on Nutrient Agar Medium (Ninawe and Kuhad, 2006). The organism was identified using molecular methods (Accession no. AY232254 for 16S rRNA sequence) and is deposited with Microbial Type Culture Collection and Gene bank, Institute of Microbial Technology, Chandigarh, India (Accession no. MTCC 7060). For xylanase production, S. cyaneus SN32 was cultivated in 250 ml Erlenmeyer flasks containing 50 ml of optimized production medium containing wheat bran (3% w/v), peptone (1% w/v), KH2PO4 (0.1% w/v), MgSO4 Æ 7H2O (0.01% w/v); pH 9.0 under shaking (200 rpm) conditions at 42 C for 48 h (Ninawe and Kuhad, 2005). The contents of the flasks were filtered through Whatman No. 1 filter paper and the filtrate was centrifuged at 10,000g for 30 min to obtain cell free culture fluid. 2.3. Purification of xylanase The purification of xylanase (cell free supernatant) was carried out in two steps. The first step involved ammonium sulphate precipitation (0–60% saturation) of 500 ml cell free supernatant. The saturated solution was left overnight at 4 C, centrifuged and precipitates were dissolved in 35 ml of 50 mM Tris–HCl buffer (pH 7.5). The dialyzed fraction of concentrated proteins (40 ml) was then loaded on to the anion-exchange DEAE–Sepharose column
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(0.9 cm · 20 cm; Pharmacia LKB Biotechnology, Uppsala, Sweden) with a with a bed volume of 40 ml was pre-equilibrated with Tris–HCl buffer. The column was operated at a flow rate of 30 ml/h. A continuous NaCl gradient (0–1 M) was applied and fractions (1.5 ml each) were collected. 2.4. Electrophoresis of protein samples SDS–PAGE of the samples from above steps was carried out by using BIORAD electrophoresis apparatus. Stacking gel (5%) and resolving gel (12%) with prescribed compositions were used (Sambrook and Russel, 2001). Protein bands in the gel were visualized by Coomassie Brilliant Blue R-250 staining. Molecular weight of test protein(s) was compared with standard LMW protein marker (Amersham Pharmacia, Biotech, UK). Native PAGE of the samples at different steps of purification was carried out with the aim to study the zymographic pattern of purified xylanase. The procedure followed was mostly similar to SDS–PAGE described above with few modifications. 250 ll of 1% (w/v) birchwood xylan was added to 1.75 ml of water during the preparation of gel. SDS and mercaptoethanol were not added in the buffer/tracking dye solution. The step of boiling samples during protein sample preparation was eliminated. The samples were loaded in duplicate sets. After completion of electrophoresis, the gel was cut in two halves, each with one set of samples. One half was stained with Coomassie Brilliant Blue solution to locate the position of the purified protein and the other portion was used for zymogram analysis. The gel for activity staining was incubated in 0.2 M citrate–phosphate buffer, pH 6.0 (stock solutions for 1 M buffer consist of 192 g/l of citric acid and 141.9 g/l Na2HPO4) for 2 min at 60 C followed by staining the gel in Congo-Red solution (0.5% w/v Congo-Red and 5% v/v ethanol in distilled water) for 15 min. The gel was de-stained with 1 M NaCl to visualize the clearing zone of hydrolysis. The gel was further exposed to 5% acetic acid to increase the colour contrast between the hydrolysis zone and the remaining portion of the gel (Chadha et al., 1999). Iso-electric focusing of purified xylanase was carried out using a mini 111 IEF apparatus (Amersham Pharmacia, Sweden) with ampholytes in the pH range of 3–11. Polyacrylamide gel was casted on the hydrophobic surface of the gel support film. 2 ll of each protein sample and marker were loaded on to the gel. Focusing was carried out under constant voltage conditions in a step-wise manner (100 V for 15 min, 200 V for 15 min and 450 V for 60 min). The gel was stained with Coomassie Brilliant Blue solution to locate the position of the purified xylanase protein. pI for the purified xylanase was determined with respect to pI marker proteins in the gel. 2.5. Characterization of xylanase The optimal pH for xylanase activity was determined by using different buffers (0.2 M) ranging between 3.0 and 11.0
