Enzymatic Deracemization of (R,S)-Ibuprofen Ester via Lipase

Article pubs.acs.org/IECR

Enzymatic Deracemization of (R,S)‑Ibuprofen Ester via Lipasecatalyzed Membrane Reactor Lau Sie Yon,† Fadzil Noor Gonawan,‡ Azlina Harun Kamaruddin,‡ and Mohamad Hekarl Uzir*,‡ †

Department of Chemical Engineering, Curtin University, Sarawak Campus, CDT 250, 98009 Miri, Sarawak, Malaysia School of Chemical Engineering, Engineering Campus, Universiti Sains Malaysia, Seri Ampangan, 14300 Nibong Tebal, Seberang Prai Selatan, Pulau Pinang, Malaysia

‡

ABSTRACT: Ibuprofen (isobutyl-propanoic-phenolic acid) is a chiral drug well-known for its analgesic, antipyretic, and antiinflammatory effects. In the present work, an enzymatic membrane reactor (EMR) has been developed for the production of optically pure (S)-ibuprofen acid. The EMR is equipped with a multitubular fixed bed racemization unit to enhance the enzymatic deracemization of (R,S)-ibuprofen ester. Several process parameters such as enzyme loading, racemization catalyst loading, reaction temperature, buffer pH, as well as the flow rate were investigated. These experimental results were then compared with the proposed process model. It was found that the experimental data were in good agreement with the theoretical results. The optimum condition of the lipase-catalyzed dynamic kinetic resolution (DKR) using the EMR could give 96−98% conversion with 97−99% of product enantiomeric excess (eep).

1. INTRODUCTION In today’s pharmaceutical industry, there is an increasing demand in obtaining optically pure active drugs, whose sole purpose is to prevent any side effects from using racemic mixtures. Intensive pharmacological research works that reported the importance of optically pure (S)-ibuprofen as a major therapeutic donor have boosted up the demand to produce and market the single enantiomer chiral drugs.1−7 Due to the dose-limiting toxicities and pharmacokinetics, a number of methods to prepare optically pure compounds have been proposed. These methods include the resolution of diastereomeric salts, resolution of racemates, supercritical fluid chromatography, asymmetric syntheses using chiral auxiliaries and chiral catalysts.8−15 Among these approaches, enzymemediated kinetic resolution is the most valuable way of obtaining a pure enantiomer due to its ability to discriminate between enantiomers. Resolution is the most widely used approach for the production of optically pure fine chemicals and pharmaceutical ingredients, which includes chromatography, diastereomer formation, kinetics, and dynamic and enzymatic routes.16−23 Resolutions are classified into enzymatic and inorganic/ chemical-catalyzed approaches. Either an enzyme or chemical resolving agent with enantiomeric differentiation capability has been widely applied in chemical transformation of the two enantiomers at substantially different rates. In the report prepared by Eijiro and co-workers, the team developed a novel supported liquid membrane (SLM) encapsulating the surfactant-enzyme complex in the liquid membrane phase and provided a highly enantioselective separation for the optically active compounds, that is, (S)-ibuprofen and L-phenylalanine from their racemic mixtures.24 Later, De Los and co-workers continued a similar work, where a lipase-catalyzed reaction was combined with the SLM, based on the characteristics of ionic liquids to achieve the selective separation of a racemic ibuprofen. In their work, the performance of the system for © 2013 American Chemical Society

the kinetic resolution of racemic 1-phenylethanol by transesterification with a vinyl ester catalyzed by a commercially immobilized Candida antarctica lipase B was investigated.25 Then, the racemic ibuprofen ester has also been kinetically resolved using surfactant-coated porcine pancreatic lipase (PPL) in an incubator shaker.26 These results showed that the enzyme has a potential in catalyzing the esterification and hydrolysis of racemic ibuprofen.27 Besides, Long and co-workers compared the catalytic behavior of lipase-catalyzed kinetic resolution of two different ibuprofen esters in a polymeric hollow fiber membrane. The optimum operating conditions to obtain an optically pure single enantiomer have been identified.19 In addition, lipase-catalyzed enantioselective esterification of racemic ibuprofen with various alcohols has also been conducted by a team of Korean researchers.28 A review article also reported and compared the results from different methods for producing enantiomerically pure ibuprofen. It also explained some important parameters in order to achieve a highly stereoselective resolution process, such as optimum temperature, substrate concentration, enzymatic activity and lifetime, pressure, and water content.29 The use of enzymes as biocatalysts has become a valuable tool for fine chemical synthesis. Presently, a large number of biotransformations were carried out at the industrial scale mainly for the production of fine and commodity chemicals. The enzymatic approach is preferable since it provides better opportunities and benefits in obtaining a safer substance and a shorter synthesis route.30,31 In general, enzymatic method or biocatalytic resolution is a process that makes use of the selectivity of enzymes, which react only with one of the Received: Revised: Accepted: Published: 9441

March 11, 2013 June 12, 2013 June 13, 2013 June 13, 2013 dx.doi.org/10.1021/ie400795j | Ind. Eng. Chem. Res. 2013, 52, 9441−9453

Industrial & Engineering Chemistry Research

Article

Figure 1. Schematic diagram of the lipase-catalyzed membrane reactor for DKR of ibuprofen ester.

