Cloning and characterization of a new β-Glucosidase from a metagenomic library of Rumen of cattle feeding with Miscanthus sinensis

Background: The study on the second generation bio-fuel is a hot area of current research of renewable energy. Among series of key points in this area, the role of beta-glucosidase in the degradation of intermediate gluco-oligosaccharides limits the rate of the complete saccharification of lignocellulose. Results: In this study, a new beta-glucosidase gene, unglu135B12, which was isolated from a metagenomic library of rumen of cattle feeding with Miscanthus sinensis by the function-based screening, encodes a 779 amino acid polypeptide that contains a catalytic domain belonging to glycoside hydrolase family 3 (GH3). It was recombinantly expressed, purified and biochemically characterized. The recombinant beta-glucosidase, unglu135B12, displayed optimum enzymatic activity at pH 5.0 at 38 degrees C, and showed the highest specific activity of 2.5 x 10(3) U/mg under this optimal condition to p-nitrophenyl-beta-D-glucopyranoside (pNPG), and its Km and Vmax values were 0.309 mmol/L and 7.292 mu mol/min, respectively. In addition, the presence of Ca2+, K+, Na+ slightly improved beta-glucosidase activity of unglu135B12 by about 5%, while about 10 similar to 85% loss of beta-glucosidase activity was induced by addition of Mn2+, Fe3+, Zn2+, Cu2+. Interestingly, unglu135B12 was activated by glucose at the concentration lower than 40 mM. Conclusions: Our findings indicate that unglu135B12 is a new beta-glucosidase derived from rumen of cattle, and it might be a potent candidate for saccharification of lignocellulose in industrial application.