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 Streptomyces sp. N174 by fusing it with the Escherichia coli 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 Å3). When applied to fungal chitosan extracted from Aspergillus niger 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.