Efficient enzymatic synthesis of cephalexin in suspension aqueous solution system.

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.

Pulsed Laser-Driven Molecular Self-assembly of Cephalexin: Aggregation-Induced Fluorescence and Its Utility as a Mercury Ion Sensor.

A fluorescent self-assembly of cephalexin is obtained by pulsed laser irradiation process. An intense fluorescence emission is found in the self-assembled form due to occurrence of a typical aggregation-induced emission in cephalexin molecules. It is observed that fluorescence quenching of the self-assembled fluorescent nanostructures occurs in the presence of extremely low Hg(++) ions concentrations (10(-7) m) as compared to other heavy metal ions e.g. Ferrous (Fe(++) ), Manganese (Mn(++) ), Magnesium (Mg(++) ), Cobalt (Co(++) ), Nickel (Ni(++) ) and Zinc (Zn(++) ) at the same concentrations.

Synthesis and application of cephalexin imprinted polymer for solid phase extraction in milk.

Molecular imprinted polymer (MIP) against cephalexin was synthesized by co-polymerization of functional monomer, cross-linker, radical initiator, along with target molecule (cephalexin) in a porogenic material. Binding of cephalexin towards prepared MIP was studied in different solvents (water, methanol, 1M NaCl, acetone and acetonitrile) and best binding was observed in methanol. Partition coefficient and selectivity of prepared imprint and non-imprint was also studied. Cross reactivity in terms of binding efficiency was also assessed with other antibiotics. Chromatographic study of MIP was carried out by packing prepared imprint into glass column. MIP was used as matrix in solid phase extraction (SPE) for recovery of cephalexin from spiked milk samples for further estimation by high performance liquid chromatography. No interference was observed from milk components after elution of cephalexin from MIP, indicating selectivity and affinity of MIP. On the other hand, interference was observed in eluate obtained from C18 SPE column.

Synthesis, characterization and antibacterial activity of a Schiff base derived from cephalexin and sulphathiazole and its transition metal complexes.

Metal(II) coordination compounds of a cephalexin Schiff base (HL) derived from the condensation of cephalexin antibiotic with sulphathiazole were synthesized. The Schiff base ligand, mononuclear [ML(OAc)(H2O)2] (M(II)=Mn, Co, Ni, Zn) complexes and magnetically diluted trinuclear copper(II) complex [Cu3L(OH)5] were characterized by several techniques, including elemental and thermal analysis, molar conductance and magnetic susceptibility measurements, electronic, FT-IR, EPR and (1)H NMR spectral studies. The analytical and molar conductance values indicated that the acetate ions coordinate to the metal ions. The Schiff base ligand HL behaves as a monoanionic tridentate NNO and tetradentate NNOO chelating agent in the mono and trinuclear complexes respectively.

Batch reactor performance for the enzymatic synthesis of cephalexin: influence of catalyst enzyme loading and particle size.

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.

Synthesis of cephalexin in aqueous medium with carrier-bound and carrier-free penicillin acylase biocatalysts.

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.

Penicillin acylase-catalyzed synthesis of beta-lactam antibiotics in highly condensed aqueous systems: beneficial impact of kinetic substrate supersaturation.

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.).

Enzyme distribution derived from macroscopic particle behavior of an industrial immobilized penicillin-G acylase.

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 with the reactive copolymer of N-isopropylacrylamide: a step toward a thermosensitive industrial biocatalyst.

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).

Enzyme reaction engineering: effect of methanol on the synthesis of antibiotics catalyzed by immobilized penicillin G acylase under isothermal and non-isothermal conditions.

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.

Integrated reactor concepts for the enzymatic kinetic synthesis of cephalexin.

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.

Advantages of using non-isothermal bioreactors for the enzymatic synthesis of antibiotics: the penicillin G acylase as enzyme model.

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.

A two-step, one-pot enzymatic synthesis of cephalexin from D-phenylglycine nitrile.

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.

[Studies on enzymatic synthesis of cephalexin by immobilized penicillin acylase by polyacrylonitrile fibres].

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.

Equal allergenic potency of beta-lactam antibiotics produced by chemical or enzymatic manufacturing--mouse IgE test.

