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

) 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.