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.