Biocatalytic properties of a recombinant aldo-keto reductase with ...

Appl Microbiol Biotechnol (2011) 89:1111–1118 DOI 10.1007/s00253-010-2941-4

BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS

Biocatalytic properties of a recombinant aldo-keto reductase with broad substrate spectrum and excellent stereoselectivity Yan Ni & Chun-Xiu Li & Hong-Min Ma & Jie Zhang & Jian-He Xu

Received: 11 August 2010 / Revised: 7 October 2010 / Accepted: 9 October 2010 / Published online: 28 October 2010 # Springer-Verlag 2010

Abstract In the screening of 11 E. coli strains overexpressing recombinant oxidoreductases from Bacillus sp. ECU0013, an NADPH-dependent aldo-keto reductase (YtbE) was identified with capability of producing chiral alcohols. The protein (YtbE) was overexpressed, purified to homogeneity, and characterized of biocatalytic properties. The purified enzyme exhibited the highest activity at 50°C and optimal pH at 6.5. YtbE served as a versatile reductase showing a broad substrate spectrum towards different aromatic ketones and keto esters. Furthermore, a variety of carbonyl substrates were asymmetrically reduced by the purified enzyme with an additionally coupled NADPH regeneration system. The reduction system exhibited excellent enantioselectivity (>99% ee) in the reduction of all the aromatic ketones and high to moderate enantioselectivity in the reduction of α- and β-keto esters. Among the ketones tested, ethyl 4,4,4-trifluoroacetoacetate was found to be reduced to ethyl (R)-4,4,4-trifluoro-3-hydroxy butanoate, an important pharmaceutical intermediate, in excellent optical purity. To the best of our knowledge, this is the first report of ytbE gene-encoding recombinant aldo-keto reductase from Bacillus sp. used as biocatalyst for stereoselective reduction of carbonyl compounds. This study provides a useful guidance for further application of this enzyme in the asymmetric synthesis of chiral alcohol enantiomers.

Y. Ni : C.-X. Li (*) : H.-M. Ma : J. Zhang : J.-H. Xu (*) Laboratory of Biocatalysis and Bioprocessing, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, People’s Republic of China e-mail: [email protected] J.-H. Xu e-mail: [email protected]

Keywords YtbE . Recombinant aldo-keto reductase . Bacillus sp. . Asymmetric reduction . Chiral alcohols

Introduction Aldo-keto reductases (AKRs) are a growing superfamily of more than 140 widely distributed individual members belonging to 15 families, which are widely distributed in plants, animals, and microorganisms (Jez and Penning 2001; Ellis 2002; Hyndman et al. 2003; www.med.upenn. edu/akr/). They catalyze the NADPH-dependent reduction of a variety of substrates including aliphatic and aromatic aldehydes/ketones, monosaccharides, steroids, prostaglandins, isoflavinoids, polyketides, and so forth (Bohren et al. 1989; Jez et al. 1997; Petrash 2007). Chiral alcohols are frequently required as important and valuable intermediates in the synthesis of numerous pharmaceuticals and other fine chemicals. Asymmetric reduction of prochiral ketones is an effective and promising route for producing chiral alcohols, which has an inherent advantage of achieving up to 100% theoretical yield (Kataoka et al. 2003; Woodley 2008). Biocatalytic transformation using isolated enzymes or whole cell systems offers some advantages in comparison with chemical methods, such as mild and environmentally benign reaction conditions and remarkable chemo-, regio-, and stereoselectivity (Nakamura et al. 2003; Kroutil et al. 2004; Goldberg et al. 2007). In addition to the conventional biocatalyst screening, bioinformatics seems to increase the possibility of discovering useful biocatalysts. Screening of a library of overexpressed enzymes from microorganisms with known wealth of data that has accrued from sequencing whole genomes is a potential tool to search a catalyst for desired reactions (Yamamoto

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et al. 2003; Moore et al. 2007; Matsuda et al. 2009). In fact, 18 key reductases from bakers’ yeast have been overexpressed in Escherichia coli and tested for the ability of reduction (Kaluzna et al. 2004; Kaluzna et al. 2005; Hammond et al. 2007). Recently, we isolated a ketone reductase-producing Bacillus sp. ECU0013 with excellent stereoselectivity and high substrate concentration tolerance (Xie et al. 2010). In this study, in the search for oxidoreductases with activity and high stereoselectivity towards prochiral ketones, we cloned 11 genes encoding oxidoreductases from the strain and expressed them heterologously in E. coli BL21(DE3). The identification of an aldo-keto reductase (YtbE) with capability of producing alcohols was achieved by screening of these recombinant reductases. Purification of YtbE was performed, and the purified enzyme was characterized of enzymatic properties, especially with respect to its substrate specificity and stereospecificity.

