Purification and characterization of cellulase produced by Bacillus sp. HMM

Abstract

Cellulose is long-chain glucose polymer, which is linked by β-1,4-glycosidic bond. Its degradation relies on the synergistic action of multi-component cellulase system, including endoglucanase, exoglucanase and β-glucosidase. Cellulase hydrolyzes cellulose to produce glucose through the sequential reactions. While the biodegradation of lignocellulosic wastes has been extensively studied, researches on cellulase production with the mixed agricultural wastes as carbon sources remain limited. To address this gap, this study innovatively employed wheat bran (WB), sugarcane bagasse (SB) and rice hull (RH) to prepare the mixed carbon sources for cellulase production by Bacillus sp. HMM. Bacillus sp. HMM in culture medium with WB showed the highest cellulase activity (3.93 ± 0.13 U/mL) and reducing sugar content (0.97 ± 0.02 mg/mL) at 12 h. Cellulase was purified by (NH₄)₂SO₄ and DEAE-52 column, and investigated by SDS-PAGE. Mw of purified cellulase was between 50 and 70 kDa with purification fold of 23.10. This cellulase had endoglucanase, exoglucanase and β-glucosidase activities, and the wide substrate specificity, which was conducive to the complete degradation of lignocellulose. CMCase activities showed the high resistance under stress conditions such as metal ions, surfactants and inhibitors. CMCase activities still retained over 90% under the treatment of most additives for 4 h, especially DTT greatly promoted CMCase activity, reaching 129.2% of control. Bacillus sp. HMM could efficiently utilize wheat bran to produce cellulase for the conversion of lignocellulosic biomass.

Keywords: Bacillus; CMCase activity; Cellulose-degrading bacterium; Rice hull; Sugarcane bagasse; Wheat bran.