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

Abstract

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.

The “life-span” of lytic polysaccharide monooxygenases (LPMOs) correlates to the number of turnovers in the reductant peroxidase reaction

“…Since LPMOs acting on crystalline surfaces have evolved a flat, solvent-exposed active site architecture, off-pathway reoxidation of the active site copper may be an unavoidable side reaction. Reoxidation by O 2 to generate H 2 O 2 has been studied quite well and it has been speculated that the reductant oxidase activity of LPMO may serve as a source of H 2 O 2 to be used by LPMOs ( 25 , 49 ) or by other H 2 O 2 consuming enzymes like lignin peroxidases ( 64 , 65 ). On the other hand, less is known about reoxidation by H 2 O 2 , whereas this reaction actually is crucial because it may lead to the irreversible enzyme inactivation of LPMO.…”

Enhanced Fenton Reaction for Xenobiotic Compounds and Lignin Degradation Fueled by Quinone Redox Cycling by Lytic Polysaccharide Monooxygenases

“…Kracher et al also identified that GMC oxidoreductases, such as glucose dehydrogenase (GDH), can regenerate the hydroquinone form of the redox couple (redox mediator) to reduce the LPMO active-site copper for cellulose oxidation . Hydroquinone derivatives, such as gallic acid and 2,3-dihydroxybenzoic acid, are also able to effectively drive the LPMO reaction under certain conditions. − These studies showed the interplay between LPMOs and hydroquinone or hydroquinone derivatives. The involvement of quinone redox cycling in the Fenton reaction has been proven in many organisms. , Many studies have also demonstrated that ligninolytic enzymes such as laccase and versatile ligninolytic peroxidase can enhance the Fenton reaction by fueling the oxidation of hydroquinone into semiquinones or quinones. , In this case, the hydroxyl radicals are mainly derived from a semiquinone-driven Fenton reaction, as the semiquinones radicals were the main agents accomplishing Fe 3+ reduction .…”

Lytic polysaccharide monooxygenases promote oxidative cleavage of lignin and lignin–carbohydrate complexes during fungal degradation of lignocellulose

“…The LPMO reaction could be driven by both molecular oxygen and hydrogen peroxide, and the reduction of active‐site LPMO‐Cu(II) to LPMO‐Cu(I) is necessary for substrate catalysis, which can be carried out by a broad range of external electron donors (Meier et al ., 2017; Chylenski et al ., 2019). Interestingly, hydroquinone and hydroquinone derivatives, a type of lignin‐derived metabolite participating in the fungus‐induced Fenton reaction (H 2 O 2 + Fe 2+ → HO • + OH − + Fe 3+ ), were recently found to reduce LPMOs as natural electron donors (Guillen et al ., 1997; Kracher et al ., 2016; Li et al ., 2021). The Fenton reaction, as a form of oxidation, could be activated by hydrogen peroxide and Fe(II) in the environment and yield hydroxyl radical (HO • ).…”

“…Interestingly, hydroquinone and hydroquinone derivatives, a type of lignin-derived metabolite participating in the fungus-induced Fenton reaction (H 2 O 2 + Fe 2+ ! HO • + OH À + Fe 3+ ), were recently found to reduce LPMOs as natural electron donors (Guillen et al, 1997;Kracher et al, 2016;Li et al, 2021). The Fenton reaction, as a form of oxidation, could be activated by hydrogen peroxide and Fe(II) in the environment and yield hydroxyl radical (HO • ).…”