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KINETICS, CATALYSIS, AND REACTION ENGINEERING Nonconventional Reactor for Enzymatic Synthesis of Semi-Synthetic β-Lactam Antibiotics Andrea L. O. Ferreira Departamento de Engenharia Quı´mica, UniVersidade Federal do Ceara´ , Campus do Pici, Bloco 709, S/N, Pici, Fortaleza, Ceara´ , Brazil, 60455-760
Raquel L. C. Giordano and Roberto C. Giordano* Departamento de Engenharia Quı´mica, UniVersidade Federal de Sa˜ o Carlos, c.p. 676, Sa˜ o Carlos, SP, Brazil 13.565-905
The enzymatic synthesis of β-lactam semi-synthetic antibiotics has been receiving increasing attention as a green-chemistry alternative for the industrial production of these drugs, because mild reaction conditions may be used. A nonconventional fed-batch reactor is presented here, using a bi-disperse gel matrix for immobilization of the enzyme penicillin G acylase (PGA) [EC 3.5.1.11]. The catalyst particles are suspended within Taylor-Couette vortices, performing the kinetically controlled synthesis of ampicillin (AMP) from phenylglycine methyl ester (PGME) and 6-aminopenicillanic acid (6-APA). This is a serial-parallel set of reactions, where the desired product (AMP) is the intermediate species, and a high selectivity is essential for the process economics. With this objective, AMP should be precipitated, withdrawing the antibiotic from the liquid phase and reducing its hydrolysis. One key point is to protect the physical integrity of the catalyst within this environment. To avoid damages to the catalyst particle caused by conventional impellers, while preserving a good mixing, Taylor-Couette flow was used. In addition, a convenient biocatalyst matrix was developed, to allow easy separation between the crystals and the enzyme support. A bench-scale (50 mL) Taylor vortex flow reactor (VFR), with a radius ratio of η ) 0.27 and operating in fed-batch mode, was used for proof of the concept. To sustain homogeneous fluidization of the biocatalyst, the VFR operated with a rotational Reynolds number of Re ) 5605, within the turbulent Taylor vortices flow region. With this reactorcatalyst ensemble, 100% activity and complete physical integrity of the particles were sustained after 200 h of operation. Introduction The isolation of 6-aminopenicillanic acid (6-APA) has made the development of synthetic penicillins possible. One of these, ampicillin (6-(D(-)R-aminobenzyl penicillin), which was introduced in 1961, was the first semi-synthetic penicillin that was effective orally, and its clinical use as a broad-spectrum penicillin has been reported extensively.1 Ampicillin is a semisynthetic β-lactam antibiotic, which is a classification that also encompasses amoxicillin, cephalexin, cefadroxil, and cefazolin, among several other molecules. β-lactams account for 65% of the world production of antibiotics,2 and their industrial manufacture started during the 1960s (Beecham patented ampicillin in 1961). The conventional route to produce β-lactam antibiotics in industry is chemical. It involves the protection of reactive groups (mainly the amino group in the side chain), uses organochloride solvents and low temperatures (approximately -30 °C), and produces significant quantities of non-recyclable waste.3,4 In 1969, Cole5 presented the possibility of an enzymatic route for the synthesis of β-lactam antibiotics, using E. coli penicillin G acylase [EC 3.5.1.11] (PGA, the same enzyme that is used in industry for the hydrolysis of microbial penicillin G). * To whom correspondence should be addressed. E-mail: roberto@ ufscar.br.
