Effects of ethylene glycol on the synthesis of ampicillin using immobilized penicillin g acylase

Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 78:431–436 (online: 2003) Effects of ethylene glycol on the synthesis
of ampicillin using immobilized penicillin G
acylase
Dong-Zhi Wei* and Liu Yang
State Key Laboratory of Bioreactor Engineering, Institute of Biochemistry, East China University of Science and Technology, Shanghai
200237, People’s Republic of China
Abstract: The effects of organic cosolvents on the synthesis of ampicillin from phenylglycine methylester (PGME) and 6-amino penicillanic acid (6-APA) using immobilized Bacillus megateriumpenicillin G acylase have been examined. Several cosolvents were tested for their influence on theenzyme in terms of enzyme stability and hydrophobicity. Among the cosolvents tested, ethylene glycolwas found to increase the yield of ampicillin by 39–50%. The effects of ethylene glycol on the pK of PGME, the hydrolysis of ampicillin and PGME, and synthetase/amidase and esterase/amidase ratioswere also studied. Experimental data indicated that ethylene glycol inhibited more the hydrolysis of theampicillin than the hydrolysis of the PGME and the synthetase/amidase ratio varied from 0.2 to 0.88when the concentration (v/v) of the cosolvent increased from 0 to 40%. The enhancement of thesynthesis yield was mainly caused by the reduction in the hydrolysis of acyl donor (PGME) and product(ampicillin) in the water–cosolvent system.
# 2003 Society of Chemical Industry Keywords: penicillin G acylase; ampicillin; ethylene glycol; enzymatic synthesis INTRODUCTION
catalyzed by penicillin G acylase. Therefore, preven- Ampicillin is a broad spectrum antibiotic and one of tion of the hydrolysis of the acyl donor substrate the most widely used b-lactam antibiotics. It is often (PGME) and product (ampicillin) is desirable for the used in combination with clavulanic acid, an inhibitor of the b-lactamase produced by microorganisms as a With this strategy, organic solvents have often been resistance mechanism to b-lactam antibiotic.1 Ampi- added to aqueous medium to suppress the enzymatic cillin can be produced by chemical synthesis, in which hydrolysis reactions. Usually, the effects of organic the protection of the a-amino group of phenylglycine solvents on yields do not change the equilibrium posi- (PG), the use of highly reactive derivatives of PG, low tion in an enzymatic reaction (experimental conditions temperature, anhydrous conditions, and the use of commonly are far from thermodynamically favorable highly toxic compounds (pyridine, dimethylaniline, ones).9–11 Rather, the solvents directly affect the and dichloromethane) are all required. Accordingly, enzyme’s catalytic properties, which depend dramati- the enzymatic synthesis approach becomes an attrac- cally on the tertiary structure of the enzyme. Small tive alternative to the production of ampicillin because conformational changes in enzyme structures may of its moderate operational conditions.
modify the shape of the active center and then affect Enzymatic synthesis of ampicillin has been achieved the catalytic properties of the enzyme.12,13 Thus with penicillin G acylase (EC 3.5.1.11; also known as organic solvents can reduce the production yield by penicillin amidase).2–8 Synthesis of b-lactam anti- inhibiting the catalytic activity of the enzyme or by biotics catalyzed by penicillin G acylase is usually demolishing the stability of the three-dimensional carried out by using a kinetically controlled strategy.
structure of the enzyme. Considering these two In kinetically controlled synthesis, it is necessary to opposing effects of organic solvents, a well-advised use an activated acyl donor (ester or amide). As shown design of the reaction medium is required to obtain a in Scheme 1, penicillin G acylase catalyzes not only the higher yield of the product in water–solvent mixtures.
synthesis of ampicillin (synthetase activity) but also the Some results have been reported about enzymatic hydrolytic reactions of PGME (esterase activity) and synthesis of b-lactam antibiotics in reaction media ampicillin (amidase activity). The yield of ampicillin other than pure aqueous solutions.4,5,11,14–23 In the depends on the three different kinetic processes presence of suitable organic solvents or additives, * Correspondence to: Dong-Zhi Wei, State Key Laboratory of Bioreactor Engineering, Institute of Biochemistry, East China University ofScience and Technology, Shanghai 200237, People’s Republic of ChinaE-mail: dzhwei@ecust.edu.cnContract/grant sponsor: Shanghai Key Discipline(Received 5 June 2002; revised version received 7 October 2002; accepted 11 October 2002) # 2003 Society of Chemical Industry. J Chem Technol Biotechnol 0268–2575/2003/$30.00 Scheme 1. Reaction mechanism for penicillin G acylase-catalyzed synthesis of ampicillin.
