Semirational Engineering of Chitinase Chi1 to Enhance the Catalytic Activity for Efficient Chitin Degradation

Efficient enzymatic depolymerization of chitin is often limited by the high crystallinity and low accessibility of the native substrates. Here, a semirational protein design was applied to improve the catalytic efficiency of the GH18 Chitinase Chi1 from Chitinibacter sp. GC72 by targeting residues in the substrate-binding channel. Four residues (Lys440, Trp467, Tyr499, and Arg527) were identified as potential functional hotspots and subjected to alanine scanning and site-saturation mutagenesis. Among the resulting variants, mutant Y499C exhibited significantly enhanced catalytic performance, showing 42% and 83% higher specific activities toward colloidal chitin and crystalline chitin, respectively, compared to the wild type. Kinetic analysis revealed that the V_max and catalytic efficiency (k_cat/K_m) of Y499C increased to 1.57-fold and 2.29-fold relative to the wild-type enzyme. In addition, Y499C achieved higher conversion than that of the wild type, reaching 29.5% for crystalline chitin and 20.45% for colloidal chitin. Molecular simulations indicated that the Y499C substitution reshaped the substrate-binding channel, stabilizing the enzyme-substrate complex and potentially improving the substrate accessibility within the catalytic cleft. These findings demonstrate that substrate-channel hotspot engineering is an effective strategy for enhancing GH18 chitinases and provide insights for developing efficient biocatalysts for chitin valorization.