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Synthesis of l -homophenylalanine via integrated membrane bioreactor: Influence of pH on yield
Biochemical Engineering Journal 52 (2010) 296–300
Contents lists available at ScienceDirect
Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Short communication
Synthesis of l-homophenylalanine via integrated membrane bioreactor: Influence of pH on yield A.L. Ahmad ∗ , P.C. Oh, S.R. Abd Shukor School of Chemical Engineering, Engineering Campus, Universiti Sains Malaysia, Seri Ampangan, 14300 Nibong Tebal, Penang, Malaysia
a r t i c l e
i n f o
Article history: Received 17 March 2010 Received in revised form 19 August 2010 Accepted 20 August 2010
Keywords: l-homophenylalanine Membrane bioreactor Biocatalysis Biotransformation Reductive amination pH
a b s t r a c t l-homophenylalanine can be used as a precursor for production of angiotensin-converting enzyme (ACE) inhibitor, which possesses significant clinical application in the management of hypertension and congestive heart failure (CHF). Pure chiral l-homophenylalanine can be synthesized efficiently by employing enzymatic methods. The biocatalysts used for biotransformation must be retained and reused due to their costs. Conventional processes for l-homophenylalanine production utilized dialysis bag for biocatalysts retention. Nevertheless, this could only be efficiently adopted for laboratory scale processes which possess low scale-up potential. In order to overcome this problem, integrated membrane bioreactor is highlighted for sustainable biocatalytic synthesis of l-homophenylalanine in environmentally friendly conditions which has not been explored by other researchers. The yield of biocatalytic reactions is greatly affected by the pH of the reaction solution, as enzymes are highly sensitive to pH change. Thus in this study, the influence of pH on the product yield at ambient temperature and atmospheric pressure was evaluated. By using l-phenylalanine dehydrogenase and formate dehydrogenase as the biocatalysts, it was found that pure l-homophenylalanine could be synthesized with >80% yield and an enantiomeric excess of over 99% in the integrated membrane bioreactor by adjusting the pH of the reaction solution. The optimal pH was found to be 8.5, when independent of other parameters. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Over the past decade, the production of angiotensin-converting enzyme (ACE) inhibitor drug intermediates has become increasingly important in the pharmaceutical industry. This is mainly due to the fact that ACE inhibitors have achieved wide-spread usage as one of the first line drugs for treatment of hypertension and congestive heart failure [1–3]. Virtually all ACE inhibitors with therapeutic significance such as enalapril, delapril, lisinopril, quinapril, ramipril, trandolapril, cilazapril and benzapril [4,5], refer to l-homophenylalanine as a common building block, due to the presence of l-homophenylalanine moiety as the central pharmacophore unit [6–8]. Thus far, there exist many conceivable methods of producing enantiomerically pure ACE inhibitor drug precursors, more commonly via chemical or biocatalytic routes by way of kinetic resolution [9] or asymmetric synthesis [10]. Today, compound biotransformation which commonly deals with biocatalysts is an extremely important research area for pharmaceutical drug discovery and development [11]. Biocatalysis is a relatively green and stereoselective technology compared to chemical processes which
∗ Corresponding author. Tel.: +60 4 5941012; fax: +60 4 5941013. E-mail addresses: [email protected], [email protected] (A.L. Ahmad). 1369-703X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2010.08.010
generally suffer from process complexity, high cost, and environmental pollution. Enzyme-catalyzed reactions are often highly stereoselective [12], can be carried out at ambient temperature and atmospheric pressure thus avoiding the use of more extreme conditions. This minimizes problems of isomerization, racemization, epimerization or rearrangement [13]. Nevertheless, biocatalysts used in biotransformation need to be retained for simple downstream processing, and improve process sustainability. It is possible to prepare l-homophenylalanine from the corresponding 2-oxo-4-phenylbutanoic acid [14,15] via various stereoselective methods including enzymatic reduction. To date, asymmetric reduction of prochiral ketone remains one of the most investigated methods for production of chiral l-homophenylalanine. Many successful advances have been published thus far, with reproducible results of satisfactory yield. One of the most established methods for synthesizing lhomophenylalanine on a laboratory scale was carried out via enzyme-catalyzed asymmetric synthesis of keto acids [16,17]. In this method, prochiral ketone was converted via reductive amination to enantiopure products [18] with bulky side chains by addition of biocatalysts such as l-phenylalanine dehydrogenase [19] in the presence of cofactor. Since the biological cofactor is too expensive to be used in stoichiometric amounts, efficient and cost-effective in-situ regeneration of the compound is prerequisite in order to meet the
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Fig. 1. Synthesis of l-homophenylalanine from 2-oxo-4-phenylbutanoic acid catalyzed by l-phenylalanine dehydrogenase coupled to NADH regeneration catalyzed by formate dehydrogenase.
