Biotransformations Catalyzed by Multimeric Enzymes: Stabilization of

Biomacromolecules 2001, 2, 95-104

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Biotransformations Catalyzed by Multimeric Enzymes: Stabilization of Tetrameric Ampicillin Acylase Permits the Optimization of Ampicillin Synthesis under Dissociation Conditions Roberto Ferna´ ndez-Lafuente,† Odette Herna´ ndez-Ju´ stiz,† Cesar Mateo,† Marco Terreni,† Gloria Ferna´ ndez-Lorente,† Miguel A. Moreno,‡ Jorge Alonso,§ Jose L. Garcıá-Lo´ pez,§ and Jose M. Guisan*,† Departamento de Biocata´ lisis, Instituto de Cata´ lisis, CSIC, Campus Universidad Autonoma, 28049 Madrid, Spain, Centro de Investigaciones Biolo´ gicas, CSIC, Vela´ zquez 144, 28006 Madrid, Spain, and Antibio´ ticos S.A, Leon, Spain Received July 18, 2000; Revised Manuscript Received October 12, 2000

The importance of the stabilization of the quaternary structure of multimeric enzymes has been illustrated using a model reaction with great industrial relevance: the enzymatic synthesis of ampicillin from 6-amino penicillanic acid (6APA) and phenylglycine methyl ester (PGM) catalyzed by the tetrameric enzyme R-amino acid ester hydrolase from Acetobacter turbidans. The stabilization of the multimeric structure of the enzyme was achieved by multi-subunit immobilization of the enzyme followed by its further solid-phase chemical intersubunit cross-linking with polyfunctional macromolecules (dextran-aldehyde). This stabilized derivative has permitted the study of the reaction under conditions where nonstabilized enzyme molecules tended to dissociate (e.g., absence of phosphate ions). Synthetic yields improved from around 65%, under conditions where the nonstabilized derivative was stable, to around 85% in conditions where only the stabilized derivative could be utilized (40% methanol and absence of phosphate ions). When using high concentrations of PGM, a significant worsening of the reaction performance was detected with a significant decrease in the yields (below 55%, using 50 mM 6APA and PGM). This problem has been sorted out by using a fed-batch reaction system. By addition of PGM continuously to the reaction mixture (to maintain the concentration between 0.5 and 3 mM), 95% of 6-APA could be transformed to antibiotic (47.5 mM) by only using a 20% excess of acylating ester. Introduction Modern chemical industry has among their most relevant goals the low cost (including both ecological and economical considerations) production of bioactive molecules (e.g., pharmacological and agrochemical active compounds). Most of these molecules have very complex structures and are very difficult to produce using traditional chemical routes. Many of such sustainable industrial chemical processes might be performed by using immobilized enzymes, taking advantage of their good activity in aqueous systems and their high selectivity and specificity.1 Enzymes are quite complex and relatively flexible structures, very sensitive to any change in their environment. These features enable the easy modulation of the enzyme properties by the reaction conditions, but it also makes it quite easy to destroy its catalytic activity. This is highly relevant, bearing in mind that many of the chemical processes that the researcher wants to perform using enzymes as * To whom correspondence may be addressed. Fax: 34 91 585 47 60. Tel: 34 91 585 48 09. E-mail: [email protected]. † Departamento de Biocata ´ lisis, Instituto de Cata´lisis, CSIC. ‡ Antibio ´ ticos S.A. § Centro de Investigaciones Biolo ´ gicas. CSIC.

catalysts are far apart from the natural pathways where enzymes are involved (considering the type of substrates, experimental conditions, etc.).1 In many cases, the optimal conditions for the selectivity, thermodynamic yields, reaction rate, and stability of an enzyme may not be coincident. In fact, the lack of enzyme stability under interesting practical conditions (organic cosolvents, drastic pH values, or temperatures) has prevented implementation of many relevant industrial biotransformations.2-5 In this sense, the preparation of very stable enzyme derivatives might extend the range of experimental conditions where a reaction can be designed. Thus, the design of stabilized enzyme derivatives becomes a critical point in the design of many interesting organic chemistry reactions. This stability problem reaches a special relevance when using complex enzymes, as multimeric proteins, composed of several subunits that must be assembled to yield catalytic activity. These enzymes are mainly inactivated by subunit dissociation, and this phenomenon may be accelerated under certain experimental conditions (a certain pH, low or high ionic strength, high temperature, etc.).6 On the other hand, it is possible that small changes in the exact assembling of the subunits may alter the shape of the active center and,

10.1021/bm000072i CCC: $20.00 © 2001 American Chemical Society Published on Web 12/02/2000

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Biomacromolecules, Vol. 2, No. 1, 2001

