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Brazilian Journal of Chemical Engineering

Print version ISSN 0104-6632On-line version ISSN 1678-4383

Braz. J. Chem. Eng. vol.17 n.4-7 São Paulo Dec. 2000

http://dx.doi.org/10.1590/S0104-66322000000400046 

A SIMPLIFIED KINETIC MODEL FOR THE SIDE REACTIONS OCCURRING DURING THE ENZYMATIC SYNTHESIS OF AMPICILLIN

 

A.L.O.Ferreira1, L.R.B.Gonçalves1,2, R.C.Giordano1*, R.L.C.Giordano1
1Departamento de Engenharia Química, Universidade Federal de São Carlos, Via
Washington Luiz, km 235, C.P.676, CEP 13565-905, São Carlos - SP, Brazil
E - mail: roberto@deq.ufscar.br)
2Departamento de Engenharia Química, Universidade Federal do Ceará,
Campus do Pici, Bloco 710, S.32, Fortaleza - CE, Brazil
E - mail:luciana@deq.ufc.br)

 

(Received: November 12, 1999 ; Accepted: April 18, 2000)

 

 

Abstract - This work presents a kinetic study of the side reactions of the ampicillin enzymatic synthesis, from phenylglycine methyl ester and 6-aminopenicillanic acid using penicillin G acylase immobilized on agarose. A Michaelis-Menten model with competitive inhibition was fitted to initial rates of ester and antibiotic hydrolysis, at pH 6.5 and 25ºC. Inherent kinetic parameters were estimated for low enzymatic loads, to assure that diffusional resistance was not important. It was observed that ampicillin inhibits the hydrolysis of PGME, but the inhibitory effect of the ester on ampicillin hydrolysis was almost negligible. The obtained parameters were: kcat1= 0.025 mM/UI min, Km1 = 155.4mM, KAE = 16.18mM, kcat2= 4.67x10-3 mM/UI min, Km2 = 11.47, KEA = 0.68 mM. Parameter values are in the range reported in the literature, except for Km1, which is much higher. The large confidence interval for this parameter denotes that the model presents low sensitivity with respect to it.
Keywords: Ampicillin hydrolysis, Penicillin G acylase, Immobilized enzyme, Kinetic model

 

 

INTRODUCTION

Ampicillin (6-[2-amino-2-phenylacetamide] penicillanic acid) is a semi-synthetic b-lactamic antibiotic which is very stable at acidic conditions, well absorbed and effective against a wide variety of Gram-positive and Gram-negative microorganisms, with low minimal inhibitory concentration (Hou and Poole, 1969). Nowadays it is manufactured in industry through a chemical route. For instance, an amino b-lactam, such as 6-aminopenicilanic acid (6-APA), having its carboxyl group protected, reacts with an activated side-chain derivative (D-phenylglycine acidchloride, to produce ampicillin). The protecting group is than removed by hydrolysis. These reactions typically involve costly steps, such as very low temperatures (about –30°C) and the use of toxic organic solvents like methylene chloride and silylation reagents (Kaasgaard et al., 1996).

Enzymatic synthesis is an interesting alternative process since it has high selectivity, specificity and activity in mild reaction conditions (aqueous medium, neutral pH and moderate temperatures). However, none of the known enzymatic methods have yet been upscaled to industrial applicability, due to the high costs caused by a low yield. Penicillin G acylase (PGA), for instance, can act as a hydrolase as well as a transferase, which means that the same enzyme catalyzes the synthesis of ampicillin as well as the hydrolysis of the activated acyl donor and the hydrolysis of the newly formed antibiotic (see Figure 1).

 

 

In this paper, a simplified kinetic model is presented to represent the side, undesired reactions that compete with the enzymatic synthesis of ampicillin from phenylglycine methyl ester (PGME) and 6-APA. The catalyst was PGA immobilized on gel of agarose.

In order to estimate reliable kinetic parameters, it was necessary to guarantee that no mass transport effects were masking the reaction rate data. Experimental conditions that eliminate mass transfer delays where previously investigated, and reported elsewhere (Ferreira et al., 1999): high-speed mechanical agitation (600rpm) and low enzymatic load (30 IU/ml of agarose gel).

