Identification and Characterization of a Previously Uncharacterized Metagenome-Derived Chitinase and Its Application in N -Acetyl- d -Glucosamine Production from Crayfish Shell Waste

Abstract The generation of agricultural and food-processing byproducts has increased rapidly, highlighting the importance of their high-value utilization. In this study, a putative chitinase gene was identified from a metagenome-assembled genome. The enzyme plays a key role in chitin hydrolysis and was heterologously expressed in Escherichia coli as a recombinant protein with a molecular weight of approximately 39.2 kDa. The chitinase exhibited optimal activity toward colloidal chitin at pH 6.0 and 40 °C. Enzyme activity was significantly enhanced by Mn2+, Mg2+, and Sr2+, remained largely unaffected by Ca2+, and was strongly inhibited by Co2+ and Fe3+, while Zn2+ and Cu2+ completely abolished activity. Site-directed mutagenesis confirmed the conserved catalytic motif G-DG-D-D-E. Furthermore, the enzyme was applied to the direct hydrolysis of crayfish shell waste without conventional acid–alkali pretreatment, resulting in the formation of N-acetyl-d-glucosamine at a concentration of 3.26 mg/g. This approach eliminates the requirement for conventional acid–alkali pretreatment and enables the efficient valorization of crustacean shell waste. The developed enzymatic strategy provides a sustainable route for producing functional amino sugars with potential applications in food and related industries.