Improving the thermostability of Cyclobacterium marinum chitin deacetylase by disulfide bond engineering

Chitin deacetylase (CDA) is a promising biocatalyst for synthesizing glucosamine (GlcN), widely used in food and biomedical industries. The main issue limiting its industrial application is its instability. In this study, disulfide bridges were used to enhance the thermostability of Cyclobacterium marinum chitin deacetylase (CmCDA), which specifically and efficiently hydrolyzes N-acetylglucosamine (GlcNAc) to GlcN. Compared to the wild-type CmCDA, variants S44C/D68C and T138C/W279C exhibited less residual activity, whereas variant A58C/ F133C demonstrated a 57% improvement after thermo-treatment at 50 degrees C. At 50 degrees C, the half-life value of variant A58C/F133C was 2.6 times that of the wild-type, and half-inactivation temperature T3050 was a 7.5 degrees C higher than that of the wild-type. Dynamic simulation (MD) analysis was used to explain the improved thermostability of variant A58C/F133C. After 48 h, A58C/F133C produced approximate 1.23-fold of GlcN. This study preliminarily investigated the effect of disulfide bridge in thermostable chitin deacetylase and suggested a feasible way to improve the thermostability of chitin deacetylases, encouraging their use in biocatalytic glucosamine synthesis.