economic constraints [20]. For this reaction, NADH was regenerated in parallel using formate dehydrogenase which catalyzes the oxidation of formate to CO2 with the concomitant reduction of NAD to NADH [21–23]. The reaction equilibrium strongly favors l-homophenylalanine synthesis as the byproduct CO2 which is not obnoxious to the drug precursors can be easily removed. In this instance, l-phenylalanine dehydrogenase and formate dehydrogenase used in the reductive amination process are preferentially retained in order to improve the sustainability of the process. Membrane-based systems have since then become the opted technology for retention of biocatalysts, both on laboratory and industrial scales. Biocatalysts used for biotransformation and biochemical synthesis must be separated from the product effluent and recycled due to their cost. For the reductive amination process, the study of l-phenylalanine dehydrogenase retention has previously been carried out by using dialysis bag [16]. Nevertheless, this process could only be efficiently adopted for laboratory scale process with low scale-up potential. In order to overcome this problem, membrane bioreactors (MBRs) are highlighted in the current research, pertaining to their potential to retain the biocatalysts that are found in biotransformation reaction medium, and for the synthesis of high purity pharmaceutical products. The goal of this work was to investigate the production of vital pharmaceutical precursor namely l-homophenylalanine via enantioselective reduction of 2-oxo-4-phenylbutanoic acid by the NADH-dependant l-phenylalanine dehydrogenase in an integrated membrane bioreactor set up using the formate/formate dehydrogenase system for coenzyme regeneration (Fig. 1). In addition, the optimum pH for the synthesis of l-homophenylalanine using an
integrated membrane bioreactor for biotransformation was studied, simultaneously evaluating the biocatalysts retention in the membrane bioreactor. 2. Materials and methods 2.1. Enzymes l-phenylalanine dehydrogenase (recombinant in E. coli) from Rhodococcus sp. M4 (EC 1.4.1.20), was obtained from Jülich Chiral Solutions GmbH, Germany; formate dehydrogenase from Candida boidinii (EC 1.2.1.2) was obtained from Sigma–Aldrich, Malaysia; and NADH was purchased from Roche Diagnostic, Germany. 2.2. Chemicals 2-Oxo-4-phenylbutanoic acid could be synthesized according to the method proposed by Chen et al. [24]; sodium formate was obtained from Sigma–Aldrich, Malaysia; and 1,4-dithiothreitol was purchased from Merck, Malaysia. 2.3. Apparatus The biotransformation and separation were carried out in an integrated membrane bioreactor system operated in semi-batch mode where the substrates were pumped into the membrane bioreactor prior to reaction. The set up of the system is illustrated in Fig. 2. A 1 L reactor module is coupled to an in-situ separation unit for the removal of products while retaining the biocatalysts.
Fig. 2. Schematic flow diagram of the integrated membrane bioreactor system for simultaneous reaction and retention of biocatalysts coupled to an acidification device for l-homophenylalanine production.
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Substrates and coenzyme were pumped into the membrane bioreactor at 5 mL/min flow rate, with the product leaving the membrane bioreactor unit through an ultrafiltration membrane with varying molecular weight cut-off (MWCO) for different enzymes. The membrane bioreactor unit was equipped with an overhead stirrer, pH electrode, temperature sensor, and the internal atmosphere was kept inert with argon gas. The product enriched solution from the membrane bioreactor was pumped into the subsequent acidification vessel system. 2.4. Membrane The Spectra/Por® 2 semi-permeable ultrafiltration membrane used in this work was purchased from Chemopharm and was made of regenerated cellulose. This type of membrane was discovered to be effective for the separation of biocatalysts and the membrane satisfy the chemical nature requirements that will provide the desired retention of biocatalysts. The membrane also presents adequate mechanical and thermal stability. The MWCO of the flat sheet regenerated cellulose membrane used was 12–14,000 Da with an active surface area of 133 cm2 and a nominal thickness of 0.002 in.. 2.5. Synthesis and isolation of l-homophenylalanine The synthesis was preferably conducted in a reaction mixture containing 2-oxo-4-phenylbutanoic acid (1.2%w/v), 1,4dithiothreitol (5.6 mmol), sodium formate (56 mmol), formate dehydrogenase (595 U), NADH (0.14 g) and l-phenylalanine dehydrogenase (945 U) in deionized water (840 mL) (Fig. 1) [19,21]. The solution was stirred at 30 ◦ C and in an inert atmosphere with the addition of 1N ammonium hydroxide to maintain the pH at a constant value. Upon retention of the biocatalysts after a mean residence time of 3–5 days in which the conversion level is stable, the product solution was removed from the membrane bioreactor by exerting a pressure of 1.5 bar and acidified to pH 5.5 with dilute HCl. The resulting white precipitate was collected by filtration, washed with cold water and dried in vacuum to yield l-homophenylalanine with >95% purity. 