t-Butyl-6-cyano-(3R,5R)-dihydroxyhexanoate is an advanced chiral precursor for the synthesis of the side chain pharmacophore of cholesterol-lowering drug atorvastatin. Herein, a robust carbonyl reductase (LbCR) was newly identified from Lactobacillus brevis, which displays high activity and excellent diastereoselectivity toward bulky t-butyl 6-cyano-(5R)-hydroxy-3-oxo-hexanoate (7). The engineered Escherichia coli cells harboring LbCR and glucose dehydrogenase (for cofactor regeneration) were employed as biocatalysts for the asymmetric reduction of substrate 7. As a result, as much as 300 g L–1 of water-insoluble substrate was completely converted to the corresponding chiral diol with >99.5% de in a space–time yield of 351 g L–1 d–1, indicating a great potential of LbCR for practical synthesis of the very bulky and bi-chiral 3,5-dihydroxy carboxylate side chain of best-selling statin drugs.

A new imine reductase from Stackebrandtia nassauensis (SnIR) was identified, which displayed over 25- to 1400-fold greater catalytic efficiency for 1-methyl-3,4-dihydroisoquinoline (1-Me DHIQ) compared to other imine reductases reported. Subsequently, an efficient SnIR-catalyzed process was developed by simply optimizing the amount of cosolvent, and up to 15 g L–1 1-Me DHIQ was converted completely without a feeding strategy. Furthermore, the reaction proceeded well for a panel of dihydroisoquinolines, affording the corresponding tetrahydroisoquinolines (mostly in S-configuration) in good yields (up to 81%) and with moderate to excellent enantioselectivities (up to 99% ee).

A novel non-heme chloroperoxidase (SvGL) with promiscuous (−)-γ-lactamase activity towards Vince lactam was identified from Streptomyces viridochromogenes by genome data-mining. SvGL possesses high activity and excellent thermal stability and enantioselectivity. Furthermore, it is able to tolerate extremely high substrate concentrations (4.0 M, 436.5 g L−1). Using the newly discovered (−)-γ-lactamase as a biocatalyst, an efficient and environmentally benign process for the production of (+)-γ-lactam was developed. The process allowed an enantioselective resolution of 436.5 g L−1 racemic γ-lactam with only 0.2 g L−1 lyophilized cell-free extract, affording an extremely high substrate/catalyst ratio of 2183 (g g−1), a space-time yield of 458 g L−1 d−1, and a very low E factor (environmental factor) of 5.7 (kg waste per kg product) even when the process water is included.

A new esterase gene abmbh, encoding a benzoate hydrolase which can enantioselectively hydrolyze l-menthyl benzoate to l-menthol, was recently identified from the genomic library of a soil isolate Acinetobacter sp. ECU2040. The abmbh gene contains a 1080-bp open reading frame encoding a protein of 360 amino acids with a calculated molecular mass of 40.7 kDa. The corresponding enzyme AbMBH was functionally expressed in Escherichia coli BL21 (DE3), purified, and characterized. The AbMBH displayed the maximum activity towards p-nitrophenyl butyrate at 50 °C, and an optimum pH of 8.5. A KM of 2.6 mM and a kcat of 0.26 s−1 were observed towards dl-menthyl benzoate. The AbMBH exhibited a moderate enantioselectivity (E = 27.5) towards dl-menthyl benzoate. It can also catalyze the enantioselective hydrolysis of a variety of racemic menthyl esters, including dl-menthyl acetate, dl-menthyl chloroacetate, and dl-menthyl butyrate.

A new bacterial nitroreductase has been identified and used as a biocatalyst for the controllable reduction of a variety of nitroarenes with an electron-withdrawing group to the corresponding N-arylhydroxylamines under mild reaction conditions with excellent selectivity (>99%). This method therefore represents a green and efficient method for the synthesis of arylhydroxylamines.

A new NADH-dependent carbonyl reductase RhCR capable of efficiently reducing the ε-ketoester ethyl 8-chloro-6-oxooctanoate (ECOO) to give ethyl (S)-8-chloro-6-hydroxyoctanoate [(S)-ECHO], an important chiral precursor for the synthesis of (R)-α-lipoic acid, was identified from Rhodococcus sp. ECU1014. Using recombinant Escherichia coli cells expressing RhCR and glucose dehydrogenase used for the regeneration of cofactor, 440 g L−1 (2 M) of ECOO were stoichiometrically converted to (S)-ECHO in a space–time yield of 1580 g L−1 d−1 without the external addition of any expensive cofactor.Ethyl (S)-8-chloro-6-hydroxyoctanoateC10H19ClO3[α]D22 = +19.8 (c 1.0, CHCl3)Source of chirality: enzymatic reductionAbsolute configuration: (S)

Optically pure 1-(3’,4’-methylenedioxyphenyl) ethanol is a key chiral intermediate for the synthesis of Steganacin and Salmeterol. A para-nitrobenzyl esterase cloned from Bacillus amyloliquefaciens (BAE) was employed to hydrolyze 1-(3’,4’-methylenedioxyphenyl) ethyl ester for the production of (R)-1-(3’,4’-methylenedioxyphenyl)ethanol. Initially, a moderate enantioselectivity (E = 35) only was obtained at 30°C. Some reaction conditions such as reaction temperature and additive approach were investigated in order to improve the enantioselectivity of the BAEcatalyzed reaction.. As a result, the enantioselectivity was improved significantly to 140 under addition of Tween-80 and a decreasing reaction temperature to 0°C. The result was confirmed in a decagram-scale preparative bioresolution also. The optimized enzymatic hydrolysis conditions provide a more effective process for the (R)-1-(3’,4’-methylenedioxyphenyl) ethanol bioproduction.