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(citrate–phosphate, pH 3.0–6.5; phosphate buffer, pH 6.0– 7.5; Tris–HCl, pH 7.5–9.2; glycine–NaOH, pH 8.5–10.5 and carbonate–bicarbonate, pH 9.0–11.0) and the optimum temperature for xylanase activity was determined between 4 and 70 C. The pH stability was determined by incubating equal volume of enzyme solution with different buffers ranging between 4.0 and 11.0 at 60 C for 1 h. For thermostability determination, the enzyme was incubated at 50 and 65 C up to 4 h at pH 6.0, and the residual activities were determined periodically. The rate of substrate hydrolysis, Km and Vmax values for birch wood xylan were determined using standard methods. 2.6. Analysis of hydrolytic products
acids of N-terminal of the enzyme was determined using automatic gas phase Edman degradation on Procise sequencer. 2.9. Xylanase assay The xylanase activity was determined by measuring the release of reducing sugars from birch wood xylan (1% w/ v) by dinitrosalicylic acid method (Miller, 1959). One unit of xylanase was defined as amount of enzyme required to release 1 lmol of xylose from birch wood xylan in 1 min under standard assay conditions (60 C, 0.2 M glycine– NaOH buffer, pH 9.0). 2.10. Protein estimation
Hydrolytic product analysis was carried out by TLC. Purified xylanase (50 ll) was added to 450 ll (1% w/v) birch wood xylan (pH 6.0, 0.2 M citrate–phosphate buffer) and incubated at 60 C. At regular time intervals, aliquots were withdrawn, centrifuged at 10,000g for 10 min at 4 C and the supernatant obtained, were spotted on the silica gel plates. A solvent system comprising of chloroform:acetic acid:water (6:7:1 v/v) was used to separate the end products. The Chromatogram was developed by spraying a solution of concentrated H2SO4:C2H5OH (5:95 v/v) and incubating the plate at 100 C for 15 min (Lopez et al., 1997). 2.7. Shelf life of xylanase The shelf life stability of xylanase was studied at room temperature (25 C) and under refrigeration (4 C) conditions. Enzyme samples were withdrawn at different time intervals up to 52 weeks to monitor residual xylanase activity under the standard assay conditions.
The extracellular protein was precipitated with 10% (w/ v) trichloroacetic acid at 4 C. After centrifugation at 10,000g for 10 min, the pellet was dissolved in 0.1 N NaOH solution. Thereafter, the protein content was estimated by Lowry’s method (Lowry et al., 1951) using bovine serum albumin (BSA) as standard. 3. Results 3.1. Purification of xylanase The xylanase from S. cyaneus SN32 was precipitated using ammonium sulphate (0–50% saturation) to yield an active pellet. The active pellet was dialyzed and used as a starting material for further purification using DEAE– sepharose ion-exchange chromatography. A 2.25-fold purification was achieved with 43.6% recovery of xylanase activity, yielding a specific activity of 893.56 IU/mg protein. The results of xylanase purification are summarised in Table 1.
2.8. N-terminal sequence of the pure xylanase 3.2. SDS–PAGE, native PAGE and IEF analysis Electro-blotting of the xylanase protein from unstained SDS–PAGE gel on to poly-vinyl difluoride (PVDF) membrane was carried out at constant voltage of 37 V (100 mA) for 60 min at room temperature (Matsudaira, 1987). The membrane was removed from the trans-blotting sandwich and saturated with 100% methanol followed by staining with Coomassie Brilliant Blue R-250. The blotted membrane was de-stained with 50% methanol and the band of xylanase protein was excised. The sequence of amino
The purified xylanase preparation was represented in the form of a single band on SDS–PAGE (Fig. 1a). The molecular weight of purified xylanase was estimated to be 20.5 kDa by SDS–PAGE. Zymogram revealed the presence of a zone of hydrolysis that corresponded with Commassie stained band of purified xylanase on native PAGE, confirming the purified protein as xylanase (Fig. 1b). The xylanase exhibited a pI value of 8.5.
