Cloning, Expression And Characterization Of The Chi36 Gene From Bacillus Thuringiensis 010

chi36 was amplified by PCR from total DNA of Bacillus thuringiensis 010 as templates. PCR product was purified, cloned into pMD18-T and sequenced. It included an open reading frame of 1083 bp, encoding a protein of 360 residues with a calculated molecular weight of 39.0 kDa and a predicted pI value of 6.52. BLAST comparisons showed that the nucleotide sequence of Bt 010 chi36 had 97%, 99%, 99%, 93% homology with Bt 15A3, Bc CH, Bc 28-9, Bc 6E1 respectively. ScanProsite analysis revealed that Chi36 belong to family 18 of glycosyl hydrolases, classified as an exochitinase,and only contained catalytic domain, without FLDs and CBD。The recombinant expression plasmid pGEX-chi36 was constructed by insertion of chi36 gene into the expression vector pGEX-KG, and then was transformed into E. coli BL21 (DE3) to express the Chi36 under the induction of 1.0 mmol/L IPTG. SDS-PAGE showed that the molecular weight of the expressed fusion protein was about 65 kDa, including 26 kDa GST carrier protein. Bioactivity assay indicated that the expressed fusion protein extracted from the culture medium can digest chitin, with the optimal pH and temperature at 5.0 and 55℃respectively.