Crop protection against a variety of pests using chemical pesticides is a common practice in agriculture to maintain the quality and increase the yield of crops. However, the development of insecticide resistance and non-specific toxic effects associated with chemical pesticides and insecticides make it challenging to fulfil the goals of sustainable agriculture [1]. The continuous use of chemical insecticides leads to their accumulation in different environmental niches resulting in toxic concentrations that pose harmful effects to non-target organisms including humans. The utilization of biopesticides or bioinsecticides has been procured as an effective approach to deal with this toxicity and mediate effective pest control [2,3].

At present 390 biopesticide active ingredients are in agricultural use which are categorized as biochemical pesticides (natural substances for pest control having a non-toxic nature), plant-incorporated protectants (substances produced by genetically modified plants), and microbial pesticides (microbes as active ingredients) [4]. Several bacterial and fungal strains have been identified and utilized as biopesticides or bioinsecticides that infect specific insects without posing any harmful off-target effects. These microbial strains live in symbiotic association with crops, providing protection to crops and procuring nutrition from them [2,3].

However, several microbial strains and their enzymes have been identified as effective biopesticides, but their low efficiency poses a great challenge. Though some studies have identified potential mutations in biopesticide enzymes that can enhance their virulence against insects for the development of the engineered enzymes or strains that can be used as efficient biopesticides [5,6], such engineered biopesticides have not yet landed in agricultural fields.

Chitin, an β-1,4-linked anhydro-2-acetamido-2-deoxy-D-glucose or N-acetyl glucosamine (NAG)amino sugar polysaccharide that forms fibrillar structures, is a major component of insect cuticle [7]. Beauveria bassiana, the second most common microbial pesticide after Bacillus thuringiensis is effective against around 200 insect species [5]. The mechanism of infection is initiated by penetration of appressoria, the infection structures produced by fungal hyphae, into the insect cuticle by utilizing enzymes that can degrade the cuticle such as proteases, lipases, and chitinases. This is followed by amplification of infection structures and secretion of toxins or secondary metabolites like beauvericin, beauverolides, bassianin, tenellin, oxalic acid, bassianolide, oosporein, and calcium oxalate crystals [8].

Chitinases play a key role in degrading the chitin present in insect cuticles and, thus are effective candidates in mediating plant protection against insects. Different strains of Beauveria bassiana species contain chitinases majorly classified under GH-18 family proteins containing TIM barrel or (α/β)8 barrel fold. These chitinases are further classified into A, B, and C subgroups on the basis of differences in substrate-binding cleft architecture, which contribute towards their catalytic potential [6]. The amino acids mediating the substrate binding are located on the loops emerging from the barrel fold. The catalytic mechanism includes the glutamate residue, which protonates the glycosidic bond of the substrate and the nucleophile oxygen in −1 sugar moiety N-acetyl group that forms the intermediate oxazolinium ion. Thus, binding of the sugar residue at the −1 position of chitin in chitinase is a determining step in the catalysis of chitin cleavage and decides the efficiency of the insecticidal effect mediated by different Beauveria bassiana strains containing different chitinases [8,9]. B. bassiana strain ARSEF 2860 is a commercially used biopesticide against planthoppers, aphids, and spider mites and contains 23 chitinases or chitinase-like proteins, out of which J5JGB8 chitinase is a member of a major group of chitinases having significant sequence similarity [10].

In the present study, we used an in-silico site-directed mutagenesis, molecular docking, and molecular dynamic simulation approach to identify the best mutation, Ala127Ser, which increases the binding affinity for chitin to enhance the catalytic activity of the J5JGB8 chitinase of B. bassiana strain ARSEF 2860. The Ala127 residue is conserved among 5 chitinases present in a major chitinase group of 7 enzymes (consisting of J5JGB8 chitinase) lacking a well-defined chitin-binding domain. This study provides the best mutant chitinase to develop an engineered enzyme-based bio-insecticide or the mutated chitinase containing B. bassiana strain for utilization in agriculture fields to enhance the insecticidal effect and promote sustainable agriculture.