Partial purification and characterization of chiIO8, a novel antifungal chitinase produced by Bacillus cereus IO8

Abstract

To investigate the distribution of chitinase IO8 in Bacillus cereus strains, the enhancing effects of the chitinase-producing B. cereus strains on biocontrol potential by dual culture assay and in vivo assay against Botrytis cinerea and also the enhancing effects of the chiIO8 on disinfectant properties against seed-borne diseases. Moreover, the application of chiIO8 treatment was also observed to improve the germinative energy. The purification steps included ammonium sulfate precipitation, with columns of DEAE-Sepharose anion-exchange chromatography and Sephacryl S-400 high-resolution gel chromatography. The method gave a 5.8-fold increase in the specific activity and had a yield of 17%. The molecular weight of the partially purified chitinase chiIO8 was found to be around 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and optimal temperature of the partially purified chitinase were pH 6.5 and 65°C, respectively. The thermostable chitinase still retained the activity after incubation for 100 min at 65°C, and it was increased about 1.25 times than that of the control (before heating) when the enzyme solution heated at 65°C for 60 min. The partially purified chitinase chiIO8 displays a wide inhibitory spectrum towards all phytopathogenic fungi tested. chiIO8 also exhibited effective disinfectant properties against seed-borne diseases. The present investigation emphasizes the potential of chitinase-producing micro-organism as promising biocontrol agents of fungal plant pathogens with chitinous cell wall. The novel chitinase chiIO8 proved an efficient, environmentally safe and user-friendly solution. This is the first investigation devoted exclusively to analyse the distribution of chitinase in B. cereus. It infers that the chitinase produced by B. cereus might play a role in the activity of the biopesticide.