Characteristics of chitinase B from Bacillus thuringiensis and its potential in antagonism and insecticidal enhancing effects

Abstract:

Abstract: [Objective] In this study we investigated characteristics of ChiB (chitinase B) from Bt 15A3(Bacillus thuringiensis subsp. colmeri 15A3) and evaluated its potential in antagonism and insecticidal enhancing effects. [Methods] The ChiB was expressed in E.coli(Escherichia coli). Expression product was purified by ammonium sulfate precipitation, dialysis and Sephadex G-200 gel filtration chromatography. The molecular mass of ChiB was directly estimated by zymogram after SDS-PAGE(sodium dodecyl sulfate-polyacryamide gel electroresis). The proteins with different molecular weights were identified by MS(mass spectrum) and analyzed through bio-informatics. We studied some kinds of metal ions’ impacts on the chitinase activity; the optimal temperature, pH and its stability in different temperature and pH. We also studied the inhibition effects on sporangia germination of fungi and the synergistic effects of ChiB on larvicidal activity. [Results] The molecular mass of ChiB was estimated 70 kDa by zymogram. The protein with 64 kDa was the product of C-terminal degradation of ChiB in E.coli. The chitinase activity was improved by Fe3+ and inhibited by Zn2+, Ag2+. The optimal temperature for ChiB was 60 ℃, whereas, the optimal pH was 5.0. The enzyme was quite stable at temperature less than 60 ℃ and pH between 4.0 and 8.0. ChiB inhibited sporangia germination and the IC50 (50 % inhibited concentration) was about 4 μg/mL. Moreover, the bioassays showed that ChiB could reduce the LC50 (50 % lethal concentration) of the crystal protein of Bt. against S.exigua and H.armigera larvae by approximately 26 % and 30 % respectively.[Conclution] ChiB not only had stable characteristics, but also had good antagonism activity and insecticidal enhancing effects.