surface. This is to overcome the possible enzyme inhibition, which caused the reduction in the production rate for the EMR in the previous work.17 It was reported that the ibuprofen enantiomers are highly stable in the acidic medium but unstable and ionize in the basic environment.38 This observation led to the discovery of base-catalyzed keto−enol tautomerism, which successfully recovers the excess amount of the unwanted enantiomer in the classic kinetic resolution. Therefore, the performance of the two potential racemization agents, that is, trioctylamine (TOA) and Amberlyst A26 hydroxide (OH− resins), were evaluated using the proposed racemization unit. The behavior of enzymatic membrane reactors has been investigated from a theoretical point of view in several articles.39−42 Long and co-workers numerically solved for reactant concentration profiles in a finite hollow fiber system over a wide range of process parameters to investigate their effects on chiral separation.43 In our previous work, a mathematical model for the DKR of an enzyme-catalyzed reaction in a hollow fiber membrane was developed and tested by formulating a set of differential equations on the diffusion, reactant consumption or product formation at each location.44 The equations were numerically solved with appropriate boundary conditions based on the imposed operating conditions. The same process model has been applied to evaluate the present DKR system in the EMR. This article serves to highlight the deracemization process of the racemic ibuprofen via lipase-catalyzed membrane reactor. The experimental results obtained from the EMR were validated by the process model described in the previous

enantiomers of a chiral molecule to produce the desired optically pure product/intermediate.32−36 However, based on the racemate as a starting substrate, the classic kinetic resolution could only achieve a maximum of 50% yield of the desired enantiomer. The disadvantage was then overcome by the introduction of dynamic kinetic resolution (DKR), which has been proven to be a potentially efficient process to give a theoretical yield of 100% of the desired product.16,37 The process efficiency of DKR is affected by several factors, which include the availability of substrate, the cost of chemical used such as solvents and biocatalyst/enzyme, the ease of racemization of the unwanted isomer and the total number of steps taken, including the downstream processing. In order to encounter these limitations, the EMR was designed in order to incorporate the enzyme and membrane technologies in the enantio-separation of the chiral drugs. The EMR combines the product separation and enzymatic reaction into an integrated reaction-separation unit and thus reduces several downstream processing steps. The application of DKR in the membrane reactor has been reported as a more efficient way to achieve substrate recovery and product separation.17 In the present EMR design, a highly improved deracemization system was introduced. A high efficiency in situ racemization unit was placed at the organic loop in order to recover the unreacted (R)-ibuprofen ester. The main function of this independent racemization unit is to provide a targetspecific racemization for the unreacted enantiomer and prevent the mixture of base catalyst with the enzyme on the membrane 9442

dx.doi.org/10.1021/ie400795j | Ind. Eng. Chem. Res. 2013, 52, 9441−9453


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article.44 The advantages of the process model include: (i) estimation of the process variables for scaling-up of the design capacity and (ii) process parameters validation as well as to determine the optimum operational parameters for the enzymatic deracemization process of the EMR.

ibuprofen ester (organic phase) from the shell-side of the membrane module enters the racemization unit and interacts with the OH− resins due to the mild turbulence created by the slow agitation. The racemized substrate (light phase) exits from the upper port of the racemization unit and enters the organic tank. The DMSO (heavy phase) was then drained from the draining port upon completion of the process. 50 mM organic substrate was prepared by diluting pure (R,S)-ibuprofen ester in isooctane. The organic phase was continuously circulated in the membrane shell-side. Transmembrane pressure (TMP) from the shell to lumen was set in the range of 0.35−0.45 bar before the aqueous pump was switched on. The aqueous phase consists of 50 mM phosphate buffer solution at a desired pH for extracting the product into the lumen side of the membrane. In the preliminary study, the screening of two base catalysts; (i) trioctylamine (TOA) and (ii) Amberlyst A26 hydroxide (OH− resins) was carried out at the same operating conditions. The performance of the base catalysts was then compared. Two ml of both aqueous and organic samples were collected hourly and the concentrations of the products were measured. In order to retain the total volume of aqueous phase, 2 mL of fresh aqueous buffer solution of the same pH was added to the buffer tank. The reaction rate was determined from the sample analysis using HPLC chiral column with a UV detector, by monitoring both substrate and product (ibuprofen acid). 2.4. Immobilization of Lipase on Membrane Reactor. Prior to the enzymatic reaction, 5 g/L Candida rugosa lipase solution was prepared using the phosphate buffer at the desired pH values. The enzyme was then immobilized into the spongy layer of the membrane through ultrafiltration of the lipase solution across the asymmetric membranes from the shell-side to the fiber lumen. The transmembrane pressure across the shell to inner lumen was maintained at 0.35 bar by manipulating the back-pressure valve. After 80% filtration of lipase solution, ultrafiltration was continued for 1 L phosphate buffer solution to reload the residual amount of free enzyme on the membrane pores as well as to wash out the unbound lipase. The amount of lipase immobilized on the membrane was calculated based on the difference between the protein content of lipase solutions before and after the immobilization procedure. The protein concentration was determined by BCA standard test tube protocol (Pierce, Rockford, IL). 2.5. Operational Stability of Immobilized and Free Lipases. The stability of immobilized lipase in EMR and free lipase in aqueous/organic emulsion were investigated at 40 °C using 50 mM (R,S)-ibuprofen ester as the substrate for a period of 120 h. The hydrolysis of racemic ester in EMR involved 1 L aqueous phase (50 mM phosphate buffer) at pH 8.0 and organic phase with 50 mM racemic ibuprofen ester in 1 L isooctane (without racemization). The lipase concentration of 2 g/L was used in this study. The aqueous and organic flow rates were fixed at 300 mL/min and 100 mL/min respectively. The batch runs were carried out at constant orbital stirring speed of 150 rpm. The samples were withdrawn from aqueous and organic phases every 5 h and analyzed by HPLC. 2.6. HPLC Analysis. Liquid chromatography analysis was performed using an ultrafast liquid chromatography (Model: Shimadzu Prominence UFLC, Japan) equipped with a UV/vis detector (SPD-20A/20AV) and operated at 254 nm UV detection setting. The concentrations of the optically pure (R)and (S)-enantiomers were determined using a special type of chiral stationary phase, i.e. (R,R)-Whelk-O1 Chiral Column (Regis Pirkle, USA). The column is capable of separating the

