Looking at LPMO reactions through the lens of the HRP/Amplex Red assay

Lytic polysaccharide monooxygenases (LPMOs) are unique redox enzymes capable of disrupting the crystalline surfaces of industry-relevant recalcitrant polysaccharides, such as chitin and cellulose. Historically, LPMOs were thought to be slow enzymes relying on O₂ as the co-substrate, but it is now clear that these enzymes prefer H₂O₂, allowing for fast depolymerization of polysaccharides through a peroxygenase reaction. Thus, quantifying H₂O₂ in LPMO reaction set-ups is of a great interest. The horseradish peroxidase (HRP)/Amplex Red (AR) assay is one of the most popular and accessible tools for measuring hydrogen peroxide. This assay has been used in various types of biological and biochemical studies, including LPMO research, but suffers from pitfalls that need to be accounted for. In this Chapter, we discuss this method and its use for assessing the often rate-limiting in situ formation of H₂O₂ in LPMO reactions. We show that, after accounting for multiple potential side reactions, quantitative data on H₂O₂ production obtained with the HRP/Amplex Red assay provide useful clues for understanding the catalytic activity of LPMOs, including the impact of reductants and transition metal ions.