Biotechnology Techniques, Vol 12, No 1, January 1998, pp. 35–37
A simple method for obtaining a highly enantioselective enzymatic preparation from baker’s yeast for the reduction of b-keto esters Reginaldo R. Menezes*, Alexandre S. Santos, Enrique G. Oestreicher and Gerson F. Pinto. Departamento de Bioqu´ımica, Instituto de Qu´ımica, Universidade Federal do Rio de Janeiro, 5o Andar Bloco A, Centro de Tecnologia, Ilha do Fundao, ˜ Rio de Janeiro, 21945-900, FAX (55-21)290-4746, Brasil, E-mail:
[email protected]. Baker’s yeast b-keto ester oxidoreductase was partially purified using anion exchange chromatography (DEAESephacell) and was enantioselective for the production of ethyl (S)-(a)-b-hydroxybutanoate and suitable for kinetic studies.
Introduction Chiral b-hydroxy esters are recognized to be valuable building blocks in asymmetric organic synthesis because of their bifunctional character and as so, they have been used as starting material for the synthesis of chiral molecules with biological activities (Hungerbuhler ¨ et al., 1981; Seebach et al., 1984). Since the chemical synthesis of enantiomerically pure bhydroxy esters is difficult and expensive, microbiological methods have been shown to be convenient alternatives (MacLeod et al., 1964; Seebach et al., 1984). Specifically, in the case of these compounds, Baker’s yeast has been the microorganism of choice to perform the enantioselective reduction of various b-keto esters and b-keto acids to the corresponding b-hydroxy compounds with variable but rather high enantiomeric excess (Seebach et al., 1984). This phenomenon is explained by the fact that yeast contains several enzymes that can reduce these compounds with different stereospecificities (Sieh et al., 1985; Heidlas et al., 1988; Nakamura et al., 1991). These keto ester oxidoreductases have been previously isolated and purified by chromatographic methods involving several steps of anion exchange, hydrophobic interaction and gel filtration column chromatography (Heidlas et al., 1988; Nakamura et al., 1991; Ishihara et al. 1994). In the present paper we report the isolation and partial purification of an oxidoreductase NADP-dependent from Baker’s yeast that enantioselectively catalyzes the reduction ethyl acetoacetate. The isolation procedure is rapid and © 1998 Chapman & Hall
simple because it just involves anion exchange chromatography. Materials and methods Materials Commercial baker’s yeast (freshly packed) was obtained from Fleischmann & Royal Ltda, Rio de Janeiro, Brazil. Glucose, Glucose dehydrogenase from Bacillus megaterium (GDH), Trizma base, NADP, NADPH, protamine sulfate, bovine serum albumin (Fraction V) (BSA), 2mercaptoethanol and phenylmethylsulfonyl fluoride were product of Sigma Chem.Co. ethyl acetoacetate, rac-ethyl3-hydroxybutanoate, ethyl (R)-(2)-3-hydroxybutanoate and ethyl (S)-(1)-3-hydroxybutanoate were purchase from Aldrich Chem. Co. DEAE-Sephacell was a product from Pharmacia Biotech, Uppsala, Sweden. All other chemicals were of analytical or chromatographic grade. Methods Enzyme assays were conducted in 3 ml quartz cuvets with 1-cm light path at 25°C by following the NADPH absorbance decrease at 340 nm with a Beckman DU 70 recording spectrophotometer as described by Heidlas et al. (1988). The enzyme activity was expressed in terms of International Units (IU) (One IU being the amount of enzyme oxidizing 1 mmole of NADPH/min at 25°C and pH 7.2). Protein concentration was determined by using a method suitable for the detection of low protein content (Hartree, 1972). Biotechnology Techniques ⋅ Vol 12 ⋅ No 1 ⋅ 1998