Production of l-menthol by bioprocesses attracts increasing attention nowadays. Herein, we attempted to develop a bioresolution process for production of l-menthol through enantioselective hydrolysis of dl-menthyl benzoate using a newly isolated bacterium from soil samples. Among 129 active soil isolates screened rapidly by thin-layer chromatography, an outstanding bacterial strain numbered ECU2040, which was subsequently identified as Acinetobacter species, was finally selected as our target enzyme producer due to its highest activity and the best enantioselectivity toward l-substrate as confirmed by chiral gas chromatography. The catalytic performance of the cell-free extract from Acinetobacter sp. ECU2040 was preliminarily examined, indicating that its optimal pH and temperature for the reaction were 7.5 and 37 °C, respectively. Under the optimal conditions, the enzymatic reaction was performed on a 1-L scale, affording l-menthol in 48 % yield and 71 % ee.

(R)-o-Chloromandelic acid is the key precursor for the synthesis of Clopidogrel®, a best-selling cardiovascular drug. Although nitrilases are often used as an efficient tool in the production of α-hydroxy acids, there is no practical nitrilase specifically developed for (R)-o-chloromandelic acid. In this work, a new nitrilase from Labrenzia aggregata (LaN) was discovered for the first time by genomic data mining, which hydrolyzed o-chloromandelonitrile with high enantioselectivity, yielding (R)-o-chloromandelic acid in 96.5% ee. The LaN was overexpressed in Escherichia coli BL21 (DE3), purified, and its catalytic properties were studied. When o-chloromandelonitrile was used as the substrate, the Vmax and Km of LaN were 2.53 μmol min−1 mg−1 protein and 0.39 mM, respectively, indicating its high catalytic efficiency. In addition, a study of substrate spectrum showed that LaN prefers to hydrolyze arylacetonitriles. To relieve the substrate inhibition and to improve the productivity of LaN, a biphasic system of toluene–water (1:9, v/v) was adopted, in which o-chloromandelonitrile of 300 mM (apparent concentration, based on total volume) could be transformed by LaN in 8 h, giving an isolated yield of 94.5%. The development of LaN makes it possible to produce (R)-o-chloromandelic acid by deracemizing o-chloromandelonitrile with good ee value and high substrate concentration.

•Thermostability of BSE was successfully enhanced by random mutagenesis.•Increased T5015 value and optimum temperature of up to 4.5 and 5.0 °C.•The total turn-over number of BSEV4 was 1.1–1.5 folds higher with 1.0 M of substrate loading for each batch.•Increased ionic bonds, hydrogen bonds and hydrophobic interactions contribute to stabilize the protein.Bacillus subtilis esterase (BSE) exhibits high activity, extraordinary substrate/product tolerance and excellent enantioselectivity in the production of l-menthol through enantioselective hydrolysis of dl-menthyl acetate. However, rapid inactivation of wild-type BSE at elevated temperatures often hampers its applications. In this work, directed evolution was used to create thermostable mutants of BSE. After screening and recombination of beneficial mutations, BSEV4 was chose for the best mutant. The BSEV4 had half-lives of 462, 248 and 0.34 h at 30, 40 and 50 °C, respectively, which were 5.6, 4.1 and 2.0 folds longer than those of BSEWT. Moreover, BSEV4 showed an increase of 4.5 °C in T5015 and a higher temperature optimum compared with the wild-type enzyme. In the kinetic resolution of dl-menthyl acetate at 1.0 M substrate loading, BSEV4 displayed improvements in operational stability than BSEWT, leading to a 1.5-fold higher total turnover number at 45 °C. The model structure of BSEV4 with four mutations, built with a highly homologous p-nitrobenzyl esterase (PDB ID: 1QE3) as the template, revealed that the newly formed hydrogen bonds and ionic bonds were beneficial for enhancing the thermostability of BSE.The thermostability of an esterase from Bacillus subtilis was enhanced by random mutagenesis and combination of beneficial mutations. The hydrolysis of racemic menthyl acetate by BSEV4 at elevated temperature indicates that better thermostability could reduce the enzyme dosage or reaction time. The modeled structure demonstrates the addition of ionic bonds, hydrogen bonds and/or hydrophobic interactions will contribute mostly to the structure stability.Download full-size image

