Adaptation of a Spectrophotometric Assay for Pectinmethylesterase to a Kinetic Microplate Reader

A continuous spectrophotometric assay for pectinmethylesterase (PME) has been adapted to a kinetic microplate reader. Linear standard curves could be constructed from 0 to 50 nMol galacturonic acid and remained linear for 15 min. Activity estimates from commercial orange peel PME were accurate from 0.009 to 0.015 units. Comparison of microplates with differing protein binding efficiencies indicated that low and medium binding plates showed significantly higher activity levels than high binding plates. The utility of the assay was demonstrated on two commercial mixtures of pectolytic enzymes and two commercial PMEs. The assay is extremely rapid; 24 samples replicated three times each could be performed in less than 1 hr. Additionally, it requires very small volumes of substrate (final volume per well was 200 μL) and sample (less than 10 μL per well).