Study On Chitinase From Bacillus Thuringiensis

Bacillus thuringiensis (Bt) is the most important insecticidal microbe in the field of biological control. In 1990's, B. thuringiensis-based products represent more than 90% of the biopesticides used worldwide. Chitinase can hydrolyze chitin, the main component of the wall of cells of fungi and peritrophic membrane of insects. Study on Bt chitinase is important to Bt preparation for it can increase the effect of biopesticides. 4 Bt strains producing chitinase,WB?,WB?6,WB--32 and WB?0, were screened in our lab by chitin plate. WB?0 can produce more chitinase than the others. The results of the study on its synthetic conditions showed that the most chitinase was synthesized when WB?0 was cultured in a basic medium containing 2. 0% chitin and 1. 0% yeast extract for 72 hours (22Orpm).The optimal pH and temperature are 7.0 and 300C, respectively. The gene of the Bacillus cereus?chitinase B and Bt subsp. pakistani?chitinase in GenBank, named chiB and chi respectively, showed high homology. With a pair of primers designed according to these sequence,a part of chitinase gene of WB?0 was obtained by PCR. The product of PCR,864bp in length, was purified by the kit from Boehringer Mannheim Company. Sequence analysis by the software of Blast on line showed that the nucleotide sequence homology between it and the corresponding part of cbiB was more than 99%.Using the specific primers , 18 of 22 Bt strains were found harboring the chitinase gene . The QCL1 and QCL2, primers designed for full-length chitinase gene,have a site for Baizkl I. The full-length chitinase gene, named chiA, was obtained by PCR with QCL1 & QCL2 and was proved by Southern hybridization. A recombinant plasmid, named pT-CHT, was constructed by the vector pGEM-T and c/i/A. A prokaryotic expression plasmid p2T-CHl containingthech/A and vector pGEX?T, which produced fusion proteins of 118KD (ChiA' GST) in E. coli, was obtained0...