2. MATERIALS AND METHOD 2.1. Enzymes and Chemicals. Lipase from Candida rugosa EC 3.1.1.3 (Type VII, 724 units per mg solids) and Amberlyst A26 hydroxide were supplied by Sigma-Aldrich (MI). The enzyme was used as purchased without further purification. BCA protein assay was obtained from Pierce (IL). Trioctylamine was purchased from Merck, Germany. The standard reference of (S)-ibuprofen acid 99% was purchased from Acros (Belgium), and the (R,S)-ibuprofen acid was purchased in bulk quantity (25 kg/drum) from Shasun Company, India. Isooctane 99%, sodium dihydrogen phosphate (KH2PO4), disodium hydrogen phosphate dehydrate (Na2HPO4·2H2O) and dimethyl sulfoxide (DMSO) were supplied by Fisher Chemicals (UK). Other chemicals and reagents used were of analytical grade. 2.2. Substrate Preparation. The racemic substrate for the hydrolysis reaction was prepared by chemical esterification using 2-ethoxyethanol as the acyl acceptor. The reaction mixture composed of (R,S)-ibuprofen (75 mM) and 2ethoxyethanol (100 mM) were dissolved in 100 mL isooctane without the addition of water. The reaction was started by adding 0.5 g of p-toluenesulfonic acid (catalyst) and refluxed at 95 °C for 8 h using a sealed Dean−Stark apparatus. After the reaction, the ester was purified in order to remove the excess reactants. Five percent sodium hydroxide (NaOH) solution was added to the ester and followed by several cycles of washing with deionized water. The mixture was left inside a separating funnel until two distinct translucent layers were formed. The aqueous phase was drained out as waste. The solvent (isooctane) was removed from the organic phase using a rotary evaporator at 95 °C in a water bath system. The substrate produced via the esterification was stored for the DKR process using the specially designed EMR. 2.3. Enzymatic Dynamic Kinetic Resolution in Lipasecatalyzed Membrane Reactor. A lipase-catalyzed membrane reactor system (Figure 1) was specially designed for the DKR process. The EMR consists of hollow fiber membrane module, a temperature-controlled incubator, a racemization unit and two tanks. A membrane module is located inside a custommade incubator and connected to the organic and aqueous streams respectively. The hollow fiber membrane module employed was the GE Healthcare ultrafiltration (UF) membrane model UFP-50-E-4 × 2MA (GE Healthcare BioSciences Corp., USA) constructed of hydrophilic polyacrylonitrile material (PAN) with an apparent retention character of 50 kDa of molecular weight cutoff (MWCO), fiber ID 1.1 mm and 50 fibers and effective surface area of 0.085 m2. The hollow fibers were fabricated in the form of a shell and tube configuration. The chemical compatibility of the PAN membrane makes it an ideal choice when the membrane is required to handle the concentrated aqueous solutions and solvent. A multitubular fixed bed racemization unit was designed for immobilizing the OH− resins. The OH− resins were added into six vertical fine-mesh columns prior to the resolution process. DMSO was also added into the racemization unit as a cosolvent in order to enhance the racemization rate.16 The unreacted (R)9443



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Figure 2. Activity of lipase in EMR and batch systems for hydrolysis of 50 mM substrate taking place at 40 °C, 2g/L lipase concentration, pH 8 buffer solution.

inhibition constant for 2-ethoxyethanol and KuS1 is the substrate (ibuprofen ester) inhibition constant. The enzymatic hydrolysis of racemic ibuprofen ester took place at the membrane porous matrix where lipase is immobilized. The substrate flows into the membrane module from the shell side. The reaction layer represents the boundary between water and the organic phase at which lipase is most active. The model equations involving the enzymatic hydrolysis incorporated with in situ racemization in the EMR are presented in eqs 3 and 4.44 Several assumptions were made during the development of the model equations, and these include; (i) constant temperature and fluid properties; (ii) steady state operation; (iii) no phase changes; (iv) the enzyme is evenly distributed on the membrane surface; (v) only radial convection are considered due to negligible axial gradient;43 (vi) the effective diffusivity of the substrate and product is constant; and (vii) the accumulation of byproduct (2ethoxyalcohol) is negligible.44 These assumptions then led to the derivation of the equations given below

(R)-and (S)- enantiomers of both acid and ester derivatives. The mobile phase used was an isocratic solvent mixture, which consists of hexane:isopropanol:acetic acid with the ratio of 98:2:0.5 at a flow rate of 1.0 mL/min. 2.7. Calculation of Optical Purity and Enantiomeric Ratio. The performance of the EMR was evaluated based on the conversion (X) and optical purity obtained from the DKR process. X represents the overall conversion for the racemic substrate. The optical purity of the desired product was expressed in terms of the enantiomeric excess of the product (eep); whereas, that of the remaining substrate was expressed in terms of the enantiomeric excess of the remaining less reactive substrate (ees). Both of them can be calculated from the molar fraction of each enantiomer using the equations reported previously.45 The enantiomeric ratio (E) represented by eq 1, characterizes the enantioselectivity of a particular enzyme and is defined as the ratio of pseudofirst order rate constants for the two enantiomers:11,46 ⎡ 1 − ee ⎤ ln⎢ 1 + ee / see ⎥ ⎣ s p⎦ E= ⎡ 1 + ees ⎤ ln⎢ 1 + ee / ee ⎥ ⎣ s p⎦

d2SA dR2

(

[I ] K nI1

+

[S*] K uS1

)

