Biochemical Characterization of a GH46 Chitosanase Provides Insights into the Novel Digestion Specificity

Endo-chitosanases (EC 3.2.1.132) are generally considered to selectively release functional chito-oligosaccharides (COSs) with degrees of polymerization (DPs) ≥ 2. Although numerous endo-chitosanases have been characterized, the digestion specificity of endo-chitosanases needs to be further explored. In this study, a GH46 endo-chitosanase OUC-CsnPa was cloned, expressed, and characterized from Paenibacillus sp. 1-18. The digestion pattern analysis indicated that OUC-CsnPa could produce monosaccharides from chitotetraose [(GlcN)₄], the smallest recognized substrate, in a random endo-acting manner. Especially, the enzyme specificities during chitosan digestion including the regulation of product abundance through a transglycosylation reaction were also evaluated. It was hypothesized that an insertion region in OUC-CsnPa may form a strong force to be involved in stabilizing (GlcN)₄ at its negative subsite for efficient hydrolysis. This is the first comprehensive report to reveal the digestion specificity and subsite specificity of monosaccharide production by endo-chitosanases. Overall, OUC-CsnPa described here highlights the previously unknown digestion properties of the endo-acting chitosanases and provides a unique example of possible structure-function relationships.