2.6. Analytical methods 2.6.1. Determination of the enantiomeric excess The enantiomeric excess of l-homophenylalanine was determined using chiral HPLC on an Astec Chirobiotic T column (Orbiting Scientific Co.) using ethanol/water, 10/90 (v/v) as mobile phase at 1 mL/min flow rate with detection at 210 nm. 2.6.2. Determination of yield of l-homophenylalanine l-homophenylalanine yield was determined after collection of the white precipitate via filtration. For HPLC analysis of the components in the effluent of the membrane bioreactor, a C18 column was used with gradient elution from an initial solution of 10% of eluent B + 90% of eluent A to a final solution of 100% of eluent B in 20 min, wherein eluent A contains 5% acetonitrile in 0.1% trifluoroacetic acid and eluent B contains 95% acetonitrile in 0.1% trifluoroacetic acid, at 1 mL/min flow rate with detection at 214 nm. 2.6.3. Determination of optimum pH The effect of pH on the activity of l-phenylalanine dehydrogenase and formate dehydrogenase were determined for reductive amination. The optimum pH for the reaction was subsequently obtained. The reaction solution was maintained at a constant pH value with the addition of 1N ammonium hydroxide. Five sets of experiment were carried out at different pH values due to the pH-
Fig. 3. Dependence of enzyme activity on pH for l-phenylalanine dehydrogenase and formate dehydrogenase.
dependence of the biocatalysts, and the yield of the product was observed for each experiment. 2.6.4. Biocatalysts assay and retention Assays were performed by sampling aliquots of the product solutions and equilibrating in a cuvette at 30 ◦ C, with the addition of NADH and ammonium chloride in Tris buffer. The reaction was started if the presence of residual l-phenylalanine dehydrogenase was detected in the product solutions. The reaction progress was monitored spectrophotometrically and the change in absorbance at 340 nm (A340 nm ) was recorded for determination of the extent of biocatalysts retention. The enzyme activity was calculated using the following equation:
Units/mL enzyme =
A340nm / min sample − A340nm / min blank (V ) (df ) (ε) (v)
(1)
where: A340 nm = change in absorbance at 340 nm; V = total volume of assay (mL); v = volume of enzyme (mL); df = dilution factor; ε = 6.22 = millimolar extinction coefficient of -NADH at 340 nm. 3. Results and discussion Prior to the determination of optimal reaction solution pH for synthesis of l-homophenylalanine via biotransformation, the effect of pH on the activity of biocatalysts used in the biotransformation namely l-phenylalanine dehydrogenase and formate dehydrogenase were observed and the results obtained were fitted to polynomials. As shown in Fig. 3, pH of 9.2 was optimal for l-phenylalanine dehydrogenase. The pH-dependence of formate dehydrogenase showed broader maxima from pH 7 to 9 with an optimum value of pH 8. It is observed that the enzyme activity for both l-phenylalanine dehydrogenase and formate dehydrogenase deteriorates at pH 10, suggesting the deactivation of enzymes at high pH. From the observed optimal pH range for both enzymes, the reaction was subsequently carried out at several pH values to determine the optimal pH for the overall reaction, independent of other parameters. The reductive amination of 2-oxo-4-phenylbutanoic acid using l-phenylalanine dehydrogenase and formate dehydrogenase as biocatalysts proceeded in yields which were dependant on the pH of reaction solution. As can be seen from Fig. 4, the optimum working pH was 8.5, giving the highest yield. For all the experiments, the biocatalysts were fully retained within the membrane bioreactor, monitored by using spectrophotometric methods. Biocatalysts were not detected in the effluent of the membrane bioreactor under all reaction conditions. This shows that the biocatalysts were effectively retained due to size exclusion
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4. Conclusions The asymmetric synthesis of l-homophenylalanine in an integrated membrane bioreactor is a good alternative to the conventional approaches which utilize dialysis tubing for retention of biocatalysts. The advantage of this process was largely due to its high selectivity in rendering an enantiomeric excess of more than 99%, besides avoiding the formation of side products which incur extra costs in downstream processing. The operated membrane bioreactor allowed retention of the investigated biocatalysts due to size exclusion. In order to operate the membrane bioreactor for more than two cycles, biocatalysts need to be added in small amount to cater for the activity loss. l-homophenylalanine could be synthesized in sufficient yield by adjusting the pH of the solution, where the optimal pH was determined to be at 8.5. Other operations conditions such as effect of temperature, substrate concentration and residence time could be further studied to increase the conversion and yield of the product. Fig. 4. Influence of pH on yield of l-homophenylalanine.