Appl Microbiol Biotechnol (2011) 89:1111–1118

Expression and purification of recombinant carbonyl reductases Recombinant protein was expressed in E. coli BL21 cells transformed with pET28a. Cultures were grown at 37°C in LB medium containing 50 μg/ml kanamycin. When the OD600 of the culture reached 0.6, IPTG was added to a final concentration of 0.5 mM. The culture was incubated at 25°C for a further period of 12 h. Cells were harvested by centrifugation, washed twice and resuspended in buffer A (20 mM sodium phosphate buffer, pH 7.4, 500 mM NaCl, 10 mM imidazole). Cell suspension was disrupted by sonication and the cell lysate was centrifuged at 10,000×g for 20 min. The clear supernatant was loaded onto a Ni– NTA column equilibrated with buffer A. The column was washed with buffer A, and the retained proteins were eluted with an increasing gradient from 10 to 500 mM of imidazole in buffer A at a flow rate of 1 ml/min. The purity of fractions was assessed by SDS–PAGE. The fractions containing the pure protein were pooled and dialyzed against sodium phosphate buffer for desalting.

Materials and methods Protein analysis Bacterial strains and vectors E. coli DH5α and E. coli BL21 (DE3) were routinely grown in Luria–Bertani (LB) medium and were used as the cloning and expression hosts, respectively. Ampicillin (100 μgml−1) and kanamycin (50 μgml−1) were used for the selection of recombinant strains in E. coli. Bacillus sp. ECU0013 was isolated and identified in our laboratory (Xie et al. 2010). The strain was deposited in China General Microbiological Cultures Center, with an accession number of CGMCC No. 2549. Cells were cultured as previously described. Plasmid pMD18-T for the direct cloning of PCR products was from TaKaRa (Dalian, China) and plasmid pET28a (+) for heterogeneous expression studies was obtained from Novagen (Shanghai, China). Cloning and construction of expression plasmids The genomic DNA of Bacillus sp. ECU0013 was extracted and purified using the TIANamp Bacteria DNA Kit from Tiangen (Shanghai, China). DNA fragments containing the different oxidoreductases-encoding genes were amplified by polymerase chain reaction (PCR) using primers with the BamHI and XhoI restriction sites. The amplified DNAs were purified and directly cloned into the TA cloning site of pMD-18T and then introduced into DH5α. The desired genes were subcloned into expression plasmid pET28a (+) using standard methods and then transformed into E. coli BL21 (DE3) cells.

Gel electrophoresis was performed on 15% SDS– polyacrylamide gel with Tris–glycine buffer system. Protein bands were visualized by staining the gel with silver stain. The apparent molecular masses were determined by gel filtration chromatography using a TSK gel 2000SWxl column (Tosoh, Japan) connected to an HPLC system equilibrated with 100 mM sodium phosphate buffer (pH 6.7) containing 0.1 M Na2SO4 at a flow rate of 0.4 ml/min. Protein molecular weight standards were horse heart cytochrome c (12.4 kDa), bovine carbonic anhydrase (29 kDa), bovine serum albumin (66 kDa), yeast alcohol dehydrogenase (150 kDa), and sweet potato β-amylase (200 kDa) from Sigma (Shanghai, China). Enzyme assays The reductase activity was assayed spectrophotometrically at 30°C by monitoring the decrease in the absorbance of NADPH at 340 nm. The standard assay mixture (1 ml) was composed of 50 mM sodium phosphate buffer (pH 7.0), 2.0 mM ethyl pyruvate, 0.05 mM NADPH, and an appropriate amount of enzyme. One unit of enzyme activity was defined as the amount of enzyme catalyzing the oxidation of 1 μmol NADPH per minute. The kinetic parameters of the purified reductase were determined by assaying (in triplicate) the activity on the substrate ethyl pyruvate of different concentrations (1–30 mM) at a fixed NADPH concentration. The maximal reaction rate (Vmax) and apparent Michaelis–Menten constant (Km) of the


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The optimum pH was determined by standard activity assay at different pH (4.0–10.0), with sodium citrate for a pH range from 4.0 to 6.0, sodium phosphate for pH range from 6.0 to 8.5, and Gly–NaOH for pH range from 8.5 to 10.0. The optimum temperature was determined under standard conditions at different temperatures in the range of 20– 65°C. The stability was studied by incubating the purified enzymes (0.1 mg·ml−1) in the same buffer at 30°C, 40°C, and 50°C, and the residual activity was assayed as described above.