Table 1 Summary of the purification steps of an extracellular xylanase produced by S. cyaneus SN32 Purification step
Volume (ml)
Total enzyme activity (IU)
Total soluble protein (mg)
Specific enzyme activity (IU/mg)
Yield (%)
Purification fold
Culture fluid Concentrated with ammonium sulfate (60% saturation) DEAE–sepharose chromatography
500 40
358,000 210,934
903.33 359.51
396.31 586.73
100 58.92
1.000 1.480
240
156,159
174.76
893.56
43.62
2.254
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Fig. 1. (a) SDS–PAGE of extracellular proteins from S. cyaneus SN32 (Lane 1: LMW protein marker; Lane 2: Culture fluid; Lane 3: Concentrated enzyme (precipitated by ammonium sulfate); Lane 4: Purified xylanase after chromatography on DEAE–Sepharose). (b) Confirmation of the purified protein as xylanase from S. cyaneus SN32 ((I) Native PAGE of purified xylanase; (II) Zymogram stained with Congo-Red followed by treatment with 5% acetic acid). A – ammonium sulphate precipitated xylanase, C – crude xylanase, P – purified xylanase.
3.3. Characterization of N-terminal sequence
3.4. Characterization of xylanase
BLAST homology search of 15 amino acid N-terminal sequence of the purified xylanase from S. cyaneus SN32 determined by automatic gas phase Edman degradation method revealed its similarity with peptide sequences of endoxylanases from Streptomyces sp. (Table 2).
The purified xylanase was active between pH 5.0 and 8.0 and retained more than 60% of its activity. It exhibited pH optima at 6.0 (Fig. 2). The xylanase was stable between pH 4.0 and 9.5, at 60 C for 1 h, retaining 80% of its activity (Fig. 2). S. cyaneus SN32 xylanase exhibited temperature
Table 2 Sequences indicating significant alignment with N-terminal of xylanase of S. cyaneus SN32
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optima of 60–65 C at pH 6.0 (Fig. 3). The half-lives of xylanase at 50 and 65 C were approximately 200 and 120 min, respectively (Fig. 4). The xylanase exhibited Km and Vmax values of 11.1 mg/ml and 45.45 lmol/min/mg. TLC analysis of hydrolysates formed by the action of purified xylanase on birch wood xylan was characterized by an initial release of xylo-oligosaccharides (Xoligo > X2) that was followed by further hydrolysis to release xylobiose and xylose as the end products, respectively. The mode of action of xylanase from S. cyaneus SN32 was found to be endo-type as it produced xylobiose (X2) as the predominant end product form birch wood xylan along with higher xylo-oligosaccharides as intermediates. The xylanase retained 100% of its activity for up to 20 weeks at 4 C. Fig. 4. Thermostability profile of xylanase from S. cyaneus SN32 Thermostability was determined at temperatures of 50 C (d) and 65 C (m) as a function of time. 100% xylanase activity was equivalent to 500.0 IU/mL.
4. Discussion
Fig. 2. Effect of pH (d) and pH stability (j) on xylanase activity assayed at 65 C for 10 min. The xylanase activity was assayed in the pH range 4.0–10.0 using different buffers (Citrate–phosphate, pH 4.0–6.0; Tris–HCl, pH 7.0–9.0 and Glycine–NaOH, 9.0–10.0). 100% xylanase activity was equivalent to 500.0 IU/mL.
Fig. 3. Effect of assay temperature on activity of xylanase. 100% xylanase activity was equivalent to 500.0 IU mL 1.
