A new isolate Bacillus stearothermophilus JY144 expressing a novel esterase with high enantioselectivity to (R)-ketoprofen ethyl ester: strain selection and gene cloning

In order to obtain novel strains that hydrolyzed the rac-ketoprofen ethyl ester to an enantiomer of ketoprofen, we enriched strains from broad ecological niches in which the thermostable enzymes were ubiquitously found. The isolated strains were first analyzed to detect the ester-hydrolyzing activity by using a selective plate, and then further screened by activity on the (R,S)-ketoprofen ethyl ester. Twenty-six strains were screened primarily and further compared for the ethyl ester-hydrolyzing activity in terms of conversion yield and enantioselectivity. Consequently, a strain JY144 was isolated as a novel strain that produced a (R)-stereospecific esterase with a high stability and systematically identified as a Bacillus stearothermophilus JY144. The enzyme was indeed stable at a broad range of pH (6.0–9.5) and temperature, up to 65 °C. The optimal temperature and pH for enzymatic conversion were 50 °C and 9.0, respectively. Since the wild-type strain showed a poor cell growth and enzyme activity, we further attempted the cloning of esterase gene into Escherichia coli, with a simple and rapid strategy for selecting the positive clones, and determined its primary structure. The cloned gene had an open reading frame (250 amino acids) with a calculated molecular mass of 27.4 kDa. The primary structure showed a relatively high homology (38–52%) to probable esterases unidentified to date, and thus presumed as a novel family enzyme. In whole cell conversions, the recombinant enzyme had a superior potential than the parent strain, and it could completely convert the rac-ketoprofen ethyl ester to (R)-ketoprofen, with an optical purity of 99%.