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Cloning and functional characterization of a novel endo-β-1,4-glucanase gene from a soil-derived metagenomic library

Author:
Liu, Juan, Liu, Wei-dong, Zhao, Xiao-li, Shen, Wen-jing, Cao, Hui, Cui, Zhong-li
Source:
Applied microbiology and biotechnology 2011 v.89 no.4 pp. 1083-1092
ISSN:
0175-7598
Subject:
DNA, Escherichia coli, bacterial artificial chromosomes, buffers, carboxymethylcellulose, cellulose, citrates, clones, databases, endo-1,4-beta-glucanase, genes, genomic libraries, open reading frames, pH, pancreatin, pepsin, salt concentration, screening, soil, China
Abstract:
A metagenomic library consisting of 3,024 bacterial artificial chromosome clones was prepared in Escherichia coli DH10B with high-molecular-weight DNA extracted from red soil in South China. A novel cellulase gene with an open reading frame of 1,332 bp, cel5G, encoding an endo-β-1,4-glucanase was cloned using an activity-based screen. The deduced enzyme, Cel5G, belongs to the glycosyl hydrolase family 5 and shares <39% identity with endoglucanases in the GenBank database. cel5G was expressed in E. coli BL21, and the recombinant enzyme Cel5G was purified to homogeneity for enzymatic analysis. Cel5G hydrolyzed a wide range of β-1,4-, β-1,3/β-1,4-, or β-1,3/β-1,6-linked polysaccharides, amorphous cellulose, filter paper, and microcrystalline cellulose. Its highest activity was in 50 mM citrate buffer, pH 4.8, at 50°C. Cel5G is stable over a wide range of pH values (from 2.0 to 10.6) and is thermally stable under 60°C. It is highly tolerant and active in high salt concentrations and is stable in the presence of pepsin and pancreatin. The K m and V max values of Cel5G for carboxymethyl cellulose are 19.92 mg/ml and 1,941 μmol min−1 mg−1, respectively. These characteristics indicate that Cel5G has potential for industrial use.
Agid:
544374