Scheme 1. Possible Effects of Experimental Conditions on Enzyme Stability and Catalytic Behavior

consequently, alter (with possible enhancement of) their catalytic properties. Thus, it may be possible that a particular condition, where the enzyme might exhibit the most adequate selectivity, coincides with conditions where that multimeric enzyme tends to dissociate, therefore becoming inactive (Scheme 1). From this point of view, the development of general strategies to stabilize the quaternary structure of multimeric enzymes may lead to the design of bioprocesses and biotransformations with interest in organic chemistry. The enzyme R-amino acid ester hydrolase from Acetobacter turbidans is an example of such interesting multimeric enzymes. This tetrameric enzyme7,8 has been found to have Scheme 2. Kinetically Controlled Synthesis of Antibiotics

Ferna´ ndez-Lafuente et al.

a potential interest for the synthesis of some relevant semisynthetic antibiotics (e.g., ampicillin) using the ester of the side chain (e.g., phenylglycine methyl ester) and the antibiotic nucleus (e.g., 6-amino penicillanic acid).9 In this process, the enzyme may catalyze three different reactions (synthesis of the antibiotic and hydrolyses of the antibiotic and the ester)10 (Scheme 2). The yields are determined by the ratio between the rates of synthesis and both hydrolyses reactions; that is, they are directly defined by the properties of the enzyme preparation. The interest of this particular enzyme is that its hydrolytic activity on ampicillin is negligible; therefore the yields are determined directly by the esterase and synthetase activities of the enzyme9 (Scheme 2). The yields obtained using this synthetic strategy depend on the enzyme properties (specificity, kinetic ratios, adsorption of the nucleophile by the enzyme active center, etc.).9-14 Therefore, this reaction not only presents a great industrial relevance but may be a good test to study the possibilities of altering the enzyme catalytic properties via engineering of the reaction medium. In this paper, we present a sequential study involving (i) the development of a protocol to purify the enzyme R-amino acid ester hydrolase from Acetobacter turbidans, (ii) a new strategy to stabilize its quaternary structure by multi-subunit covalent attachment onto activated supports and further crosslinking with polyfunctional reagents (aldehyde-dextran)15 (Scheme 3), (iii) utilizing the derivative to evaluate the effect of many different experimental conditions on the synthetic properties of the enzyme, including conditions quite far from those where the free enzyme was stable, and (iv), finally, the synthesis of ampicillin pursuing industrially interesting values (yields, concentrations, etc.). Materials and Methods Crude protein extract (20 mg/mL) from a native strain of A. turbidans (ATCC 9325) containing R-amino acid ester

Biomacromolecules, Vol. 2, No. 1, 2001 97

Biotransformations Catalyzed by Multimeric Enzymes Scheme 3. Strategy of Immobilization and Stabilization of the Quaternary Structure of Multimeric Enzymes

hydrolase (purity under 0.2% by SDS-PAGE), 6-amino penicillanic acid (6APA), penicillin G acylase from E. coli (PGA), and D-phenylglycine methyl ester (D-PGM) were kindly donated by Antibioticos S.A. (Leon, Spain). 4% crosslinked amino agarose beads (MANAE) activated with 5 or 40 µmol/mL were a kind gift from Hispanagar S.A. (Burgos, Spain) and produced as previously described.16 All other reagents were from Sigma Chemical Co. (St. Louis, MO). Aldehyde dextran (Mw 18 000) was prepared by its full oxidation with sodium periodate as previously described.17 Carboxymethyl-cellulose (CM-52) was from Whatman, and phenyl sepharose was from Pharmacia Biotech. PGA from E. coli was immobilized onto glyoxyl agarose as previously described.18 Glutaraldehyde-activated agarose was prepared as previously reported elsewhere.19 Purification of the Enzyme r-Amino Acid Ester Hydrolase from Acetobacter turbidans. Ten milliliters of enzyme extract was diluted with 90 mL of 5 mM sodium phosphate at pH 6.5, and 5 mL of CM-52 equilibrated with that buffer was added. The resulting suspension was stirred for 3 h, and the gel was filtered and washed using 3 volumes of the same buffer. To elute the enzyme, the gel with the adsorbed enzyme was washed with 4 volumes of 50 mM sodium phosphate (pH 5) containing 0.5 M KCl. The eluted solution was collected and offered to 1.5 mL of phenyl sepharose, keeping the solution under gentle stirring for 3 h. The support with the adsorbed enzyme was then filtered and washed with 3 volumes of 50 mM sodium phosphate (pH 5) containing 0.5 M KCl. To elute the enzyme, the support was washed with 4 volumes of 2 mM sodium phosphate (pH 7) and collected in a vessel containing 0.6 mL of 1 M sodium phosphate (pH 7). Activity was measured by assaying the hydrolysis of 10 mM PGM in 25 mM phosphate buffer at 4 °C and pH 6.5. This reaction was followed by HPLC as described in the synthetic experiments. All the steps (adsorptions, desorptions, washings, elutions) were performed at 4 °C.