1997) for genes encoding oxidoreductases resulted in 11 open reading frames as candidates to design specific primers for discovering synthetically useful biocatalysts. With standard cloning techniques and the genomic DNA of Bacillus sp. ECU0013 as template, E. coli overexpression plasmids for the 11 proteins of interest were created. During the screening process, a ytbE gene-encoding reductase (YtbE) was found to be able to reduce 2-chloroacetophenone to (R)2-chloro-1-phenylethanol, and was chosen as a potential biocatalyst for further studies. The ytbE nucleotide sequences identified in this study have been submitted to GenBank (accession no. HM590486). A BLAST search revealed that the deduced amino acid sequence of ytbE gene is practically identical to that of a putative aldo-keto reductase from B. subtilis 168 (Genbank accession no. CAB14865).

Effect of metal ions and additives on activity

Protein purification

Influence of various metal ions and additives on enzyme activity was investigated by pre-incubating the enzyme with different compounds in 50 mM sodium phosphate buffer (pH 7.0) for 20 min at 30°C. The enzyme activity was estimated using standard assay protocol. Relative activity was expressed as a percentage of the activity in the absence of any test compound.

The N-terminal His-tagged recombinant enzyme, which was fully soluble, was purified to electrophoretic homogeneity by immobilized metal affinity chromatography. The specific activity of enzyme after purification was 1.62 U mg−1, corresponding to a 1.3-fold improvement in purity compared to the crude extract. Samples of the crude extract and target fraction after purification were analyzed by SDS–PAGE (Fig. 1), which revealed a single band with an apparent molecular size of 35.7 kDa, corresponding to the theoretical value of the enzyme (Fig. 1, lane 3). Additionally, the purified protein eluted from the TSK gel column as a single peak with an elution volume corresponds to an apparent molecular mass of 31.5 kDa, suggesting a monomeric structure.

purified reductase were calculated from Lineweaver–Burk plot. pH and temperature optima and thermostability

Enantioselectivity The enantioselectivity was determined by examining the reduction of aromatic ketones and keto esters using an NADPH regeneration system consisting of the purified reductase and glucose dehydrogenase (GDH). The reaction mixture containing 0.5 mM NADP+, 2 mM aromatic ketones or 10 mM keto esters, 0.8 U of the purified reductase, 0.4 U of GDH, and 5% glucose in 0.4 ml of 50 mM potassium phosphate buffer (pH 7.0) was incubated at 30°C with shaking for 12 h. After the reaction, each reaction mixture was extracted twice with ethyl acetate. The enantiomeric excess (ee) of the product and the level of conversion were determined by GC or HPLC analysis. GC analyses were performed using a CP-Chirasil-DEX CB (Varian, USA) or Beta-DEX 120 column (Supelco, USA). HPLC analyses were performed using Chiralcel OD-H columns (Daicel Co., Japan; 250 mm×Φ4.6 mm).

Results Screening of the recombinant reductases Our approach was to find new reductases with capability of reducing prochiral ketones to corresponding chiral alcohols. Analysis of the Bacillus subtilis 168 genome (Kunst et al.

Substrate specificity Substrate specificity of the enzyme was assessed rapidly by using spectrophotometric assays. The ability of the enzyme to catalyze the reduction of different aromatic ketones and keto esters was determined (Table 1). Acetophenone Fig. 1 SDS–PAGE analysis of the purified YtbE. Lane 1 protein markers, 2 crude extract, 3 purified enzyme. Protein bands were visualized by silver staining

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Table 1 Substrate specificity of the reductase

4′-Aminoacetophenone 2-Acetylpyridine 3-Acetylpyridine 4-Acetylpyridine Keto esters Ethyl acetoacetate Ethyl 4-chloroacetoacetate Ethyl 4,4,4-trifluoroacetoacetate Ethyl benzoylacetate Ethyl 3-oxo-pentanoate Ethyl pyruvate Ethyl 3-methyl-2-oxo-butyrate Methyl benzoylformate Ethyl 2-oxo-4-phenylbutyrate

9.0 100 40.3 140 0 64.2 32.8 0 43.3 13.4 34.3

Effect of various metal ions and additives 145 8,776 74.6 13.4 16.4 7,821 29.5 309 576

