[Rational design and molecular engineering of a chitosanase for preparing specific chito-oligosaccharides with degree...

To achieve efficient enzymatic preparation of specific high-degree-of-polymerization chito-oligosaccharides (degree of polymerization≥5, DP≥5), rational molecular engineering of chitosanase Csn46-Mut4 was performed in this study. Through site-directed mutagenesis coupled with DNS enzyme activity-HPLC product distribution screening, a superior mutant, C41A/T47A/V144A, was obtained. Under conditions of 55℃ and pH 5.5, the catalytic efficiency and thermostability of this enzyme were remarkably enhanced, reaching 2.56-fold and 1.67-fold those of Csn46-Mut4, respectively. Peak area normalization analysis revealed that the relative content of chitopentaose (DP5) increased to 22.8%, representing a 12-fold increase compared with Csn46-Mut4, while the total proportion of chitooligosaccharides with DP≥5 reached 27.33%, corresponding to a 6.68-fold increase. Moreover, the total yield of DP≥5 chitooligosaccharides by the mutant reached 64.18% (5.776 g/L), which was 3.14-fold that of Csn46-Mut4 (20.41%, 1.837 g/L). Further molecular dynamics simulations revealed that the three mutation sites acted synergistically via the C41A and T47A substitutions in the catalytic flexible region and the V144A substitution in the substrate-binding channel region. This synergy strengthened the global hydrogen-bonding network and improved the structural rigidity while inducing characteristic dynamic changes in the substrate-binding pocket. In the initial simulation stage, pocket expansion facilitated the entry of long-chain substrates, whereas in the later stage, the enhanced hydrogen-bonding network, combined with the introduction of local critical flexibility, drove the accumulation of medium- and long-chain products, particularly leading to a notable increase in chitopentaose. This study not only yields a novel chitosanase capable of efficiently and specifically producing high-DP chitooligosaccharides (DP≥5) but also offers a new strategy for optimizing enzyme catalytic performance through multi-site synergistic design across different protein regions.