However, this “green chemistry” approach still has an important drawback: it must be competitive with the fine-tuned conventional processes in the production of inexpensive, “almostcommodity” drugs. Hence, the optimization of the bioreactor design and operation is essential for the success of the “environmental-friendly” synthesis of these antibiotics.6 Ampicillin was chosen as a case study for the proof of concept of a nonconventional reaction system (biocatalyst plus reactor configuration) that could contribute to this task. Currently, after continuous progress of the techniques for the immobilization and stabilization of enzymes, running parallel with extensive studies concerning reactor configuration and operation modes,7-14 the enzymatic synthesis of β-lactams has become more attractive. The industrial implementation of this process has been reported for cephalexin.15 Penicillin G Acylase (PGA) from E. coli is a heterodimer.16 Subunit R has 209 amino acids, and subunit β has 566. This enzyme promotes a nucleophilic attack to the carbonyl carbon of amide or ester bonds. The active site is located at the bottom of a conic depression formed by residues of the two subunits, which are tightly intertwined. Several papers have reported on the crystal structure of PGA, complexed with different sidechain ligands.17-23 The terminal serine residue essential for catalytic activity is in the β subunit. The formation of the covalent acyl-enzyme complex occurs in this position. The
10.1021/ie0614071 CCC: $37.00 © 2007 American Chemical Society Published on Web 10/13/2007
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Scheme 1. Enzymatic Synthesis of Ampicillin (Kinetically Controlled)a
a
PGME ) D-phenylglycine methyl ester, 6-APA ) 6-aminopenicillanic acid, PG ) D-phenylglycine, and PGA ) penicillin G acylase.
nucleophilicity of the Ser β1 Oγ is enhanced by its own R-amino group.17 PGA is catalytically active for hydrolysis and condensation reactions when small side-chain substitutes in the R-position are present.24-26 However, the thermodynamically controlled synthesis of β-lactam antibiotics by PGA is not favored.3 Hence, in the pH range where the enzyme is active, the only possible enzymatic route is the kinetically controlled reaction, which uses the acyl donor in an activated formseither as an ester or an amide.27 Scheme 1 shows the kinetically controlled synthesis of ampicillin (AMP) from phenylglycine methyl ester (PGME) and the β-lactam nucleus, 6-aminopenicillanic acid (6-APA). Two other undesired reactions, which also are catalyzed by PGA, compete with the synthesis of AMP: phenylglycine (PG) and methanol are the products of the hydrolysis of PGME, whereas PG and 6-APA are produced via the hydrolysis of AMP. This is indeed a classical reaction engineering problem: a series-parallel set of reactions, with the intermediate being the desired product. AMP yields in aqueous solution are highly dependent on the initial concentrations of PGME and 6-APA.28 We have studied different 6-APA/PGME ratios for the synthesis of AMP.29,30 In particular, Ribeiro et al.30 mapped the selectivity of the enzymatic synthesis of ampicillin for a large range of substrate concentrations. Their results showed that increasing concentrations of 6-APA led to smaller acylation rates, but affected the hydrolysis of PGME more intensively than the AMP synthesis, thus improving the overall selectivity (assumed here to be the molar ratio between the products AMP and PG, S/H). In other words, the conditions that led to the highest selectivity were different from those that provided the highest productivity. Selectivity improved when the ratio of 6-APA to PGME was increased. The highest productivity was obtained when both reactant concentrations were high. One way to reduce AMP hydrolysis is to transfer the antibiotic to the solid phase. That can be achieved by operating the synthesis in a pH where AMP solubility is low, so that all antibiotics produced, beyond the supersaturation limit, will crystallize and, thus, will not be attacked by the enzyme.11,13,31,32 The use of immobilized PGA to catalyze the synthesis is very important, to reduce costs. However, if ampicillin were to precipitate, antibiotic crystals and the biocatalyst must be easily separated at the end of each batch, and the growing crystals of product should not damage the enzyme matrix support. On the other hand, the catalyst must bear long runs, in the presence of crystals of product and under convenient agitation. In addition, it must retain high enzymatic activity and physical integrity after several operation cycles.
