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In the present study, we carried out a comprehensive investigation of glycoside hydrolase (GH) 46 model-chitosanases based on cleavage specificity classification to understand their unknown bifunctional activity. We for the first time show that GH46 chitosanase CsnMHK1 from Bacillus circulans MH-K1, which was previously thought to be strictly exclusive to chitosan, can hydrolyze both chito- and cello-substrates. We determined the digestion direction of bifunctional chitosanase CsnMHK1 from class III and compared it with class II chitosanase belonging to GH8, providing insight into unique substrate specificities and a new perspective on its reclassification. The results lead us to challenge the current understanding of chitosanase substrate specificity based on GH taxonomy classification and suggest that the prevalence from the common bifunctional activity may have occurred. Altogether, these data contribute to the understanding of chitosanase recognition and hydrolysis toward chito- and cello-substrates, which is valuable for future studies on chitosanases.

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Chitooligosaccharides (COSs) and D-glucosamine (GlcN), the most valuable biomolecules due to their various physiological functions, can be available inexpensively from chitosan by bio-enzymatic degradation, in which chitosanases play a key role. On the other hand, pretreatment of chitosan is the necessary procedure in the bioproduction by enzymatic hydrolysis. Therefore, the green and efficient pretreatment strategy as well as superior enzyme activity are the most critical aspects in scaled up production. This study investigated environmentally friendly and efficient enzymatic hydrolysis of powdery chitosan (PC) pretreated by ball-milling. First, heterologous expression yielded OUC-CsnPT, a glycoside hydrolase family 46 chitosanase with a predominant specific activity of 5346.56 U/mg at pH 6.0°C and 45°C. The endo-type chitosanase OUC-CsnPT hydrolyzed chitosan to produce GlcN and chitobiose [(GlcN) 2 ]. Then, ball-milling was employed to pretreat PC to promote chitosanase OUC-CsnPT hydrolysis, and the effectiveness was characterized by scanning electron microscope, X-ray diffraction, and FTIR-attenuated total reflection. Through the development of the novel route, the yield of COSs and GlcN for ball-milling powdery chitosan increased by 10.8-folds with a concentration of 14.5 μmol/ml. Hence, this technique presents a promising strategy suited to be a straightforward and environmentally friendly option for oligosaccharides production, broadening the theoretical basis for PC biodegradation.

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.

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