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KMID : 0364820160520030336
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Korean Journal of Microbiology
2016 Volume.52 No. 3 p.336 ~ p.343
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Molecular cloning and characterization of β-mannanase B from Cellulosimicrobium sp. YB-43
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Yoon Ki-Hong
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Abstract
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A mannanase gene was cloned into Escherichia coli from Cellulosimicrobium sp. YB-43, which had been found to produce two kinds of mannanase, and sequenced completely. This mannanase gene, designated manB, consisted of 1,284 nucleotides encoding a polypeptide of 427 amino acid residues. Based on the deduced amino acid sequence, the ManB was identified to be a modular enzyme including two carbohydrate binding domains besides the catalytic domain, which was highly homologous to mannanases belonging to the glycosyl hydrolase family 5. The N-terminal amino acid sequence of ManB, purified from a cell-free extract of the recombinant E. coli carrying a Cellulosimicrobium sp. YB-43 manB gene, has been determined as QGASAASDG, which was correctly corresponding to signal peptide predicted by SignalP4.1 server for Gram-negative bacteria. The purified ManB had a pH optimum for its activity at pH 6.5~7.0 and a temperature optimum at 55°C. The enzyme was active on locust bean gum (LBG), konjac and guar gum, while it did not exhibit activity towards carboxymethylcellulose, xylan, starch, and para-nitrophenyl-β-mannopyranoside. The activity of enzyme was inhibited very slightly by Mg2+, K+, and Na+, and significantly inhibited by Cu2+, Zn2+, Mn2+, and SDS. The enzyme could hydrolyze mannooligosaccharides larger than mannobiose, which was the most predominant product resulting from the ManB hydrolysis for mannooligosaccharides and LBG.
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KEYWORD
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Cellulosimicrobium sp. YB-43, cloning, mannanase, purification
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