Enhancing Chitosanase Activity via Directed Evolution and Its Molecular Mechanisms

Directed evolution is a powerful strategy to enhance enzyme performance. In this study, we optimized the rationally designed chitosanase SsCsn46 (mSsCsn46) from <i>Streptomyces</i> sp. N174 by fusing it with the <i>Escherichia coli</i> OmpA signal peptide and subjecting it to directed evolution. The resulting variant M1, carrying 18 mutations, and the enzyme activity reached 1680 U/mg, an 812-fold increase over mSsCsn46. With continuous substrate feeding for 3 h, M1 produced 142 g/L of chitooligosaccharides (COS) at 89.9% conversion. Molecular dynamics simulations revealed that the mutations stabilized Asn157/GlcN-3 interactions and expanded the substrate-binding pocket (1483.8 ų). When applied to fungal chitosan extracted from <i>Aspergillus niger</i> ATCC 1015, M1 converted chitosan to COS within 30 min with 95.1% conversion. This work demonstrates how directed evolution can remodel chitosanase for highly efficient COS production, providing an effective enzymatic tool for industrial production.