Combining Signal Peptide Optimization with Directed Evolution to Enhance Chitosanase Activity

Directed evolution is a powerful approach for enhancing chitosanase performance, but can be constrained by inefficient screening systems. Here, we strategically engineered the native signal peptide of Csn, a chitosanase from <i><i>Bacillus subtilis</i></i> strain 168 (ATCC 23857, hereafter referred to as Csn168), using site-saturation mutagenesis to attenuate protein secretion. This modification enabled high-resolution screening of superior variants based on hydrolytic zone formation. Following iterative directed evolution, we identified the M8 variant, which produced a 28 mm hydrolytic zone within 33 h and exhibited a 10-fold improvement in catalytic activity compared with Csn168. M8 also showed markedly enhanced substrate affinity, as evidenced by a reduction in <i>K</i>_m from 25.56 to 11.75 g/L. Molecular dynamics simulations indicated that M8 facilitates more rapid product dissociation after substrate binding, resulting in accelerated catalytic turnover. Collectively, these findings establish that coupling signal peptide turning with directed evolution constitutes an effective strategy for rapid discovery of high-performance chitosanase variants.