Cloning Of A Chitosanase Genetic Fragment And Construction Of The Expression Vector

Chitosanases (EC 3.2.1.132) could catalyse the hydrolysis of glycosidic bonds in chitosan to obtain chitosan oligomers which is widely used in medicine and dietary supplement, food and other industrial areas. In this paper two chitosanase-producing strains were screened from Dalian sea waters mud and a chitosanase gene was amplified, its expression vector in E. coli was also built. The main conclusions include:Two chitosan enzyme producing strains were screened from Dalian sea waters mud and one strain was determined as bacillus genus by 16S rDNA comparison and it has close relatives with bacillus anthracis.A pair of specific primers was designed; one 500 bp DNA fragment was amplified from Bacillus anthracis W-2 genome. After lignated with pMD20-T and transformed into the competent E.coli DH 5a cells,the recombinant plasmid was identified and sequenced, the fragment was 486 bp, compared with bacillus genus chitosanase sequence alignment analysis coding area published on NCBI, and it was considered as chitosanase gene fragment. The combined plasmid was named pMD20-T-chiW. The chiW gene was cloned into pET28a and transformed into E.coli BL21(DE3) to express chitosanase which induced by IPTG. The SDS-PAGE electrophoresis showed the expressed target protein was between 66.4KD to 43.3KD.The sequence of chiW was also analyzed by blasting with other coding sequences from Bacillus.sp. Results indicated that it showed 41.75% sequence identity with coding sequence of Bacillus subtilis and 45.93% with Bacillus sp. KFB-CO4, and 52.91% with coding Bacillus sp.No.S-1. Thus we concluded that the fragment was similar to chitosanase gene.the characterics of the putative protein of chiW were:its formula was C809H1181N201O209S13, Molecular weight was 17,483.2Da, Theoretical pI was 8.63, The N-terminal of the sequence considered was M (Met),The putative protein was unstable, hydrophilic, and had 21.48% Helix,38.93% Strand and 39.06% Loop.