penicillin acylase-catalyzed synthesis of b-lactam anti- dmÀ3 sodium-phosphate buffer (pH7.8) together with biotics such as ampicillin,4,5 cephaloglycin,9 cepha- 2.5 cm3 of the soluble enzyme. The suspension, in a lothin,14,23 cephalexin,15 and cefazolin19 can achieve vial, was agitated for 48 h in a temperature-controlled higher yields than in aqueous buffer solutions.
incubator (HZ-9310K, China) at 150 rpm at pH7.8 Here we describe the effects of organic solvents on and 34 °C. The immobilized enzyme was collected the synthesis of ampicillin from 6-amino penicillanic after filtration and washed with a concentration of acid (6-APA) and PGME using Bacillus megaterium 100 mmol dmÀ3 sodium-phosphate buffer at pH 7.8.
penicillin G acylase. This study shows that the syn- The volume of the washing solution was approxi- thesis yield of ampicillin can be significantly improved mately three times the volume of the enzyme. Prior to in the presence of ethylene glycol. In addition, the use, the immobilized enzyme was washed with effects of ethylene glycol on the synthesis of ampicillin 50 mmol dmÀ3 sodium-phosphate buffer (with the same pH as applied in the actual experiment) andfiltered through sinted glass again.
MATERIALS AND METHODS
Chemicals
The soluble penicillin G acylase from Bacillus mega- PGME and ampicillin were kindly donated by North terium, provided by the Institute of Biochemistry of China Pharmaceutical Co. Organic solvents and all CAS (Chinese Academy of Science), was immobilized other reagents were of analytical grade and obtained on epoxyacrylic resin. The active values of immo- from Shanghai Chemicals Supply Co, China.
bilized and soluble PGA were 220 IU/gÀ1 (wet) and1060 IU/cmÀ3, respectively. A unit of penicillin G Enzyme reactions
acylase was defined as the amount of enzyme required Enzymatic reactions were carried out at pH6.3 and to produce 1 mmol of 6APA minÀ1 in a 4% (w/v) 25 °C in stirred bioreactors with jackets for water solution of penicillin G at pH 7.8. The enzyme activity circulation. The pH value was monitored throughout was determined by a spectrophotometric assay with the reaction by using a pH controller (LM-4HC, p-dimethylaminobenzaldehyde (PDAB) as a colori- Japan) using 20 mmol dmÀ3 ammonia hydroxide and metric substrate.24 One enzymatic activity unit was 10 mmol dmÀ3 hydrochloric acid. The reaction tem- expressed as the amount of enzyme that hydrolyzed perature was controlled by a constant temperature 1 mmol of ampicillin minÀ1 (amidase) or PGME minÀ1 circulation water bath. For enzymatic reactions in an (esterase) or synthesized 1 mmol of ampicillin minÀ1 aqueous solution, 50 mmol dmÀ3 sodium-phosphate (synthetase). Amidase or esterase activities were buffer (pH6.3) was used as a standard reaction measured as the initial hydrolysis rate of ampicillin medium, the synthesis of ampicillin was carried out or PGME under the conditions of 10 mmol dmÀ3 using 100 mmol dmÀ3 6APA and 200 mmol dmÀ3 ampicillin or 100 mmol dmÀ3 PGME, 100 mmol PGME, respectively. The hydrolysis of PGME and dmÀ3 sodium-phosphate buffer, pH6.3 and 25 °C, ampicillin was carried out at 100 mmol dmÀ3 PGME and synthetase activity was measured as the initial and 10 mmol dmÀ3 ampicillin, respectively. The en- synthesis rate of ampicillin in reaction solution con- zymatic reaction began with the addition of 1 g taining 20 mmol 6-APA dmÀ3 and 40 mmol PGME immobilized penicillin G acylase to 50 cm3 of the reaction solution. Each 30 min during the reaction a To immobilize penicillin G acylase, 5g (wet) of sample of 0.2 cm3 was taken out for HPLC analysis.
epoxyacrylic resin was suspended in 40 cm3 of 1 mol Enzymatic reactions in water–solvent or water–polyol J Chem Technol Biotechnol 78:431–436 (online: 2003) mixtures were done under the same conditions as sorbitol (20% w/v), the yields of ampicillin and employed in an aqueous solution expect that organic cephalexin increased by 28% and 20%, respectively.