It was observed that conventional impellers gradually damaged the catalyst beads. Debris of the gel matrix (with the costly enzyme immobilized on it) were withdrawn from the reactor, together with the crystals of product during harvesting. This work presents a new reactor-biocatalyst setup, which keeps the performance of the system intact during long-term journeys with crystallization of products. This is done by combining Taylor-Couette vortex flow with a proper biocatalyst configuration, using immobilized and stabilized PGA. Taylor-Couette vortices are a secondary flow pattern that emerges above a critical rotation in the gap between an inner rotating cylinder and an outer (rotating or stationary) cylinder. It is probably one of the most widely studied phenomena in the field of fluid dynamics, ever since the remarkable work of Taylor.33 Applications of Taylor-Couette flow to improve the performance of chemical and biochemical reactors (usually known as vortex flow reactors, or VFRs) have been reported by several authors. The reactor may be operated either in batch/ semi-continuous mode or in continuous mode; in the last case, an axial Poiseuille annular flow is superimposed to the Taylor flow. These ideas have been applied to homogeneous and heterogeneous (two- or three-phase) reactors, and studies concerning the mass-transfer characteristics of VFRs, for either case, are also widespread. The same apparatus also may be used as an adsorption unit, with the fluidization of suspended particles of adsorbent sustained by the vortices. A comprehensive review of all applications is beyond our scope here; however, some examples may be found elsewhere.34-45 Nevertheless, the application of a VFR for an enzymatic synthesis with simultaneous crystallization of the products has not been reported yet, to the best of our knowledge. Here, we present a VFR that integrates enzymatic reactions catalyzed by an immobilized-stabilized enzyme and crystallization of the product.46,47 Initial concentrations of the reactants were selected to favor selectivity (high S/H). Results at two pHs, two temperatures, and using two distinct fed-batch strategies (either feeding only PGME, or feeding the two substrates simultaneously) are presented to illustrate the potential of the system. Materials and Methods 1. Materials. Phenylglycine methyl ester (PGME) was obtained from Aldrich Chemical Co. (USA), 6-aminopenicilanic acid (6-APA) and ampicillin (AMP) were obtained from Winlab (U.K.), and D-phenylglycine was obtained from Bachem (Switzerland). Penicillin G Acylase (PGA) [EC 3.5.1.11] from Escherichia coli was donated by Antibioticos S.A. (Spain), and Agarose 10 BCL was donated by Hispanagar S. A. (Spain).
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All other chemicals were laboratory grade and obtained from different commercial suppliers. 2. Methods. 2.1. Determination of Dissociation Constants. The pK values of phenylglycine acid (PG), AMP, PGME, and 6-APA were determined at 25 °C, using a Titrine pHstat Metrohm instrument. The compounds (5-10 mM) were dissolved in water and titrated with 0.1 N NaOH or 0.1 N HCl. All pK and pI values were obtained from the average of at least three replicates. 2.2. Preparation of the Biocatalyst. Multipoint immobilization of PGA on agarose gel (Aga) was performed, following the methodology of Fernandez-Lafuente et al.49 The activation of agarose gel (Aga-O-CH2-CHOH-CH2OH) was obtained after etherification with glycidol (2,3-epoxypropanol) and oxidation with sodium periodate, resulting in glyoxyl gels (Aga-OCH2-CHO). Further control of the PGA (amine)-agarose (aldehyde) multipoint attachment was achieved by reaction at pH 10 (bicarbonate buffer, 50 mM), in the presence of 100 mM of phenylacetic acid (PAA) for 3 h at 20 °C. Final reduction of the Schiff bases formed by the reaction of the lysine amino groups of the enzyme with aldehyde groups in the support was obtained with sodium borohydride (1 mg/mL of solution), for 30 min at room temperature. After each step, the gel was filtered and washed with distilled water. The enzymatic load of agarose-glyoxyl was 400 × 106 IU/(m3 gel). The agaroseglyoxyl-PGA derivative had an average diameter of 0.25 ( 0.06 mm. These agarose particles were then wrapped in pectin gel, as described elsewhere.46,47 The final bi-dispersed biocatalyst had an average diameter of 2.4 ( 0.2 mm. 2.3. Scanning Electron Microscopy (SEM) of the Biocatalyst. The biocatalyst samples were cooled first to 5 °C, then to -5 °C. Finally, water was withdrawn via liofilization. The biocatalyst was