Φ2SA

(1 + )(1 + φξ SA ΘA

IP

+ SBξIS) (3)

d2SB 2

dR

+

⎡ S − SB ⎤ dS 1 (1 + B0 ) B = γ ⎢ A ⎥ R dR ⎣ SA + SB ⎦

(4)

where SA or SB, ΘA and B0 represent the dimensionless parameters of substrate concentration ((S)- or (R)- enantiomer), Michaelis constant and Bodenstein number, respectively. The Thiele modulus for enzymatic hydrolysis of ibuprofen ester and in situ racemization constant are designated as γ and Φ2. Meanwhile ξIS and ξIP are the dimensionless inhibition constants for the substrate and byproduct (alcohol) respectively. Furthermore, a dimensionless group namely Bodenstein number (B0) was used to evaluate the flow rate of the system. B0 is normally used to describe the diffusion phenomena in reactors. It is defined in the following form

νmax[S] (K m + [S]) 1 +

dS 1 (1 + B0 ) A = R dR

(1)

2.8. Modeling of Dynamic Kinetic Resolution in the Membrane Matrix. An enzymatic reaction mechanism for the conversion of substrate (S) to product (P′) via DKR process has been proposed elsewhere.44 The reaction rate constant was obtained from the kinetic studies, which incorporated the substrate ((R)- and (S)-ibuprofen esters) and noncompetitive product (2-ethoxyethanol) inhibitions. Three assumptions were made during the development of the enzymatic model, and these include; (i) the enzyme is directly in contact with the organic substrate; (ii) constant reaction temperature, and (iii) constant enzyme activity. The modified rate equation for the DKR process is given by eq 2 ν=

+

(2)

where vmax and Km are the maximum rate and the Michaelis constant respectively. [S], [S*] and [I] are the substrate, inhibitor and byproduct concentrations; KnI1 is the product

B0 = 9444

F 2π ln Deff

(5)



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where, F, L, N and Deff represent the flow rate, length of hollow fiber, number of hollow fiber and effective diffusivity, respectively. The dimensionless boundary conditions for the solution of eqs 3 and 4 are

38.70 mM of (S)-ibuprofen, which is the highest amount produced with conversion of 96.25 and 98.40% eep among all cases. DKR catalyzed by TOA produced a slightly lower amount of (S)-ibuprofen (34.04 mM) with 86.67 and 94.86% conversion and eep respectively within 10 h reaction time. In this case, TOA is a weak amine while the Amberlyst Hydroxide (OH− resin) is a strong basic polymeric resin for use in the nonaqueous media. The result indicates that both OH− resin and TOA could give twice the production as compared to the system without catalytic racemization. Moreover, stronger base (OH−) has considerably increased the performance of the enzymatic deracemization process. The OH− resin is an excellent racemization catalyst for in situ racemization due to its macro reticular matrix, which provides sponge-like structure and results in a high surface area per unit volume.The large pore structure of Amberlyst OH− resin allows large molecule, that is, (R)-ibuprofen ester to enter and interact with the entrapped hydroxyl ions in the matrix. This feature combined with its strong basicity, permits the rapid ketol-enol tautomerism in the matrix. The rate of racemization for OH− resins was also observed higher than that of TOA. The racemization proceeds through a base-mediated hydrogen abstraction, forming an intermediate enolate species. The subsequent readdition of the hydrogen to the enolate completed the catalytic cycle to give the racemized substrate.47,48 In this process, OH− resins contributed to the resonance-stabilized enolization, which then stabilizes the enolate structure and, thus, enhanced the racemization rate. The presence of stationary OH− resins packing in the racemization unit also protected the immobilized lipase on the membrane module from the substrate and product inhibitions. The substrate inhibition was significantly reduced since most of the unreacted substrates were racemized in the racemization unit rather than at the membrane surface. In addition, the presence of hydroxyl ions has also shown the possibility to absorb ethanol produced during the hydrolysis reaction and reduced the contamination in the aqueous phase.49 TOA also showed a good performance in the production of (S)-ibuprofen acid, which indicated that a simultaneous racemization of (R)-ibuprofen enantiomer has taken place during the DKR process. Nevertheless, the calculated enantiomeric excess and conversion achieved does not give an excellent result as compared to the OH− resins. This was apparently due to the basicity of the base catalyst. The weaker amine gave a lower affinity for the proton compared to the OH− conjugate base. Hence, it was concluded that the OH− resin was chosen as racemization catalyst for the subsequent deracemization process in the EMR system. 3.3. Effect of Amberlyst Hydroxide Loading. The effect of OH− resin loading in the DKR was examined. The addition of OH− resins into the reaction medium showed a significant effect toward improving the hydrolysis rate and enhancing the formation of (S)-ibuprofen acid. Thus, further investigation was carried out in order to monitor such improvement by varying the loading amount of OH− resin from 0 to 12 g/column. The experimental and simulated results are presented in Figure 3. Low conversion and eep were observed for DKR without adding the Amberlyst resins. The conversion achieved was 41.6%, which resulted in 80.2% eep. However, the conversion and eep increased with the increasing amount of the OH− resin. The conversion and eep value was above 90% and remained constant for the resin loading above 8 g/column. In general, the

dS dSA b = 0; B = 0, at r = and a dR dR B0 B dS B dSA c = = 0 (1 − SB), at r = (1 − SA ); R R a dR dR

Collocation technique of ordinary differential equation (ODE) was employed in one-dimensional boundary value problem and implemented in MATLAB for numerical simulation. Both eqs 3 and 4 were solved simultaneously in order to simulate the concentration profiles of the complete enzymatic DKR system. The performance of EMR in carrying out the DKR of (R,S)-ibuprofen ester was studied based on several dimensionless analyses of the model.

