Journal of Microbiology

1 "function-based screening strategy"

Filter

Article category

Keywords

Publication year

Research Support, Non-U.S. Gov't

Characterization of a Novel β-Glucosidase-Like Activity from a Soil Metagenome: Chengjian Jiang , Gefei Ma , Shuangxi Li , Tingting Hu , Zhiqun Che , Peihong Shen , Bing Yan , Bo Wu; J. Microbiol. 2009;47(5):542-548. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-009-0024-y

Abstract: We report the cloning of a novel β-glucosidase-like gene by function-based screening of a metagenomic library from uncultured soil microorganisms. The gene was named bgl1C and has an open reading frame of 1,443 base pairs. It encodes a 481 amino acid polypeptide with a predicted molecular mass of about 57.8 kDa. The deduced amino acid sequence did not show any homology with known β-glucosidases. The putative β-glucosidase gene was subcloned into the pETBlue-2 vector and overexpressed in E. coli Tuner (DE3) pLacІ; the recombinant protein was purified to homogeneity. Functional characterization with a high performance liquid chromatography method demonstrated that the recombinant Bgl1C protein hydrolyzed D-glucosyl-β-(1-4)-D-glucose to glucose. The maximum activity for Bgl1C protein occurred at pH 8.0 and 42°C using p-nitrophenyl-β-D-glucoside as the substrate. A CaCl2 concentration of 1 mM was required for optimal activity. The putative β-glucosidase had an apparent Km value of 0.19 mM, a Vmax value of 4.75 U/mg and a kcat value of 316.7/min under the optimal reaction conditions. The biochemical characterization of Bgl1C has enlarged our understanding of the novel enzymes that can be isolated from the soil metagenome.

Search