Rational Engineering of Chitosanase for Producing Higher-Degree-of-Polymerization Chitooligosaccharides and Evaluating Their Antibacterial Properties.

High-degree of polymerization (DP) chitooligosaccharides (COS) possess enhanced biological activities. Here, a rational design strategy was developed to increase the DP of COS by introducing three negatively charged mutations (L117E/G21D/T50E) into chitosanase Csn168 (a chitosanase from Bacillus subtilis 168). The engineered mutant shifted product distribution from predominantly low-DP COS, with (GlcN)2 and (GlcN)3 accounting for 92.4% in wild-type Csn168, to high-DP COS, with (GlcN)4 and (GlcN)5 reaching 85.3%. The resulting high-DP COS exhibited potent antibacterial activity against Staphylococcus aureus, achieving complete growth inhibition at 8 g/L. Molecular dynamics simulations indicated that enhanced membrane interactions of (GlcN)4/(GlcN)5 may contribute to the improved antibacterial activity. This study offers a rational design strategy for modulating COS polymerization degree and advancing the production of functional COS.