3. RESULTS AND DISCUSSION 3.1. Operational Stability of Lipase. The operational stability of EMR is one of the principal factors affecting reactor productivity for immobilized enzyme systems. In order to justify the advantages of the EMR process over the free lipase system, experiments were carried out in a batch system and they were compared in terms of their lipase activities. Figure 2 shows the lipase activity of EMR and batch systems for a period of 120 h operation at a constant temperature of 40 °C. The initial lipase activity in EMR system was 141.2 mmol/m2.h.gprotein and gradually decreased with time. However, the activity of immobilized lipase only reduced 53.8% after 120 h compared to that of 88.9% reduction in the batch system. Although the batch system exhibited higher initial activity (157.4 mmol/m2.h.g-protein) than that in the EMR, there was a rapid decrease in the lipase activity after 10 h of reaction. The average hourly reduction of lipase activity for the free enzyme and immobilized systems were 1.61 and 0.89 mmol/m2.gprotein, respectively. The result shows that the immobilized system could contribute to a relatively stable catalytic condition versus that of the free lipase. This is due to the low mobility of immobilized lipase in the membrane that could preserve the structural conformation for a prolonged operation as compared to the free lipase. The overall lipase activity exhibited by the immobilized lipase was higher than the free lipase; thus, it was concluded that the EMR is a more practical and viable system during a long-term operation. 3.2. Effect of Catalytic Racemization. The experimental study apparently shows a significant improvement in terms of conversion and yield of the (S)-ibuprofen by adding the racemization catalyst. It can be observed in Table 1 that both conversion and enantiomeric excess are above 50%. However, the choice of base catalyst has different effects on the (S)ibuprofen production in DKR process. Amberlyst resin gave Table 1. Effect of Racemization Catalyst on the DKR of Racemic Ibuprofen Ester in the EMR type of catalyst parameter

OH− resin

TOA

no catalyst

Conversion, X% (S)-Ibuprofen acid, mM Enantiomeric excess of product, eep%

96.25 38.70 98.40

86.67 34.04 94.86

44.71 17.98 92.18 9445



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and eep values were closely dependent on the amount of enzyme loading. The ees decreased from 23.37 to 9.03% when lipase loading was increased from 0.87 to 3.76 g-protein/m2. However, the eep is directly proportional to the lipase loading where an increase in lipase loading from 0.87 to 3.76 g-protein/ m2 significantly increased the eep. The result also shows that the immobilization capacity on the spongy layer is within the satisfactory range, although the enzyme was physically immobilized on the hydrophilic membrane. There was an average of 76.43% of lipase, successfully immobilized on both spongy layer and membrane surface. Knežević and co-workers reported that the protein binding capacity was affected by the electrostatic charge distribution and the number of potential coupling sites in the lipase molecules.50 Figure 4 apparently shows that the enzyme could achieve its optimum productivity at only a moderate enzyme loading. An average of 4.26 g-product/h.g-protein was obtained at an enzyme loading of 1.69 g-protein/m2. However, the immobilized lipase becomes less stable as the reaction proceeds whenever the loading increases. This might be due to the fact that large amount of lipase was aggregated throughout the fixed membrane space when the loading capacity was increased, which then resulted in a limited surface area for it to adsorb. Lipase in its aggregated form is less stable due to the unavailability of its “open structure”. When the membrane is loaded with a higher enzyme concentration, the protein molecules can be packed in layers, forming an additional barrier to transport and therefore obstructing the enzyme on the rear layer. It was reported that lipase would form inactive dimers when present at high concentration, thus reducing its catalytic activity.51 The experimental results also agreed with model simulations as well as observations made by other researchers, that higher enantioselectivity was observed when the reaction was carried out at lower enzyme concentrations.11 The high cost of enzymes however, makes high dosage impractical for this type of application. For the aforementioned reason, an enzyme loading of 1.69 g-protein/m2 at 2.0 g/L lipase solution was selected for the remaining experiments. 3.5. Effect of Temperature. The operating temperature is an important parameter that affects conversion of the substrate and lipase activity. Figure 5 shows the conversion of racemic ibuprofen ester under the temperature range of 25−60 °C at the 10th hour of reaction. The conversion of the substrate increased as the reaction temperature increased until it reached an optimum reaction temperature of 45 °C. The conversion obtained at this temperature was 95.9 ± 3.5%, which gave the highest conversion among other evaluated temperatures. However, the average conversion decreased rapidly when the temperature was further elevated. There was only 78.6 ± 1.8% conversion attainable at 60 °C. The desired temperature, which gave a high level of conversion, was in the range of 40−50 °C. The actual optimum temperature for a reaction is a temperature at which the enzyme exhibits a constant activity over a period of time. The effect of the desired temperature on the immobilized lipase stability in the membrane reactor was evaluated at three different temperatures (40, 45, and 50 °C) at fixed pH 8 with constant flow rate for a period of 100 h. Figure 6 shows that the lipase obtained the highest initial hydrolysis rate at 50 °C as compared to that of 40 and 45 °C. Nonetheless, the lipase activity started to reduce greatly after 30 h of operation. Conversely, the EMR, which operates at 40 °C,