by using the flat sheet membrane with 12–14,000 Da MWCO. Thus, it is proven that the integrated membrane bioreactor module could be utilized for retention of l-phenylalanine dehydrogenase and formate dehydrogenase enzymes, and could replace the conventional process which utilized dialysis bag. l-homophenylalanine which precipitated from the reaction mixture was analyzed using Nuclear Magnetic Resonance (NMR). The synthesized l-homophenylanaine had the following physical properties: 1 H NMR (D O): 2.05 ppm (m, 2H), 2.63 ppm (m, 2H), 3.65 ppm 2 (t, 1H) 7.19 ppm (m, 5H). The results showed that pure l-homophenylalanine was obtained for all experiments without the presence of impurities by comparing with a standard. The enantiomeric excess of l-homophenylalanine was ascertained using a chiral T column. Reduction of 2-oxo-4phenylbutanoic acid to l-homophenylalanine was achieved with an enantiomeric excess of over 99% for all experiments since only a single peak was eluted (Fig. 5). The synthesized l-homophenylalanine had a retention time of 7.34 min, where no d-antipode which had a retention time of 10.12 min could be observed. The product was optically pure as determined by optical rotation and compared to an authentic sample and literature ◦ values [˛]20 D = +48 (c1, 1N HCl).
Fig. 5. HPLC Chromatogram showing the enantiomeric excess of the enzymatically synthesized l-homophenylalanine using a Chiral T Column.
Acknowledgements This work was supported by Universiti Sains Malaysia under the Research University Postgraduate Research Grant Scheme No. 8041013, and Research University Grant No. 814002. P.C. Oh gratefully acknowledges the National Science Fellowship (NSF) received from the Malaysian Ministry of Science, Technology and Innovation (MOSTI) for her Ph.D program. References [1] A. Liese, U. Kragl, H. Kierkels, B. Schulze, Membrane reactor development for the kinetic resolution of ethyl 2-hydroxy-4-phenylbutyrate, Enzyme Microb. Technol. 30 (2002) 673–681. [2] A.J. Trevor, B.G. Katzung, S.B. Masters, Katzung & Trevor’s Pharmacology, McGraw-Hill Companies, Inc, New York, 2002. [3] M.A. Ondetti, D.W. Cushman, Inhibition of the renin-angiotensin system: a new approach to the therapy of hypertension, J. Med. Chem. 24 (1981) 355–361. [4] A. Liese, K. Seelbach, C. Wandrey, Industrial Biotransformations, Wiley-VCH Verlag GmbH, Weinheim, 2000. [5] A.L. Johnson, W.A. Price, P.C. Wong, R.F. Vavala, J.M. Stump, Synthesis and pharmacology of the potent angiotensin-converting enzyme-inhibitor N-[1(S)(ethoxycarbonyl)-3-phenylprophyl]-(S)-alanyl-(S)-pyroglutamic acid, J. Med. Chem. 28 (1985) 1596–1602. [6] B. Larissegger-Schnell, S.M. Glueck, W. Kroutil, K. Faber, Enantiocomplementary deracemization of (+/−)-2-hydroxy-4-phenylbutanoic acid and (+/−)-3-phenyllactic acid using lipase-catalyzed kinetic resolution combined with biocatalytic racemization, Tetrahedron 62 (2006) 2912– 2916. [7] Y.L. Bai, S.T. Yang, Biotransformation of R-2-hydroxy-4-phenylbutyric acid by d-lactate dehydrogenase and Candida boidinii cells containing formate dehydrogenase coimmobilized in a fibrous bed bioreactor, Biotechnol. Bioeng. 92 (2005) 137–146. [8] A.L. Ahmad, P.C. Oh, S.R.A. Shukor, Sustainable biocatalytic synthesis of lhomophenylalanine as pharmaceutical drug precursor, Biotechnol. Adv. 27 (2009) 286–296. [9] M.C. Yen, W.H. Hsu, S.C. Lin, Synthesis of l-homophenylalanine with immobilized enzymes, Process Biochem. 45 (2010) 667–674. [10] B.K. Cho, J.H. Seo, T.W. Kang, B.G. Kim, Asymmetric synthesis of lhomophenylalanine by equilibrium-shift using recombinant aromatic l-amino acid transaminase, Biotechnol. Bioeng. 83 (2003) 226–234. [11] W.N. Wu, L.A. McKown, Recent advances in biotransformation of CNS and cardiovascular agents, Curr. Drug Metab. 1 (2000) 255–270. [12] S. Ahuja, Chiral Separations: Application and Technology, American Chemical Society, Washington, 1997. [13] R.N. Patel, A. Goswami, L. Chu, M.J. Donovan, V. Nanduri, S. Goldberg, R. Johnston, P.J. Siva, B. Nielsen, J.Y. Fan, W.X. He, Z.P. Shi, K.W. Wang, R. Eiring, D. Cazzulino, A. Singh, R. Mueller, Enantioselective microbial reduction of substituted acetophenones, Tetrahedron: Asymmetry 15 (2004) 1247–1258. [14] H.H. Lo, W.D. Lin, S.K. Hsu, W.H. Hsu, Asymmetrically simultaneous synthesis of l-homophenylalanine and N-6-protected-2-oxo-6-amino-hexanoic acid by engineered Escherichia coli aspartate aminotransferase, Biotechnol. Prog. 25 (2009) 1637–1642. [15] J.Y. Hwang, J. Park, J.H. Seo, M. Cha, B.K. Cho, J. Kim, B.G. Kim, Simultaneous synthesis of 2-phenylethanol and l-homophenylalanine using aromatic transaminase with yeast Ehrlich pathway, Biotechnol. Bioeng. 102 (2009) 1323–1329.