This simple two-step purification protocol gives a high purification factor (around 400) with a fairly good yield (over 60%) (see Table 1). Preparation of r-Amino Acid Ester Hydrolase Derivatives from Acetobacter turbidans. Ten milliliters of agaroseglutaraldehyde activated with 5 or 40 µmol/mL was added to 90 mL of a solution containing 20 international units of enzyme (around 0.5 mg of purified enzyme) in 100 mM sodium phosphate (pH 7) (activity was determined by the hydrolysis of 10 mM D-PGM in 25 mM sodium phosphate (pH 6.5) at 4 °C). After the desired immobilization time frame (with stirring) at 4 °C, 500 mL of 200 mM sodium bicarbonate (pH 8.5) and 800 mg of solid sodium borohydride were added to the immobilization solution in order to reduce the remaining (nonreacted) aldehyde groups and the glutaraldehyde-enzyme bonds.20 The resulting suspension was stirred for an extra 1 h, and the derivative was washed with an excess of distilled water. To chemically cross-link the derivative with aldehyde dextrans, 10 mL of derivative prepared on agarose-glutaraldehyde activated with 40 µmol/mL was added to 10 mL of 200 mM sodium phosphate (pH 7)/20 mL of the aldehydedextran solution (1.8 mM) at 4 °C.15 After 24 h of gentle stirring at 4 °C, 500 mL of cold (4 °C) 200 mM sodium bicarbonate (pH 8.5) and 800 mg of solid sodium borohydride were added to the immobilization solution in order to reduce the remaining (nonreacted) aldehyde groups and the amine-aldehyde bonds.17 This suspension was stirred for an extra 1 h, and the derivative was finally washed with an excess of distilled water. Study of the Stabilization of the Quaternary Structure of the Different Derivatives To check the stabilization of the quaternary structure of the protein, different enzyme derivatives (containing 2 mg of protein/mL) were boiled in one volume of 2% SDS.21 All the bonds established between the enzyme and the support or the dextran are secondary amino bonds, and these bonds may stand even 110 °C in 6 M HCl.22 In this way, any molecule that was not covalently attached to the support (directly or via cross-linking with one subunit already covalently attached to the support) is released to the medium. Then, SDS-PAGE analysis of the supernatant was performed and the gel was stained with Coomassie and analyzed by densitometry. Kinetically Controlled Synthesis of Ampicillin. Experiments were performed by using a thermostated jacketed column containing 10 mL of enzyme derivative. Reactions were followed by HPLC analyses of samples withdrawn from

Table 1. Purification of the Enzyme R-Amino Acid Ester Hydrolase from A. Turbidansa enzyme preparation diluted crude CM-52 phenyl-sheparose

volume (mL)

[protein] (mg/mL)

total activity (U)

specific activity (U/mg)

purification factor

yield (%)

100 20 6.6

2.0 0.432 0.050

20.0 18.2 12.5

0.1 2.3 42.0

1 23 420

100 90 62

aExperiments were carried out as described in Methods. Activity is expressed in µmoles of D-PGM hydrolyzed per minute and milligram of protein under standard conditions (see Methods).

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the column, and recording the absorbance at 254 nm. The mobile phase was constituted by 10% MeOH and 90% of 100 mM ammonium phosphate (pH 5). The HPLC column was a kromasil C8 (150 mm × 4.6 mm) produced by Ana´lisis Vinicos (Spain). At a flow rate of 2 mL/min, retention times were 1.25 min for phenylglycine, 3 min for 6APA, 11 min for D-PGM, and 22 min for ampicillin. Identification and evaluation of the concentration of substrates and products were performed by comparison with standard solutions of authentic samples. Batch Reactions. After the desired amounts of 6-APA and D-PGM were dissolved, the pH was adjusted at the indicated value by adding concentrated NaOH. One hundred ten milliliters of reaction mixture was passed through the column at a flow rate of 2 mL/min to equilibrate the derivative, and the remaining 90 mL was placed in a thermostated external reservoir, from which the reaction mixture was recirculated. pH control was performed in the external reservoir using 2 M NaOH under vigorous stirring. Occasionally, some additives were added to the reaction mixture (viz. ethanol, methanol, glucose, phosphate, Ca2+, Mg2+, Mn2+, etc.) to study possible effects of these compounds on the synthetic properties of the enzyme. When organic cosolvents were used, pH values are given without corrections as pHapp. Fed-Batch Reactions. In a standard experiment, a solution of 150 mL of 100 mM 6APA was prepared. One hundred ten milliliters of this solution was passed through the column to equilibrate it. Then, the remaining 40 mL of substrate solution was recirculated as in the previous case from a reservoir tank (total initial reaction volume equal to 50 mL). To this reservoir, 1.5 mL of 100 mM D-PGM was added and the reaction started (initial concentration of D-PGM was around 3 mM). New additions of ester were performed when its concentration was under 0.5 mM, to rase it to 3 mM. When 50 mL of this D-PGM solution was added (that is, a final 50 mM/50 mM “theoretical concentration” of both substrates and a volume of 100 mL), the addition of D-PGM was stopped and the reaction allowed to proceed until total consumption of the ester. The relation of volumes was maintained in all experiments. Results and Discussion (A) Stabilization of the Quaternary Structure of r-Amino Acid Ester Hydrolase from Acetobacter turbidans via Immobilization and Postimmobilization Techniques. (1) Functional Stabilization. The stability of this tetrameric enzyme in soluble form is quite low and decreased with decreasing enzyme concentration (Figure 1), suggesting that some dissociative process may be the key step of the enzyme inactivation. The immobilization of the enzyme onto supports with low activation degree has a negligible effect on its stability, even when using longer immobilization times (e.g., 24 h). When using these low-activated supports, the dependence of the immobilized enzyme stability on the protein concentration was quite similar to that of the soluble counterpart (see Figure 1). On the contrary, when using highly activated supports, the thermal stability of the enzyme was significantly


