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Appl Microbiol Biotechnol. 2011 Feb;89(4):1083-92. doi: 10.1007/s00253-010-2828-4. Epub 2010 Oct 12.

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

Author information

1
Key Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, College of Life Sciences of Nanjing Agricultural University, 6 Tongwei Road, Nanjing, Jiangsu 210095, People's Republic of 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.

PMID:
20938774
DOI:
10.1007/s00253-010-2828-4
[Indexed for MEDLINE]

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