solvent or polyol solution was added to the standard Additionally, Illanes and Fajardo5 carried out ampi- aqueous medium composed of sodium-phosphate cillin synthesis by E coli penicillin G acylase in the presence of ethylene glycol and optimized the reactionconditions for the synthesis. Fernandez-Lafuente et Enzyme stability assays
al 17 synthesized cephaloglycin by penicillin G acylase The stability of the penicillin G acylase derivative was from E coli and observed that the yield was increased in determined in monophasic water/organic solvent mixtures. The enzyme derivative (1 g) was suspended In order to enhance the yield of ampicillin catalyzed in 20 cm3 of the buffered organic solvent mixture by penicillin G acylase, some water-miscible organic composed of 50% (v/v) of solvent in pH 7.8 sodium- solvents (30%, v/v) or polyols (20%, w/v) were used.
phosphate buffer at 25 °C. The stability of immo- These organic cosolvents and polyols were chosen in bilized penicillin G acylase was determined by light of the published literature. Since enzymes usually measuring its residual activity after 24 h incubation.
undergo inactivation in the presence of organicsolvents, the stability of penicillin G acylase in organic Kinetic studies
solvents and polyols was also investigated.
Kinetic parameters were determined by measuring the As shown in Table 1, the stability of penicillin G initial reaction rates at different substrate concentra- acylase in polyols was high; the polyols probably prevent the unfolding of protein by strengthening the weaver–Burk plots. The initial velocities of PGME hydrogen bonds in the hydrophilic interactions.25 hydrolysis and ampicillin synthesis were obtained by Since the stability of penicillin G acylase in methanol examination of the HPLC data. Ampicillin hydrolysis and isopropanol was poor, the amount of penicillin G was determined following the release of 6-APA with acylase should be very high in the synthesis reaction.
Ethylene glycol, glycerol, methanol, sucrose, sorbitoland mannitol were found to increase the yield of DpK of PGME
ampicillin compared with those obtained in an The DpK was defined as the difference of the pK aqueous buffer solution. The increase reached as high values in the water–cosolvent mixture and a fully as 50% in the presence of ethylene glycol, and it did aqueous environment, respectively. About 50 cm3 of little damage to penicillin G acylase. Hereby, ethylene 20 mmol dmÀ3 PGME was dissolved in water or in glycol was selected as cosolvent in reaction medium for water–cosolvent mixtures and the solution was ad- justed to pH 8.0 by adding dilute aqueous NaOH.
HCl (20 mmol dmÀ3) was used to lower the pH value Effect of ethylene glycol concentration on the
of solutions by virtue of a pH controller.
synthesis of ampicillin
The time course of the yield was studied with different
Analysis
concentrations of ethylene glycol to determine the Substrates and products were identified and analyzed optimal concentration of ethylene glycol for ampicillin by using a Shimadzu Class-VP HPLC (Japan) synthesis. As shown in Fig 1, although the rates of equipped with a Shimadzu UV detector, a NSPD- 10AVP detector (Japan) and a C18 column (5 mm; amounts of ethylene glycol in the reaction medium, 250 Â 4.6 mm; Waters). After injection, the columnwas consecutively eluted with 88% (v/v) sodiumacetate buffer (50 mmol dmÀ3, pH5.0) and 12% Table 1. Effect of organic solvents on the yield of ampicillin synthesis
(v/v) acetonitrile at a flow rate 1.0 cm3 minÀ1 and the solutes were detected by the UV detector at 254 nm at RESULTS AND DISSCUSSION
Selection of organic solvents
For enzymatic synthesis of b-lactam antibiotics with kinetic control, the yield of product may increase in the presence of organic solvents. For synthesis of cefazolin by E coli penicillin G acylase, Park et al 19 found that the yield increased by 65% and 56% in the presence of ethyl acetate (30% v/v) and carbon tetrachloride (30% v/v), respectively. Aguirre et al 4 also used penicillin G a Hydrophobicity of organic solvent.
acylase but from Bacillus megaterium for the synthesis b Relative maximum yield: maximum yield in water–cosolvent mixture (Y) of ampicillin and cephalexin. In the presence of normalized to that obtained in an aqueous buffer solution (Y 0).
J Chem Technol Biotechnol 78:431–436 (online: 2003) the maximum synthetic yield increased with increasesin the cosolvent volume ratio. The yield of ampicillinvaried from 40% (in the absence of cosolvent) to 52%at 40% (v/v) cosolvent, and the further addition ofethylene glycol to the reaction medium did not have asignificant effect on it. Considering the rate ofsynthesis, 40% (v/v) ethylene glycol in the reactionmixture was selected as the optimal concentration.