Purification and characterization of the enzyme are important pre-requisites for its successful biotechnological application. Purification of xylanases from microorganisms generally requires precipitation of the active protein followed by two to three chromatographic steps (Sunna and Antranikian, 1997). A single step gel filtration for purification of xylanase from Aspergillus ochraceus with a 50% recovery of the active protein has been reported earlier (Biswas et al., 1990). Li et al. (2000) have obtained 40% xylanase yield using ethanol precipitation and a single anion-exchange chromatography. One step affinity purification of xylanase from Aspergillus sp. (Gawande and Kamat, 1999) and Bacillus sp. strain K-1 (Ratanakhanokchai et al., 1999) using Eudragit S100 and affinity adsorption–desorption on insoluble xylan, respectively, has also been reported. In the present study, ammonium sulfate precipitated xylanase from S. cyaneus SN32 was purified to homogeneity following the single step anion exchange chromatography (DEAE–Sepharose). As ion exchange chromatography can be easily scaled up in comparison to gel filtration or alcohol precipitation based procedures, the current procedure aim at high through put purification of the xylanases for commercial application. Molecular weight of the purified xylanase of S. cyaneus SN32 was found to be 20.5 kDa and is well within the average range of molecular weight for xylanases i.e. 11–85 kDa (Wong et al., 1988; Beg et al., 2001). Comparative analysis of molecular weight (20.5 kDa) and pI (8.5) of the purified enzyme from S. cyaneus SN32 indicated that it follows dichotomous pattern and falls under Group 1 xylanases (low molecular weight – basic proteins) based on the classification suggested by Wong et al. (1988). Presence of only one band on zymogram of xylanase on native gel indicated the presence of a single active xylanase. However, native-
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PAGE of partially purified xylanases from Aspergillus foetidus strain and subsequent activity staining revealed three clear and distinct bands, indicating the presence of multiple forms of xylanases (Shah and Madamwar, 2005). The purified xylanase was optimally active at pH 6.0 and retained more than 80% of its activity in a wide range of pH (4.0–9.5). There are very few reports of xylanases optimally active under alkaline conditions (Gessesse and Mamo, 1998; Gupta et al., 2000; Khandeparkar and Bhosle, 2006). The uses of alkaline xylanases, which are operationally stable at higher temperature, are more beneficial because of savings in cooling cost and time. In this regard, the present xylanase is expected to operate under conditions close to those of most mills, i.e. high pH and temperature. It may also find application in waste management programs, where xylanases can be used to hydrolyze xylan in industrial and municipal waste. The pH stability of xylanases between 6.5 and 10.0 (40 C), 5.0–11.0 (50 C), 3.0–8.0 (30 C), and 7.5 and 9.2 (50 C) have been reported from Micrococcus sp. AR-135 (Gessesse and Mamo, 1998), Acidobacterium capsulatum (Inagaki et al., 1998) and Streptomyces chattanogenesis CECT 3336 (Fernandez et al., 1998) and Staphylococcus sp. SG-13 (Gupta et al., 2000), respectively. S. cyaneus SN32 xylanase exhibited temperature optima of 60–65 C at pH 9.0 (Fig. 3). A similar temperature optima of 60–65 C has been reported earlier (Beg et al., 2000; Basaran and Hang, 2000; Sa-Pereira et al., 2002; Anthony et al., 2003; Heck et al., 2006). The half-lives of S. cyaneus SN32 xylanase at 50 and 65 C were approximately 200 and 50 min, respectively. Anthony et al. (2003) reported that Aspergillus fumigatus xylanase was stable up to 40 C over long period of incubation and at 50 C it lost activity gradually after 30 min. At high temperatures a rapid loss of activity was observed with half-life for deactivation of 17, 12, and 7 min at 55, 60, and 70 C, respectively. Purified xylanase from Bacillus sp. was capable of retaining full activity after incubation at 50 C for more than 23 h (Sapre et al., 2005). However, xylanase from alkalophilic Micrococcus sp. AR-135 was very stable up to 40 C followed by a rapid loss of activity above 45 C (Gessesse and Mamo, 1998). The purified