Figure 3. Comparison of conversion and eep for various Amberlyst resin loading (50 mM substrate, 2 g/L lipase, 40 °C, pH 8 buffer solution, 200 mL/min aqueous flow rate and 100 mL/min organic flow rate).

simulated conversion and eep trends matched those of the experimental results. The optimum performance was obtained with Amberlyst loading range between 6−8 g/column since larger amount of resins provide no significant improvement in the conversion and eep. However, lower resin loading of 6 g/column is adequate for catalyzing the DKR process. This may be due to the fact that only a small amount of OH− is required at physiological pH range of 7.0−7.3. Generally, the proton is transferred to the base in the transition state of the slow step.48 Since the DKR under investigation involves both hydrolysis and racemization, which also incorporates the enzyme as a biocatalyst, thus, an extreme condition should be avoided in order to obtain the best reactor performance. In a nutshell, high concentration of base is favored for the racemization process. However, in considering the cost and performance, 6 g/column of OH− resin was chosen as the optimum loading in the DKR process. 3.4. Effect of Enzyme Loading. The optimal concentration of enzyme is very important in order to ensure that the enzyme is distributed as a mono layer and catalyzes under reaction-limited condition.11 In order to secure the efficiency of lipase enzyme, the effect of enzyme loading on the enzymatic hydrolysis reaction in the EMR was investigated. A 50 kDa PAN membrane was chosen due to its capability to retain Candida rugosa lipase (molecular weight of 50−70 kDa) from passing through the membrane micropores. The amount of immobilized lipase on the PAN membrane and that the ee values obtained at different enzyme loadings are presented in Table 2. It can be seen from the table that the PAN membrane could provide considerably high enzyme loadings and different values of ee were obtained at different loading capacities. As observed in the tabulated results, the ees Table 2. ees and eep Values at Different Enzyme Loadings enantiomeric excess, ee (%) lipase solution (g/L)

lipase immobilized on membrane (g-protein/m2)

substrate, ees

product, eep

1.0 2.0 3.0 5.0

0.87 1.69 2.13 3.76

23.37 19.28 15.06 9.03

89.6 93.7 96.5 98.7 9446



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Figure 4. Enzyme productivity at different enzyme loadings on spongy layer (50 mM substrate, 6 g/column OH− resin, 40 °C, pH 8 buffer solution, 200 mL/min aqueous flow rate and 100 mL/min organic flow rate).

maximum value of 159.7 mmol/h.m2.g-protein in a pH 8 buffer solution and slowly dropped with the increase in the pH value. As a result, buffer solution at pH 8.0 was chosen as the optimum aqueous phase owing to the rapid extraction of (S)ibuprofen as well as preserving the lipase activity at its highest level. In most enzymatic chiral resolution processes, it has been suggested that the variation in pH of buffer solution might influence the chiral selectivity, since the conformation of an enzyme depends on its ionization state.53,54 At any given pH, the intact lipase may contain both positively and negatively charged groups. Such an ionizable group often constitutes part of the active site and frequently involves in an acid−base catalysis. However, a catalytically active lipase tends to exist in only one particular ionization state. Usually, the catalytic activity of lipase changes with the pH in a bell-shaped fashion, thus, yielding a maximum rate in the stability range.55 In addition, lipase from Candida rugosa showed a good performance in the hydrolysis process at a slightly basic condition of pH 8.0. The pH of the medium resulted in the change of ionic form of the active site and the activity of the enzyme, thus enhancing the rate of reaction. The results obtained was in agreement with the work reported by Long and co-workers, which indicated that pH 8.0 favored the hydrolysis of ester using Candida rugosa lipase.19 3.7. Effect of Flow Rates. 3.7.1. Aqueous Flow Rate. The productivity of DKR was found to be greatly affected by the volumetric flow rate of the aqueous phase.56 Therefore, the effect of aqueous flow rate was investigated with flow rates ranging from 50 to 350 mL/min at fixed organic flow rate of 100 mL/min. Figure 8 shows the product concentration of (S)ibuprofen acid as a function of time. It was observed that the higher aqueous phase flow rate gave a higher concentration of optically pure (S)-ibuprofen acid. The DKR of racemic ibuprofen ester produced 144.7 ± 3.7 mM/m2 product at 350 mL/min aqueous flow rate within 10 h of operation, whereas there was only 72.3 ± 2.4 mM/m2 of (S)-ibuprofen formed at 50 mL/min. In the DKR process, the product (S)ibuprofen acid is a polar component and could easily diffuse

Figure 5. Average of the 10th hour conversion at various reaction temperatures (50 mM substrate, 6 g/column OH− resin, 1.69 g-lipase/ m2, pH 8 buffer solution, 200 mL/min aqueous flow rate and 100 mL/ min organic flow rate).

has a propensity to give a higher lipase stability but lower initial hydrolysis rate. A sharp decay in hydrolysis rate at 50 °C was mainly due to the exposure of enzyme above its optimum temperature for a long period of time. The accumulated heat could result in partial deactivation and denaturation of enzyme structure. It was observed that the higher reaction temperature resulted in an increase of the initial lipase activity but consequently reduced its stability. This result agreed with other reported findings that the stability of enzyme decreased considerably with the elevated temperatures, although the initial reaction rate is high.52 Therefore, based on the results obtained, the operating temperature of 45 °C was selected for the EMR system, for which both rapid hydrolysis and high enzyme sustainability were essential for an optimum operation. 3.6. Effect of pH. In the lipase-catalyzed EMR, the pH effect on lipase activity was also carefully investigated. Figure 7 shows that the catalytic activity of Candida rugosa lipase is relatively low (32.8 mmol/h.m2.g-protein) in acidic condition (pH 6.0). However, the activity tremendously increased to a 9447



