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An efficient method for the enzymatic synthesis of cephalexin (CEX) from 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and d-phenylglycine methyl ester (PGME) using immobilized penicillin G acylase (IPGA) as catalyst in a suspension aqueous solution system was developed, where the reactant 7-ADCA and product CEX are mainly present as solid particles. The effects of key factors on the enzymatic synthesis were investigated. Results showed that continuous feeding of PGME was more efficient for the synthesis of CEX than the batch mode. Under the optimized conditions, the maximum 7-ADCA conversion ratio of 99.3% and productivity of 200 mmol/L/H were achieved, both of which are much superior to the homogeneous aqueous solution system. Besides, IPGA still retained 95.4% of its initial activity after 10 cycles of enzymatic synthesis, indicating the excellent stability of this approach. The developed approach shows great potential for the industrial production of CEX via an enzyme-based route., (© 2020 International Union of Biochemistry and Molecular Biology, Inc.)

A mathematical model is presented for the kinetically controlled synthesis of cephalexin that describes the heterogeneous reaction-diffusion process involved in a batch reactor with glyoxyl-agarose immobilized penicillin acylase. The model is based on equations considering reaction and diffusion components. Reaction kinetics was considered according to the mechanism proposed by Schroën, while diffusion of the reacting species was described according to Fick's law. Intrinsic kinetic and diffusion parameters were experimentally determined in independent experiments. It was found that from the four kinetic constants, the one corresponding to the acyl-enzyme complex hydrolysis step had the greatest value, as previously reported by other authors. The effective diffusion coefficients of all substances were about 5×10(-10)m(2)/s, being 10% lower than free diffusion coefficients and therefore agreed with the highly porous structure of glyoxyl-agarose particles. Simulations made from the reaction-diffusion model equations were used to evaluate and analyze the impact of internal diffusional restrictions in function of catalyst enzyme loading and particle size. Increasing internal diffusional restrictions decreases the Cex synthesis/hydrolysis ratio, the conversion yield and the specific productivity. A nonlinear relationship between catalyst enzyme loading and specific productivity of Cex was obtained with the implication that an increase in catalyst enzyme loading will not increase the volumetric productivity by the same magnitude as it occurs with the free enzyme. Optimization of catalyst and reactor design should be done considering catalyst enzyme loading and particle size as the most important variables. The approach presented can be extended to other processes catalyzed by immobilized enzymes., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The use of very high substrate concentrations favors the kinetically controlled synthesis of cephalexin with penicillin acylase (PA) not only by Michaelian considerations, but also because water activity is depressed, so reducing the rates of the competing reactions of product and acyl donor hydrolysis. Commercial PGA-450, glyoxyl agarose immobilized (PAIGA) and carrier-free cross-linked enzyme aggregates of penicillin acylase (PACLEA) were tested in aqueous media at concentrations close to the solubility of nucleophile and at previously determined enzyme to nucleophile and acid donor to nucleophile ratios. The best temperature and pH were determined for each biocatalyst based on an objective function considering conversion yield, productivity, and enzyme stability as evaluation parameters. Stability was higher with PAIGA and specific productivity higher with PACLEA, but best results based on such objective function were obtained with PGA-450. Yields were stoichiometric and productivities higher than those previously reported in organic medium, which implies significant savings in terms of costs and environmental protection. At the optimum conditions for the selected biocatalyst, operational stability was determined in sequential batch reactor operation. The experimental information gathered is being used for a technical and economic evaluation of an industrial process for enzymatic production of cephalexin in aqueous medium.

Advantages of performing penicillin acylase-catalyzed synthesis of new penicillins and cephalosporins by enzymatic acyl transfer to the beta-lactam antibiotic nuclei in the supersaturated solutions of substrates have been demonstrated. It has been shown that the effective nucleophile reactivity of 6-aminopenicillanic (6-APA) and 7-aminodesacetoxycephalosporanic (7-ADCA) acids in their supersaturated solutions continue to grow proportionally to the nucleophile concentration. As a result, synthesis/hydrolysis ratio in the enzymatic synthesis can be significantly (up to three times) increased due to the nucleophile supersaturation. In the antibiotic nuclei conversion to the target antibiotic the remarkable improvement (up to 14%) has been gained. Methods of obtaining relatively stable supersaturated solutions of 6-APA, 7-ADCA, and D-p-hydroxyphenylglycine amide (D-HPGA) have been developed and syntheses of ampicillin, amoxicillin, and cephalexin starting from the supersaturated homogeneous solutions of substrates were performed. Higher synthetic efficiency and increased productivity of these reactions compared to the heterogeneous "aqueous solution-precipitate" systems were observed. The suggested approach seems to be an effective solution for the aqueous synthesis of the most widely requested beta-lactam antibiotics (i.e., amoxicillin, cephalexin, cephadroxil, cephaclor, etc.)., (Copyright 2004 Wiley Periodicals, Inc.)

