Two-step purification and characterization of chitinase from Bacillus cereus V5331: evaluation of antagonistic potential of enzyme against fruit juice contaminant Saccharomyces cerevisiae.

Abstract

Chitinases are gaining importance as biocatalysts for environmentally friendly food preservation and biocontrol. The present study reports on the purification and biochemical characterization of a chitinase from Bacillus cereus V5331 and its anti-yeast activity in grape juice. The enzyme was purified by Sephadex G-100, leading to 10.3-fold purity and a specific activity of 2951 U mg^-1. The molecular weight of ∼54 kDa was confirmed by SDS-PAGE and zymogram analysis. The chitinase showed optimum activity at pH 7.0 °C and 37 °C. Substrate specificity tests exhibited highest activity toward swollen chitin. Kinetic analysis also revealed the lowest K_m (0.023) and V_max of 77.5 for swollen chitin, showing good substrate affinity and catalysis. Metal ions, halogens, organic solvents, and essential oils greatly influenced enzyme activity, indicating the enzyme's sensitivity to environmental factors. A high-performance liquid chromatography (HPLC) study showed NAG as the major hydrolysis product of chitin. Purified chitinase was found to be effective in suppressing the growth of Saccharomyces cerevisiae in grape juice, resulting in a ∼5-log decline in viable cell counts. This was followed by a time-dependent rise in NAG release as a marker of increasing cell wall degradation. The obtained results suggest the possible applications of B. cereus V5331 chitinase as a natural biocontrol agent in food preservation systems. Furthermore, the availability of a functionally active enzyme increases its potential for industrial and agricultural applications, such as long-term bioconversion of chitinous waste, and simplifies downstream processing.