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Figure 6. Time-course hydrolysis rate at 40, 45, and 50 °C (50 mM substrate, 6 g/column OH− resin, 1.69 g-lipase/m2, pH 8 buffer solution, 200 mL/min aqueous flow rate and 100 mL/min organic flow rate).

into the flowing aqueous stream. The permeability of a membrane not only depends on its pore size, structure and transmembrane pressure, but also on its hydrophilicity. As a result, the unreacted substrate ((R)-ester) in the organic phase was retained at the shell-side due to its nonpolar characteristics. The effect of aqueous flow rate on the (S)-ibuprofen production rate is depicted in Figure 9. The figure clearly shows that the aqueous flow rates of 50 and 100 mL/min resulted in stable (S)-ibuprofen production rates with average values of 7.44 mmol/h.m2 and 12.77 mmol/h.m2 respectively. The higher average production rates of 16.42 and 17.81 mmol/h.m2 were obtained when the flow rates were increased to 200 and 350 mL/min, respectively. However, the production rate for the higher aqueous flow rates tends to decrease throughout the process. This phenomenon might be due to the formation of aqueous turbulence flow near the enzyme-immobilized membrane interface and consequently caused the conformational change to the enzyme active site. Therefore, a fixed aqueous flow rate of 200 mL/min was chosen for the EMR

Figure 7. Effect of buffer solution pH on lipase activity (50 mM substrate, 6 g/column OH− resin, 1.69 g-lipase/m2, 45 °C, 200 mL/ min aqueous flow rate and 100 mL/min organic flow rate).

Figure 8. Effect of various aqueous flow rates on (S)-acid concentration with lipase immobilized on spongy layer (50 mM substrate, 6 g/column OH− resin, 1.69 g-lipase/m2, 45 °C and 100 mL/min organic flow rate). 9448



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Figure 9. Effect of various aqueous phase flow rates on membrane productivity with lipase immobilized on spongy layer (50 mM substrate, 6 g/ column OH− resin, 1.69 g-lipase/m2, 45 °C and 100 mL/min organic flow rate).

Figure 10. Effect of various organic flow rates on (S)-ibuprofen acid concentration (50 mM substrate, 6 g/column OH− resin, 1.69 g-lipase/m2, 45 °C and 200 mL/min aqueous flow rate).

unreacted substrate in the organic phase then enters the in situ racemization unit for the substrate recovery. It is important to determine the optimum organic flow rate, which could ensure the couple reaction of hydrolysis and racemization to perform efficiently. Hence, the EMR was evaluated as a function of the organic flow rate, which is in the range between 50−200 mL/ min at a fixed aqueous flow rate of 200 mL/min. Figure 10 shows the overall concentration profiles for all the organic flow rates which exhibited a proportional relationship with the reaction time. However, it was observed that the increase in the organic flow rate resulted in lower overall product concentration profiles. For instance, the EMR operated at organic flow rate of 50 mL/min could give a higher product concentration (161.17 mM/m2) compared to that of 200 mL/min, which only produced 52.9 mM/m2 product concentration. The result also shows that although the EMR operated at the organic flow rate of 50 mL/min resulted in a higher concentration at the beginning of the reaction, it however,

operations so as to ensure operational stability and maintaining a satisfactory productivity. In general, the production rate of (S)-ibuprofen acid increases with the increase in the aqueous flow rate. This is due to the rapid and continuous replacement of fresh aqueous solution for the extraction of polar product, that is, (S)ibuprofen through the hydrophilic membrane. In addition, the hydrophilicity of membrane and transmembrane pressure gradient could also enhance the product diffusion in the resolution process. The hydrophilic hollow fiber membranes separate the DKR system into organic region (membrane external surface) and aqueous region (inner lumen). During the hydrolysis of racemic ester, diffusion of the polar molecules from organic region to aqueous region was driven by the convective flux of transmembrane pressure gradient. 3.7.2. Organic Flow Rate. In the EMR operation, the substrate-enriched organic phase is continuously flowing on the lipase-immobilized surface for the hydrolysis of ester. The 9449



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Figure 11. Effect of various organic phase flow rates on (S)-acid production rate with lipase immobilized on spongy layer (50 mM substrate, 6 g/ column OH− resin, 1.69 g-lipase/m2, 45 °C and 200 mL/min aqueous flow rate).

