Computer-Based Design to Improve Bacillus thuringiensis Chitinase for Industrial Applications

Chitin serves as a vital biopolymer across multiple industrial biotechnology sectors, and its extraction from seashell waste through enzymatic bioconversion offers an efficient and environmentally sustainable approach. Chitinase plays a key role in this process; however, strategies to enhance its catalytic performance remain limited. In this study, in silico site-directed mutagenesis was employed to improve the enzyme's activity while preserving its structural stability. Mutation sites were identified within the catalytic domain based on high mutability scores and functional relevance. Six hotspot mutations V215A, V215F, S262G, R264H, F288L, and G291A were selected for further evaluation. Interaction analyses revealed that the beneficial mutants demonstrated stronger substrate affinity compared to the native enzyme. Furthermore, molecular orbital analysis indicated that substrate binding to these mutants, particularly the G291A variant, exhibited enhanced nucleophilic and electrophilic characteristics. Notably, the G291A mutant showed reduced activation energy of 49.53 kcal/mol, in contrast to 97 kcal/mol for the native enzyme, signifying improved catalytic efficiency. Overall, this study highlights the potential of computationally guided site-directed mutagenesis as a rational approach for optimizing chitinase activity and advancing enzyme engineering for bioconversion applications.