Microplate-Based Detection of Lytic Polysaccharide Monooxygenase Activity by Fluorescence-Labeling of Insoluble Oxidized Products

Most existing methods for screening the activity of lytic polysaccharide mono-oxygenases (LPMOs) on polysaccharides are based on the detection of soluble oxidized sugars. This approach might underestimate the total performance of LPMOs since oxidation events that do not lead to oligosaccharide release are not detected. Using PcLPMO9D as a model enzyme, a microplate-based method has been developed to detect C1-oxidizing LPMO activity by covalently linking a water-soluble fluorophore to oxidized positions within the cellulose fiber. This fluorescence method was validated using X-ray photoelectron spectroscopy and then combined with high-performance anion-exchange chromatography to track total PcLPMO9D activity.