An assay for mandelate racemase using high-performance liquid chromatography

Mandelate racemase (EC 5.1.2.2) catalyzes the interconversion of the two stereoisomers of mandelic acid. A fixed-time assay for the quantification of mandelate racemase activity has been developed. The assay involves enzymatic conversion of R-mandelate to S-mandelate (or the reverse reaction) followed by separation and detection of the substrate and product using isocratic reversed-phase high-performance liquid chromatography on a Sumichiral OA-6100 column and absorbance detection. This method offers an economical and efficient alternative to the existing circular dichroism-based and coupled assays.