title: Purification and characterization of an alkaline cellulase produced by Bacillus subtilis (AS3).
abstract: An extracellular alkaline carboxymethycellulase (CMCase) from Bacillus subtilis was purified by salt precipitation followed by anion-exchange chromatography using DEAE-Sepharose. The cell-free supernatant containing crude enzyme had a CMCase activity of 0.34 U/mg. The purified enzyme gave a specific activity of 3.33 U/mg, with 10-fold purification and an overall activity yield of 5.6%. The purified enzyme displayed a protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular size of 30 kDa, which was also confirmed by zymogram analysis. The enzyme displayed multisubstrate specificity, showing significantly higher activity with lichenan and β-glucan as compared to carboxymethylcellulose (CMC), laminarin, hydroxyethylcellulose, and steam-exploded bagasse, and negligible activity with crystalline substrate such as Avicel and filter paper. It was optimally active at pH 9.2 and temperature 45°C. The enzyme was stable in the pH range 6-10 and retained 70% activity at pH 12. Thermal stability analysis revealed that the enzyme was stable in temperature range of 20°C to 45°C and retained more than 50% activity at 60°C for 30 min. The enzyme had a Km of 0.13 mg/ml and Vmax of 3.38 U/mg using CMC as substrate.
authors: Centre for the Environment, Indian Institute of Technology Guwahati, Guwahati, Assam, India.
authors: Deepmoni
authors: D
authors: Deka
authors: Mohammad
authors: M
authors: Jawed
authors: Arun
authors: A
authors: Goyal
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D002266:Carboxymethylcellulose Sodium / Q000302:isolation & purification* / Q000378:metabolism*; D002482:Cellulose / Q000031:analogs & derivatives / Q000378:metabolism; D002852:Chromatography, Ion Exchange / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005936:Glucans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D011134:Polysaccharides / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D047071:beta-Glucans / Q000378:metabolism
chemical_list: D005936:Glucans; D011134:Polysaccharides; D047071:beta-Glucans; D002482:Cellulose; C002283:hydroxyethylcellulose; C027433:bagasse; C008247:laminaran; D002266:Carboxymethylcellulose Sodium