Characterization of a Novel Acid-Stable Chitosanase from Lentinula edodes Suitable for Chitooligosaccharide Preparation

As high-value chitosan derivatives, chitooligosaccharides (COSs) with biodegradable, biocompatible, nontoxic, antimicrobial, and antioxidant activities have been widely applied in food-related fields. Chitosanases can hydrolyze chitosan to produce COSs. Herein, a chitosanase (<i>Le</i>Cho1) from <i>Lentinula edodes</i> was successfully expressed in <i>Escherichia coli</i> and was then purified and characterized. <i>Le</i>Cho1 had a low sequence identity with other chitosanases reported from the GH75 family. The recombinant protein showed a molecular mass of 27 kDa on SDS-PAGE. <i>Le</i>Cho1 preferentially hydrolyzed chitosan with a high degree of deacetylation (DDA) and exhibited maximal activity (71.88 U/mg) towards 95% DDA chitosan at pH 3.0 and 50 °C. It possessed good stability at pH 2.0-6.0 and temperatures below 45 °C. Its hydrolytic activity was remarkably enhanced by the metal ion Mn²⁺ at 1 mM, while it was totally inhibited by 1 mM Fe³⁺ or 10 mM EDTA. Its <i>K</i>_m and <i>V</i>_max values were 0.04 μM and 76.81 μmol·min^-1·mg^-1, respectively, indicating good substrate affinity. <i>Le</i>Cho1 degraded chitosan into COSs with degrees of polymerization (DPs) of 2-5, while it had no action on COSs with DPs of less than 5, revealing its endo-chitosanase activity. This study proved that chitosanase <i>Le</i>Cho1 is a promising candidate in the industrial preparation of COSs due to its excellent properties.