covered by gold and scanned for observation via scanning electron microscopy (SEM), using an accelerating voltage of 15 kV. 2.4. Enzyme Activity. The enzyme activity was evaluated by colorimetric analysis of the 6-APA that was released during hydrolysis of penicillin G. 6-APA reacted with p-dimethylaminobenzaldehyde (PDAB) in 10 mM phosphate buffer, pH 8.50 The difference between enzymatic activities of the supernatant (free enzyme) before and after immobilization was used to assess the enzymatic load of the gel. A measure of 1 IU (international unit) of enzyme was defined as the amount of enzyme that hydrolyzes 1 µmol of penicillin G (5% mass/unit volume) per minute at pH 8.0 and 37 °C. 2.5. Analysis. Concentrations of PGME, AMP, 6-APA, and PG were determined using high-performance liquid chromatography (HPLC). The HPLC analysis utilized the following: a C18 column (Waters Nova-Pack, C18, 60 Å, 4 µm, 3.9 mm × 150 mm) and an eluent that contained 35% acetonitrile, 2% lauryl sodium sulfate (SDS), 10 mM H3PO4, and 5 mM K2H2PO4, with a flow of 1 mL/min at 25 °C and a wavelength of λ ) 225 nm. 2.6. Taylor Vortex Flow Reactor. The enzymatic jacketed reactor had an inner rotating cylinder (composed of stainless steel) within an outer, stationary one (composed of glass). The VFR characteristics are given in Table 1. 2.7. Ampicillin Synthesis Experiments. A jacketed reactor was used in all bench-scale experiments. The same amount of biocatalyst was used in all assays (16 × 106 IU/(m3 reactor)). The pH during the enzymatic syntheses was controlled through the addition of concentrated sodium hydroxide (NaOH). The total reactor volume was 50 × 10-6 m3. Samples with a volume of 10 µL were taken from the reaction mixture, diluted in the
Table 1. Vortex Flow Reactor (VFR) Dimensions and Operational Characteristics parameter
value
dimensions inner cylinder radius outer cylinder inner wall radius total height inner cylinder rotation rate geometric characteristics radius ratio (η ) Ri/Ro) aspect ratio (Γ ) L/d) Re Re/Reca
9.5 × 10-3 m 3.5 × 10-2 m 7.3 × 10-1 m 800 rpm 0.271 1.62 5605 70.3
a Rec ) (1 / 0.15562)[ (1 + η)2 / 2ηx(1 - η)(3 + η)], calculated after Esser and Grossmann.48
mobile phase (using 990 µL), and analyzed via HPLC. To analyze the dissolved compounds, another sample (100 µL) was taken, using a 0.20 µm filter. The reaction medium and biocatalyst were kept in the annular space between the inner rotating cylinder and the outer, stationary one. Stirring was promoted by Taylor-Couette vortices, which appeared in the annular space for rotation rates above a critical value. In the present case, the critical Reynolds number (Rec) for the onset of the vortices would be 79.7, according to the equation of Esser and Grossman.48 Our VFR operated at a Reynolds number of Re ) 5605 (where Re ) ωRid/ν), thus, with Re/Rec ) 671. The kinematic viscosity of the reaction medium was 0.91 × 10-6 m2/s at 25 °C, and the rotation rate was 800 rpm (measured in a Brooksfield model LV-DVIII+ viscometer, with concentric cylinders). Hence, the VFR flow regime was well inside the turbulent vortices region (which starts at Re/Rec ) 30).51 In this region, the reactor presents ideal mixing, without noticeable gradients of concentration within it.38,41 It is known that the yield of AMP synthesis decreases when the pH increases.3,52 The range of pH for the present assays was previously bracketed by Ferreira et al.29 The effect of temperature on the yield of AMP also was studied. The solubility of the compound slightly decreases at 4 °C. Most of the solubility values of the pure component at 4 °C were ∼15% lower than those at 25 °C. However, operation of the system at 4 °C significantly reduces its productivity and demands refrigeration (thus enhancing costs). Hence, 25 °C was selected as the working temperature. Results and Discussion Any optimization of the enzymatic reactor must consider the productivity in ampicillin (P) and the global reaction selectivity (S/H), which are defined as follows:
P)
CAMP E∆t
S/H )
CAMP CFG
(1) (2)
Equation 2 is the integral selectivity, in contrast with the differential (or instantaneous) one (the ratio between the rates of synthesis and hydrolysis at each time). The yield of AMP, with respect to PGME or 6-APA, may also be included in the reactor cost function, because the system stoichiometry provides two degrees of freedom. However, overall optimization of the fed-batch reactor is not within the scope in the present work. Thus, we selected productivity and selectivity as the process indices to provide a picture of the system.
