Polarimetric Assay for the Medium-Throughput Determination of α-Amino Acid Racemase Activity

摘要

A polarimetric assay has been developed for the identification of alpha-amino acid racemase activity. The setup consists of a microcuvette polarimeter (40 microL volume) connected to a pipetting robot for microtiter plates, a pump, and data processing. It could be demonstrated for a glutamate racemase from Lactobacillus fermentii, expressed in Escherichia coli, serving as model enzyme, that its activity can be determined from the time-dependent change of the optical rotation using l-glutamate as substrate. Thus, the specific activity was determined to 111.4 mdeg/min which corresponds to 45.7 micromol/min per mg purified enzyme. Moreover, a protocol was developed that allows the measurement of racemase activity from 96-well microtiter plates using purified enzymes. Thus, the method described can be used to determine racemase activity in an automatic manner. It should be also applicable for the screening of enzyme libraries created by directed evolution.