The macroscopic kinetic behavior of an industrially employed immobilized penicillin-G acylase, called Assemblase, formed the basis for a discussion on some simple intraparticle biocatalytic model distributions. Assemblase catalyzes the synthesis of the widely used semisynthetic antibiotic cephalexin. Despite the obvious advantages of immobilization, less cephalexin and more of the unwanted byproduct d-(-)-phenylglycine are obtained due to diffusional limitations when the immobilized enzyme is employed. To rationally optimize Assemblase, the parameters particle size, enzyme loading, and enzyme distribution, which severely determine the macroscopic particle performance, were studied on the basis of macroscopic observations. Laser diffraction measurements showed that the particle sizes in Assemblase vary as much as 100-fold. The relative and total enzyme loadings in Assemblase and fractions thereof of different sizes were determined by initial-rate d-(-)-phenylglycine amide hydrolysis, cephalexin synthesis experiments, and active-site titration. These experiments revealed that the loading of penicillin-G acylase in Assemblase was inversely correlated with the particle diameter. Apart from enzyme loadings, estimates on the intraparticle enzyme distribution came from cephalexin synthesis experiments, where mass-transport limitations were present. Although this method cannot provide the level of detail of specific labeling experiments, it is simple, fast, and cheap. Within the set of simple model predictions, a heterogeneous enzyme distribution with most biocatalyst present in the outer region of the particle (within the outer 100 microm) gave the best description of the observed behavior, although no exact correlation was established. Highly detailed determination of intraparticle enzyme distributions must come from immunolabeling.

Conjugation of penicillin acylase (PA) to poly-N-isopropylacrylamide (polyNIPAM) was studied as a way to prepare a thermosensitive biocatalyst for industrial applications to antibiotic synthesis. Condensation of PA with the copolymer of NIPAM containing active ester groups resulted in higher coupling yields of the enzyme (37%) compared to its chemical modification and copolymerization with the monomer (9% coupling yield) at the same NIPAM:enzyme weight ratio of ca. 35. A 10-fold increase of the enzyme loading on the copolymer resulted in 24% coupling yield and increased by 4-fold the specific PA activity of the conjugate. Two molecular forms of the conjugate were found by gel filtration on Sepharose CL 4B: the lower molecular weight fraction of ca. 10(6) and, presumably, cross-linked protein-polymer aggregates of MW > 10(7). Michaelis constant for 5-nitro-3-phenylacetamidobenzoic acid hydrolysis by the PA conjugate (20 microM) was found to be slightly higher than that of the free enzyme (12 microM), and evaluation of V(max) testifies to the high catalytic efficiency of the conjugated enzyme. PolyNIPAM-cross-linked PA retained its capacity to synthesize cephalexin from d-phenylglycin amide and 7-aminodeacetoxycephalosporanic acid. The synthesis-hydrolysis ratios of free and polyNIPAM-cross-linked enzyme in cephalexin synthesis were 7.46 and 7.49, respectively. Thus, diffusional limitation, which is a problem in the industrial production of beta-lactam antibiotics, can be successfully eliminated by cross-linking penicillin acylase to a smart polymer (i.e., polyNIPAM).