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[16] C.W. Bradshaw, C.H. Wong, W. Hummel, M.R. Kula, Enzyme-catalyzed asymmetric-synthesis of (S)-2-amino-4-phenylbutanoic acid and (R)-2-hydroxy-4-phenylbutanoic acid, Bioorg. Chem. 19 (1991) 29– 39. [17] P. Busca, F. Paradisi, E. Moynihan, A.R. Maguire, P.C. Engel, Enantioselective synthesis of non-natural amino acids using phenylalanine dehydrogenases modified by site-directed mutagenesis, Org. Biomol. Chem. 2 (2004) 2684–2691. [18] A.S. Bommarius, M. Schwarm, K. Drauz, Biocatalysis to amino acid-based chiral pharmaceuticals – examples and perspectives, J. Mol. Catal. B: Enzym. 5 (1998) 1–11. [19] Y. Asano, A. Yamada, Y. Kato, K. Yamaguchi, Y. Hibino, K. Hirai, K. Kondo, Enantioselective synthesis of (S)-amino acids by phenylalanine dehydrogenase from Bacillus sphaericus: use of natural and recombinant enzymes, J. Org. Chem. 55 (1990) 5567–5571.
[20] U. Kragl, W. Kruse, W. Hummel, C. Wandrey, Enzyme engineering aspects of biocatalysis: cofactor regeneration as example, Biotechnol. Bioeng. 52 (1996) 309–319. [21] M. Politino, M.M. Cadin, P.M. Skonezny, J.G. Chen, Process for Preparing Dipeptidyl IV Inhibitors and Intermediates Therefor, The Patent Cooperation Treaty (PCT). U.S. Patent, WO 2005/106011 A3, 2005. [22] R. Hanson, M.J. Donovan, S. Goldberg, P.A. Jass, W.S. Li, R. Patel, K. Ramig, L.J. Szarka, J.J. Venit, Stereoselective Reductive Amination of Ketones, U.S. Patent 6,140,088, 2000. [23] Z. Shaked, G.M. Whitesides, Enzyme-catalyzed organic-synthesis – NADH regeneration by using formate dehydrogenase, J. Am. Chem. Soc. 102 (1980) 7104–7105. [24] S.T. Chen, M.J. Tseng, T. Kao, B. Sookkheo, T. Surat, Facile Synthesis of l-Homophenylalanine By Equilibrium Shift Enzymatic Reaction Using Engineered Tyrosine Aminotransferase, U.S. Patent 6,146,859, 2000.
