Cloning and characterization of a new ß-glucosidase from a metagenomic library of rumen of cattle feeding with Miscanthus sinensis.

ABSTRACT

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 ß-glucosidase in the degradation of intermediate gluco-oligosaccharides limits the rate of the complete saccharification of lignocellulose. RESULTS: In this study, a new ß-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 ß-glucosidase, unglu135B12, displayed optimum enzymatic activity at pH 5.0 at 38°C, and showed the highest specific activity of 2.5 × 10(3) U/mg under this optimal condition to p-nitrophenyl-ß-D-glucopyranoside (pNPG), and its Km and Vmax values were 0.309 mmol/L and 7.292 µmol/min, respectively. In addition, the presence of Ca2+, K+, Na+ slightly improved ß-glucosidase activity of unglu135B12 by about 5%, while about 10~85% loss of ß-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 ß-glucosidase derived from rumen of cattle, and it might be a potent candidate for saccharification of lignocellulose in industrial application.