Development of a high-throughput screening method for racemase activity and its application to the identification of alanine racemase variants with activity towards l-arginine

摘要

A high-throughput solid phase screening method has been developed for alanine racemase. Conversion of an l-amino acid to the d-amino acid is detected by a d-amino acid oxidase, which is utilised as a reporter enzyme along with horseradish peroxidase and a colourimetric dye. This system has successfully been used to identify a mutant with increased activity towards arginine. Kinetic analysis and modelling studies have been performed to understand the effect of the mutations found.