Enzyme Cascade Reaction Involving Lytic Polysaccharide Monooxygenase and Dye-Decolorizing Peroxidase for Chitosan...

In situ H₂O₂ generation systems are efficient for H₂O₂-dependent biocatalytic oxidation reactions. Here, we report that lytic polysaccharide monooxygenases (LPMOs), copper-dependent polysaccharide monooxygenases, can efficiently supply H₂O₂ in situ to dye-decolorizing peroxidases (DyPs) using substrate gallic acid (GA) for chitosan functionalization. The maximum grafting ratio induced by the cascade reaction was significantly higher than that achieved by a reaction with initial exogenous H₂O₂. The maximum grafting ratio was obtained with 12 g/L GA, 5.6 mg/L DyP, 20-30 mg/L LPMO, and pH 4.5-5.0. UV-vis, Fourier transform infrared (FT-IR), and nuclear magnetic resonance (¹H NMR) spectroscopy confirmed GA grafting onto chitosan. X-ray diffraction (XRD) analysis and thermogravimetric analysis (TGA) indicated that GA-chitosan conjugates had lower thermal stability and crystallinity than chitosan. The GA-chitosan conjugates had significantly higher antioxidant activity than chitosan. This study supplies a green and high-efficiency approach to achieve an enzymatic cascade reaction for chitosan functionalization and has potential applications in H₂O₂-dependent biocatalytic oxidation reactions.