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KMID : 0545120080180010160
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Journal of Microbiology and Biotechnology
2008 Volume.18 No. 1 p.160 ~ p.166
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Gene Cloning, Expression, and Characterization of a Novel β-Mannanase from Bacillus circulans CGMCC 1416
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Li Yanan
Yang Peilong
Meng Kun
Wang Yaru
Luo Huiying
Wu Ningfeng
Yao Bin
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Abstract
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A DNA fragment containing 2,079 base pairs from Bacillus circulans CGMCC 1416 was cloned using degenerate PCR and inverse PCR. An open reading frame containing 981 bp was identified that encoded 326 amino acids residues, including a putative signal peptide of 31 residues. The deduced amino acid sequence showed the highest identity (68.1%) with endo-β-1,4-D-mannanase from Bacillus circulans strain K-1 of the glycoside hydrolase family 5 (GH5). The sequence encoding the mature protein was cloned into the pET-22b(+) vector and expressed in Escherichia coli as a recombinant fusion protein containing an N-terminal hexahistidine sequence. The fusion protein was purified by Ni2+ affinity chromatography and its hexahistidine tag cleaved to yield a 31-kDa β-mannanase having a specific activity of 481.55 U/mg. The optimal activity of the purified protein, MANB48, was at 58oC and pH 7.6. The hydrolysis product on substrate locust bean gum included a monosaccharide and mainly oligosaccharides. The recombinant MANB48 may be of potential use in the feed industry.
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KEYWORD
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β-Mannanase, Bacillus circulans, gene expression, characterization
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