Fusion expression of carbohydrate-binding module and linker peptide improves the product specificity of chitosanase

The efficacy of chitooligosaccharides (COS) is closely related to their degree of polymerization (DP), with higher-DP COS offering significant bioactivities and stability advantages. The objective of this study is to enhance the product specificity of chitosanase for producing higher-DP COS. In this study, we proposed a novel strategy to enhance the affinity and activity of the chitosanase Csn75 through the fusion of a carbohydrate-binding module CBM32 (specifically its DD1 subdomain) using rigid or flexible linkers of varying lengths. The fusion chitosanase with a 10-amino-acid rigid linker (Csn-E2-DD1) demonstrated optimal activity at 50 °C and pH 5.5, exhibiting a 60% increase in crude enzyme activity compared to the native Csn75. Kinetic analysis revealed its higher substrate affinity (K_m = 0.668 mg·mL^-1) and catalytic efficiency. The hydrolysis product profile shifted from DP 2-5 to DP 2-6, and a final yield exceeding 95% was obtained. This strategy offers a potential alternative for the bioproduction of desirable COS to meet the requirements of application in food systems, providing an efficient strategy for the targeted production of high-DP COS.