Figure 1. Thermal inactivation of different derivatives of R-amino acid ester hydrolase from A. turbidans: solid lines and solid symbols, concentration of protein was 1 µg/mL; dashed lines and empty symbols, concentration of protein was 10 µg/mL; triangles, soluble enzyme and immobilized in low-activated glutaraldehyde supports; squares, enzyme immobilized on highly activated glutaraldehdye supports; rhombus, previous derivative further modified with polyaldehyde-dextran. Inactivation was carried out in 25 mM phosphate at 25 °C and pH 6.5, using purified enzyme.

Figure 2. Effect of phosphate concentration on the stability of R-amino acid ester hydrolase from A. turbidans: rhombus, stabilized enzyme derivative at 0 or 100 mM of sodium phosphate; empty triangles, soluble enzyme in 100 mM phosphate; solid triangles, soluble enzyme in 100 mM NaCl or water. Inactivations were performed at pH 6.5 and 25 °C with a fixed concentration of protein (5 µg/mL).

improved. Such stabilization increased when the enzymesupport reaction time increased, reaching a maximum after 24 h of contacting the enzyme with the support. In this derivative, the dependence of the enzyme stability on the protein concentration becomes smaller than that in the soluble enzyme, but it is still quite significant. The modification of such an immobilized and partially stabilized derivative with aldehyde-dextran promoted a further stabilization of the enzyme (see Figure 1). Moreover, now the stability of the enzyme derivative did not depend at all on the concentration of protein. This derivative was much more stable than the soluble enzyme or the enzyme immobilized onto low-activated supports (see Figure 1). These results suggest that dissociation of the enzyme subunits may be responsible for the low stability of the soluble enzyme. On the other hand, Figure 2 shows that the stability of the soluble enzyme extremely depended on the phosphate concentration. The enzyme stability was very low in the absence of phosphate, and the effect of the protein concentration was even more relevant than that in 100 mM


Biotransformations Catalyzed by Multimeric Enzymes

Table 2. Effect of Temperature upon the Synthetic Yields and Reaction Rates in the Synthesis of Ampicillin Catalyzed by R-amino Acid Ester Hydrolase from A. Turbidansa

T (°C)

rel. activity

Vs/Vh1

synthetic yields (%)

4 15 22 30

10% 25% 60% 100%

3.6 3.2 2.2 1.5

78 76 69 60

a Experiments were carried out using 2.5 mM D-PGM and 50 mM 6APA at pH 6.5. Other specifications were as described in Methods. Vs/Vh1 refers to synthesis/hydrolysis ratio.

Table 3. Effect of pH Value upon the Synthetic Yields and Reaction Rates in the Synthesis of Ampicillin Catalyzed by R-amino Acid Ester Hydrolase from A. Turbidans. Figure 3. Desorption of noncovalently immobilized protein subunits from the support in the prepared derivatives: 1, crude extract; 2, fraction of purified enzyme; 3, enzyme immobilized on agaroseglutaraldehyde poorly activated, 1 h of immobilization; 4, enzyme immobilized on agarose-glutaraldehyde highly activated, 24 h of immobilization; 5, previous derivative modified with polyaldehydedextrans. Desorption was carried out by boiling the derivatives in the presence of SDS.

phosphate. However, the immobilized and chemically crosslinked derivative remained fully active even in the absence of phosphate ions for very long incubation times (see Figure 2). These results suggest that the phosphate ions may be somehow related to the maintenance of the quaternary structure of the enzyme. The immobilization and modification of the enzyme promoted only a slight decrease in its activity (ca. 15-20%) while increasing the operational stability of the diluted enzyme by several orders of magnitude. (2) Structural Stabilization. Figure 3 shows the SDSPAGE gel obtained after boiling the different derivatives in the presence of SDS, offering in all cases similar amounts of protein. Lane 2 shows the purified enzyme with a main band corresponding to a molecular weight of around 70 000 (corresponding to the molecular weight of each monomer of this enzyme).7 After being subjected to the same treatment, the derivative prepared in low-activated glutaraldehyde support still produced that band, with an intensity of ca. 70% of that observed for the purified enzyme. This result indicated that only one of the four protein subunits has been covalently attached to the support and may explain why the stability of this derivative was almost identical to that of the soluble enzyme (the possibilities of subunit dissociation would be almost identical). When the derivative prepared using highly activated agarose-glutaraldehyde was analyzed, after 24 h of enzyme support multi-interaction, some protein could be still released from the support by the desorption treatment, but the relative intensity of the band is now around 30-40% (regarding the pure enzyme), suggesting that at least two to three of the four subunits of the enzyme have been covalently attached to the support. However, some of the subunits were not yet covalently attached to the support and can dissociate from the whole protein. These results may explain the significant increase in the enzyme stability and the significant decrease of the dependence of the enzyme stability on the enzyme concentration found when studying this derivative. The derivative that had been chemically cross-linked with

