A rapid throughput assay for screening (R)-2-(4-hydroxyphenoxy)propionic acid producing microbes

Abstract

(R)-2-(4-hydroxyphenoxy)propionic acid ((R)-HPOPA) is an important intermediate for the synthesis of optically pure aryloxyphenoxypropionic acid herbicides. Regioselective hydroxylation of (R)-2-phenoxypropionic acid ((R)-POPA) by microbes is one of the most useful methods for the industrial production of (R)-HPOPA. In this study, we designed and optimized a rapid throughput assay for screening (R)-HPOPA producing bacterial/fungal strains which can regioselectively hydroxylate (R)-POPA. (R)-HPOPA could react with 4-aminoantipyrine (4-AAP) in the presence of potassium hexacyanoferrate (K₃[Fe(CN)₆]) to form indoxyl antipyrine, an orange-red chromophore, that can easily spectrophotometrically be determined at 550 nm. During the verification of the assay we observed an average recovery rate of between 97.3% and 104.5%. Apart from the rapid throughput, no obvious differences in detection (R)-HPOPA in the culture broth samples were found between our rapid throughput multiplate assay and a high-performance liquid chromatography method. Our optimized assay method is simple, rapid and accurate with high repeatability. It has the potential for high throughput screening (about 3000-5000 samples/day) of the (R)-HPOPA producing strains.

Keywords: (R)-2-(4-hydroxyphenoxy) propionic acid; 4-Aminoantipyrine spectrophotometry; Microplate assay; Rapid throughput screening.