ZYMOGRAPHIC IDENTIFICATION AND BIOCHEMICAL CHARACTERIZATION OF CHITINASE AGAINST PHYTOFUNGAL PATHOGENS

Abstract

An endospore forming Gram positive bacterium (MBCU4) was isolated from a vermicompost amended soil, and confirmed as Bacillus subtilis through the 16S rRNA sequence analysis. An extracellular chitinase was detected from this strain of B. subtilis under specific environmental condition. An attempt was made to purify the enzyme by ammonium sulfate precipitation followed by DEAE sepharose CL-6B column chromatography. The purified enzyme was demonstrated as a single band, having the molecular weight 31kDa on SDS PAGE analysis and its activity in the gel was determined by clear zone on zymogram. Further characterization of the isolated enzymes has showed that this enzyme is most active at pH 6.0 and at the optimized temperature of 50 0C. The purified chitinase exhibited high degree of antifungal activity particularly by degrading their cell wall components of plant pathogens Macrophomina phaseolina (69.0%) and Rhizoctonia solani (52.0%). It infers that the chitinase produced by B. subtilis could play an important role for biopesticidal activity.