Chitin deacetylase hydrolyzes N-acetyl-D-glucosamine in chitin and chito-oligosaccharides, showing strong potential for industrial chitosan and chito-oligosaccharide production. Although a heat-resistant chitin deacetylase from Saccharomyces cerevisiae (ScCDA₂) has been previously reported, the molecular basis of its thermostability remains unclear. Here, we systematically investigated the thermal stabilization mechanisms of ScCDA₂ using variable temperature circular dichroism, site-direct mutagenesis, and differential scanning calorimetry. The results showed that ScCDA₂ retained about 50 % of its catalytic activity after heating, and circular dichroism analysis revealed incomplete refolding rather than full restoration of the native secondary structure. Removal of individual N-glycosylation sites significantly impaired both enzymatic activity and thermostability, with the most pronounced effects observed for N181 and N199. A parallel trend was noted upon disruption of a key salt bridge via R210 mutagenesis: while catalytic activity was retained in the mutant, its structural stability was markedly reduced. Intriguingly, even non-glycosylated sites such as N142 significantly affected ScCDA₂ performance, with N142Q showing severe destabilization and activity loss, highlighting its potential structural significance. The above experimental results indicate that both N-glycosylation and salt bridges are play important roles in maintaining the thermal stability of ScCDA₂. This work provides mechanistic insight into ScCDA₂ stability and offers a foundation for rational engineering of thermostable CDAs for industrial applications.

• PMID: 41475645
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Journal Article

Authors

Wei H, Xu Z, Song X, Zhu X, Liu Y, Li T, Yin H

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