Highly Thermostable Alkaline Cellulase-Free Xylanase from Thermoalkalophilic Bacillus sp. JB 99 Suitable for Paper and Pulp Industry: Purification and Characterization

• Published in: Applied biochemistry and biotechnology 2010-11, Vol.162 (7), p.2049-2057
• Main Authors: Shrinivas, Dengeti, Savitha, Gunashekaran, Raviranjan, Kumar, Naik, Gajanan Ramchandra
• Format: Article
• Language: English
• Subjects: Alkalinity; Amino Acid Sequence; Amino acids; Bacillus; Bacillus - chemistry; Bacillus - enzymology; Bacillus - genetics; Bacteria; Biochemistry; Biological and medical sciences; Biotechnology; Chemistry; Chemistry and Materials Science; Endo-1,4-beta Xylanases - chemistry; Endo-1,4-beta Xylanases - genetics; Endo-1,4-beta Xylanases - isolation & purification; Endo-1,4-beta Xylanases - metabolism; Enzyme Stability; Fundamental and applied biological sciences. Psychology; Hydrolysis; Industrial Microbiology; Kinetics; Molecular Sequence Data; Molecular Weight; Pulp & paper industry; Sequence Alignment; Substrate Specificity; Thin layer chromatography; Xylans - chemistry; Xylans - metabolism
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-010-8980-6

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.