Contents lists available at ScienceDirect
Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Short communication
Synthesis of l-homophenylalanine via integrated membrane bioreactor: Influence of pH on yield A.L. Ahmad ∗ , P.C. Oh, S.R. Abd Shukor School of Chemical Engineering, Engineering Campus, Universiti Sains Malaysia, Seri Ampangan, 14300 Nibong Tebal, Penang, Malaysia
a r t i c l e
i n f o
Article history: Received 17 March 2010 Received in revised form 19 August 2010 Accepted 20 August 2010
Keywords: l-homophenylalanine Membrane bioreactor Biocatalysis Biotransformation Reductive amination pH
a b s t r a c t l-homophenylalanine can be used as a precursor for production of angiotensin-converting enzyme (ACE) inhibitor, which possesses significant clinical application in the management of hypertension and congestive heart failure (CHF). Pure chiral l-homophenylalanine can be synthesized efficiently by employing enzymatic methods. The biocatalysts used for biotransformation must be retained and reused due to their costs. Conventional processes for l-homophenylalanine production utilized dialysis bag for biocatalysts retention. Nevertheless, this could only be efficiently adopted for laboratory scale processes which possess low scale-up potential. In order to overcome this problem, integrated membrane bioreactor is highlighted for sustainable biocatalytic synthesis of l-homophenylalanine in environmentally friendly conditions which has not been explored by other researchers. The yield of biocatalytic reactions is greatly affected by the pH of the reaction solution, as enzymes are highly sensitive to pH change. Thus in this study, the influence of pH on the product yield at ambient temperature and atmospheric pressure was evaluated. By using l-phenylalanine dehydrogenase and formate dehydrogenase as the biocatalysts, it was found that pure l-homophenylalanine could be synthesized with >80% yield and an enantiomeric excess of over 99% in the integrated membrane bioreactor by adjusting the pH of the reaction solution. The optimal pH was found to be 8.5, when independent of other parameters. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Over the past decade, the production of angiotensin-converting enzyme (ACE) inhibitor drug intermediates has become increasingly important in the pharmaceutical industry. This is mainly due to the fact that ACE inhibitors have achieved wide-spread usage as one of the first line drugs for treatment of hypertension and congestive heart failure [1–3]. Virtually all ACE inhibitors with therapeutic significance such as enalapril, delapril, lisinopril, quinapril, ramipril, trandolapril, cilazapril and benzapril [4,5], refer to l-homophenylalanine as a common building block, due to the presence of l-homophenylalanine moiety as the central pharmacophore unit [6–8]. Thus far, there exist many conceivable methods of producing enantiomerically pure ACE inhibitor drug precursors, more commonly via chemical or biocatalytic routes by way of kinetic resolution [9] or asymmetric synthesis [10]. Today, compound biotransformation which commonly deals with biocatalysts is an extremely important research area for pharmaceutical drug discovery and development [11]. Biocatalysis is a relatively green and stereoselective technology compared to chemical processes which
∗ Corresponding author. Tel.: +60 4 5941012; fax: +60 4 5941013. E-mail addresses: [email protected], [email protected] (A.L. Ahmad). 1369-703X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2010.08.010
generally suffer from process complexity, high cost, and environmental pollution. Enzyme-catalyzed reactions are often highly stereoselective [12], can be carried out at ambient temperature and atmospheric pressure thus avoiding the use of more extreme conditions. This minimizes problems of isomerization, racemization, epimerization or rearrangement [13]. Nevertheless, biocatalysts used in biotransformation need to be retained for simple downstream processing, and improve process sustainability. It is possible to prepare l-homophenylalanine from the corresponding 2-oxo-4-phenylbutanoic acid [14,15] via various stereoselective methods including enzymatic reduction. To date, asymmetric reduction of prochiral ketone remains one of the most investigated methods for production of chiral l-homophenylalanine. Many successful advances have been published thus far, with reproducible results of satisfactory yield. One of the most established methods for synthesizing lhomophenylalanine on a laboratory scale was carried out via enzyme-catalyzed asymmetric synthesis of keto acids [16,17]. In this method, prochiral ketone was converted via reductive amination to enantiopure products [18] with bulky side chains by addition of biocatalysts such as l-phenylalanine dehydrogenase [19] in the presence of cofactor. Since the biological cofactor is too expensive to be used in stoichiometric amounts, efficient and cost-effective in-situ regeneration of the compound is prerequisite in order to meet the
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Fig. 1. Synthesis of l-homophenylalanine from 2-oxo-4-phenylbutanoic acid catalyzed by l-phenylalanine dehydrogenase coupled to NADH regeneration catalyzed by formate dehydrogenase.