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Figure 1. Ampicillin (AMP) synthesis with crystallization of phenylglycine (PG) and AMP at 25 °C and pH 6.5. Vortex flow reactor (VFR) batch assay with equimolar initial substrate concentrations: 6-APA (250 mM) and phenylglycine methyl ester (PGME) (250 mM): (a) concentrations of substrates and products (the bars are standard deviations of triplicate measurements) and (b) global selectivity (S/H) along the reaction course.
Figure 1 shows typical results of a batch run, for equimolar initial concentrations of substrates, at 25 °C and pH 6.5. The evolution of crystallized AMP (and PG) may be followed in Figure 2a, and the global selectivity (S/H) is shown in Figure 2b. The S/H value constantly increased during the run, reaching a value of 2.2 after 11 h. Crystallization of the AMP within the reactor is essential for improving the selectivity, because the antibiotic in the solid phase cannot be reached by the enzyme. Thus, a fed-batch reactor, which constantly feeds the substrates while the product precipitates, is a natural choice for this system. Separation between crystals of product and the biocatalyst is an important issue in this reactor. The biocatalyst described by Giordano et al.46,47 was designed for this purpose and could be easily separated from precipitated material, at the end of the run, using a sieve at the bottom of the reactor. AMP and PG crystals were drained, filtrated, and proceeded to be used in the next downstream operation: recrystallization. At pH 6.5, AMP is slightly more soluble than PG. The solubility of AMP increases with pH, and its isoelectric point is 4.95. The solubility of PG does not present a significant change with changes in pH. PG has pI 5.49. In this way, it is feasible to separate AMP from PG by modulating the pH of the system.
Figure 2. AMP synthesis with crystallization of PG and AMP at 25 °C and pH 6.5: (a) concentrations of substrates and products (precipitate plus dissolved) and (b) global selectivity (S/H) along the reaction course. Conditions for panel a: VFR batch-fed assay, with initial substrate concentrations of 300 mM 6-APA and 60 mM PGME; only one substrate was added (PGME), and the bars are standard deviations of triplicate measurements.
However, when using conventional stirred-tank reactors, with different impellers, the beads of catalyst were slowly worn out, and their debris were washed out, together with the crystals of product. To overcome this drawback, Taylor-Couette flow was used to promote the mixing within the reactor. A VFR that is operating in the region of the turbulent vortices will have the same mixing pattern of an ideal stirred tank.36,37 However, the lower shear stresses, which are characteristic of Taylor flow and the absence of impeller blades, are expected to contribute to preserving the integrity of the gel beads. There are indications in the literature that a lower ratio between PGME and 6-APA favors selectivity toward synthesis of the antibiotic. A detailed mechanistic discussion about this topic, for AMP, may be found in Ribeiro et al.30 Gonc¸ alves et al.,53 who studied the synthesis of amoxicillin, observed a similar behavior. These observations suggest a reaction mechanism where the occurrence of synthesis would be favored by the previous adsorption of 6-APA on the active site. When the acyl-enzyme complex is formed before the adsorption of the β-lactam nucleus (6-APA in the present case), acyl transfer to water would be favored, and the PGME hydrolysis would be preponderant. Based on these remarks, a fed-batch assay, with the addition of PGME and a high initial concentration of 6-APA, was performed, at the same temperature and pH (25 °C and pH 6.5)
Ind. Eng. Chem. Res., Vol. 46, No. 23, 2007 7699 Table 2. Performance of the Enzymatic Synthesis of Ampicillin under Different Operational Conditions Value parameter
at pH 6.5
T ) 25 °C (PGME/6-APA)0 ) 250/250 ) 1.00a conditions at maximum productivity (5 h)
P ) 3.67 × 10-3 mM/IU/min, S/H ) 1.86 P ) 2.22 × 10-3 mM/IU/min, S/H ) 2.18
conditions at maximum AMP concentration (11 h)
Fed-Batch Feed: PGME