BACKGROUND: Cephalexin and amoxicillin are semisynthetic beta-lactam antibiotics with a broad spectrum of antibacterial activity against gram-positive and gram-negative microorganisms. Both antibiotics are produced by a new 'green' process in which enzyme technology is used to combine the intermediate structure and the side chain in an aqueous medium to yield cephalexin or amoxicillin, thus avoiding the use of several chemical reagents and volatile organic solvents. As a result of the enzyme technology a new residual protein impurity has been identified. To check for the sensitizing capacity of the residual protein, a mouse IgE test was used to detect differences in the production of specific IgE by chemical or enzymatic preparations of the antibiotics. METHODS: Balb/c female mice were immunized intraperitoneally with alum and conjugates of different amoxicillins or cephalexins with ovalbumin (OVA). After 16 days, the amoxicillin mice were injected with one half the original amount of antigen. After 19-23 days, the sera were tested for specific IgE by the passive cutaneous anaphylaxis assay in Sprague-Dawley rats. The greatest dilution of sera which resulted in a positive response was the titer of specific IgE. RESULTS: No significant differences were found between the titers of specific IgE caused by the chemically and enzymatically produced beta-lactam antibiotics, indicating that the antibiotics are equal in allergenicity. CONCLUSIONS: The data show that a residual level of 35 ppm protein did not affect the allergenic potency of these beta-lactam antibiotics as determined by the mouse allergenicity model.

Modeling of the enzymatic kinetic synthesis of cephalexin--influence of substrate concentration and temperature.

During enzymatic kinetic synthesis of cephalexin, an activated phenylglycine derivative (phenylglycine amide or phenylglycine methyl ester) is coupled to the nucleus 7-aminodeacetoxycephalosporanic acid (7-ADCA). Simultaneously, hydrolysis of phenylglycine amide and hydrolysis of cephalexin take place. This results in a temporary high-product concentration that is subsequently consumed by the enzyme. To optimize productivity, it is necessary to develop models that predict the course of the reaction. Such models are known from literature but these are only applicable for a limited range of experimental conditions. In this article a model is presented that is valid for a wide range of substrate concentrations (0-490 mM for phenylglycine amide and 0-300 mM for 7-ADCA) and temperatures (273-298 K). The model was built in a systematic way with parameters that were, for an important part, calculated from independent experiments. With the constants used in the model not only the synthesis reaction but also phenylglycine amide hydrolysis and cephalexin hydrolysis could be described accurately. In contrast to the models described in literature, only a limited number (five) of constants was required to describe the reaction at a certain temperature. For the temperature dependency of the constants, the Arrhenius equation was applied, with the constants at 293 K as references. Again, independent experiments were used, which resulted in a model with high statistic reliability for the entire temperature range. Low temperatures were found beneficial for the process because more cephalexin and less phenylglycine is formed. The model was used to optimize the reaction conditions using criteria such as the yield on 7-ADCA or on activated phenylglycine. Depending on the weight of the criteria, either a high initial phenylglycine amide concentration (yield on 7-ADCA) or a high initial 7-ADCA concentration (yield on phenylglycine amide) is beneficial.

Beta-lactam antibiotics functionalized with gem-bisphosphonates.

Acylation of amoxycillin and cephalexin with acids III, V and VII, and with isocyanate VIII furnished the corresponding beta-lactam antibiotics (X and XIII-XV, respectively). The antibacterial activity of these new antibiotic analogues against Helicobacter pylori was found to be identical with those of amoxycillin, Augmentin, erythromycin and ciprofloxacin.

Enzymatic synthesis of beta-lactam antibiotics using penicillin-G acylase in frozen media.

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.

Synthesis and antimicrobial activity of a dihydroampicillin and a dihydrocephalexin.

A dihydroampicillin (3) and a dihydrocephalexin (4) have been synthesized by condensing alpha-(cyclohexa-1, 3-dienyl)glycyl chloride with 6-aminopenicillanic acid (1) and 7-deacetoxycephalosporanic acid (2) respectively. The two antibiotics obtained show enhanced antimicrobial activity towards certain bacterial strains compared with ampicillin and cephalexin.