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R.R. Menezes et al. The crude extract from yeast cells was prepared by washing the cells with cold distilled water and then resuspending them in buffer I (50 mM Tris/HCl buffer, pH 7.2, containing 5 mM of 2-mercaptoethanol and 2 mg of phenylmethylsulfonyl fluoride/l to make a 50% (wet weight/volume) suspension. This suspension was passed through a French pressure cell with 1 inch diameter piston (14,200 PSI). The supernatant fluid resulting after centrifugation of the homogenate at 8,000xg for 10 min was centrifuged at 100,000xg for 90 min. The supernatant fluid obtained from this latter centrifugation (referred to as crude extract) was treated with 10% volume of 15mg of protamine sulfate/ml aqueous solution in order to remove nucleic acids. After centrifugation at 10,000xg for 20 min, the resulting supernatant fluid was adjusted to pH 7.2 to initiate the chromatographic procedure. 50 ml of the crude extract (1.7 g of protein), were applied to a DEAE-Sephacell column (25 3 2.6 cm internal diameter) equilibrated with buffer I. The column was first eluted with buffer I, when a large non-adsorbed protein fraction containing approximately 12% of the total b-keto ester oxidoreductase present in the crude extract, was eluted. The elution was continued using a linear gradient concentration of 0–0.8 M NaCl in buffer I (300 ml). A second peak of this enzymatic activity was eluted at 0.2 M NaCl. These fractions, accounting for about 88% of the total enzymatic activity, were pooled, dialyzed overnight against 50 volumes of buffer I and rechromatographed on a DEAE-Sephacell column (4 3 2.5 cm internal diameter). The experimental conditions of this chromatographic step were the same of the former one, both being performed at room temperature (20°C). Reduction of ethyl acetoacetate, in a preparative scale of 78 mg was performed in a jacketed batch reactor continuously stirred of 50 ml. The reaction medium contained in a total volume of 20 ml: 0.2 M Tris-HCl buffer, pH 7.2, 30 mM ethyl acetoacetate, 180 mM glucose, 42 IU of GDH, 2 IU of keto ester oxidoreductase, 1% (w/v) BSA and 1% (w/v)of sodium azide. The reaction mixture was maintained under continuos magnetic stirring and the temperature was kept at 25°C by circulating water through the jacket of the reactor with a thermocirculating bath. Reaction was started by adding 0.1 mM NADP. The extent of reaction
was determined in 50 ml aliquots removed from the reaction mixture at different times. These samples were diluted 20 times with a mixture of methanol/acetonitrile (20:80) and after centrifugation at 15000xg for 1 min, both the residual concentration of ethyl acetoacetate and the concentration of ethyl 3-hydroxybutanoate produced were determined by HPLC by using a C18 reverse phase column with UV detection (210 nm) and eluted isocratically with acetonitrile:H2O 30:70 (v/v). The enantiomeric excess and absolute configuration of the product were determined by chiral high resolution gas chromatography (HRGC) which was performed on a capillary column (20 m 3 0.3 mm internal diameter) coated with 2,3-bi-O-methyl-6-O-tbuthyl-dimethyl-silaneb-cyclodextrin (TBCD/SE-54), isothermally at 70°C by using a HP-5890 (Series II) chromatograph. Ethyl (R)(2)-3-hydroxybutanoate, ethyl (S)-(1)-3-hydroxybutanoate and ethyl rac-3-hydroxybutanoate were used as standards. Results and discussion Table 1 is a summary of the purification protocol devised to obtain Baker’s yeast b-keto ester oxidoreductase. The specific activity of the enzyme preparation was 0.386 IU/mg of protein, which represents a purification of 21 fold with a yield of 40% referred as percentage of the total a-keto ester oxidoreductase present in the crude extract (Table 1). In order to test the quality of this enzyme preparation for performing kinetic experiments, the preparation was submitted to further purification by using a HPLC system equipped with a diode array detector (PDA) and a gel filtration SW 300 column (Protein-pak, 7.8 3 300 mm, Waters, Millipore Co.,U.S.A.) .The column was equilibrated and eluted with a 0.1 M phosphate buffer (pH 7.0) containing 0.1 M KCl. The sample was fractionated into ten peaks of protein, only one of which contained the enzymatic activity. This peak contained only 16% of the total protein of the sample. The kinetic parameters of the Michaelis-Menten equation were estimated and the values obtained with this enzyme preparation as well as those obtained with the enzyme not submitted to this extra purification step were compared. It was found that both enzyme preparation presented an identical value of KMapp
