Discovery of an Escherichia coli Esterase with High Activity and Enantioselectivity toward 1,2-O-Isopropylideneglycerol Esters
ABSTRACTEscherichia colihas been widely used as an expression host for the identification of desired biocatalysts through screening or selection assays. We have previously usedE. coliin growth selection and screening assays for identification ofBacillus subtilislipase variants (located in the periplasm) with improved activity and enantioselectivity toward 1,2-O-isopropylideneglycerol (IPG) esters. In the course of these studies, we discovered thatE. coliitself exhibits significant cytoplasmic esterase activity toward IPG esters. In order to identify the enzyme (or enzymes) responsible for this esterase activity, we analyzed eightE. coliknockout strains, in which single esterase genes were deleted, for their ability to hydrolyze IPG butyrate. This approach led to the identification of esterase YbfF as the majorE. colienzyme responsible for the hydrolytic activity toward IPG esters. The gene coding for YbfF was cloned and overexpressed inE. coli, and the corresponding protein was purified and characterized for its biocatalytic performance. YbfF displays a high level of activity toward IPG butyrate and IPG caprylate and prefers theR-enantiomer of these substrates, producing theS-enantiomer of the IPG product with high enantiomeric excess (72 to 94%ee). The enantioselectivity of YbfF for IPG caprylate (E= 40) could be significantly enhanced when using dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) as cosolvents in kinetic resolution experiments. The enzyme also shows high enantioselectivity toward 1-phenylethyl acetate (E≥ 200), giving the chiral product (R)-1-phenylethanol with >99%ee. The high activity and enantioselectivity of YbfF make it an attractive enzyme for organic synthesis.