In order to describe the functional characteristics of new esterases by evaluating their capacity of releasing alcohols from the corresponding esters, and to build “visiting cards” which might guide more widespread application in other reactions, an array of 20 acetates of structurally diverse alcohols were designed and synthesized for rapidly fingerprinting the activities of newly discovered lipases or esterases. At first, two well-known commercial lipases/esterases, Candida rugosa lipase (CRL) and pig liver esterase (PLE), were employed as models to verify the feasibility of this method. Subsequently, four home-made new enzymes were tested to compare their substrate fingerprints. Three parameters were adopted for quantitative characterization of the fingerprints of enzymes. Among them, the Shannon–Wiener index was adopted for the first time to quantitatively describe the breadth of substrate spectrum, making it easy and comprehensive to evaluate the breadth of substrate spectrum of homologous enzymes.Graphical abstractShannon–Wiener index was adopted for the first time to quantitatively evaluate the breadth of substrate spectrum of newly discovered lipases or esterases, using an array of 20 acetates of structurally diverse alcohols.Download full-size imageHighlights► An array of 20 acetates of structurally diverse alcohols was designed and synthesized for rapidly fingerprinting the activities. ► Different preference of esterase and lipase to acyl groups were compared. Different acyl groups for fingerprinting the activities of esterase and lipase were recommended. ► The Shannon–Wiener index was adopted for the first time to describe the breadth of substrate spectrum of enzymes in a quantitative mode.

•A novel process was developed to produce a key precursor for (R)-α-lipoic acid.•Increase E value by designing the reacted site (OH group) on the chiral center.•Redesigned and optimized the producing process.•Succeeded in repetitive use of catalyst for producing the target products.A new and efficient process was developed by lipase-catalyzed transacylation to resolve ethyl 8-chloro-6-hydroxy octanoate (ECHO) to produce an important chiral precursor for the synthesis of (R)-α-lipoic acid. After optimization of biocatalyst, solvent, acyl donor, temperature and enzyme loading, (S)-O-acetylated ECHO was achieved in 94% ee, 35% isolated yield and 38 g L−1 d−1 space-time yield using Novozym 435 as biocatalyst. Subsequently, the enzymatic resolution reaction was successfully repeated for 7 batches, retaining over 40% conversions.A new and efficient process was developed by lipase-catalyzed transacylation to resolve ethyl 8-chloro-6-hydroxy octanoate (ECHO) to produce an important chiral precursor for the synthesis of (R)-α-lipoic acid.Download full-size image

The whole cells of recombinant Escherichia coli BL21 overexpressing a Bacillus subtilis esterase (BsE) were utilized to sequentially hydrolyze the dicarboxyester of 1-phenyl-1,2-ethanediol for production of (S)-1-phenyl-1,2-ethanediol (PED), exhibiting high hydrolytic activity, excellent regio- and enantioselectivities towards the dicarboxyester of PED. Among the dicarboxyesters with different acyl chains (e.g., acetyl, n-butyl, and n-hexyl), the best enantioselectivity (E = 176) was observed when PED diacetate was employed as the initial substrate. Various reaction conditions were systematically investigated for enantioselective hydrolysis of PED diacetate. Under the optimal reaction conditions, kinetic resolution of 100 mM PED diacetate resulted in 49% conversion within 1 h, affording (S)-PED in 96% ee. A 150-ml scale reaction was performed, affording (S)-PED in 49% yield and 95% ee. After recrystallization in chloroform, the optical purity of (S)-PED was improved up to >99% ee, with a total yield of 45%. These results imply that this recombinant esterase (BsE) is a potentially promising biocatalyst for bioproduction of (S)-PED, an important chiral building block with wide application in pharmaceutical industry and liquid-crystal display materials.Graphical abstractDownload full-size imageHighlights► A recombinant Bacillus subtilis esterase was producted (S)-1-phenyl-1,2-ethanediol. ► The BsE exhibited the best enantioselectivity (E = 176) towards diacetate of PED. ► The hydrolysis reaction gave a 49% conversion within 1 h, and (S)-PED in 96% ee. ► The BsE is a potentially promising biocatalyst for bioproduction of (S)-PED.

A new bacterial nitroreductase has been identified and used as a biocatalyst for the controllable reduction of a variety of nitroarenes with an electron-withdrawing group to the corresponding N-arylhydroxylamines under mild reaction conditions with excellent selectivity (>99%). This method therefore represents a green and efficient method for the synthesis of arylhydroxylamines.

A novel non-heme chloroperoxidase (SvGL) with promiscuous (−)-γ-lactamase activity towards Vince lactam was identified from Streptomyces viridochromogenes by genome data-mining. SvGL possesses high activity and excellent thermal stability and enantioselectivity. Furthermore, it is able to tolerate extremely high substrate concentrations (4.0 M, 436.5 g L−1). Using the newly discovered (−)-γ-lactamase as a biocatalyst, an efficient and environmentally benign process for the production of (+)-γ-lactam was developed. The process allowed an enantioselective resolution of 436.5 g L−1 racemic γ-lactam with only 0.2 g L−1 lyophilized cell-free extract, affording an extremely high substrate/catalyst ratio of 2183 (g g−1), a space-time yield of 458 g L−1 d−1, and a very low E factor (environmental factor) of 5.7 (kg waste per kg product) even when the process water is included.