xylanase of S. cyaneus SN32 retained almost 100% of its activity up to 20 weeks, when stored at 4 C. Xylanase from A. foetidus retained full activity when stored in deep freeze up to 6 months. At refrigeration temperature no loss of activity was found up to 2 weeks but after 4 weeks a marginal decrease (5–10%) was found (Shah and Madamwar, 2005). Further characterization of a purified xylanase through analysis of N-terminal sequence confirmed it as an endoxylanase. Comparison of the N-terminal sequence of xylanase from S. cyaneus SN32 with other known sequences through BLAST search indicated its similarity with xylanases from Genus – Streptomyces. The homologous proteins from Streptomyces sp. EC3, S. roseiscleroticus and S. coelicolor A3 (2) (Accession numbers – S47512, A57001 and T37005) are extracellular having higher molecular weight
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as compared to the purified xylanase from S. cyaneus SN32. Low molecular weight xylanases as bleaching agents in pulp and paper industry are desirable, since they can easily penetrate into the re-precipitated xylan on the surface of kraft pulp. This alleviates the problem of xylan barrier on the surface of lignin containing pulp during subsequent chemical bleaching steps (Kuhad et al., 1997). In the present study, purification of xylanase from S. cyaneus SN32 was a single step process. Moreover, S. cyaneus SN32 needs economically viable nutritional requirements for xylanase production to the magnitude of 720 IU/ml, the highest production reported so far using actinomycete strain (Ninawe and Kuhad, 2005). These characteristics of xylanase from S. cyaneus SN32 enhance its industrial potentials. Acknowledgements Dr. Suchita Ninawe thankfully acknowledges Department of Biotechnology, Ministry of Science and Technology, Government of India, New Delhi for granting study leave to undertake this work and Department of Microbiology University of Delhi for providing research facilities. References Anthony, T., Chandra Raj, K., Rajendran, A., Gunasekaran, P., 2003. High molecular weight cellulase-free xylanase from alkali-tolerant Aspergillus fumigatus AR1. Enzyme Microb. Technol. 32, 647–654. Bajpai, P., 1997. Microbial xylanolytic enzyme system: properties and applications. Adv. Appl. Microbiol. 43, 141–194. Bajpai, P., 2004. Biological bleaching of pulps. Crit. Rev. Biotechnol. 24, 1–58. Basaran, P., Hang, Y.D., 2000. Purification and characterisation of acetyl esterase from Candida guilliermondii. Lett. Appl. Microbiol. 30, 167– 171. Bedford, M.R., Classen, H.L., 1992. The influence of dietary xylanase on intestinal viscosity and molecular weight distribution of carbohydrates in rye-fed broiler chick. In: Visser, J., Beldman, G., Vansomenen, A.K., Voragen, A.G.J. (Eds.), Xylans and Xylanases. Elsevier, Amsterdam, pp. 361–370. Beg, Q.K., Bhushan, B., Kapoor, M., Hoondal, G.S., 2000. Production and characterisation of thermostable xylanase and pectinase from Streptomyces sp. QG-11-3. J. Ind. Microbiol. Biotechnol. 24, 396–402. Beg, Q.K., Kapoor, M., Mahajan, L., Hoondal, G.S., 2001. Microbial xylanases and their industrial applications: a review. Appl. Microbiol. Biotechnol. 56, 326–338. Biswas, S.R., Jana, S.C., Mishra, A.K., Nanda, G., 1990. Production, purification and characterization of xylanase from a hyperxylanolytic mutant of Aspergillus ochraceus. Biotechnol. Bioeng. 35, 244–251. Chadha, B.S., Jaswinder, K., Rubinder, K., Saini, H.S., Singh, S., 1999. Xylanase production by Thermomyces lanuginosus wild and mutant strains. World J. Microbiol. Biotechnol. 15, 195–198. Fernandez, C.L., Rodreiguez, J., Ball, A.S., Patino, J.L.C., Leblic, M.I.P., Arias, M.E., 1998. Application of affinity binding of xylanase to oat spelt xylan in the purification of endoxylanase CM-2 from Streptomyces chattanogenesis CECT 3336. Appl. Microbiol. Biotechnol. 50, 284–287. Gawande, P.V., Kamat, M.Y., 1999. Purification of Aspergillus sp. xylanase by precipitation with an anionic polymer Eudragit S100. Process Biochem. 34, 577–580. Gessesse, A., Mamo, G., 1998. Purification and characterization of an alkaline xylanase from alkalophilic Micrococcus sp. AR-135. J. Ind. Microbiol. Biotechnol. 20, 210–214.
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