pH

rel. activity

Vs/Vh1


6.0 6.5 7.0 8.0

40% 75% 90% 100%

4.0 3.6 2.6 1.3

80 78 72 55

a Experiments were carried out using 2.5 mM D-PGM and 50 mM 6APA at 4 °C. Other specifications were as described in Methods. Vs/Vh1 refers to synthesis/hydrolysis ratio.

aldehyde-dextran, after boiling in SDS, did not release any detectable protein to the supernatant. Therefore, this treatment seems to be able to fully stabilize the quaternary structure of this complex tetrameric enzyme (see Figure 3, lane 5). This explains the nondependence of the stability of the enzyme on the enzyme concentrations, when analyzing this derivative, and also explains the high stability of such enzyme derivative in the absence of any phosphate ions. Thus, this simple two-step treatment is able to fully stabilize the quaternary structure of the enzyme. (B) Optimization of the Enzymatic Synthesis of Ampicillin. A great excess of nucleophile has been employed to carry out these studies. In this way, it has been possible to consider that its concentration during the reaction course was constant, to simplify the analysis of the results. (1) Effect of Experimental Conditions. The dependence of the ampicillin synthetic yields and the enzyme activity on some experimental conditions is shown in Tables 2 and 3. The temperature has a significant influence in the synthetic yields. The ampicillin synthetic yields decreased when increasing the reaction temperature. For example, synthetic yields decreased from 78% to 60% when the temperature increased from 4 to 30 °C. Also, the pH value had a great influence in the synthetic yields. The synthetic yields were 80% at pH 6 and 55% at pH 8. However, the synthetic activity was quite low at pH 6 and 4 °C (optimal conditions considering only the ampicillin synthetic yield); for these reasons, pH 6.5 (at which activity was almost doubled when compared with that at pH 6) was chosen as a compromise solution to get good enzyme activity and yield (Table 2). Also, the solubility and stability of 6APA and the stability of PGM were better at this (more neutral) pH value. Several additives were added to the reaction medium to study their possible effects on the enzyme synthetic properties. The presence in the reaction medium of glucose (1030% (w/v)), ethanol (10-30%), NaCl (1 M), and ammonium

100


Figure 4. Effect of the presence of phosphate upon the synthetic yields and reaction rates in the synthesis of ampicillin catalyzed by optimal derivative of R-amino acid ester hydrolase from A. turbidans: triangles, synthetic activity; squares, synthetic yields. pH value was 6.5, temperature was 4 °C, and the concentrations of D-PGM and 6APA were 2.5 and 50 mM, respectively.

Figure 5. Effect of methanol on the synthetic yields (squares) and reaction rates (triangles) in the synthesis of ampicillin catalyzed by the optimized derivative of R-amino acid ester hydrolase from A. turbidans. Experiments were performed in the absence of phosphate at 4 °C and pH 6.5 using 50 mM 6APA and 2.5 mM D-PGM.

sulfate (0.5-2 M) have slightly negative effects on the ampicillin synthetic yields (results not shown). Significant effects were not detected by using 25 mM of different metal cations (Na+, K+, Ca2+, Mg2+, Mn2+). However, a clear decrease in the yields was found when increasing the concentration of sodium phosphate. While the yield in the absence of phosphate buffer was 78%, it dropped to 62% in the presence of 100 mM sodium phosphate (see Figure 4). On the other hand, addition of small amounts of methanol significantly enhanced the synthetic yields (see Figure 5). Thus, yields improved from 78% to 86% when increasing the methanol concentration from 0 to 50% (v/v) (even although the pH had to be increased to 6.8, when using 3050% MeOH, to allow full solubilization of the 6APA). Such an effect of methanol in the yields is not related to a shift in the synthetic equilibrium of the reaction. In both cases the concentration of ampicillin at the thermodynamic equilibrium of this reaction is negligible.23-26 In fact, other organic cosolvents with higher effects on this equilibrium (e.g., dimethylformamide)27-30 had a negative effect on the synthetic yields. The effect of the presence of methanol on the synthetic yields might be caused by several facts, e.g., a conformational change of the enzyme,12 the possible recycling of the activated acyl donor,31 or the reinforcement of


Figure 6. Effect of reaction conditions on the synthetic yields at different concentrations of 6APA in the synthesis of ampicillin catalyzed by the optimized derivative of R-amino acid ester hydrolase from A. turbidans: squares, 100 mM phosphate pH 7.5 at 22 °C; triangles, 40% MeOH at 4 °C and pH 6.8 and in absence of phosphate. D-PGM concentration was 2.5 mM.