T ) 20 °C (PGME/6-APA)0 ) 25/300 ) 0.08a time at which maximum productivity occurs conditions at maximum productivity time at which maximum AMP concentration occurs conditions at maximum AMP concentration T ) 25 °C (PGME/6-APA)0 ) 60/300 ) 0.20a time at which maximum productivity occurs conditions at maximum productivity time at which maximum AMP concentration occurs conditions at maximum AMP concentration T ) 25 °C (PGME/6-APA)0 ) 25/300 ) 0.08a time at which maximum productivity occurs conditions at maximum productivity
5h P ) 3.83 × 10-3 mM/IU/min, S/H ) 4.50 10 h P ) 3.31 × 10-3 mM/IU/min, S/H ) 3.56
3.5 h P ) 3.90 × 10-3 mM/IU/min, S/H ) 3.21 7.5 h P ) 2.87 × 10-3 mM/IU/min, S/H ) 3.21
3.5 h P ) 6.33 × 10-3 mM/IU/min, S/H ) 4.82 8h P ) 4.12 × 10-3 mM/IU/min, S/H ) 3.50
2.5 h P ) 5.60 × 10-3 mM/IU/min, S/H ) 2.32 4h P ) 3.76 × 10-3 mM/IU/min, S/H ) 1.48
Fed-Batch Feed: PGME and 6-APA
time at which maximum AMP concentration occurs conditions at maximum AMP concentration a
at pH 7.5
Batch
4h P ) 6.63 × 10-3 mM/IU/min, S/H ) 3.69 7h P ) 5.23 × 10-3 mM/IU/min, S/H ) 1.75
1.5 h P ) 4.05 × 10-3 mM/IU/min, S/H ) 1.26 2.5 h P ) 3.17 × 10-3 mM/min, S/H ) 0.93
Conditions at t ) 0 h.
Figure 3. Repeated batch-fed arrays: AMP synthesis with the crystallization of PG and AMP at 25 °C and pH 6.5. Initial substrate concentrations are 300 mM 6-APA and 25 mM PGME, with the addition of PGME.
(see Figures 2a and 2b). Both productivity and selectivity increased, and the AMP concentration attained a value of 270 mM. With respect to the stability of 6-APA, Grant et al.54 reported that it was stable at 23 °C for 3 days in solutions with AMP. Thus, the run depicted in Figures 2a and 2b started with 300 mM of 6-APA and PGME was gradually fed into the mixture over 8 h. As may be seen, the selectivity was improved considerably (see Figure 2b). Its decrease along the reaction course is another indication that a higher 6-APA/PGME ratio is important for improving the selectivity. The enzyme is immobilized on a porous support; therefore, an intraparticle pH gradient is expected to occur within the biocatalyst bead. At pH 7.5, higher reactions rates are expected, but the selectivity would be reduced.3,29 Nevertheless, because the pH at the center of the bead would be lower than in the
Figure 4. Instantaneous selectivity during the course of the reaction for different operational strategies. Here, S/H denotes the rate of synthesis, relative to the rate of hydrolysis; this is equivalent to the rate of formation of AMP, relative to the rate of formation of PG.
bulk medium, we have tried assays that controlled the pH of the solution at a value of 7.5. Table 2 summarizes some typical results, which confirm that expectation. The spans of process time always decrease at pH 7.5, when compared to the same conditions at pH 6.5, but the cost is high: the S/H ratios became considerably lower. In an attempt to favor the crystallization of AMP, the reactor was operated at 20 °C. Indeed, the selectivity values were higher than those at 25 °C, as the data in Table 2 show; however, the productivity decreased consistently for both pHs, as expected. An assay was performed with the specific objective of monitoring the integrity of the biocatalyst after longer runs. Figure 3 shows the results of three repeated fed-batch runs (10 h each), using the Taylor VFR. It should be stressed that
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To ascertain whether the biocatalyst was really intact, SEM photomicrographs were taken. Figures 5a and 5b show the biocatalyst before and after a 30-h synthesis assay, respectively. No difference could be observed in the surface structure. Crystals of the product, still deposited on the surface, can be observed in Figure 5b. Visually, the biocatalyst was uniformly distributed along the reactor (see Figure 6), as reported elsewhere.37,38,40,44,45 Conclusions
Figure 5. Scanning electron microscopy (SEM) photomicrographs of a sample biocatalyst particle (a) before and (b) after a 30-h synthesis assay. Agarose spheres, with multipoint immobilized PGA, are wrapped by pectin gel. Crystals of AMP and PG can be observed on the surface of the particle in panel b.
