β-N-acetylglucosaminease mutants with improved thermal stability and catalytic performance, their screening methods and applications

Abstract

This invention provides β-N-acetylglucosaminease mutants with improved thermal stability and catalytic performance, along with their screening methods and applications, relating to the field of enzyme engineering. This invention comprehensively utilizes X-ray crystal structure, AlphaFold2 and AlphaFold3 predicted structures, combined with ΔΔG... fold Computational analysis, sequence conservation analysis, B-factor flexible residue screening, and disulfide bond engineering were used to screen and combine beneficial mutations, resulting in the combinatorial mutant M9. Compared to the cbHEX synergistic system, the M9 synergistic system showed a 26.7-fold increase in monosaccharide yield and a 28% increase in crystalline chitin degradation rate. The strategy described in this invention can significantly improve the thermal stability and catalytic performance of the target enzyme and can provide a methodological reference for the rational modification of enzyme molecules.

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