Biochemical and Functional Characterization of a Novel GH46 Chitosanase for Efficient Chitooligosaccharide Synthesis

ABSTRACT Chitosan, a derivative of the abundant biopolymer chitin, holds significant biotechnological potential but is limited by its poor solubility. Its oligomeric forms, chitooligosaccharides (COSs), exhibit superior bioactivity and solubility, driving demand for efficient enzymatic production methods. This study reports the biochemical and functional characterization of a novel chitosanase, SlCsn46A, identified from Streptomyces lydicus M01 and classified into the glycoside hydrolase family 46 (GH46). The enzyme was successfully heterologously expressed in Escherichia coli and purified. SlCsn46A demonstrated high catalytic efficiency, with an optimal activity at 60°C and pH 6.0 and a maximum specific activity of 957.80 U·mg − 1 . It exhibited broad pH stability and significantly enhanced activity in the presence of Mn 2 + and Tween 80. Kinetic analysis revealed a low Michaelis constant ( K m = 0.61 mg·mL − 1 ), indicating strong substrate affinity. Product analysis confirmed its endo‐type action mode, specifically hydrolyzing chitosan to yield chitobiose [(GlcN) 2 ] and chitotriose [(GlcN) 3 ] as the predominant end products. These properties collectively establish SlCsn46A as an efficient and specific biocatalyst, demonstrating great potential for the targeted and efficient synthesis of low‐degree‐of‐polymerization COS for applications in the food, agricultural, and biomedical industries.