A nonconventional, fed-batch reactor for the integrated enzymatic synthesis of ampicillin (AMP) from 6-aminopenicillanic acid (6-APA) and phenylglycine methyl ester (PGME), using immobilized penicillin G acylase (PGA), was presented. To improve selectivity, the desired product must precipitate during the reaction. Within this scope, the catalyst/reactor assemble must conform to two requirements: easy separation between the crystals of product and catalyst at the end of the run, and preservation of the catalyst activity and physical integrity. With this purpose, the enzyme was multipoint-immobilized on agarose gel spheres (∼0.24 mm of diameter), which were wrapped by pectin gel, generating beads ∼2.5 mm in diameter. This biocatalyst proved to be stable (maintaining its activity after long journeys) and was easily separable from the crystals of product at the end of each fed-batch run. However, its fragility, with respect to shear stresses, made the use of Taylor vortices necessary, instead of conventional propellers, to promote the mixing within the reactor. The bi-disperse biocatalyst preserved unaltered enzymatic activity after 200 h of operation, with the gel beads remaining intact, and separation from the product crystals was straightforward, using a sieve. The vortex flow, operating in the turbulent region, was able to sustain an homogeneous distribution of the biocatalyst suspended in the medium, even when the products precipitated. Although the reactor operational conditions are still not optimized (especially with respect to the reactant feed profiles and crystals seeding), promising results were obtained, with selectivity ranging from ∼15 (at startup) to ∼3.6 (after 10 h) for the best set of operational conditions among those that were tested. Nomenclature AbbreViations
Figure 6. Photograph of the VFR with crystallized products and suspended biocatalyst particles.
the activity of the biocatalyst was always assessed after each run, and no deactivation was observed after 30 h of operation. Actually, in summary of all the experiments, the biocatalyst preserved its overall integrity after 200 h of operation. Figure 3 shows that the system had good reproducibility, confirming that the biocatalyst was very stable. The separation of precipitated products was simple, as already explained. The catalyst beads were neither affected by the crystallization of the reaction products nor by the stirring promoted by the Taylor-Couette vortices. Figure 4 summarizes the behavior of the reactor, with respect to the selectivity. It can be seen that, for pH 6.5, T ) 25 °C, with a feed of PGME only, the instantaneous selectivity (the ratio between the production rates of AMP and PG) is in the range of 4-13.
AMP ) ampicillin 6-APA ) 6-aminopenicillanic acid PDAB ) p-dimethylaminobenzaldehyde PGA ) penicillin G acylase PGME ) phenylglycine methyl ester VFR ) Taylor vortex flow reactor Variables CAMP ) ampicillin concentration (mM) CPG ) PG concentration (mM) d ) annular gap width; d ) Ro - Ri (m) L ) axial length of the reactor (m) P ) productivity (mM/min) Ri ) inner (rotating) cylinder radius (m) Ro ) outer (stationary) cylinder radius (m) Re ) rotational Reynolds number; Re ) ωRid/ν Rec ) critical Reynolds number for the onset of vortices S/H ) global selectivity; S/H ) CAMP/CPG at time t