Integrated process concepts for enzymatic cephalexin synthesis were investigated by our group, and this article focuses on the integration of reactions and product removal during the reactions. The last step in cephalexin production is the enzymatic kinetic coupling of activated phenylglycine (phenylglycine amide or phenylglycine methyl ester) and 7-aminodeacetoxycephalosporanic acid (7-ADCA). The traditional production of 7-ADCA takes place via a chemical ring expansion step and an enzymatic hydrolysis step starting from penicillin G. However, 7-ADCA can also be produced by the enzymatic hydrolysis of adipyl-7-ADCA. In this work, this reaction was combined with the enzymatic synthesis reaction and performed simultaneously (i.e., one-pot synthesis). Furthermore, in situ product removal by adsorption and complexation were investigated as means of preventing enzymatic hydrolysis of cephalexin. We found that adipyl-7-ADCA hydrolysis and cephalexin synthesis could be performed simultaneously. The maximum yield on conversion (reaction) of the combined process was very similar to the yield of the separate processes performed under the same reaction conditions with the enzyme concentrations adjusted correctly. This implied that the number of reaction steps in the cephalexin process could be reduced significantly. The removal of cephalexin by adsorption was not specific enough to be applied in situ. The adsorbents also bound the substrates and therewith caused lower yields. Complexation with beta-naphthol proved to be an effective removal technique; however, it also showed a drawback in that the activity of the cephalexin-synthesizing enzyme was influenced negatively. Complexation with beta-naphthol rendered a 50% higher cephalexin yield and considerably less byproduct formation (reduction of 40%) as compared to cephalexin synthesis only. If adipyl-7-ADCA hydrolysis and cephalexin synthesis were performed simultaneously and in combination with complexation with beta-naphthol, higher cephalexin concentrations also were found. In conclusion, a highly integrated process (two reactions simultaneously combined with in situ product removal) was shown possible, although further optimization is necessary., (Copyright 2002 Wiley Periodicals, Inc.)

The effect of methanol on the kinetically controlled synthesis of cephalexin by free and immobilized penicillin G acylase (PGA) was investigated. Catalytic and hydrophobic membranes were obtained by chemical grafting, activation, and PGA immobilization on hydrophobic nylon supports. Butyl methacrylate (BMA) was used as graft monomer. Increasing concentrations of methanol were found to cause a greater deleterious effect on the activity of free than on that of the immobilized enzyme. Methanol, however, improved the kinetic stability of cephalexin synthesized by free PGA, resulting in higher maximum yields. By contrast, immobilized PGA reached 100% yields even in the absence of the cosolvent. Cephalexin synthesis by the catalytic membrane was also performed in a non-isothermal bioreactor. Under these conditions, a 94% increase of the synthetic activity and complete conversion of the limiting substrate to cephalexin were obtained. The addition of methanol reduced the non-isothermal activity increase. The physical cause responsible for the non-isothermal behavior of the hydrophobic catalytic membrane was identified in the process of thermodialysis.

A cascade of two enzymatic transformations is employed in a one-pot synthesis of cephalexin. The nitrile hydratase (from R. rhodochrous MAWE)-catalyzed hydration of D-phenylglycine nitrile to the corresponding amide was combined with the penicillin G acylase (penicillin amidohydrolase, E.C. 3.5.1.11)-catalyzed acylation of 7-ADCA with the in situ-formed amide to afford a two-step, one-pot synthesis of cephalexin. D-Phenylglycine nitrile appeared to have a remarkable selective inhibitory effect on the penicillin G acylase, resulting in a threefold increase in the synthesis/hydrolysis (S/H) ratio. 1,5-Dihydroxynaphthalene, when added to the reaction mixture, cocrystallized with cephalexin. The resulting low cephalexin concentration prevented its chemical as well as enzymatic degradation; cephalexin was obtained at 79% yield with an S/H ratio of 7.7., (Copyright 2002 Wiley Periodicals, Inc.)

A new hydrophobic and catalytic membrane was prepared by immobilizing Penicillin G acylase (PGA, EC.3.5.1.11) from E. coli on a nylon membrane, chemically grafted with butylmethacrylate (BMA). Hexamethylenediamine (HMDA) and glutaraldehyde (Glu) were used as a spacer and coupling agent, respectively. PGA was used for the enzymatic synthesis of cephalexin, using D(-)-phenylglycine methyl ester (PGME) and 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) as substrates. Several factors affecting this reaction, such as pH, temperature, and concentrations of substrates were investigated. The results indicated good enzyme-binding efficiency of the pre-treated membrane, and an increased stability of the immobilized PGA towards pH and temperature. Calculation of the activation energies showed that cephalexin production by the immobilized biocatalyst was limited by diffusion, resulting in a decrease of enzyme activity and substrate affinity. Temperature gradients were employed as a way to reduce the effects of diffusion limitation. Cephalexin was found to linearly increase with the applied temperature gradient. A temperature difference of about 3 degrees C across the catalytic membrane resulted into a cephalexin synthesis increase of 100% with a 50% reduction of the production times. The advantage of using non-isothermal bioreactors in biotechnological processes, including pharmaceutical applications, is also discussed., (Copyright 2002 Wiley Periodicals, Inc.)

