Microtuning of the enzyme active pocket has led to a smart library of epoxide hydrolase variants with an expanded substrate spectrum covering a series of typical β-blocker precursors. Improved activities of 6- to 430-fold were achieved by redesigning the active site at two predicted hot spots. This study represents a breakthrough in protein engineering of epoxide hydrolases and resulted in enhanced activity toward bulky substrates.

Chiral secondary alcohols with additional functional groups are frequently required as important and valuable synthons for pharmaceuticals, agricultural and other fine chemicals. With the advantages of environmentally benign reaction conditions, broad reaction scope, and high stereoselectivity, biocatalytic reduction of prochiral ketones offers significant potential in the synthesis of optically active alcohols. A CmCR homologous carbonyl reductase from Pichia guilliermondii NRRL Y-324 was successfully overexpressed. Substrate profile characterization revealed its broad substrate specificity, covering aryl ketones, aliphatic ketones and ketoesters. Furthermore, a variety of ketone substrates were asymmetrically reduced by the purified enzyme with an additionally NADPH regeneration system. The reduction system exhibited excellent enantioselectivity (>99% ee) in the reduction of all the aromatic ketones and ketoesters, except for 2-bromoacetophenone (93.5% ee). Semi-preparative reduction of six ketones was achieved with high enantioselectivity (>99% ee) and isolation yields (>80%) within 12 h. This study provides a useful guidance for further application of this enzyme in the asymmetric synthesis of chiral alcohol enantiomers.

In order to search for oxidoreductases suitable for the preparation of methyl (R)-o-chloromandelate [(R)-CMM], the key intermediate for clopidogrel, the homologous proteins of Gre2p were expressed in Escherichia coli, among which CgKR1 showed the most satisfactory activity and stereoselectivity towards methyl o-chlorobenzoylformate (CBFM). Using the crude enzyme of CgKR1 and glucose dehydrogenase (GDH), as much as 300 g⋅L⁻¹ of CBFM was almost stoichiometrically converted to (R)-CMM with excellent enantiomeric excess (98.7% ee). More importantly, the reaction could be performed without external addition of an expensive cofactor. The substrate profile indicates that keto esters serve as the most suitable substrate, which was confirmed by gram-scale preparations. Homology modeling and docking analysis revealed the molecular basis for the high stereoselectivity of CgKR1. These demonstrate not only the feasibility of in silico mining of novel enzymes based on sequence homology but also the applicability of this new reductase for the practical production of optically active (R)-CMM.

A novel epoxide hydrolase (BMEH) with unusual (R)-enantioselectivity and very high activity was cloned from Bacillus megaterium ECU1001. Highest enantioselectivities (E>200) were achieved in the bioresolution of ortho-substituted phenyl glycidyl ethers and para-nitrostyrene oxide. Worthy of note is that the substrate structure remarkably affected the enantioselectivities of the enzyme, as a reversed (S)-enantiopreference was unexpectedly observed for the ortho-nitrophenyl glycidyl ether. As a proof-of-concept, five enantiopure epoxides (>99% ee) were obtained in high yields, and a gram-scale preparation of (S)-ortho-methylphenyl glycidyl ether was then successfully performed within a few hours, indicating that BMEH is an attractive biocatalyst for the efficient preparation of optically active epoxides.

A β-ketoacyl-ACP reductase (FabG) gene from Bacillus sp. ECU0013 was heterologously overexpressed in Escherichia coli and the encoded protein was purified to homogeneity. The recombinant reductase could reduce a broad spectrum of prochiral ketones including aromatic ketones and keto esters and showed the highest activity in the asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate (OPBE). Using E. coli cells coexpressing both FabG and glucose dehydrogenase (GDH) genes, as much as 620 g⋅L⁻¹ of OPBE was almost stoichiometrically converted to ethyl (S)-2-hydroxy-4-phenylbutyrate [(S)-HPBE] with excellent (>99%) enantiomeric excess. More importantly, the process could be performed smoothly without external addition of an expensive cofactor as usually done and could be scaled up very easily. All these positive features demonstrate the applicability of this reductase for the large-scale production of optically active α-hydroxy acids/esters.

A facile chemo-enzymatic process has been developed for producing stereoisomers of 4-substituted 2-hydroxy-4-butyrolactones with good to excellent enantioselectivity. This process involves an easy separation of the diastereoisomers by column chromatography and efficient enzymatic resolution by whole cells of Escherichia coli JM109 expressing Fusarium proliferatum lactonase gene. This biocatalyst shows strong tolerance towards different substrate structures and at least three out four possible isomers could be obtained in excellent enantiomeric purity. Different substrate concentrations (10 mM–200 mM) were examined, giving a substrate to catalyst ratio of up to 26:1. This general and efficient enzymatic process provides access to stereoisomers of 4-substituted 2-hydroxy-4-butyrolactones readily and cost-effectively. The stereochemical assignments were conducted systematically based on NMR, X-ray diffraction and circular dichroism, leading to further understanding of the enzyme’s stereoselectivity.