the adsorption of the nucleophile onto the active center of the enzyme.10 Bearing in mind substrate solubilities, enzyme activity, and synthetic yields, we have chosen as the optimal conditions to perform the synthesis of ampicillin as catalyzed by this new enzyme derivative: pH 6.8, 4 °C, absence of phosphate buffer, and presence of 40% MeOH. Another important feature of an enzyme as a catalyst for a kinetically controlled synthesis is its capacity to adsorb the nucleophile, that is, the concentration of 6APA that it is necessary to reach the maximum values of yield. This value has a great relevance in the course of the reaction when using an excess of the ester (condition closer to the industrial requirements). The saturation of the enzyme by 6APA was studied under optimal and standard (those where soluble enzyme was stable) conditions (see Figure 6). Saturation conditions seemed to be achieved at a moderately low 6APA concentration (under optimal conditions, ca. 20 mM), while saturation under standard conditions was not achieved even at much higher 6APA concentrations. (2) Effect of the Enzyme Derivative. Figure 7 shows the reaction courses observed when using stabilized and nonstabilized derivatives of this enzyme. Under conditions where both enzyme derivatives were stable (100 mM sodium phosphate and absence of methanol), both reaction courses were almost identical, and both gave only moderate synthetic yields (around 65%). From these results, it seems that the stabilization protocol has not altered the enzyme properties as a catalyst of kinetically controlled synthesis of ampicillin. However, when the reaction were performed in the absence of phosphate ions and in the presence of methanol, the yields significantly increased when using the stabilized derivative, while the reaction stopped after a few moments when using the nonstabilized derivative, very likely reflecting the low stability of this enzyme under these conditions (see Figure 2). In this way, only the stabilized derivative permits the achievement of high yield synthesis of ampicillin. The stabilized derivatives could be reused for 50 reaction cycles in the different conditions described in this paper without significant changes in the reaction rates or in the synthetic yields (results not shown).



Figure 7. Effect of the stabilization of enzymatic derivatives on the improvement of synthetic yield of Ampicillin synthesis: triangles, nonstable derivative; squares, optimized derivative; dashed line and empty symbol, 100 mM phosphate without methanol; solid line and solid symbol, 40% MeOH, absence of phosphate ions. Experiments were performed in 40% MeOH at 4 °C and pH 6.8 using 50 mM 6APA and 2.5 mM D-PGM.

Figure 8. Comparison between the synthetic yield of Ampicillin obtained with derivatives of different enzymes: triangles, stabilized derivative of penicillin G acylase from E. coli; squares, stabilized derivative of acylase from A. turbidans. Experiments were performed at 4 °C and pH 6.0 using 50 mM 6APA and 2.5 mM D-PGM.

(3) Effect of the Enzyme Source. The most popular enzyme utilized to catalyze this process is the penicillin G acylase from E. coli.13 For this reason, both enzymes have been compared under optimal conditions. Figure 8 shows that the yield of ampicillin reached with the derivative of penicillin G acylase from E. coli at pH 6.0 (around 40%) is much lower than that achieved using this new enzyme derivative (around 80%). Moreover, while the enzyme from E. coli hydrolyzed very rapidly the antibiotic, the enzyme from A. turbidans left the antibiotic untouched long after consuming all the ester (see Figure 8). Therefore, this new immobilized and stabilized preparation of the enzyme from A. turbidans seems to have much better prospects to catalyze this reaction than the enzyme derivative of PGA from E. coli. Optimization of the Reaction Using High Substrate Concentrations. From a practical point of view, the synthesis of ampicillin should be carried out at high and next to

Figure 9. Effect of the D-PGM concentration on the yields of synthesis of Ampicillin at different concentrations of 6APA in the reaction catalyzed by the optimized derivative of R-amino acid ester hydrolase from A. turbidans: squares, 20 mM 6APA; triangles, 100 mM 6APA. Yields were calculated regarding the first 2.5 mM of D-PGM consumed during the reaction. Experiments were performed at pH 6.8 in 40% MeOH and 4 °C.

equimolecular concentrations of both substrates (PGM and 6APA). When the behavior of the new biocatalyst in the presence of increasing concentrations of PGM was studied, it was found that the enzyme esterase activity increased while the synthetic one decreased. This promoted a significant drop in the yields even in the first stages of the reaction (see Figure 9) while slightly increased the reaction rate. Similar results were found under other conditions and using derivatives with different purification degrees. The decrease in the esterase/hydrolase ratio presented a similar magnitude at low or high concentrations of 6APA (see Figure 9). Thus, the negative effect of D-PGM upon the yields did not seem to be a competitive inhibition of this reagent to the 6APA adsorption onto the active site of the enzyme. Moreover, the production of D-PGM dimers was not observed when the enzyme acted on 100 mM D-PGM in the presence or in the absence of 6-APA. Similar changes

