Chitooligosaccharides (COS) is a kind of functional carbohydrates with great application potential as its various biological functions in food, cosmetics, and pharmaceutical fields. Exploring the relationship between structure and function of chitosanase is essential for the controllable preparation of chitooligosaccharides with the specific degree of polymerization (DP). GsCsn46A is a cold-adapted glycosyl hydrolase (GH) family 46 chitosanase with application potential for the controllable preparation of chitooligosaccharides. Here, we present two complex structures with substrate chitopentaose and chitotetraose of GsCsn46A, respectively. The overall structure of GsCsn46A contains nine α-helices and two β-strands that folds into two globular domains with the substrate between them. The unique binding positions of both chitopentaose and chitotetraose revealed two novel sugar residues in the negatively-numbered subsites of GH family 46 chitosanases. The structure–function analysis of GsCsn46A uncovers the substrate binding and catalysis mechanism of GH family 46 chitosanases. Structural basis mutagenesis in GsCsn46A indicated that altering interactions near +3 subsite would help produce hydrolysis products with higher DP. Specifically, the mutant N21W of GsCsn46A nearly eliminated the ability of hydrolyzing chitotetraose after long-time degradation.
Chitosanase plays an important role in the production of chitooligosaccharides (CHOS), which possess various biological activities. Herein, a glycoside hydrolase (GH) family 46 chitosanase-encoding gene,csnB, was cloned from marine bacteriumBacillussp. BY01 and heterologously expressed inEscherichia coli. The recombinant chitosanase, CsnB, was optimally active at 35 °C and pH 5.0. It was also revealed to be a cold-adapted enzyme, maintaining 39.5% and 40.4% of its maximum activity at 0 and 10 °C, respectively. Meanwhile, CsnB showed wide pH-stability within the range of pH 3.0 to 7.0. Then, an improved reaction condition was built to enhance its thermostability with a final glycerol volume concentration of 20%. Moreover, CsnB was determined to be an endo-type chitosanase, yielding chitosan disaccharides and trisaccharides as the main products. Overall, CsnB provides a new choice for enzymatic CHOS production.Keywords:chitosanase;Bacillussp. BY01;cold-adaption
危险品化学品经营许可证（带存储）