A Continuous Fluorometric Assay for Pectin Methylesterase

A continuous, fluorometric assay for pectin methylesterase (PME) activity is described. In this assay, methanol produced by PME hydrolysis of pectin methyl esters is oxidized to formaldehyde by alcohol oxidase, and the formaldehyde is continuously reacted with 4-amino-3-penten-2-one to create a stable, fluorescent product. The increase in fluorescence intensity is linearly proportional to PME activity. The assay can be used in crude plant or fungal extracts with relatively little interference with chemicals or buffers commonly used in PME purification. The fluorescence assay has a useful pH range, from pH 5.0 to 6.5, which overlaps pH optima for many bacterial and fungal PMEs, but which limits its usefulness in assaying plant PMEs with alkaline pH optima. Nevertheless, the method is valuable for rapid assay of plant PMEs during their purification or for comparison of plant tissue PME activities.