Characterization of cold adapted and ethanol tolerant β-glucosidase from Bacillus cellulosilyticus and its application for directed hydrolysis of cellobiose to ethanol.

A β-glucosidase gene from Bacillus cellulosilyticus was expressed in Escherichia coli BL21 and characterized. The recombinant enzyme (BcBgl1A) showed the optimal activity at 40°C, and the half-life of BcBgl1A activity at 40°C was 24h. BcBgl1A was sensitive to heat and exhibited obvious cold-adapted activity, retained 27.2% and 46.5% of the optimal activity at 4 and 20°C, respectively. The best activity was observed at pH 7.0 and stable over the range of pH 6.0-8.0. The activity was significantly enhanced by Fe2+, Fe3+ and Mn2+, also showed a high level of NaCl tolerance. BcBgl1A exhibited high activity using both pNPG and cellobiose as substrates, the Vmax for pNPG and cellobiose was 66.2 and 151.5U/mg of protein, respectively, and its Km values were 2.97 and 10.4mM, respectively. Catalysis efficiency (Kcat/Km) of 45.8s-1 (cellobiose) and 70.3s-1 (pNPG). It displayed high tolerance to glucose and ethanol, the Ki and IC50 value was 200mM and 15% (v/v), respectively. Additionally, the BcBgl1A was found to be highly efficient for conversion of cellobiose (100g/L) to ethanol by simultaneous saccharification and fermentation process, achieved an ethanol content of 47.2g/l corresponding to 87.9% of the theoretical ethanol yield. The results suggest that BcBgl1A has a potential for biotechnological applications in the bioconversion of lignocellulosic materials.