The reductase activity was estimated using the standard assay protocol. The activity for 2-chloroacetophenone was taken as 100% and those for others were represented as percentages of that for 2-chloroacetophenone

derivatives and acetyl pyridines were accepted by the reductase, except for propiophenone and 4′-aminoacetophenone. The reductase showed the strongest activity on 2,2,2trifluoacetophenone among the substituted acetophenones tested. Meanwhile, both α- and β-keto esters served as suitable substrates for the reductase. The obviously high activity was observed on ethyl 4-chloroacetoacetate and ethyl pyruvate. In the present work, ethyl pyruvate was chosen as the standard substrate to assay the enzyme activity during the enzyme characterization and to calculate kinetic parameters of the purified reductase. The maximal reaction rate (Vmax) and apparent Michaelis–Menten constant (Km) were found to be 2.66±0.14 Umg−1 protein and 4.47±0.23 mM, respectively. pH and temperature effects and thermostability The effect of pH on the enzyme activity was examined in the range of pH 4.0–10.0. As demonstrated in Fig. 2a, the optimal activity was observed at pH 6.5 and the enzyme showed good activity (>75%) at pH ranging from 6 to 8. The optimum temperature of carbonyl reductase activity

The effect of various compounds on the purified reductase was assessed by incubating the enzyme with salts of different metal ions or additives at a final concentration of 1 mM (Table 2). The enzyme displayed a slightly higher 120

A Relative activity (%)

Aromatic ketones Acetophenone 2-Chloroacetophenone 2-Bromoacetophenone 2,2,2-Trifluoroacetophenone Propiophenone 4′-Chloroacetophenone 4′-Bromoacetophenone

Relative activity (%)

100 80 60 40 20 0

3

4

5

6

7

8

9

10

11

pH 120


Substrate (2 mM)

was determined by measuring the enzyme activity at 20– 65°C (Fig. 2b). The maximum activity was observed at around 50°C, being 150% higher than that under standard assay conditions (30°C). Thermostability of the purified carbonyl reductase was examined at temperatures of 30°C, 40°C, and 50°C (Fig. 3). The enzyme was quite labile at higher temperatures (40°C and 50°C) but more stable at 30°C. The half-lives of the enzyme measured at 30°C, 40°C, and 50°C were 268, 27, and 10 min, respectively. This suggests that higher temperatures will result in a rapid loss of the reductase activity. The relatively low protein concentration of the purified enzyme sample (ca. 0.1 mg·ml−1) might be responsible for the poor thermostability.

B 100 80 60 40 20 0 10

20

30

40

50

60

70

Temperature (oC) Fig. 2 Effects of pH and temperature on activity of the purified enzyme. a To ascertain the pH optima of the purified enzyme, enzyme assay was performed using standard assay procedure in the following buffers of 50 mM: (1) citrate (pH 4.0–6.0), (2) phosphate (pH 6.0– 8.5), and (3) Gly–NaOH (pH 8.5–10.0). b The optimum temperature for the carbonyl reductase was estimated at various temperatures (20– 65°C) in phosphate buffer (50 mM, pH 7.0) using the standard assay procedure. Relative activity was expressed as a percentage of maximum activity under the experimental conditions


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tration were also subjected to bioreduction by the whole cells of recombinant E. coli under the same conditions, and the optical purities of the corresponding alcohols were extremely satisfactory (>99% ee).

Residual activity (%)

100 80 60 40

Discussion 20 0

0

60

120

180

240

300

Time (min) Fig. 3 Thermostability of the purified enzyme at different temperatures. Diamond 50°C, square 40°C, triangle, 30°C. Samples were withdrawn at different time intervals to estimate the residual activity using the standard assay protocol. Residual activity was expressed as a percentage of the activity measured initially without any pre-incubation