economic constraints [20]. For this reaction, NADH was regenerated in parallel using formate dehydrogenase which catalyzes the oxidation of formate to CO2 with the concomitant reduction of NAD to NADH [21–23]. The reaction equilibrium strongly favors l-homophenylalanine synthesis as the byproduct CO2 which is not obnoxious to the drug precursors can be easily removed. In this instance, l-phenylalanine dehydrogenase and formate dehydrogenase used in the reductive amination process are preferentially retained in order to improve the sustainability of the process. Membrane-based systems have since then become the opted technology for retention of biocatalysts, both on laboratory and industrial scales. Biocatalysts used for biotransformation and biochemical synthesis must be separated from the product effluent and recycled due to their cost. For the reductive amination process, the study of l-phenylalanine dehydrogenase retention has previously been carried out by using dialysis bag [16]. Nevertheless, this process could only be efficiently adopted for laboratory scale process with low scale-up potential. In order to overcome this problem, membrane bioreactors (MBRs) are highlighted in the current research, pertaining to their potential to retain the biocatalysts that are found in biotransformation reaction medium, and for the synthesis of high purity pharmaceutical products. The goal of this work was to investigate the production of vital pharmaceutical precursor namely l-homophenylalanine via enantioselective reduction of 2-oxo-4-phenylbutanoic acid by the NADH-dependant l-phenylalanine dehydrogenase in an integrated membrane bioreactor set up using the formate/formate dehydrogenase system for coenzyme regeneration (Fig. 1). In addition, the optimum pH for the synthesis of l-homophenylalanine using an
integrated membrane bioreactor for biotransformation was studied, simultaneously evaluating the biocatalysts retention in the membrane bioreactor. 2. Materials and methods 2.1. Enzymes l-phenylalanine dehydrogenase (recombinant in E. coli) from Rhodococcus sp. M4 (EC 1.4.1.20), was obtained from Jülich Chiral Solutions GmbH, Germany; formate dehydrogenase from Candida boidinii (EC 1.2.1.2) was obtained from Sigma–Aldrich, Malaysia; and NADH was purchased from Roche Diagnostic, Germany. 2.2. Chemicals 2-Oxo-4-phenylbutanoic acid could be synthesized according to the method proposed by Chen et al. [24]; sodium formate was obtained from Sigma–Aldrich, Malaysia; and 1,4-dithiothreitol was purchased from Merck, Malaysia. 2.3. Apparatus The biotransformation and separation were carried out in an integrated membrane bioreactor system operated in semi-batch mode where the substrates were pumped into the membrane bioreactor prior to reaction. The set up of the system is illustrated in Fig. 2. A 1 L reactor module is coupled to an in-situ separation unit for the removal of products while retaining the biocatalysts.
Fig. 2. Schematic flow diagram of the integrated membrane bioreactor system for simultaneous reaction and retention of biocatalysts coupled to an acidification device for l-homophenylalanine production.
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Substrates and coenzyme were pumped into the membrane bioreactor at 5 mL/min flow rate, with the product leaving the membrane bioreactor unit through an ultrafiltration membrane with varying molecular weight cut-off (MWCO) for different enzymes. The membrane bioreactor unit was equipped with an overhead stirrer, pH electrode, temperature sensor, and the internal atmosphere was kept inert with argon gas. The product enriched solution from the membrane bioreactor was pumped into the subsequent acidification vessel system. 2.4. Membrane The Spectra/Por® 2 semi-permeable ultrafiltration membrane used in this work was purchased from Chemopharm and was made of regenerated cellulose. This type of membrane was discovered to be effective for the separation of biocatalysts and the membrane satisfy the chemical nature requirements that will provide the desired retention of biocatalysts. The membrane also presents adequate mechanical and thermal stability. The MWCO of the flat sheet regenerated cellulose membrane used was 12–14,000 Da with an active surface area of 133 cm2 and a nominal thickness of 0.002 in.. 2.5. Synthesis and isolation of l-homophenylalanine The synthesis was preferably conducted in a reaction mixture containing 2-oxo-4-phenylbutanoic acid (1.2%w/v), 1,4dithiothreitol (5.6 mmol), sodium formate (56 mmol), formate dehydrogenase (595 U), NADH (0.14 g) and l-phenylalanine dehydrogenase (945 U) in deionized water (840 mL) (Fig. 1) [19,21]. The solution was stirred at 30 ◦ C and in an inert atmosphere with the addition of 1N ammonium hydroxide to maintain the pH at a constant value. Upon retention of the biocatalysts after a mean residence time of 3–5 days in which the conversion level is stable, the product solution was removed from the membrane bioreactor by exerting a pressure of 1.5 bar and acidified to pH 5.5 with dilute HCl. The resulting white precipitate was collected by filtration, washed with cold water and dried in vacuum to yield l-homophenylalanine with >95% purity. 2.6. Analytical methods 2.6.1. Determination of the enantiomeric excess The enantiomeric excess of l-homophenylalanine was determined using chiral HPLC on an Astec Chirobiotic T column (Orbiting Scientific Co.) using ethanol/water, 10/90 (v/v) as mobile phase at 1 mL/min flow rate with detection at 210 nm. 2.6.2. Determination of yield of l-homophenylalanine l-homophenylalanine yield was determined after collection of the white precipitate via filtration. For HPLC analysis of the components in the effluent of the membrane bioreactor, a C18 column was used with gradient elution from an initial solution of 10% of eluent B + 90% of eluent A to a final solution of 100% of eluent B in 20 min, wherein eluent A contains 5% acetonitrile in 0.1% trifluoroacetic acid and eluent B contains 95% acetonitrile in 0.1% trifluoroacetic acid, at 1 mL/min flow rate with detection at 214 nm. 2.6.3. Determination of optimum pH The effect of pH on the activity of l-phenylalanine dehydrogenase and formate dehydrogenase were determined for reductive amination. The optimum pH for the reaction was subsequently obtained. The reaction solution was maintained at a constant pH value with the addition of 1N ammonium hydroxide. Five sets of experiment were carried out at different pH values due to the pH-
Fig. 3. Dependence of enzyme activity on pH for l-phenylalanine dehydrogenase and formate dehydrogenase.
