PMID:21764964

Citation	Godinho, LF, Reis, CR, Tepper, PG, Poelarends, GJ and Quax, WJ (2011) Discovery of an Escherichia coli esterase with high activity and enantioselectivity toward 1,2-O-isopropylideneglycerol esters. Appl. Environ. Microbiol. 77:6094-9
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.
Links	PubMed PMC3165400 Online version:10.1128/AEM.05122-11
Keywords	Alkenes/metabolism; Cloning, Molecular; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/isolation & purification; Escherichia coli Proteins/metabolism; Esterases/chemistry; Esterases/genetics; Esterases/isolation & purification; Esterases/metabolism; Esters/metabolism; Gene Expression; Gene Knockout Techniques; Glycerol/analogs & derivatives; Glycerol/metabolism; Models, Molecular; Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Stereoisomerism; Substrate Specificity
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
ECOBD:C6EHW2		GO:0016788: hydrolase activity, acting on ester bonds	ECO:0000314:		F		Figure 1 shows the esterase catalyzed effects on 1,2-ortho-isopropylideneglycerol (IPG)esters	complete

References

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