activity with the addition of Ca 2+. Metal ions that considerably activate the reductase were not found. On the other hand, the presence of Ag+, Ni2+, Pb2+, and Zn2+ considerably diminished the activity and Fe3+ totally inhibited the activity. Ni2+ could bind with His116, a catalytically important residue for the reductase activity (Lei et al. 2009; Kilunga et al. 2005), which probably resulted in its inhibitory effect on the activity. The presence of EDTA, a chelating agent, slightly affected enzyme activity, suggesting that the protein does not require metals for its activity. Enantioselectivity Enantioselectivity of the enzyme was analyzed in aqueous phosphate buffer using glucose dehydrogenase/glucose as an external NADPH regeneration system. The results were summarized in Table 3. It can be seen that YtbE catalyzes all the aromatic ketones tested affording chiral alcohols with excellent enantiomeric excess (>99%). Among them, 2,2,2trifluoroacetophenone and 2-acetylpyridine were efficiently reduced by the enzyme, affording 100% conversion. The versatility of the enzyme was further shown by using keto esters as substrates, and it was found that YtbE functions as a robust biocatalyst to reduce all of the five keto esters tested with high reactivity. The enantioselectivities were very high for the bioreduction of ethyl acetoacetate, ethyl 4,4,4trifluoroacetoacetate, and ethyl pyruvate (>99% ee), but only relatively moderate for ethyl 4-chloroacetoacetate and ethyl 2-oxo-4-phenylbutyrate, giving 62% ee and 86% ee of products, respectively. On the whole, the enantioselectivity data indicate that the hydride of NADPH is transferred to the Re-face of the carbonyl group, suggesting that the enantiopreference of this enzyme generally follows Prelog’s rule, except for the reduction of ethyl 2-oxo-4-phenylbutyrate. Furthermore, all these aromatic ketones at 10 mM concen-

This study demonstrated the utility of screening a set of recombinant enzymes created by using bioinformatics and gene cloning techniques for rapid identification of useful biocatalysts capable of transforming targeted substrates. It would also be an efficient and easy-handling approach to allow high-throughput screening of various substrates. In the screening of 11 E. coli strains expressing 11 recombinant oxidoreductases from Bacillus sp. ECU0013, the ytbE gene-encoding reductase (YtbE) was found to be able to reduce prochiral ketones. The deduced amino acid sequence of ytbE gene is practically identical to that of putative aldoketo reductase from B. subtilis 168. The YtbE enzyme from B. subtilis has been assigned as AKR5G2 of aldo-keto reductase (AKR) superfamily on the basis of sequence alignment. To date, although YtbE from B. subtilis has been studied by crystallographic researchers and found to show catalytic activity on aldehydes (Lei et al. 2009), the use of YtbE for asymmetric reduction of prochiral carbonyl compounds has not yet been reported so far. In this work, the reductase was overexpressed, purified to homogeneity, and characterized with respect to substrate specificities and prochiral selectivities. In addition to catalyzing the reduction of aldehyde or aldose, the common AKR substrates, some members of AKR superfamily were found to reduce keto esters, such as 2,5-diketo-D-gluconate reductase (YqhE) from E. coli (Q46857; Habrych et al. 2002), carbonyl reductase from Gluconobacter oxydans (YP_191077; Schweiger et al. Table 2 Effect of metal ions and additives on reductase activity

The reductase was pre-incubated with the various metal ions or additives for 20 min and the relative activity was determined using the standard assay. Relative activity was expressed as a percentage of the activity obtained in absence of any test compound

Metal ion (1 mM)


Ag+ Ca2+ Co2+ Cu2+ Fe3+ Mg2+ Mn2+ Na+ Ni2+ Pb2+ Zn2+ EDTA

3.2 104 17.1 7.2 0 101 90.4 96.3 4.8 3.2 2.4 108

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Table 3 Stereoselective reduction of different substrates by the purified reductase

Substrate

Product

1a Cl

2a Br

CF3

CF3

4a

>99 (R)

49.6 ± 1.8

>99 (S)

56.0 ± 0.7

>99 (S)

100 ± 0

>99 (S)

33.2 ± 0.6

>99 (S)

66.8 ± 2.1

>99 (S)

100 ± 0

>99 (S)

100 ± 0

62.2 ± 0.6 (R)c

100 ± 0

>99 (R)

100 ± 0

>99 (S)

OH

5a

Cl

5b

Cl

O

OH

6a

Br

6b

Br

O

OH

N

N

7b

7a O

OH

N

N

8b

8a O

OH

N

N

9a

9b

O

O

O

10a

O

11a

Cl

CF3

O

12a

11b

OH

O

CF3

O

OH

O

O

O

10b

O

O

OH

O

O Cl

12b

OH

O

O

O

13a

O

13b

OH

O

O

O O

100 ± 0 4b

O

Determined by GC analyses after acetylation of the product

>99 (R)

OH

O

c

72.8 ± 2.0 3b

3a

O

>99 (R)

OH

Br

Conversion yields and enantiomeric excesses were determined by chiral GC analysis. Configurations of products were determined by comparing the retention time with that of standard samples

74.5 ± 2.1 2b

O

b

>99 (S)

