A Highly Thermostable Alkaline Cellulase-Free Xylanase from Thermoalkalophilic Bacillus sp. JB 99 Suitable for Paper and Pulp Industry: Purification and Characterization Dengeti Shrinivas & Gunashekaran Savitha & Kumar Raviranjan & Gajanan Ramchandra Naik Received: 2 February 2010 / Accepted: 26 April 2010 / Published online: 14 May 2010 # Springer Science+Business Media, LLC 2010 Abstract A highly thermostable alkaline xylanase was purified to homogeneity from culture supernatant of Bacillus sp. JB 99 using DEAE-Sepharose and Sephadex G-100 gel filtration with 25.7-fold increase in activity and 43.5% recovery. The molecular weight of the purified xylanase was found to be 20 kDA by SDS-PAGE and zymogram analysis. The enzyme was optimally active at 70 °C, pH 8.0 and stable over pH range of 6.0–10.0.The relative activity at 9.0 and 10.0 were 90% and 85% of that of pH 8.0, respectively. The enzyme showed high thermal stability at 60 °C with 95% of its activity after 5 h. The K m and V max of enzyme for oat spelt xylan were 4.8 mg/ml and 218.6 μM min -1 mg -1 , respectively. Analysis of N- terminal amino acid sequence revealed that the xylanase belongs to glycosyl hydrolase family 11 from thermoalkalophilic Bacillus sp. with basic pI. Substrate specificity showed a high activity on xylan-containing substrate and cellulase-free nature. The hydrolyzed product pattern of oat spelt xylan on thin-layer chromatography suggested xylanase as an endoxylanase. Due to these properties, xylanase from Bacillus sp. JB 99 was found to be highly compatible for paper and pulp industry. Keywords Bacillus sp. JB 99 . Thermoalkalophilic . Xylanase . Purification . Glycosyl hydrolases Introduction Xylan is the major constituent of hemicellulose, a complex polysaccharide that is the most abundant biological carbon source in nature. Xylanase (E.C. 3.2.1.8) produced by microorganisms have attracted a great deal of attention during the past few decades because of their potential biotechnological applications in various industries including the food, feed, fuel, textile and paper and pulp industries, and in waste treatment [1]. Global Appl Biochem Biotechnol (2010) 162:2049–2057 DOI 10.1007/s12010-010-8980-6 D. Shrinivas : G. Savitha : K. Raviranjan : G. R. Naik (*) Department of Biotechnology, Gulbarga University, Gulbarga 585106 Karnataka, India e-mail: grnaikbiotech@gmail.com