Chiral liquid chromatography–mass spectrometry for high-throughput screening of enzymatic racemase activity

Abstract

In finding suitable biocatalysts for processes in chemical industry, expression libraries are constructed containing typically >10 000 clones. Search for a desired activity is done by examination of all the clones in one or more libraries using a high-throughput screening assay. Here we describe a method for the screening of the enzymatic racemase activity of clones from an expression library on α-amino-ε-caprolactam (ACL) using a fast chiral LC separation and ionspray-MS as the detection technique. After substrate incubation with S-ACL, the 96-well microplates were centrifuged to remove cell material. The conversion of S-ACL to R-ACL was monitored by quantitation of the R-ACL enantiomer. Separation of the two ACL enantiomers was performed on a Crownpak CR+ column within 1 min. A Gilson 215 autosampler with a 889 multiple injection probe was used for injecting the samples into the LC system. The total analysis time for a 96-well microplate was 56 min. The MS was operated in the positive-ion mode using selected ion monitoring at m/ z 129 [M+H] + of ACL. Using this method over 12 000 samples were analyzed without loss in performance of the system. The LC column remained stable without loss of resolution and the MS system did not show loss in sensitivity throughout the screening. Inter-day reproducibility was within 15%.