7-19: Improvement of esterase enantioselectivity by using mutagenesis targeted to the substrate binding site

Widespread application of enzymes of discriminating optical isomers depends on the stability for temperature and solvent as well as high resolution of the stereoisomers [1-2]. In order to separate optical isomers, we have examined the enantioselectivity toward to (R,S)-ketoprofen ethyl ester of various esterases and carried out gene cloning and purification of those for a long time. As a result, we isolated several esterases based on enantioselectivity; (R)-specific esterase (Esterase JY144), (S)-specific esterase (Esterase 1767), none-specific esterase (EST 25), (S)-preferential esterase (Est-Y 29) and (R)-preferential esterase (Est-AF).  In order to reveal that what makes these enzymes have different enantioselectivity, we solved the three dimensional structures of these proteins through X-ray diffraction. And, based on the results, we searched the amino acid resides that cause increase of enantioselectivity by substituted in to another amino acid, and carried out site-directed mutagenesis of enzymes and checked the enzymatic characters, most importantly enantioselectivity. As the result, several mutant esterases were obtained which showed enhanced enantioselectivity. In further study, if the 3D structure of esterase 1767 and Est CC were solved it would helpful to demonstrate the relationship between protein structure and optical specificity, then through this work, effective and improved enzymes for certain purpose will be acquired easily.

Reference

1. Bornscheuer, U.T.a.K., R.J., Hydrolases in Organic Synthesis ± Regio-and Stereoselective Biotransformations, 2nd Edition. 2005, Weinheim: . Wiley-VCH

2. Jaeger, K.E., B.W. Dijkstra, and M.T. Reetz, Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases. Annu Rev Microbiol, 1999. 53: p. 315-51.