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A continuous fluorometric assay for pectin methylesterase
1Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado, 80309-0215, USA.
Analytical Biochemistry
|May 15, 1996
Summary
A new continuous fluorescence assay accurately measures pectin methylesterase (PME) activity. This method uses methanol detection for reliable enzyme quantification in plant and fungal extracts.
Area of Science:
- Biochemistry
- Enzymology
Background:
- Pectin methylesterase (PME) is crucial in plant cell wall modification and microbial metabolism.
- Accurate and efficient assays are needed for PME characterization and purification.
Purpose of the Study:
- To develop a continuous, fluorometric assay for quantifying pectin methylesterase (PME) activity.
- To assess the assay's applicability in crude plant and fungal extracts.
Main Methods:
- Pectin hydrolysis by PME produces methanol.
- Methanol is oxidized to formaldehyde by alcohol oxidase.
- Formaldehyde reacts with 4-amino-3-penten-2-one to form a fluorescent product, measured continuously.
Main Results:
- Fluorescence intensity is directly proportional to PME activity.
- The assay shows minimal interference from common purification chemicals and buffers.
- The assay is effective within a pH range of 5.0 to 6.5.
Conclusions:
- This fluorometric assay provides a rapid and reliable method for quantifying PME activity.
- It is suitable for use with crude plant and fungal extracts, aiding enzyme purification and comparative studies.
- The pH range limits its use for PME with alkaline optima, but it remains valuable for many applications.