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Table 4. Ampicillin Synthetic Yields in the Synthesis Catalyzed by R-Amino Acid Ester Hydrolase from A. Turbidans at High Concentrations of D-PGM Using a Batch or a Fed-Batch Reaction Systema reaction conditions 50 mM 6APA/50mM D-PGM 40% MeOH, 50 mM 6APA/ 50mM D-PGM 40% MeOH, 50 mM 6APA/ 60mM D-PGM

reaction system batch fed-batch batch fed-batch batch fed-batch

donor concentration when using the enzyme from E. coli is the inhibition of the hydrolysis of the antibiotic.26


Conclusions

53 75 65 82 73 94

The results presented in this paper clearly exemplified that the design of a reaction catalyzed by a multimeric enzyme may be extremely complex, very likely because the possibilities of modulating their catalytic properties are very wide. In this way, the design of these biotransformations must consider the necessity of using derivatives able to work under conditions quite different from the physiological ones, even under conditions where the enzyme tends to dissociate. The model reaction chosen for this paper, the enzymatic synthesis of ampicillin under environmentally friendly conditions, is a very important goal for the pharmaceutical companies.31-34 The thermodynamically controlled synthesis of antibiotics having as side chain phenylglycine (using the free acid) has been found to give very poor yields by several reasons.23-26 The strategy used in this paper, kinetically controlled synthesis, has been the most used one to try to achieve an enzymatic route of synthesis of ampicillin. However, a combination of biocatalysts and conditions that allow the high yield synthesis of this interesting antibiotic are not available at the moment. Most studies related to this process have been performed using the best known and most used enzyme, the penicillin G acylase (PGA) from E. coli.31-43 However, the PGA from E. coli is a poor transferase using 6APA as nucleophile and phenylglycine as acyl donor and has a high hydrolytic activity on ampicillin compared to the synthetic activity40-42 (see Scheme 2). Therefore the ampicillin synthetic yields are very poor even using high excesses of acyl donors, usually under 50%. This has promoted an intense evaluation of many other enzyme sources44-53 including the one reported in this paper. However, the results have not been fully satisfactory, in many cases because of the limits established by the enzyme stability. The multidisciplinary strategy employed in this paper has permitted a great advance in the enzymatic synthesis of ampicillin. The design of a stabilized biocatalyst (by using a novel strategy that permits the stabilization of multimeric structures of proteins15) has been critical to take advantage of the good properties of the enzyme. Only the preparation of an enzyme derivative with the quaternary structure fully stabilized has permitted to work under conditions where the ampicillin synthetic yields reached very high values but where the enzyme subunits tend to dissociate. This fact has limited the use of this and other similar enzymes in the development of these very interesting processes.51-53 Under optimal conditions, the enzyme derivative properties seemed to be adequate to catalyze this process: saturation of the enzyme by the nucleophile is achieved at moderate concentrations of 6APA and the transferase/esterase ratio would seem to be high enough to give yields over 80% using equimolar substrate concentrations. Finally, the use of a fedbatch reaction system has permitted keeping the concentration of D-PGM under a desired value, reducing the deleterious effect of this compound upon the synthetase activity

a Experiments were carried out at pH 6.8 and 4 °C. Other specifications were as described in Methods.

in the enzyme properties were induced by phenylacetic acid,which was, however, unable to inhibit the rate of hydrolysis of ampicillin or PGM. Such a result cannot be predicted from the reaction scheme and has not been reported in any other kinetically controlled synthesis to date, where (to our knowledge) the increment of the ester is the usual way of improving the yields regarding the nucleophile.13 Thus, it seems to be related to a change in the enzyme properties somehow induced by the presence of high concentrations of the ester. Use of a Fed Batch Reaction System. After the results presented above, it is possible to conclude that the stabilized derivative of the enzyme R-amino acid ester hydrolase from A. turbidans presented some very suitable properties to catalyze the industrial synthesis of Ampicillin: (i) The hydrolysis of ampicillin was negligible. (ii) The biocatalyst was saturated by the 6APA at moderately low concentrations of this nucleophile. (iii) The transferase activity of the biocatalyst may give very good synthetic yields. Thus, the only drawback of this immobilized and stabilized enzyme was the PGM-induced reduction of the yields at high concentrations of this acylating reagent. This effect may be very interesting and deserves further research to determine its true nature. However, there is a very simple technological solution to prevent this negative effect even without knowing the exact cause: to perform the reaction under a continuous low concentration of PGM, using a fed-batch reaction system. In this way, PGM could be progressively added in order to prevent that its concentration reached levels over a fixed concentration (e.g., 3 mM). Table 4 shows that by using this reaction system, yields were significantly improved when compared to those achieved in a batch system. Under optimal conditions (40% MeOH, pH 6.8, 4 °C) and a final “theoretical concentration” of 6APA and D-PGM of 50 mM, ampicillin synthetic yields using the fed batch reactor were 82% (compared to 65% in the batch reaction) (Table 4). By use of only a 20% molar excess of PGM, yields reached very high values, with 94-95% of the 6APA being transformed into antibiotic (Table 4). Paradoxically, with reactions performed using the enzyme from E. coli, the fed-batch reaction system promotes a decrease in yields, rather than the increase observed in this case (results not shown). This is probably because one of the positive effects that promoted this increment in the acyl


