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Applied Microbiology and Biotechnology

, Volume 89, Issue 4, pp 1083–1092 | Cite as

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

  • Juan Liu
  • Wei-dong Liu
  • Xiao-li Zhao
  • Wen-jing Shen
  • Hui Cao
  • Zhong-li Cui
Biotechnologically Relevant Enzymes and Proteins

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.

Keywords

Metagenomic BAC library Cellulase Endo-β-1,4-glucanase Gene cloning Functional characterization 

Notes

Acknowledgments

We are grateful to Prof. Mei-Zhong Luo (College of Life Science and Technology, Huazhong Agricultural University) and Prof. Rod A. Wing (Arizona Genomics Institute, University of Arizona) for plasmid pCUGIBACI and constructive ideas for BAC library construction. We thank Dr. Xiao-Zhou Zhang (Senior Research Associate, Biological Systems Engineering of Virginia Polytechnic Institute and State University) for good suggestions on the enzyme study and thank Dr. Rui-fu Zhang (University of California at Davis) for critically reading the manuscript. This work was supported by the National Science Foundation of China (40871125), the New Century Talent of MOE, China (NCET-06-0490) and the National Hi-tech Development Project of China (2007AA021304).

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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  1. 1.Key Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of AgricultureCollege of Life Sciences of Nanjing Agricultural UniversityNanjingPeople’s Republic of China

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