Effect of ethylene glycol on DpK of PGME
The DpK of PGME was investigated to find out therole of ethylene glycol in enhancing the yield ofampicillin production. It is commonly known thatpenicillin G acylase exhibits catalytic activity onlytoward nonionic substrates.12 The addition of organicsolvents to the reaction media facilitates the stabiliza-tion of the nonionic substrates. As a result, a decreasein their dissociation constant occurred, which led to an Figure 2. Effect of ethylene glycol content on the hydrolysis of ampicillin.
increase in the pK . However, that effect was only detected on the acyl donor substrate.
100 mmol dmÀ3 and 10 mmol dmÀ3, respectively. The results of the hydrolysis reaction of ampicillin and buffer–ethylene glycol did not obviously increase.
PGME are shown in Figs 2 and 3, respectively.
According to the classification given by Fernandez- When 10 mmol dmÀ3 of ampicillin was hydrolyzed Lafuente et al, ethylene glycol can be considered to be by penicillin G acylase in an aqueous buffer solution, a soft monophasic solvent, which promoted a very more than 50% of ampicillin was hydrolyzed within slight increase in the pK of acyl donor and only hard 40 min. However, in the water–ethylene glycol system, cosolvents could promote a rather large increase in the hydrolysis rates of ampicillin slowed down pK values. This indicated that the increase of the yield remarkably and more than 90% of the initial ampicillin in the buffer–cosolvent system was not caused by remained when the ethylene glycol content was more stabilization of the nonionic substrate.
than 40% (v/v) in the reaction mixture after 40 min ofreaction (Fig 2).
Suppression of hydrolysis reactions in
In an aqueous buffer system, 100 mmol dmÀ3 buffer–cosolvent system
PGME was completely hydrolyzed into the corre- Penicillin G acylase catalyzed the synthesis of ampi- sponding acid, phenylglycine (PG), in about 50 min cillin from 6-APA and PGME; however, it also (Fig 3). When the same experiment was carried out in hydrolyzed PGEM, as well as the product ampicillin.
a water–ethylene glycol system, hydrolysis rates To examine the performances of ethylene glycol in decreased, and with the increase of ethylene glycol enhancing the yield of ampicillin production, the content, the hydrolytic rates declined continuously.
dependence of the hydrolysis of ampicillin and PGMEon reaction media was investigated. Hydrolysis reac-tions were carried out under the same conditions asthose used in the ampicillin synthesis reaction and theinitial concentrations of ampicillin and PGME were Figure 1. Effect of solvent content on the enzymatic synthesis of ampicillin
Figure 3. Effect of ethylene glycol content on the hydrolysis of PGME.
J Chem Technol Biotechnol 78:431–436 (online: 2003) in the synthesis,26 the ratio of esterase to amidase is aparameter strictly related to the synthesis yield. Theeffect of ethylene glycol on the esterase/amidase is alsoshown in Fig 5, and the increase in esterase/amidaseupon increasing the content of ethylene glycol can beobserved. The hydrolyses of PGME and ampicillinwere both inhibited by ethylene glycol which inhibitsmore the hydrolysis of the ampicillin than the hydroly-sis of the PGME From these results, we can deduce that the key role of ethylene glycol for improving the ampicillin synth-esis is to suppress the hydrolysis of PGME and Figure 4. Noncompetitive inhibition of ethylene glycol on PGME hydrolysis
In Fig 4, the effect of PGME concentration on the CONCLUSION
rate of hydrolysis is shown. The reaction follows In this study, we investigated the synthesis of ampicillin in water–cosolvent mixtures and found the maximum yield of ampicillin could be obtained in the and 25 °C and ethylene glycol behaved as a noncom- presence of ethylene glycol. Further experiments showed that hydrolysis of PGME and ampicillin couldbe suppressed to a great extent with the addition of Effect of ethylene glycol on the
ethylene glycol to the reaction medium. In the buffer– synthetase/amidase and esterase/amidase
ethylene glycol mixture, the ratio synthesis/hydrolysis activities ratios
of ampicillin increased, since the hydrolysis of anti- The yields in the synthesis of ampicillin catalyzed by biotic was strongly inhibited by ethylene glycol. It penicillin G acylase depended on the ratio between seems that the main reason for the increase of the yield synthetase and amidase.17 The effect of ethylene glycol in the ampicillin synthesis is the reduction of side on the ratio of the synthesis of ampicillin and the reactions (hydrolysis of PGME and ampicillin; see hydrolysis of ampicillin (synthetase/amidase) is shown Scheme 1) in the buffer–ethylene glycol system.
in Fig 5 which illustrates that the progressive increasein the ethylene glycol concentration yielded a con-tinuous increase in the synthetase/amidase ratio inampicillin synthesis. In aqueous solution, the synthe- REFERENCES
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J Chem Technol Biotechnol 78:431–436 (online: 2003)

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