New Insights into Bifunctional Chitosanases with Hydrolysis Activity toward Chito- and Cello-Substrates

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 thefirst time showthat GH46 chitosanase CsnMHK1 fromBacillus circulansMH-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 fromclass III and compared it with class II chitosanase belonging to GH8, providing insight into unique substrate specificities and a newperspective on its reclassification. The results lead us to challenge the current understanding of chitosanase substrate specificity basedon 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

Automated summary

In this study, we investigated the bifunctional activity of GH46 chitosanases based on cleavage specificity classification. We found that CsnMHK1 from Bacillus circulans MH-K1, previously thought to only hydrolyze chitosan, can also hydrolyze cello-substrates. Comparison with GH8 chitosanase provided new insights into substrate specificities and challenged the current understanding of chitosanase classification. These findings contribute to our understanding of chitosanase recognition and hydrolysis, which is valuable for future studies.