 

MATERIALS AND METHODS

Materials

Phenylglycine methyl ester (PGME) purchased from Aldrich Chem. Co., USA; Ampicillin, from Winlab, U.K.; 6-aminopenicilanic acid (6-APA), from Winlab, U.K.; Penicillin G Acylase from recombinant Escherichia coli was donated by Antibioticos S.A., Spain; Agarose 6, 8 and 10 BCL was donated by Hispanagar S. A., Spain. All other chemicals were of laboratory grade from commercial suppliers.

Methods

Preparation of PGA Derivatives (Fernandez-Lafuente, 1992)

Activation of agarose gel was performed by etherification with glycidol and oxidation with sodium periodate. Further control of the PGA (amine)-agarose (aldehyde) multi-point attachment was achieved by reaction at pH 10 (bicarbonate buffer, 50mM), in the presence of phenylacetic acid. Final reduction of the amino double bonds was performed with sodium borohydride.

Agarose Gel Characterization

Agarose gel density was determined by picnometry. An image analysis software (Image-Pro Plus, Media Cybernetics, and an optical microscope Olympus BX50, Japan) was used to estimate the average diameter and standard deviation of the agarose particles.

Enzyme Activity

Enzyme activity was evaluated by colorimetric analysis of p-nitro-analide benzoic acid, released during 6 Nitro-3 Fenilacetamide Benzoic Acid (NIPAB) 33mg/l hydrolysis in a 50mM phosphate buffer, pH 8. The difference between enzymatic activities of the supernatant before and after immobilization was used to assess the enzymatic load of the gel. 1 IU (international unit) of enzyme was defined as the quantity of enzyme which hydrolyses 1mmol of penicillin G per minute at pH 8.0 and 38°C.

Analysis

Concentrations of phenylglycine methyl ester, ampicillin, 6-APA and phenylglycine were determined using HPLC: C18 column (Waters Nova-Pack, USA, C18, 60Å, 3.9x150mm) and mobile phase with 35% acetonitrile, 2‰ SDS (Lauryl sodium sulphate); 5mM K2H2PO4 and 10mM H3PO4 at 25°C.

Kinetic Studies:

Kinetic parameters for the hydrolysis of PGME and ampicillin were obtained using the non-linear least squares algorithm of Marquardt, 1963, with 95% confidence interval for the parameter estimates. The experimental data were initial rates of hydrolysis for different initial concentrations of substrate, in the absence and in the presence of inhibitors.

Experiments

A jacketed, batch reactor with mechanic stirring was used in all the experiments described in this paper to eliminate the extra-particle mass transport resistance. Mechanical agitation was preferred rather than magnetic stirring, to avoid gel disruption and loss of enzyme activity. The pH of the solutions during the enzymatic hydrolysis and synthesis reactions was kept constant by an automatic titrator. Enzymatic hydrolysis was carried out in a phosphate buffer solution, 100mM, pH 6.5, at 25°C. The same amount of biocatalyst was used in all assays: 1.0ml of agarose with 30 IU of immobilized PGA/mlgel. This enzymatic load was low enough to avoid intra-particle diffusion effects. All experiments were performed in a batch reactor at pH 6.5, 100mM phosphate buffer and 25°C. Stirring speed was 600 rpm and the total reactor volume was 40 ml.

Kinetic Model (Gonçalves et al., 1999)

The model equations follow a Michaelis-Menten kinetics, with competitive inhibition. Equation 1 represents the antibiotic hydrolysis (vAN). The ester (AB) is assumed to be a competitive inhibitor, as it binds to the same active site occupied by the substrate. Equation 2 represents the hydrolysis of the ester (vAB). For the same reason, the antibiotic is considered a competitive inhibitor.

(1)

 

(2)

 

RESULTS AND DISCUSSION

Inhibitory effects on the hydrolysis of phenylglycine methyl ester (PGME)

PGME hydrolysis reactions were performed in the absence and in the presence of ampicillin. The parameters in equation (2) were estimated and the results are shown in Fig. 2. Table 1 shows the estimated parameter (with 95% confidence interval at 25ºC and pH 6.5) and some kinetic parameter values from other authors, for the sake of comparison. Vmax values were not directly comparable because experimental conditions, the amount of the enzyme and the reaction temperature were different; nevertheless, the reactions were similar. Ospina et al. (1996) used PGA from Escherichia coli to produce of ampicillin at 25ºC and pH 6. Gonçalves et al. (1999) used PGA immobilized in agarose to hydrolyze amoxicillin and p-hydroxyphenylglycine methyl ester at 25ºC and pH 6.5.