of the enzyme and enabling the high yield production of high concentrations of ampicillin. By use of a fed-batch system and only a 20% molar excess of D-PGM, around 95% of the 6APA was transformed to ampicillin (as far as we know, the best results published to date). Acknowledgment. Authors gratefully acknowledge a fellowship for Dr. Hernandez-Justiz, granted by the Spanish Institute of Iberoamerican Cooperation. We are grateful to Antibioticos S.A. for their kind supply of enzyme and reagents and to Hispanagar S.A. for the supply of agarose supports. We also gratefully recognize the suggestions of Dr. V. M. Balcao. References and Notes (1) Wong, C.-H.; Whitesides, G. M. Enzymes in synthetic organic chemistry; Tetrahedron organic chemistry series; Baldwin, J. E., Magnus, Eds.; Pergamon: Oxford, 1994; Vol. 12, pp 41-130. (2) Gupta, M. N. Thermostabilization of proteins. Biotechnol. Appl. Biochem. 1991, 4, 1-11. (3) Gianfreda, L.; Scarfi, M. R. Enzyme stabilization: State of the art. Mol. Cell. Biochem. 1991, 109, 97-128. (4) Klibanov, A. M. Stabilization of enzymes against thermal inactivation. AdV. Appl. Microb. 1982, 29, 1-28. (5) Stability and Stabilization of Biocatalysts. Progress in Biotechnology; Ballesteros, A., Plou, F. J., Iborra, J. L., Halling, P. J., Eds.; Elsevier: Amsterdam, 1998; Vol. 13. (6) Poltorak, O. M.; Chukhary, E. S.; Torshin, I. Y. Dissociative thermal inactivation, stability and activity of oligomeric enzyme. Biochemistry (Moscow) 1998, 63, 360-369. (7) Rhee, D. K.; Lee, S. B.; Rhee, J. S.; Ryu, D. D. Y. Enzymatic biosynthesis of cephalexin. Biotechnol. Bioeng. 1980, 22, 12371247. (8) Ryu, Y. W.; Ryu, D. D. Y. Semisynthetic lactam antibiotics synthesizing enzyme from Acetobacter turbidans: purification and properties. Enzyme Microb. Technol. 1987, 9, 339-344. (9) Hernańdez-Justiz, O.; Terreni, M.; Pagani, G.; Garcıá-Lo´pez, J. L.; Guisań, J. M.; Fernańdez-Lafuente, R. Evaluation of different enzymes as catalysts of the kinetically controlled synthesis of several lactamic antibiotics. Enzyme Microb. Technol. 1999, 25, 336-343. (10) Kasche, V. Mechanism and yields in enzyme catalysed equilibrium and kinetically controlled synthesis of beta-lactamic antibiotics, peptides and other condensation products. Enzyme Microb. Technol. 1986, 8, 2-16. (11) Fernandez-Lafuente, R.; Rosell, C. M.; Guisań, J. M. The use of stabilised penicillin G acylase derivatives improves the design of kinetically controlled synthesis. J. Mol. Catal. 1995, 101, 91-97. (12) Fernandez-Lafuente, R.; Rosell, C. M.; Guisan, J. M. The presence of methanol exerts a strong and complex modulation of the synthesis of different antibiotics by immobilized penicillin G acylase. Enzyme Microb. Technol. 1998, 23, 305-310. (13) Savidge, T. A. Enzymatic conversions used in the production of penicillins and cephalosporins. Biotechnology of industrial antibiotics. Vandamme, E. J., Ed.; Drugs and the Pharmaceutical Sciences; Marcel Decker: New York, 1984; Vol. 22, pp 177-224. (14) Justiz, O. H.; Fernandez-Lafuente, R.; Guisan, J. M.; Negri, P.; Pagani, G.; Pregnolato, M.; Terreni, M. One pot chemoenzymatic synthesis of 3′-functionalized cephalosporines (cephazolin) by three consecutive biotransformations in fully aqueous medium. J. Org. Chem. 1997, 62, 9099-9106. (15) Fernańdez-Lafuente, R.; Rodrı´guez. V.; Mateo, C.; Penzol, G.; Hernańdez-Justiz. O.; Irazoqui, G.; Villarino, A.; Ovsejevi, K.; Batista, F.; Guisań, J. M. Strategies for the stabilization of multimeric enzymes via immobilization and post-immobilization techniques. J. Mol. Catal. B: Enzym. 1999, 7, 181-189. (16) Fernandez-Lafuente, R.; Rosell, C. M.; Rodriguez, V.; Santana, M. C.; Soler, G.; Bastida, A.; Guisan, J. M. Preparation of activated supports containing low pK amino groups. A new tool for protein immobilization via the carboxyl coupling method. Enzyme Microb. Technol. 1993, 15, 546-550.

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Biotransformations Catalyzed by Multimeric Enzymes: Stabilization of

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