engleski

Improved spectrophotometric assay for lytic polysaccharide monooxygenase

Background: The availability of a sensitive and robust activity assay is a prerequisite for efcient enzyme production, purifcation, and characterization. Here we report on a spectrophotometric assay for lytic polysaccharide monooxygenase (LPMO), which is an advancement of the previously published 2, 6- dimethoxyphenol (2, 6-DMP)-based LPMO assay. The new assay is based on hydrocoerulignone as substrate and hydrogen peroxide as cosubstrate and aims toward a higher sensitivity at acidic pH and a more reliable detection of LPMO in complex matrices like culture media. Results: An LPMO activity assay following the colorimetric oxidation of hydrocoerulignone to coerulignone was developed. This peroxidase activity of LPMO in the presence of hydrogen peroxide can be detected in various bufers between pH 4–8. By reducing the substrate and cosubstrate concentration, the assay has been optimized for minimal autoxidation and enzyme deactivation while maintaining sensitivity. Finally, the optimized and validated LPMO assay was used to follow the recombinant expression of an LPMO in Pichia pastoris and to screen for interfering substances in fermentation media suppressing the assayed reaction. Conclusions: The biphenol hydrocoerulignone is a better substrate for LPMO than the monophenol 2, 6- DMP, because of a ~30 times lower apparent KM value and a 160 mV lower oxidation potential. This greatly increases the measured LPMO activity when using hydrocoerulignone instead of 2, 6-DMP under otherwise similar assay conditions. The improved activity allows the adaptation of the LPMO assay toward a higher sensitivity, diferent bufers and pH values, more stable assay conditions or to overcome low concentrations of inhibiting substances. The developed assay protocol and optimization guidelines increase the adaptability and applicability of the hydrocoerulignone assay for the production, purifcation, and characterization of LPMOs.

Activity assay, 2, 6-Dimethoxyphenol, Hydrocoerulignone, Hydrogen peroxide, Inhibitors, Lytic polysaccharide monooxygenase, Peroxidase activity

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