Screening, production and properties of a stereospecific esterase from Pseudomonas sp. S34 with high selectivity to ( S)-ketoprofen ethyl ester

Abstract

To isolate novel strains expressing an esterase that hydrolyzed the rac-ketoprofen ethyl ester to ( S)-ketoprofen in the stereospecific manner, we screened broad ecological niches and soil samples in which the activity was expected to be found. Thousands of microbial strains were tested to determine their ester-hydrolyzing activity by using an agar plate containing insoluble tributyrin as an indicative substrate, and then further screened by activity on the ( R, S)-ketoprofen ethyl ester. Twenty-eight strains were screened primarily and compared with respect to the potential to ketoprofen ethyl ester-hydrolyzing activity in terms of conversion yield and chiral specificity. Consequently, a strain S34 was isolated as a best producer and finally identified as a Pseudomonas sp. S34. We first formulated the optimal medium for the high level production of the enzyme, and as a preliminary experiment for enzymatic resolution, we characterized the fractionated enzyme. The enzyme with ketoprofen ethyl ester-hydrolyzing activity to ( S)-ketoprofen showed a high degree of enantioselectivity (>94%) and was mainly found in cell extracts, whereas no distinct activity was detected in culture broth. The optimum pH and temperature of the enzyme were 9.5 and 35 °C, respectively. The activity of the enzyme was markedly increased (four-fold) by addition of a non-ionic detergent Triton X-100 and, resultantly, a high activity toward ketoprofen ethyl ester (52 U/mg) was found. The small-scale conversion of ( R, S)-ketoprofen ethyl ester to ( S)-ketoprofen using the partially purified enzymes was completed in 28 h, with optical purity of 99% and yield of 47%.