In-silico site-directed mutagenesis and MD simulation analysis to enhance the potential of symbiont fungal chitinase of Beauveria bassiana for bioinsecticide development

The use of microbial insecticides is a promising approach to circumvent the toxic effects of chemical insecticides due to their eco-friendly nature and significant effectiveness. Beauveria bassiana strain ARSEF 2860 is a commercially used bioinsecticide that lives in a symbiont association with a variety of plants or crops. The insecticidal mechanism of this fungal strain is initiated by chitinases that degrade the chitin layer of the insects. Among these chitinases, a significant number of chitinases lack a distinct chitin-binding domain and thus have compromised catalytic efficiency. Engineering of these chitinases to enhance the chitin-binding can be a potential approach to develope high potential bioinsecticides. Present study deals with analysis of 96 mutants of the J5JGB8 chitinase of B. bassiana strain ARSEF 2860 to improve chitin-binding in the substrate binding cavity. In-silico site-directed mutagenesis revealed 30 mutations as stable, having an effective change in Gibb's free energy. Molecular docking of J5JGB8 chitinase and all stable mutants with chitin subunit proved significantly high negative binding energy of Ala127Ser mutant (−8.24 kcal/mol) compared to the wild-type enzyme (−6.75 kcal/mol). Molecular dynamic simulation analysis of Ala127Ser chitinase-chitin and wild-type chitinase-chitin complexes revealed higher number of hydrogen bonding in Ala127Ser chitinase-chitin complex, displaying high stability of chitin-binding in the substrate binding cavity of the mutant. End state free binding energy analysis showed effective change in electrostatic energy of the interactions stabilizing the binding of chitin at the substrate binding site of the Ala127Ser mutant J5JGB8 chitinase with respect to wild-type confirming improved binding of chitin with the mutant chitinase. Hence, this study provides a beneficial Ala127Ser mutant form of J5JGB8 chitinase that can itself be developed in to an effective bioinsecticide or may be used to enhance the potential of B. bassiana strain ARSEF 2860 bioinsecticide using enzyme engineering approach to encourage agricultural sustainability.