|
KMID : 0545119990090050631
|
|
Journal of Microbiology and Biotechnology
1999 Volume.9 No. 5 p.631 ~ p.636
|
|
|
Cloning and Expression of Thermostable Chitosanase Gene from Bacillus sp.KFB-C108
|
|
Kim, Hee Yun
Yoon, Ho Geun/Kim, Hye Kyung/Kim, Kyung Hyun/Hwang, Han Joon/Cho, Hong Yon
|
|
|
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 pUC18 using E. coli DH5α 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 DH5α 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.
|
|
|
KEYWORD
|
|
|
|
|
|
FullTexts / Linksout information
|
|
|
|
|
|
Listed journal information
|
|
  
|