Abstract
BackgroundThe availability of a sensitive and robust activity assay is a prerequisite for efficient enzyme production, purification, 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.ResultsAn 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 buffers 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.ConclusionsThe 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, different buffers 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, purification, and characterization of LPMOs.
Highlights
The availability of a sensitive and robust activity assay is a prerequisite for efficient enzyme production, purification, and characterization
In which Bissaro et al [6] demonstrated that hydrogen peroxide (H2O2) is a cosubstrate of lytic polysaccharide monooxygenase (LPMO), we previously developed a colorimetric assay that employs LPMO’s active site copper center in a peroxidase-like reaction to convert 2,6-DMP into the highly colored product coerulignone [7]
The conversion of 2,6-DMP by LPMO, a small phenolic compound occurring in lignin, is not unexpected considering that LPMOs can oxidize similar lignin degradation products to obtain the necessary electron for its active site copper activation [8,9,10]
Summary
The availability of a sensitive and robust activity assay is a prerequisite for efficient enzyme production, purification, and characterization. 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. Breslmayr et al Biotechnol Biofuels (2019) 12:283 fermentation and purification protocols or to simplify its biochemical characterization, e.g., deactivation studies Such an assay should detect LPMOs from various classes without being limited by the use of class-specific polysaccharide substrates. In which Bissaro et al [6] demonstrated that H2O2 is a cosubstrate of LPMOs, we previously developed a colorimetric assay that employs LPMO’s active site copper center in a peroxidase-like reaction to convert 2,6-DMP into the highly colored product coerulignone [7]. A similar effect on the efficiency of a reductant in regard to redox potential and pH has been observed for 2,3-dihydroxybenzoic acid [12]
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