Cloning and identification of novel cellulase genes from uncultured microorganisms in rabbit cecum and characterization of the expressed cellulases |
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Authors: | Yi Feng Cheng-Jie Duan Hao Pang Xin-Chun Mo Chun-Feng Wu Yuan Yu Ya-Lin Hu Jie Wei Ji-Liang Tang Jia-Xun Feng |
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Institution: | (1) Guangxi Key Laboratory of Subtropical Bioresources Conservation and Utilization, The Key Laboratory of Ministry of Education for Microbial and Plant Genetic Engineering, and College of Life Science and Technology, Guangxi University, Nanning, Guangxi, 530005, People’s Republic of China;(2) College of Life Science and Technology, Guangxi University, The Eastern Campus, 75 Xiuling Road, Nanning, Guangxi, 530005, People’s Republic of China |
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Abstract: | A metagenomic cosmid library was prepared in Escherichia coli from DNA extracted from the contents of rabbit cecum and screened for cellulase activities. Eleven independent clones expressing
cellulase activities (four endo-β-1,4-glucanases and seven β-glucosidases) were isolated. Subcloning and sequencing analysis
of these clones identified 11 cellulase genes; the encoded products of which shared less than 50% identities and 70% similarities
to cellulases in the databases. All four endo-β-1,4-glucanases and all seven β-glucosidases, respectively, belonged to glycosyl
hydrolase family 5 (GHF 5) and family 3 (GHF 3) and formed two separate branches in the phylogenetic tree. Ten of the 11 cloned
cellulases exhibited highest activities at pH 5.5 ∼ 7.0 and 40 ∼ 55°C, a condition similar to that in the rabbit cecum. All
the four endo-β-1,4-glucanases could hydrolyze a wide range of β-1,4-, β-1,4/β-1,3- or β-1,3/β-1,6-linked polysaccharides.
One endo-β-1, 4-glucanase gene, umcel5G, was overexpressed in E. coli, and the purified recombinant enzyme was characterized in detail. The enzymes cloned in this work represented at least some
of the cellulases operating efficiently in the rabbit cecum. This work provides the first snapshot on the cellulases produced
by bacteria in rabbit cecum. |
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Keywords: | Cellulase Cloning Metagenome Rabbit cecum Expression Enzyme Properties |
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