A fast and sensitive activity assay for lytic polysaccharide monooxygenase

Area of Science:

• Biochemistry
• Enzymology

Background:

• Lytic polysaccharide monooxygenases (LPMOs) degrade polysaccharides like cellulose, hemicellulose, and chitin.
• Current methods for detecting LPMO activity, such as MS/HPLC, are time-consuming and require specialized laboratories.
• A simplified assay is needed for routine screening, expression monitoring, and enzyme characterization.

Purpose of the Study:

• To develop a fast, robust, and sensitive spectrophotometric assay for measuring LPMO activity.
• To leverage a newly discovered peroxidase activity of LPMO for this assay.

Main Methods:

• Utilized 2,6-dimethoxyphenol (2,6-DMP) as a chromogenic substrate and H2O2 as a cosubstrate.
• Developed a spectrophotometric assay measuring the formation of coerulignone at 469 nm.
• Optimized the assay for a 300-second reaction time with 1 mM 2,6-DMP and 100 µM H2O2.

Main Results:

• Established a sensitive spectrophotometric assay for LPMO activity based on peroxidase activity.
• The assay is effective for LPMO concentrations ranging from approximately 0.5-50 mg/L.
• The high molar absorption coefficient of coerulignone (ε469 = 53,200 M⁻¹cm⁻¹) contributes to assay sensitivity.

Conclusions:

• The developed assay detects a novel peroxidase activity of LPMO, distinct from its polysaccharide-degrading activity.
• This assay simplifies LPMO screening, production monitoring, and characterization, including studies on binding constants and thermal stability.
• Caution is advised when using the assay with crude extracts due to potential interference from other enzymes like laccase or peroxidase.