Cloning and characterization of a new β-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 β-glucosidase in the degradation of intermediate gluco-oligosaccharides limits the rate of the complete saccharification of lignocellulose.

Results

In this study, a new β-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 β-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-β-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 Ca ²⁺, K ⁺, Na ⁺ slightly improved β-glucosidase activity of unglu135B12 by about 5%, while about 10 ~ 85% loss of β-glucosidase activity was induced by addition of Mn ²⁺, Fe ³⁺, Zn ²⁺, Cu ²⁺. Interestingly, unglu135B12 was activated by glucose at the concentration lower than 40 mM.

Conclusions

Our findings indicate that unglu135B12 is a new β-glucosidase derived from rumen of cattle, and it might be a potent candidate for saccharification of lignocellulose in industrial application.

Electronic supplementary material

The online version of this article (doi:10.1186/1472-6750-14-85) contains supplementary material, which is available to authorized users.