A fast, sensitive and fluorescent LPMO activity assay.

Area of Science:

• Biochemistry and enzymology
• Industrial biotechnology
• Microbial ecology

Background:

• Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes essential for degrading recalcitrant polysaccharides.
• These enzymes are secreted by microorganisms and play key roles in lignocellulosic biorefineries.
• Existing methods for LPMO activity assessment can be limited in sensitivity or scope.

Purpose of the Study:

• To develop a novel, sensitive fluorescence-based kinetic assay for measuring LPMO activity.
• To establish a tool for quantifying LPMO function in industrial and ecological contexts.
• To validate the assay's performance under relevant reaction conditions.

Main Methods:

• Development of a fluorescence assay utilizing the enzymatic conversion of a reduced fluorescein precursor.
• Optimization of assay conditions to maximize sensitivity and specificity for LPMO detection.
• Demonstration of the assay's ability to detect low nanomolar concentrations of LPMO and identify peroxidase activity.

Main Results:

• The developed assay can detect LPMO activity down to 1 nM under optimized conditions.
• The assay successfully distinguishes LPMO activity from peroxidase activity using the same substrate.
• Effective assay performance was confirmed at low concentrations of hydrogen peroxide and dehydroascorbate.

Conclusions:

• A sensitive and versatile fluorescence-based assay for LPMO activity has been successfully developed.
• This assay provides a valuable tool for research and industrial applications involving LPMOs.
• The assay's ability to detect low enzyme concentrations and differentiate activities enhances its utility.