The extracellular penicillin acylase from Bacillus megaterium was immobilized by coupling to derivatives of polyacrylonitrile fibres. The apparent activity of the immobilized enzyme was about 153 U/g (wet weight). The optimal pH and temperature were 6.5 and 40 degrees C for synthesis of cephalexin by penicillin acylase, respectively. Whe the concentration of 7-ADCA was 4% and the ratio of PGME and 7-ADCA and 1:2, the average velocity of synthesis reaction was highest. The optimal supplied amount of immobilized penicillin acylase was 1.125 g/g 7-ADCA.. The apparent Michaelis constant for 7-ADCA was 0.162 mol/L and for PGME was 0.364 mol/L, Vmax was 0.0462 mol.L-1.min-1 at 30 degrees C and pH6.5. The remained activity was about 83.9% after operating 50 times.

Penicillin-G acylase (EC 3.5.1.11) from Escherichia coli catalyzed the synthesis of various beta-lactam antibiotics in ice at -20 degrees C with higher yields than obtained in solution at 20 degrees C. The initial ratio between aminolysis and hydrolysis of the acyl-enzyme complex in the synthesis of cephalexin increased from 1.3 at 20 degrees C to 25 at -20 degrees C. The effect on the other antibiotics studied was less, leading us to conclude that freezing of the reaction medium influences the hydrolysis of each nucleophile-acyl-enzyme complex to a different extent. Only free penicillin-G acylase could perform transformations in frozen media: immobilized preparations showed a low, predominantly hydrolytic activity under these conditions.

Yields of kinetically controlled synthesis of antibiotics catalyzed by penicillin G acylase from Escherichia coli (PGA) have been greatly increased by continuous extraction of water soluble products (cephalexin) away from the surroundings of the enzyme. In this way its very rapid enzymatic hydrolysis has been avoided. Enzymes covalently immobilized inside porous supports acting in aqueous two-phase systems have been used to achieve such improvements of synthetic yields. Before the reaction is started, the porous structure of the biocatalyst can be washed and filled with one selected phase. In this way, when the pre-equilibrated biocatalyst is mixed with the second phase (where the reaction product will be extracted), the immobilized enzyme remains in the first selected phase in spite of its possibly different natural trend. Partition coefficients (K) of cephalexin in very different aqueous two-phase systems were firstly evaluated. High K values were obtained under drastic conditions. The best K value for cephalexin (23) was found in 100% PEG 600-3 M ammonium sulfate where cephalexin was extracted to the PEG phase. Pre-incubation of immobilized PGA derivatives in ammonium sulfate and further suspension with 100% PEG 600 allowed us to obtain a 90% synthetic yield of cephalexin from 150 mM phenylglycine methyl ester and 100 mM 7-amino desacetoxicephalosporanic acid (7-ADCA). In this reaction system, the immobilized enzyme remains in the ammonium sulfate phase and hydrolysis of the antibiotic becomes suppressed because of its continuous extraction to the PEG phase. On the contrary, synthetic yields of a similar process carried out in monophasic systems were much lower (55%) because of a rapid enzymatic hydrolysis of cephalexin., (Copyright 1998 John Wiley & Sons, Inc.)

The possible use of various procedures for estimation of the ionization constants of the Michaelis complex by the pH dependence of the maximum enzymatic reaction rate is discussed. It is shown that the procedures described in the literature for estimation of the close ionization constants of the enzyme-substrate complexes have limitations and in some cases cannot be used. The paper presents the methods for estimation of the constants and means for quantitative description of the bell-shaped pH dependence of the kinetic and equilibrium parameters of the biocatalytic reaction. The equations recommended in the paper were used in analysis of the pH dependences of the maximum rate of the reactions during the enzymatic synthesis of cefalexin catalysed with immobilized penicillinamidase (IPA) (CE 3.5. 1.11). The ionization constants of the enzyme-substrate complexes of IPA were compared during hydrolysis and synthesis of the compounds acylated with phenylacetic and aminophenylacetic acids. The effect of the nature of the leaving substrate group and added nucleophilic gent on the electrochemical state of the Michaelis complex is discussed.