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Greek Symbols Γ ) aspect ratio; Γ ) L/d η ) radius ratio; η ) Ri/Ro ν ) kinematic viscosity (m2/s) Acknowledgment Funding of the Brazilian research agencies FAPESP (State of Sao Paulo), FINEP, and CNPq (Federal) is gratefully acknowledged. Literature Cited (1) Acred, P.; Turner, D. H.; Wilson, M. J.; Brown, D. M. Pharmacology and chemotherapy of ampicillinsA new broad-spectrum penicillin. Br. J. Pharmacol. 1962, 18, 356. (2) Elander, R. P. Industrial production of β-lactam antibiotics. Appl. Microbiol. Biotechnol. 2003, 61, 385. (3) Ospina, S.; Barzana, E.; Ramirez, O. T.; Lopez-Munguia, A. Effect of pH in the synthesis of ampicillin by penicillin acylase. Enzyme Microb. Technol. 1996, 19, 462. (4) Youshko, M. I.; Langen, L. M.; Vroom, E.; Rantwijk, F.; Sheldon, R. A.; Svedas, V. K. Penicillin acylase catalyzed ampicillin synthesis using a pH gradient: a new approach to optimization. Biotechnol. Bioeng. 2002, 78, 589. (5) Cole, M. Penicillins and other acylamino compounds synthesized by cell-bound penicillin G acylase of E. coli. Biochem. J. 1969, 115, 747. (6) Giordano, R. C.; Ribeiro, M. P. A.; Giordano, R. L. C. Kinetics of beta-lactam antibiotics synthesis by penicillin G acylase (PGA) from the viewpoint of the industrial enzymatic reactor optimization. Biotechnol. AdV. 2006, 24, 27. (7) Bruggink, A.; Roos, E. C.; Vroom, E. Penicillin acylase in the industrial production of β-lactam antibiotics. Org. Process Res. DeV. 1998, 2, 128. (8) Ghosh, A. C.; Bora, M. M.; Dutta, N. N. Developments in liquid membrane separation of β-lactam antibiotics. Bioseparation 1996, 6, 91. (9) Hernandez-Ju´stiz, O.; Terrini, M.; Pagani, G.; Garcia, J. L.; Guisan, J. M.; Fernandez-Lafuente, R. Evaluation of different enzymes as catalyst for the production of β-lactam antibiotics following a kinetically controlled strategy. Enzyme Microb. Technol. 1999, 25, 336. (10) Kim, G. M.; Lee, S. B. Effect of organic solvents on penicillin acylase-catalyzed reactions: interation of organic solvents with enzymes. J. Mol. Catal. B-Enzym. 1996, 1, 181. (11) Boesten, W. H. J.; Moody, H. M.; Roos, E. C. Process for the recovery of ampicillin. U.S. Patent 5,916,762, June 29, 1999. (12) Ferreira, A. L. O.; Gonc¸ alves, L. R. B.; Giordano, R. C.; Giordano, R. L. C. A simplified kinetic model for the side reactions occurring during the enzymatic synthesis of ampicillin. Braz. J. Chem. Eng. 2000, 17, 835. (13) Youshko, M. I.; Langen, L. M.; Vroom, E.; Moody, H. M.; Rantwijk, F.; Sheldon, R. A.; Svedas, V. K. Penicillin acylase-catalyzed synthesis of ampicillin in “aqueous solution-precipitate” systems. high substrate concentration and supersaturation effect. J. Mol. Catal. B 2000, 10, 509. (14) Clausen, K. E.; Dekkers, R. M. Process for preparation of β-lactams at constantly high concentration of reactants. U.S. Patent 6,048,708, April 11, 2000. (15) Wegman, M. A.; Janssen, M. H. A.; van Rantwijk, F.; Sheldon, R. A. towards biocatalytic synthesis of β-lactam antibiotics. AdV. Synth. Catal. 2001, 343, 559. (16) Bo¨ck, A.; Wirth, R.; Schmid, G.; Shumacher, G,; Lang, G.; Buckel, P. The Penicillin acylase from Escherichia coli ATCC11105 consists of two dissimilar subunits. FEMS Microbiol. Lett. 1983, 20, 135. (17) Duggleby, H. J.; Tolley, S. P.; Hill, C. P.; Dodson, E. J.; Dodson, G. D.; Moody, P. C. E. Penicillin acylase has a single-amino-acid catalytic center. Nature 1995, 373, 264. (18) Done, S. H.; Brannigan, J. A.; Moody, P. C.; Hubbard, R. E. Ligandinduced conformational change in penicillin acylase. J. Mol. Biol. 1998, 284, 463. (19) Brannigan, J. A.; Dodson, G. G.; Done, S. H.; Hewitt, L.; McVey, C. E.; Wilson, K. S. Structural studies of penicillin acylase. Appl. Biochem. Biotechnol. 2000, 88, 313. (20) Alkema, W. B. L.; Hensgens, C. M. H.; Kroezinga, E. H. K.; de Vries, E.; Floris, R.; van der Laan, J.-M.; Dijkstra, B. W.; Janssen, D. B. Characterization of the β-lactam binding site of penicillin acylase of
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ReceiVed for reView November 2, 2006 ReVised manuscript receiVed June 20, 2007 Accepted August 27, 2007 IE0614071