dependence of the biocatalysts, and the yield of the product was observed for each experiment. 2.6.4. Biocatalysts assay and retention Assays were performed by sampling aliquots of the product solutions and equilibrating in a cuvette at 30 ◦ C, with the addition of NADH and ammonium chloride in Tris buffer. The reaction was started if the presence of residual l-phenylalanine dehydrogenase was detected in the product solutions. The reaction progress was monitored spectrophotometrically and the change in absorbance at 340 nm (A340 nm ) was recorded for determination of the extent of biocatalysts retention. The enzyme activity was calculated using the following equation:
Units/mL enzyme =
A340nm / min sample − A340nm / min blank (V ) (df ) (ε) (v)
(1)
where: A340 nm = change in absorbance at 340 nm; V = total volume of assay (mL); v = volume of enzyme (mL); df = dilution factor; ε = 6.22 = millimolar extinction coefficient of -NADH at 340 nm. 3. Results and discussion Prior to the determination of optimal reaction solution pH for synthesis of l-homophenylalanine via biotransformation, the effect of pH on the activity of biocatalysts used in the biotransformation namely l-phenylalanine dehydrogenase and formate dehydrogenase were observed and the results obtained were fitted to polynomials. As shown in Fig. 3, pH of 9.2 was optimal for l-phenylalanine dehydrogenase. The pH-dependence of formate dehydrogenase showed broader maxima from pH 7 to 9 with an optimum value of pH 8. It is observed that the enzyme activity for both l-phenylalanine dehydrogenase and formate dehydrogenase deteriorates at pH 10, suggesting the deactivation of enzymes at high pH. From the observed optimal pH range for both enzymes, the reaction was subsequently carried out at several pH values to determine the optimal pH for the overall reaction, independent of other parameters. The reductive amination of 2-oxo-4-phenylbutanoic acid using l-phenylalanine dehydrogenase and formate dehydrogenase as biocatalysts proceeded in yields which were dependant on the pH of reaction solution. As can be seen from Fig. 4, the optimum working pH was 8.5, giving the highest yield. For all the experiments, the biocatalysts were fully retained within the membrane bioreactor, monitored by using spectrophotometric methods. Biocatalysts were not detected in the effluent of the membrane bioreactor under all reaction conditions. This shows that the biocatalysts were effectively retained due to size exclusion
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4. Conclusions The asymmetric synthesis of l-homophenylalanine in an integrated membrane bioreactor is a good alternative to the conventional approaches which utilize dialysis tubing for retention of biocatalysts. The advantage of this process was largely due to its high selectivity in rendering an enantiomeric excess of more than 99%, besides avoiding the formation of side products which incur extra costs in downstream processing. The operated membrane bioreactor allowed retention of the investigated biocatalysts due to size exclusion. In order to operate the membrane bioreactor for more than two cycles, biocatalysts need to be added in small amount to cater for the activity loss. l-homophenylalanine could be synthesized in sufficient yield by adjusting the pH of the solution, where the optimal pH was determined to be at 8.5. Other operations conditions such as effect of temperature, substrate concentration and residence time could be further studied to increase the conversion and yield of the product. Fig. 4. Influence of pH on yield of l-homophenylalanine.
by using the flat sheet membrane with 12–14,000 Da MWCO. Thus, it is proven that the integrated membrane bioreactor module could be utilized for retention of l-phenylalanine dehydrogenase and formate dehydrogenase enzymes, and could replace the conventional process which utilized dialysis bag. l-homophenylalanine which precipitated from the reaction mixture was analyzed using Nuclear Magnetic Resonance (NMR). The synthesized l-homophenylanaine had the following physical properties: 1 H NMR (D O): 2.05 ppm (m, 2H), 2.63 ppm (m, 2H), 3.65 ppm 2 (t, 1H) 7.19 ppm (m, 5H). The results showed that pure l-homophenylalanine was obtained for all experiments without the presence of impurities by comparing with a standard. The enantiomeric excess of l-homophenylalanine was ascertained using a chiral T column. Reduction of 2-oxo-4phenylbutanoic acid to l-homophenylalanine was achieved with an enantiomeric excess of over 99% for all experiments since only a single peak was eluted (Fig. 5). The synthesized l-homophenylalanine had a retention time of 7.34 min, where no d-antipode which had a retention time of 10.12 min could be observed. The product was optically pure as determined by optical rotation and compared to an authentic sample and literature ◦ values [˛]20 D = +48 (c1, 1N HCl).
Fig. 5. HPLC Chromatogram showing the enantiomeric excess of the enzymatically synthesized l-homophenylalanine using a Chiral T Column.
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