Site-directed mutagenesis and semi-rational design to enhance chitinolytic activity of bacterial exochitinase

Chitin, a prevalent biopolymer found in arthropod exoskeletons and fungal cell walls, requires efficient depolymerization to provide bioactive derivatives suitable for agriculture, medicine, and wastewater treatment. Bacterial chitinases, such as the exochitinases produced by Paenibacillus barengoltzii, are potential biocatalysts for chitin recycling; however, they exhibit insufficient activity and stability for industrial use. In this study, Semi-rational engineering and site-directed mutagenesis were employed to enhance catalytic efficiency, resulting in five mutants: M1 (D344Y), M2 (D192Y), M3 (D144Y), M4 (D534G), and M5 (D192A). M3 and M5 significantly increased chitinolytic activity, boosting it by 338% and 276%, respectively, compared to the original enzyme. Further enhancement was achieved by incorporating a chitin-binding domain (CBD), resulting in a six-fold increase in activity with the M35-CBD chimaera. Molecular dynamics (MD) simulations were performed on wild-type, M3, and M5 variations to elucidate the structural basis for these enhancements. M3 (D144Y) exhibited greater structural stability, less RMSD fluctuations, and an improved hydrogen bond network in the substrate-binding region. Notably, enhanced π-stacking interactions facilitated by solvent-exposed aromatic residues, including tryptophan, improved substrate affinity. Unlike the wild-type enzyme, the produced mutants exhibited reduced conformational mobility alongside increased substrate interaction. The results demonstrate the efficacy of targeted mutagenesis and domain engineering in creating stable chitinases for industrial and environmental biotechnology applications.