Cloning, expression and antibacterial functions of ChiKJ40, a chitinase gene from Streptomyces sampsonii

Abstract:

[Background] There are very rare researches on the biocontrol genes of Streptornyces sampsonii until now. Only two chitinase gene fragments were cloned, and there is no relevant study in the complete gene sequence of the chitinase. [Objective] To clone and prokaryotic expression of the chitinase gene ChiKJ40 of S. sampsonii KJ40, then purify recombinant protein and investigating antibacterial characters. [Methods] Firstly we cloned the chitinase gene ChiKJ40 from S. sampsonii KJ40 by PCR amplification, then ligated it into vector pET-32a and expressed in Escherichia coli BL21(DE3). We purified the recombinant chitinase using the His-tagged protein microscopy kit, determined the concentration of the crude enzyme solution and the purified enzyme solution by the Bradford protein concentration assay kit, measured the chitinase activity of the crude enzyme solution and the purified enzyme solution using the chitinase kit. Finally also checked antibacterial characters of the recombinant chitinase on the Cylindrocladium scoparium, Cryphonectria parasitica, Alternaria alternata and Rhizoctonia violacea. [Results] We induced expression of the ChiKJ40 gene (accession number: MF434484) through IPTG in E. coli, the recombinant chitinase size is 42 kD. There are no significant differences in production of protein using different concentrations of IPTG at 37 °C inducing 3 h. 0.2 mmol/L IPTG inducing 16 °C overnight, recombinant chitinase mainly existed in the form of soluble supernatant, small existed in inclusions precipitation. The chitinase activities of crude protein and purified protein were 0.080 U/mL and 0.046 U/mL, respectively. The specific activity of crude protein and purified protein were 0.041 U/mg and 0.115 U/mg, the purification ratio was 2.8, the rate of 57.5%. After treating with the purified protein, mycelium cells of C. scoparium, C. parasitica, A. alternata were segmented with inflating the mycelia, and myceliums of R. violacea were broken. [Conclusion] This study of ChiKJ40 provides biocontrol background of S. sampsonii and finds a new source for the chitinase genes, and lays a theoretical foundation for its application.