Enhancing Thermostability of a Highly Active Chitinase Chi1 Through Semirational Design for Efficient Chitin Hydrolysis

In this study, a semirational design strategy integrating sequence and structural analyses was utilized to improve the thermostability of the highly active chitinase Chi1. Comprehensive screening for thermostability and enzymatic activity led to the identification of a beneficial mutant, D615S. Thermostability evaluations demonstrated that the half-life of the D615S mutant at 40 and 45 °C was prolonged by 3.6-fold and 24-fold, respectively, compared to that of the wild-type (WT) enzyme, while its melting temperature (<i>T</i>_m) was increased by 6.0 °C. Molecular dynamics (MD) simulations demonstrated that the D615S mutant significantly reduced local structural fluctuations and improved overall structural stability compared to the WT enzyme, leading to a remarkable enhancement in thermostability. Furthermore, the D615S mutant sustained the efficient enzymatic degradation of chitin powder via an integrated affinity adsorption-enzymatic catalysis approach, achieving a degradation rate of 50.3% within 120 h. Subsequent separation and purification processes yielded <i>N</i>-acetylglucosamine (NAG) and its dimer (NAG₂) with purities exceeding 95%. These findings underscore the potential of the D615S mutant for industrial applications, particularly in the field of chitin degradation.