Continuous photometric activity assays for lytic polysaccharide monooxygenase-Critical assessment and practical considerations

Lytic polysaccharide monooxygenase (LPMO) is a monocopper-dependent enzyme that cleaves glycosidic bonds by using an oxidative mechanism. In nature, they act in concert with cellobiohydrolases to facilitate the efficient degradation of lignocellulosic biomass. After more than a decade of LPMO research, it has become evident that LPMOs are abundant in all domains of life and fulfill a diverse range of biological functions. Independent of their biological function and the preferred polysaccharide substrate, studying and characterizing LPMOs is tedious and so far mostly relied on the discontinuous analysis of the solubilized reaction products by HPLC/MS-based methods. In the absence of appropriate substrates, LPMOs can engage in two off-pathway reactions, i.e., an oxidase and a peroxidase-like activity. These futile reactions have been exploited to set up easy-to-use continuous spectroscopic assays. As the natural substrates of newly discovered LPMOs are often unknown, widely applicable, simple, reliable, and robust spectroscopic assays are required to monitor LPMO expression and to perform initial biochemical characterizations, e.g., thermal stability measurements. Here we provide detailed descriptions and practical protocols to perform continuous photometric assays using either 2,6-dimethoxyphenol (2,6-DMP) or hydrocoerulignone as colorimetric substrates as a broadly applicable assay for a range of LPMOs. In addition, a turbidimetric measurement is described as the currently only method available to continuously monitor LPMOs acting on amorphous cellulose.