Discovery of an Escherichia coli Esterase with High Activity and Enantioselectivity toward 1,2- O -Isopropylideneglycerol Esters

Abstract

Escherichia coli has been widely used as an expression host for the identification of desired biocatalysts through screening or selection assays. We have previously used E. coli in growth selection and screening assays for identification of Bacillus subtilis lipase 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 that E. coli itself exhibits significant cytoplasmic esterase activity toward IPG esters. In order to identify the enzyme (or enzymes) responsible for this esterase activity, we analyzed eight E. coli knockout 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 major E. coli enzyme responsible for the hydrolytic activity toward IPG esters. The gene coding for YbfF was cloned and overexpressed in E. 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 the R-enantiomer of these substrates, producing the S-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.Hydrolases (EC 3) represent a class of enzymes that catalyze the hydrolysis of a chemical bond and are widely employed in organic synthesis (7,8,29). Depending on the chemical bond that they act on, hydrolases are grouped into several subclasses. Esterases and other hydrolytic enzymes that belong to the broad group of carboxylic ester hydrolases (EC 3.1.1) catalyze the hydrolysis and synthesis of ester bonds (5). Their broad substrate acceptance and high stability and enantioselectivity and the fact that they do not require cofactors make them attractive biocatalysts for organic synthesis (5). For example, the carboxylesterase NP from Bacillus subtilis Thai I-8 shows very high levels of activity and stereoselectivity toward esters of nonsteroidal anti-inflammatory drugs (NSAID), such as the naproxen and ibuprofen methyl esters, demonstrating its exciting potential for selective drug synthesis (6).We are interested in developing a biocatalytic process for the kinetic resolution of racemic 1,2-O-isopropylideneglycerol (IPG) esters. In previous studies, we have used Escherichia coli in directed evolution experiments for the identification of Bacillus subtilis lipase variants (located in the periplasm) with improved activity and enantioselectivity toward IPG butyrate (substrate 1) and IPG caprylate (substrate 3) (Fig. 1) ...

An Esterase with Superior Activity and Enantioselectivity towards 1,2‐O‐Isopropylideneglycerol Esters Obtained by Protein Design

“…In previous work reported by our group, [5] YbfF was identified as the major enzyme responsible for the pronounced activities and enantioselectivities in cellfree extracts prepared from E. coli cells toward substrates 1 and 3 (Scheme 1), with formation of (S)-2 in excess over (R)-2 for both substrates. YbfF exhibits moderate enantioselectivity (E = 10) towards 1 and a high enantioselectivity (E = 29) towards 3.…”

“…The carbonyl oxygen of (R)-IPG-C4 is at hydrogen bonding distance to the nitrogen atoms of Leu-25 and Met-90, suggesting that these two residues compose the oxyanion hole of YbfF and trap the carbonyl oxygen atom of the substrate, in agreement with the mechanism suggested for other hydrolases of this family. [5] Docking of substrate and minimization runs were performed using available tools in Discovery Studio 2.5.…”

“…[5] The crystal structure of YbfF has been solved and the enzyme is composed of an a/b hydrolase-fold domain with a three-helical bundle cap that is linked by a twist helix to the a/b hydrolase-fold domain. [6] The sequence motif GxSxG, characteristic for a/b hydrolase-fold superfamily of enzymes, is also conserved in YbfF.…”

“…The effect of pH and temperature on enzyme activity ( Figure S13, Supporting Information) and temperature on enzyme stability ( Figure S14, Supporting Information) of mutant W235I were found to be similar to the values determined for the YbfF wild-type. [5] Kinetic Analysis of Wild-Type and W235I…”

Carbon-dot–hydrogel for enzyme-mediated bacterial detection

“…6A) might provide an indication as to the relative contribution of esterases vs. lipases to the observed C-dot-hydrogel uorescence quenching. 45,46 Fig. 6B depicts quenching of the C-dot-hydrogel uores-cence following addition of commercially-obtained esterase extracted from B. subtilis (B. subtilis belongs to the same subgroup of Bacillus species as B. cereus).…”