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KMID : 0364820180540020126
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Korean Journal of Microbiology
2018 Volume.54 No. 2 p.126 ~ p.135
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Gene cloning of β-mannanase C from Cellulosimicrobium sp. YB-43 and characterization of the enzyme
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Yoon Ki-Hong
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Abstract
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The characteristics of enzyme and gene for mannanase B hadbeen reported from Cellulosimicrobium sp. YB-43 producingsome kind of mannanase. A gene coding for the enzyme, namedmannanase C (ManC), was expected to be located downstreamof the manB gene. The manC gene was cloned by polymerasechain reaction and sequenced completely. From this nucleotidesequence, ManC was identified to consist of 448 amino residuesand contain a carbohydrate binding domain CBM2 besides acatalytic domain, which was homologous to mannanase belongingto the glycosyl hydrolase family 5. The catalytic domain ofManC showed the highest amino acid sequence similarity of55% with the mannanases from Streptomyces sp. SirexAA-E(55.8%; 4FK9_A) and S. thermoluteus (57.6%; BAM62868). The His-tagged ManC (HtManC) lacking N-terminal signalpeptide with hexahistidine at C-terminus was produced andpurified from cell extract of recombinant Escherichia coli. Thepurified HtManC showed maximal activity at 65°C and pH 7.5,with no significant change in its activity at pH range from 7.5to 10. HtManC showed more active on konjac and locust beangum (LBG) than guar gum and ivory nut mannan (ivory nut). Vmax and Km values of the HtManC for LBG were 68 U/mgand 0.45 mg/ml on the optimal condition, respectively. Mannobioseand mannotriose were observed on TLC as major productsresulting from the HtManC hydrolysis of mannooligosacharides. In addition, mannobiose and mannose were commonlydetected as the hydrolyzed products of LBG, konjac and ivorynut.
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KEYWORD
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Cellulosimicrobium sp. YB-43, cloning, mannanase C, purification
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