Characterizations Of Chitinases From Bacillus Thuringiensis Subsp Colmeri 15A3 And Identification Of Promoter Sequence Of ChiB

Bacillus thuringensis subsp.colmeri 15A3 (Bt.15A3) constitutively expresses two chitinases, namely, ChiA and ChiB. Bt.15A3 shows high insecticidal enhancing activity against Spodoptera exigua and Helicoverpa armigera.In order to reveal the biochemical characterization and insecticidal activity of ChiA and ChiB isolated from Bt.15A3, chiA and chiB are expressed in E. coli XL-Blue. The ChiA and ChiB are precipitated with 70%(NH4)SO4 dialysis and purified using Sephadex G-200 and their molecular mass are estimated to be 36 kDa and 70kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Detections of chitinase activity on SDS-PAGE after protein renaturation of ChiA and ChiB indicate that the molecular mass of ChiA of the protein band expressing chitinase activity is approximately 72 kDa. This suggests that the dimeric form of ChiA is the enzymatically active form when glycol chitin is used as a substrate. The molecular mass of ChiB of the protein band expressing chitinase activity is approximately 70 kDa. ChiA has optimal activity at 50℃and retains most of its activity between 20 and 60℃. The optimum pH for ChiA activity is pH 5.0, and the enzyme is active between pH 4.0 and 8.0. ChiB has optimal activity at 60℃and retains most of its activity between 20 and 60℃. The optimum pH for ChiB activity is pH 5.0, and the enzyme is active between pH 4.0 and 8.0. The effects of metal ions and other reagents upon ChiA and ChiB are diverse. Their enzyme activity is all significantly inhibited by Ag+ and Zn2+. The spore germination of four species of fungi are significantly inhibited by ChiA and ChiB. The median inhibitory concentrations (IC50) of ChiA on the spore germination of Penicillium glaucum and Sclerotinia fuckelian are 11.27μg/mL and 10.57μg/mL, respectively. The median inhibitory concentrations (IC50) of ChiB on the spore germination of P. glaucum and S. fuckelian are 4.12μg/mL and 4.02μg/mL, respectively. The inhibition effect of ChiA is lower than that of ChiB. In surface contamination bioassays, the crude ChiA protein (12.6 mU) reduces the LC50 (50% lethal concentration) of the crystal protein of Bt.15A3 against the larvae of Spodoptera exigua and Helicoverpa armigera by 18% and 19.8% respectively. The crude ChiB protein (12.6 mU) reduces the LC50 of the crystal protein of Bt.15A3 against the larvae of S. exigua and H. armigera by 26% and 30% respectively.In order to reveal the C-terminal proteolytic degradation of ChiB in the culture of E. coli, the protein of 64 kDa were identified by Mass Spectrum (MS) and analyzed through bio-informatics. The protein identification is performed by peptide fragment fingerprinting. The result indicates that the protein with 64kDa is the product of C-terminal degradation of ChiB, and it lacks a part of chitin binding domain.We use the NCBI CCD database to analysis the schematic structure of ChiB. As the domain constrctions of ChiB, progressive C-terminal ChiB deleted derivatives which lacking a part or whole chitin binding domain(CBD) named Chi66, Chi64 and Chi60 with the molecular weight of 66 kDa,64 kDa and 60 kDa respectively are'constructed. The chitinase activity assay shows that Chi66, Chi64 and Chi60 exhibit catalytic activity. They can degrade colloidal chitin as ChiB does. The zymogram analysis shows that ChiB and its deletion derivatives can degrad MU-(GlcNAc)3 but can't degrad MU-(GlcNAc)2. These results indicate that ChiB is a endochitinase and the catalytic property of ChiB doesn't change with C-terminal degrations. The biochemical characterization and effects of metal ions and other reagents upon Chi66, Chi64 and Chi60 are diverse. Chi66 and Chi64 significantly inhibite the spore germination of four species of fungi. Chi60 which lacking the CBD have nearly no inhibition effects against the spore germination of P. glaucum and S. fuckelian. These results indicate that the CBD plays an important role in the antifungal activity of ChiB.In order to identify the promorter sequence of chiB, we use promoter prediction softwares on line to find out two different predicted promoter sequences:Promoter 1 and Promoter 2. To identify which promoter sequence directs the expression of chitinase gene, four deletions are constructed from the 5'end of chiB, named chiBΔ59, chiBΔ102, chiBΔ122 and chiBΔ248. These deletions are deleted 59bp, 102bp,122bp and 248bp from the 5'upstream sequence respectively. chiBΔ102 is deleted 102bp from the 5'terminal including the promoter 1. chiBΔ122 is deleted 122bp from the 5' terminal including the promoter 2. Then these deletions are cloned into T-vector and transformed into E. coli cells to express chitinase. The results of chitinase activity assay, SDS-PAGE and Northern blot analysis show that E. coli haboring chiBΔ102 express chitinase B at a low level, while both E. coli harboring chiBΔ122 and chiBΔ248 lose the expression of chiitnase. These results indicate that Promoter 2 is the right promoter region of chiB. The promoter region lies in the interval from-126bp to-166bp upstream of the translation start site (ATG is+1). The-10 region and the -35 region of the promoter region show high homology to the -10 region and the -35 region of E. coliσ70 promter.