Reprogramming chitosanase specificity via a structure-guided engineering and high-throughput screening platform

Natural chitosanases typically produce heterogeneous chitooligosaccharides (COS), which limits the preparation of structurally defined carbohydrate products and hinders structure-activity studies and product-specific applications. Here we established an integrated platform that combines homology modeling, saturation mutagenesis, in situ TLC-based high-throughput screening, and fluorescence binding analysis to reprogram Bacillus circulans MH-K1 chitosanase. Guided by subsite modeling, MNN-based biased saturation mutagenesis at A174/L175 near the (-2) subsite and insertion of an aspartate between H75/P76 yielded variant Csn-TI-D. This mutant produces chitotriose with 84-90% of detectable COS signal compared with ∼30% for the wild-type enzyme, while retaining endo-type cleavage behavior. Tryptophan fluorescence and closed-state docking suggest that weakened (-2) subsite affinity, together with a localized electrostatic gate at H75/P76, shifts the substrate-binding register to favor trimer-length alignment. These results demonstrate a scalable, structure-guided strategy for programming product length in GH46 chitosanases and support precision biocatalytic production of defined chitooligosaccharides.