Table 1 Purification of baker’s yeast β-keto ester oxidoreductase. Step Crude extract DEAE-Sephacell (25 × 2.6 cm) DEAE-Sephacell (4 × 2.6 cm)
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Total activity (IU)
Total protein (mg)
Specific activity (IU/mg)
Recovery (%)
Purification (× fold)
30.4 16.9 12.2
1700 80 31.5
0.018 0.213 0.386
100 55.6 40.1
1 12 21
Biotechnology Techniques ⋅ Vol 12 ⋅ No 1 ⋅ 1998
A simple method for obtaining a highly enantioselective enzymatic preparation from baker’s yeast for the reduction of β-keto esters Table 2 Reaction time-course for the reduction of ethyl acetoacetate into ethyl-3-hydroxybutanoate catalyzed by baker’s yeast β-keto ester oxidoreductase. Time (h) ethyl acetoacetate (mM) ethyl-3-hydoxybutanoate (mM)
0.6 23.2 4.9
1.1 18.2 8.5
for ethyl acetoacetate (1.0 mM). This result suggests that the enzyme preparation obtained with just anion exchange chromatography is appropriated for kinetic studies. Reaction time-course for the reduction of ethyl acetoacetate (78 mg) by the enzymatic preparation, using the glucose/GDH in situ NADPH regeneration system is shown in Table 2. As shown in Table 2, after approximately 4 h of reaction under the conditions detailed in Materials and Methods total conversion of the substrate into ethyl-3-hydroxybutanoate was achieved. The analysis of ethyl 3-hydroxybutanoate by chiral HRGC showed that when rac-ethyl 3-hydroxybutanoate was analyzed two peaks of the same area were obtained with retention times of 5.03 and 5.32 min, respectively. The standard of ethyl (S)-(1)-3-hydroxybutanoate presented a retention time of 5.05 min and the corresponding (R)(2)-enantiomer, a retention time of 5.36 min. When the product obtained via enzymatic reduction was analyzed, only one peak with retention time of 5.02 min was detected. Moreover when this product was co-injected with the standard of ethyl (S)-(1)-3-hydroxybutanoate just one peak with retention time of 5.00 min was detected. On the other hand, co-injection of the enzimatically obtained product with the (R)-(2)-enantiomer gave rise to two peaks with retention times of 5.03 and 5.31 min, respectively. These results clearly suggest that the product of the reduction of ethyl acetoacetate catalyzed by the enzymatic preparation described in the present paper is ethyl (S)(1)-3-hydroxybutanoate. In addition, the fact that no trace of the peak with the higher retention time (5.36 min) was detected in the sample of the enzimatically obtained
2.0 12.5 16.9
3.0 5.0 24.2
3.7 0 30
3.9 0 30
product, is indicative of an enantiomeric excess higher than 99% and that the enzymatic preparation of b-keto ester oxidoreductase, obtained with a simple purification procedure involving just anion exchange chromatography, is absolutely enantioselective and thus, suitable for utilization in asymmetric synthesis. Acknowledgments Financial support (grant N° 6426-2) from FUJB/UFRJ and (grant N° 65.96.0656.00) from FINEP is acknowledged. We wish to express our gratitude to Prof. Maria da Conceiç˜ao K.V. Ramos from LADETEC/IQ/UFRJ for running HRGC and to Prof. Jos´e Godinho S. Junior from DBQ/IQ/UFRJ for his contribution with the SW300 column chromatografic work. References Hartree, E.F. (1972). Anal. Biochem. 48, 422–427. Heidlas, J., Engel, K.H. and Tressl, R. (1988). Eur.J.Biochem. 172, 633–639. Hungerbuhler, ¨ E., Seebach, D and Wasmuth, D. (1981). Helv. Chim.Acta 64, 1467–1487. Ishihara, K., Nakajima, N., Tsuboi, S. and Utaka, M. (1994). Bull.Chem.Soc.Jpn. 67, 3314–3319. MacLeod, R., Prosser, H., Fickentscher, L., Lanyi, J. and Mosher, H.S. (1964). Biochemistry 3, 838–846. Nakamura, K., Kawai, Y., Nakajima, N. and Ohno, A. (1991). J.Org.Chem. 56, 4778–4783. Seebach, D., Sutter, M.A. Weber, R.H., Zuger, ¨ M.F. (1984) Yeast reduction of ethyl acetoacetate: (S)-(a)- 3-hydroxybutanoate. In: Organic Synthesis, G. Saucy, ed. vol. 63. pp. 1–9, New York: John Wiley and Sons, Inc. Sieh, W.-R., Gopalan, A.S. and Sih, C.J. (1985). J.Am.Chem.Soc. 107, 2993–2994.
Received: 1 September Revisions requested: 7 October Revisions received: 3 November Accepted: 4 November
1997 1997 1997 1997
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