Cloning and expression of thermostable chitosanase gene from Bacillus sp KFB-C108

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 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(pTrEB 13). The resulting plasmid exerted high chitosanase activity upon transformation of E. coli DH5a 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.