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KMID : 0364820100460020207
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Korean Journal of Microbiology
2010 Volume.46 No. 2 p.207 ~ p.212
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Cloning and Characterization of Mannanase Gene from Bacillus subtilis WL-8
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Yoon Ki-Hong
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Abstract
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A bacterium producing the extracellular mannanase was isolated from Korean soybean paste. The isolate WL-8 has been identified as Bacillus subtilis on the basis on its 16S rRNA sequence, morphology and biochemical properties. The mannanase productivity of strain WL-8 was increased in LB broth by addition of wheat bran. The maximum mannanase productivity was reached to approximately 20 U/ml in LB medium supplemented with 6% wheat bran. A gene encoding the mannanase of WL-8 was cloned into Escherichia coli and its nucleotide sequence was subsequently determined. The mannanase gene consisted of 1,086 nucleotides encoding a polypeptide of 362 amino acid residues. The deduced amino acid sequence was highly homologous with those of several mannanases from B. subtilis belonging to GH family 26. Reaction temperature and pH profiles were investigated using the culture filtrate and cell-free extract of the recombinant E. coli carrying a WL-8 mannanase gene, respectively. Optimal conditions for the two fractions occurred at pH 5.5 and 60°C.
The cell-free extract showed higher mannanase activity than the culture filtrate at above 60°C.
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KEYWORD
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B. subtilis, cloning, identification, mannanase, reaction property
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