 

 

 

Our parameters are in the range of the ones from literature, except for Km1, which is much higher in our case. The large confidence interval for this parameter, however, denotes that the model presents low sensitivity with respect to it.

Observing figure 2, it can be noticed that the response of the kinetic model in the presence of inhibitor departs systematically from the experimental data – the initial slopes of the model curves are too small in these cases. This indicates that further investigation is necessary to establish a more consistent mechanistic model.

Inhibitory Effects on the Hydrolysis of Ampicillin

Initial rate assays of ampicillin hydrolysis in the presence and in the absence of PGME were performed. The parameters of equation (1) were estimated through the same procedure and the obtained results are shown in Fig. 3. Table 2 shows these estimated parameters (with 95% confidence interval at 25ºC and pH 6.5) and some kinetic parameters available in the literature.

 

 

 

CONCLUSIONS

The simplified kinetic model is consistent and gives a reasonable response when compared to experimental data. Our parameters are close to the ones estimated by other authors’, except for Km1, which is much higher. The large confidence interval for this parameter denotes that the model presents low sensitivity with respect to it. The kinetic model response in the presence of inhibitor, for both reactions, departs systematically from the experimental data. This indicates that further investigation is necessary, for instance, other inhibitory effects must be investigated. However, these results do not invalidate the use of the simplified model to optimize and control an industrial reactor, since ampicillin concentrations used in our tests are much higher than the amounts produced during the actual synthesis of the antibiotic.

 

ACKNOWLEDGMENTS

The authors are grateful to CNPq, CAPES and FAPESP for their financial support.

 

NOMENCLATURE

AB Activated acyl donor (PGME);
CAB PGME concentration (mM);
CAN Ampicillin concentration (mM);
CEZ Enzyme activity (UI/mlgel);
HPLC High performance liquid chromatographic;
KAE Inhibition constant (mM);
kcat1 Kinetic constant (mM/UI min);
Kcat2 Kinetic constant (mM/UI min);
KEA Inhibition constant (mM);
Km1 Michaelis-Menten constant (mM);
Km2 Michaelis-Menten constant (mM);
NIPAB 6 Nitro-3 Fenilacetamide Benzoic Acid;
PGA Penicillin G acylase;
PGME Phenylglycine methyl ester;
6-APA 6-aminepenicillanic acid;
vAN Rate of ampicillin consumption (mM/min);
Vmax Maximum rate (mM/min).

 

REFERENCES

Fernandez-Lafuente, R. F. (1992), Sintesis de Antibióticos b-Lactamicos Catalizada por Derivados Inmobilizados-Estabilizados de Penicilina G Acilasa, Ph.D. thesis, Universidad Autonoma de Madrid, Madrid, Spain.        [ Links ]

Ferreira, A. L. O.; Gonçalves, L. R. B.; Giordano, R. L. C. and Giordano, R. C. (1999), Efeitos Difusivos na Síntese de Ampicilina Catalisada por Penicilina G Acilase Imobilizada, Anais do XXVII ENEMP, Campos de Jordão (in press).        [ Links ]

Gonçalves,L.R.B.; Fernadez-Lafuente, R.; Guisán, J.M. and Giordano, R.L.C.(2000), A kinetic study of the synthesis of amoxicillin using penicillin G acylase immobilized on agarose, Applied Biochemistry and Biotechnology, vol. 84-86 (in press).        [ Links ]

Hou, J. P. and Poole, J. W. (1969), the amino acid nature of ampicillin and related penicillins, Journal of Pharmaceutical Sciences, vol. 58, no 12, 1510-1515.        [ Links ]

Kaasgaard, S.G. and Veitland; U. (1996), Process for preparation of .beta.-lactams utilizing a combined concentration of acylating agent plus.beta.-lactam derivative of at least 400 mm, US Patent 5,525,483; USA.        [ Links ]

Marquardt, D. W.(1963), An algorithm for least-squares estimation of nonlinear parameters, J. Soc. Ind. Appl. Math., 11, 431-41.        [ Links ]

Ospina, S.; Barzana, E.; Ramírez, O.T. and López-Munguía, A. (1996), Effect of pH in the Synthesis of Ampicillin by Penicillin Amidase, Enzyme and Microbial Technology, 19, 462-469.        [ Links ]

 

 

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