OH Cl

Not optimized (reaction time of 12 h)

70.8 ± 0.9 1b

O

a

ee (%)b

OH

O

O

Conversion (%)a, b

14a

2010), β-keto ester reductase from Penicillium citrinum (CAD43583; Itoh et al. 2004), aldehyde reductase from Sporobolomyces salmonicolor (P27800; Yamada et al. 1990; Kita et al. 1996), and 2-methylbutyraldehyde reductase (Ypr1p) from Saccharomyces cerevisiae (Q12458; Ford and Ellis 2002). Our results showed that

100 ± 0 O

85.7 ± 0.4 (S)

14b

YtbE accepts both α- and β-keto esters. In fact, a BLAST analysis revealed that the primary structure of YtbE shows 47–34% identities to those reductases. However, since few of these aldo-keto reductases had been tested for aromatic ketone reductions, we assessed the ability of YtbE to reduce aromatic ketones. Fortunately, the result was very encour-


aging that YtbE serves as a versatile reductase with a broad substrate spectrum towards aromatic ketones in addition to keto esters. Studies on substrate specificity of the enzyme revealed that the substituents on the phenyl ring exert some effect on the enzyme activity. It seemed that the electronwithdrawing substituents enhanced the reduction of the substrate while the electron-releasing groups (like amino-) demolished the reductase activity. Chiral alcohols are important synthons for pharmaceuticals, agrochemicals, and fine chemicals, and are thus of steadily increasing interest. All the aromatic ketones tested could be reduced by YtbE with high optical purity of products (>99% ee), although the enantioselectivity was only moderate for the reduction of ethyl 4-chloroacetoacetate (62% ee) and ethyl 2-oxo-4-phenylbutyrate (86% ee). Some of the chiral alcohols produced have been used as valuable intermediates for synthesis of biologically or pharmacologically active compounds, for example, (R)-2b for antidepressant fluoxetine, tomoxetine, and nisoxetine (Ramu et al. 2002); (R)-11b for nutrient (R)-carnitine (Kitamura et al. 1988); and (R)-12b for antidepressant befloxatone (Rabasseda et al. 1999). (R)-4b is applicable to the synthesis of liquid crystals (Fujisawa et al. 1993), and optically active pyridyl alcohols [e.g., (S)-7b, (S)-8b, and (S)-9b] are used as pharmaceutical intermediates and chiral ligands in asymmetric synthesis (Okano et al. 2000). YtbE generally exhibited Prelog specificity except for the reduction of ethyl 2-oxo-4-phenylbutyrate. That might be due to the bulkiness of the substituents, which affected the enzyme’s enantiopreference. It was interesting to note that the substituent at the 4-position of ethyl 3-oxo-butyrate exerted great effect on the enzyme enantioselectivity. Ethyl 4,4,4-trifluoroacetoacetate (12a) was found to be reduced to ethyl (R)-4,4,4-trifluoro-3-hydroxybutanoate [(R)-12b] with excellent enantioselectivity. Several studies have been reported on the asymmetric reduction of 12a using Baker’s yeast (Davoli et al. 1999), Bacillus pumilus Phe-C3 (Zhang et al. 2006), and Saccharomyces uvarum SW-58 (He et al. 2007). However, the unsatisfactory enantioselectivity of these bioreactions do limit their applications for practical production. Therefore, YtbE was established to be attractive for the production of (R)12b. Further studies on asymmetric synthesis of (R)-12b with an E. coli transformant coexpressing both the ytbE and the glucose dehydrogenase (GDH) genes are still in progress. In conclusion, YtbE exhibited high stereoselectivity towards various carbonyl substrates of interest, which is one of the most important factors for determining the potential value of an enzyme. As far as we know, this is the first report of ytbE gene-encoding recombinant aldo-keto reductase from Bacillus sp. serving as a robust biocatalyst for stereoselective reduction of prochiral carbonyl com-

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pounds. The present study will provide a useful guidance for further application of this enzyme in the biocatalytic production of chiral synthons. Further improvement of enzyme functions like activity, thermostability or enantioselectivity might be achieved via enzyme engineering approaches including directed evolution, immobilization, or a combination of these techniques. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (Nos. 20773038 and 20902023), Ministry of Science and Technology, P.R. China (Nos. 2009CB724706 and 2009ZX09501-016), China National Special Fund for State Key Laboratory of Bioreactor Engineering (No. 2060204), and Shanghai Leading Academic Discipline Project (No. B505).

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