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A rapid fluorescence-based assay for detecting soluble methane monooxygenase
A R Miller1, W K Keener, M E Watwood
1Idaho National Engineering and Environmental Laboratory, Idaho Falls 83415-2203, USA. millar@inel.gov
Applied Microbiology and Biotechnology
|March 6, 2002
Summary
A new fluorescence assay uses coumarin oxidation to measure soluble methane monooxygenase (sMMO) activity. This method offers a faster, more convenient, and stable alternative for enzyme activity estimation.
Area of Science:
- Biochemistry
- Enzymology
- Microbiology
Background:
- Soluble methane monooxygenase (sMMO) is a key enzyme in methane oxidation.
- Accurate and efficient methods are needed to quantify sMMO activity.
- Existing assays, like naphthalene oxidation, have limitations.
Purpose of the Study:
- To develop a novel fluorescence-based assay for estimating sMMO activity.
- To identify a suitable substrate for a fluorescence assay.
- To compare the new assay with existing methods.
Main Methods:
- Utilized whole cells of Methylosinus trichosporium OB3b expressing sMMO.
- Screened 12 compounds for fluorescent product formation upon oxidation by sMMO.
- Identified coumarin as a substrate, producing 7-hydroxycoumarin.
- Confirmed product identity using UV-Vis spectroscopy and 13C-NMR.
- Investigated sMMO inhibition using acetylene.
- Determined apparent kinetic parameters (Km(app), Vmax(app)).
Main Results:
- Coumarin oxidation by sMMO yields fluorescent 7-hydroxycoumarin.
- The reaction rate correlates with trichloroethylene degradation and naphthalene oxidation.
- Apparent kinetic parameters: Km(app)=262 µM, Vmax(app)=821 nmol/min/mg protein.
- The fluorescence assay demonstrated greater speed, convenience, and product stability compared to naphthalene oxidation.
Conclusions:
- A novel, sensitive, and efficient fluorescence assay for sMMO activity has been developed using coumarin oxidation.
- This assay provides a valuable tool for biochemical and environmental studies involving sMMO.
- The coumarin-based assay offers significant advantages over traditional methods for sMMO activity determination.