simulated range used in the comparative study are presented in Table 3. The overall process describes the radial flow diffusion

started to reach a steady-state after 7 h of operation with a product concentration of 161.17 mM/m2. On the other hand, the product concentration for the EMR operated at 80 mL/min continued to increase and surpassed the amount obtained in the former case with 187.1 mM/m2 of (S)-ibuprofen at 10th hour of operation. This is probably due to the fact that very low flow rate promotes the accumulation of the unreacted substrate and byproduct (alcohol) at the membrane surface, which could inhibit the activity of the enzyme. Therefore, a slightly higher flow rate of 80 mL/min is preferable for a long-run DKR reaction in the EMR. The influence of organic flow rate is rather opposite to that observed with the effect of aqueous flow rate. The former parameter is related to the reactor retention time, whereas the latter emphasized on the product diffusion. This is due to the sufficient contact time between the substrate and enzyme at the reaction layer, which is only possible at a lower organic phase flow rate. In addition, the performance of EMR for hydrolysis reaction was also investigated in terms of the (S)-ibuprofen acid production rate at different organic flow rates. Figure 11 shows the organic flow rate, which resulted into a significant influence on the reactor performance. It was observed from the result that a lower organic flow rate resulted in a higher (S)-ibuprofen production rate. An average production rate of 20.5 mmol/h.m2 was obtained at the organic flow rate of 50 mL/min, but the operation of the EMR at 200 mL/min gave only an average production of 5.44 mmol/h.m2. This is probably due to the diffusion limitation of the substrate molecules in the perpendicular direction to the enzyme-immobilized membrane and resulted in a poor resolution at the increased organic flow rate. Although the highest production rate (22.3 mmol/h.m2) was observed at 50 mL/min, the production rate gradually decreased for the entire operation until 16.1 mmol/h.m2 at the 10th hour. Meanwhile, the organic flow rate of 80 mL/min portrayed a more stable production rate compared to that of the former case. Therefore, the optimum organic flow rate of 80 mL/min was chosen because of the consistent product formation and relatively high production rate. 3.8. Comparison between Simulated and Experimental Results. The DKR diffusion-reaction model equations were simulated based on the optimum operating parameters used in the experimental work. The experimental values and the

Table 3. Input Parameters for the Simulation of the Process Model properties Initial racemic substrate concentration, STo (mM) Effective diffusivity, Deff (cm2min−1) Substrate inhibition constant for (R)-ester, KuSI (mM L−1) Product inhibition constant for alcohol, KnI1 (mM L−1) Maximum reaction rate, υmax (mM h−1) Racemization reaction constant, krac (mM h−1) Michaelis constant for (S)-enantiomer, KmA (mM) Dimensionless Michaelis constant, ΘA Dimensionless Substrate inhibition constant, ξIS Dimensionless byproduct inhibition constant, ξIP Thiele Modulus, Φ2 Dimensioless racemization constant, γ

experimental value

simulated range

50

5−100

5.12× 10−4 67.48

1−10× 10−4 10−100

255.60

250−500

3.18 2.43

1−10 1−5

23.10

10−50

2.1 1.5

0.1−5 0.1−10

0.7

0.1−10

4.13−6.34 5−8

1−30 1−30

of the hydrophobic ibuprofen ester from the organic phase into the hydrophilic membrane surface. The ester then comes in contact with the immobilized lipase and undergoes in situ racemization of the unreacted enantiomer at the membrane reactor module.44 The experimental results that include; enantiomeric excess of substrate and product (ees, eep) and the overall conversion (X) were compared with the theoretical model. Figure 12 compares both the results obtained from the experimental work and simulation at fixed process parameters of Φ2 = 1.5, B0 = 26.04, γ = 5 and ΘA = 1 for the performance of enzymatic enantioseparation (ees and eep) against the conversion. It was observed that the experimental results fit well to the theoretical model within an error of ±7.25%. In general, the eep increases with conversion and approaches the steady state point between 97− 99%. Meanwhile, the ees decreases slightly and remains constant 9450



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Figure 12. Comparison between experimental and simulated results on the performance of the EMR in enantio-separation of racemic ibuprofen ester at optimum conditions (50 mM substrate, 6 g/column OH− resin, 1.69 g-lipase/m2, 45 °C, pH 8 buffer solution, 200 mL/min aqueous flow rate and 80 mL/min organic flow rate).

at 1−2% conversion. This is due to the continuous hydrolysis and racemization of the substrate in the DKR system. In terms of the enantioselective properties of the lipase enzyme, the immobilized-lipase shows an excellent performance in catalyzing the hydrolysis reaction by showing the enantiomeric ratio (E value) of 105.45 (based on ees 1.14% and eep 98.1%). This result apparently shows that the DKR has a greater capability of obtaining the optical enantiomer compared to the classic kinetic resolution, which was initially reported by Long and coworkers.57

ACKNOWLEDGMENTS

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REFERENCES

We acknowledge the financial support (USM-RU grant 1001/ PJKIMIA/814114) and research facilities provided by Universiti Sains Malaysia (USM). We also thank Dr. Subhash Bhatia for his constant help and advice throughout this research work.

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4. CONCLUSION The deracemization of racemic ibuprofen has been successfully carried out via a coupled reactor system of simultaneous enzymatic reaction and in situ racemization. The performance of this newly devised EMR system was investigated with a view toward the industrial relevance. It was observed that the highest enantioselectivity of the enzyme was obtained with 1.69 gprotein/m2 (2.0 g/L lipase solution) of immobilized enzyme, 6 g/column of OH− resins, phosphate buffer solution at pH 8.0, temperature of 45 °C and at the organic flow rate of 80 mL/ min and aqueous flow rate of 200 mL/min. The compliance of the experimental results with the theoretical model implies the feasibility and practicability of the deracemization process by adopting the new in situ racemization unit into the EMR system. The simulated results have also proven the ability of the EMR in achieving high product enantiomeric excess (eep) and conversion at optimum operating conditions. In a nutshell, the use of a lipase-catalyzed membrane reactor for DKR becomes more potent and viable as this innovative technology contributes to the achievement of 100% theoretical yield when combined with the in situ racemization process.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 604-5996464. Fax: 604-5941013. Notes

The authors declare no competing financial interest. 9451



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Enzymatic Deracemization of (R,S)-Ibuprofen Ester via Lipase

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