Structure-Assisted Design of Chitosanase Product Specificity for the Production of High-Degree Polymerization Chitooligosaccharides.

Chitosanases are valuable enzymatic tools in the food industry for converting chitosan into functional chitooligosaccharides (COSs). However, most of the chitosanases extensively characterized produced a low degree of polymerization (DP) COSs (DP = 1-3, LdpCOSs), indicating an imperative for enhancements in the product specificity for the high DP COS (DP >3, HdpCOSs) production. In this study, a chitosanase from Methanosarcina sp. 1.H.T.1A.1 (OUC-CsnA4) was cloned and expressed. Analysis of the enzyme-substrate interactions and the subsite architecture of the OUC-CsnA4 indicated that a Ser49 mutation could modify its interaction pattern with the substrate, potentially enhancing product specificity for producing HdpCOSs. Site-directed mutagenesis provided evidence that the S49I and S49P mutations in OUC-CsnA4 enabled the production of up to 24 and 26% of (GlcN) ₅ from chitosan, respectively─the wild-type enzyme was unable to produce detectable levels of (GlcN) ₅ . These mutations also altered substrate binding preferences, favoring the binding of longer-chain COSs (DP >5) and enhancing (GlcN) ₅ production. Furthermore, molecular dynamics simulations and molecular docking studies underscored the significance of +2 subsite interactions in determining the (GlcN) ₄ and (GlcN) ₅ product specificity. These findings revealed that the positioning and interactions of the reducing end of the substrate within the catalytic cleft are crucial factors influencing the product specificity of chitosanase.