Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Cloning and Expression of Thermostable Chitosanase Gene from Bacillus sp. KFB-C108

  • Yoon, Ho-Geun (Graduate School of Biotechnology, Korea University) ;
  • Kim, Hee-Yun (Department of Food Additives, Korea Food and Administration) ;
  • Kim, Hye-Kyung (Department of Food and Biotechnology, Hanseo University) ;
  • Kim, Kyung-Hyun (Graduate School of Biotechnology, Korea University) ;
  • Hwang, Han-Joon (Graduate School of Biotechnology, Korea University) ;
  • Cho, Hong-Yon (Graduate School of Biotechnology, Korea University)
  • Published : 1999.10.01
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Abstract

The thermostable endo-chitosanase gene from the isolated strain Bacillus sp. KFB-C108 was identified on the basis of a phylogenetic analysis of the 16S rRNA gene sequence, and was cloned into plasmid pUCl8 using E. coli $DH5\alpha$ as the host strain. Positive clones carrying recombinant plasmids (pKCHO I and pKCHO II) containing chitosanase activity were selected using the direct activity staining method. Detailed physical maps showed the two plasmid inserts were identical except that the KCHO II insert (2.6 kb) was 1.8 kb smaller than that of the KCHO I. The recombinant plasmids were analyzed to determine the essential region for chitosanase activity, and a 1.3-kb fragment (KCHO-6) was subcloned into pTrc99A using the EcoRI and BamHI sites to construct pTrc99A/KCHO-6(pTrEB13). The resulting plasmid exerted high chitosanase activity upon transformation of E. coli $DH5{\alpha}cells$, overproducing about 20 times more in the cloned cells than in the wild-type cells. The cloned chitosanase protein exhibited the same molecular weight and catalytic activity similar to those of Bacillus sp. KFB-C108. The cloned enzyme was an endo-type that produced a chitosan tetramer as the major reaction product; however, it produced no monomers or dimers.

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