A fast and sensitive coupled enzyme assay for the measurement of l-threonine and application to high-throughput screening of threonine-overproducing strains

Abstract

Amino acids are important fermentation products that are used in the food, animal feed, pharmaceutical, and cosmetics industries. Development of a better quantitation assay to measure amino acid levels will facilitate research and application in medical and industrial fields. Here we report the development of an assay that allows the rapid measurement of threonine concentration. l-threonine aldolase (LTA) catalyzes the cleavage of threonine to glycine and acetaldehyde. The resulting acetaldehyde is further converted into ethanol by alcohol dehydrogenase accompanying NADH reduction. Therefore, monitoring the changes in NADH concentration can serve as a readout for the amount of threonine present within certain range. This LTA assay can distinguish a 0.5mM difference in threonine concentration in some certain mediums. We successfully utilized this assay to measure l-threonine levels in industrial fermentation samples. Moreover, we were able to scale up this assay to 96-well format and use it in high-throughput screening of threonine-overproducing strains. We show this assay is simple, accurate, specific and suitable for determination of threonine concentration in large numbers of samples.