Enhancement of Enzymatic Adipyl-7-ADCA Hydrolysis

We studied enzymatic adipyl-7-ADCA hydrolysis as a new process for the production of 7-aminodeacetoxycephalosporanic acid (7-ADCA), one of the building blocks for cephalosporin antibiotics like cephalexin and cefadroxil. Adipyl-7-ADCA hydrolysis carried out with immobilised glutaryl acylase was considerably enhanced by addition of phenylglycine amide, the side-chain donor used for cephalexin synthesis; unlike reactions carried out with free enzyme. The rate enhancing effect was not specifically related to phenylglycine amide; we found a linear relationship between the reaction rate and the buffering capacity of the added substance. These observations can be explained by a pH-gradient in the immobilised enzyme, the pH inside the particle being lower (corresponding to low enzyme activity) than outside. It was concluded that the buffer reduced the pH-gradient inside the biocatalyst, and therewith, caused the reaction rate enhancing effects. Further, chloride ions decreased the reaction rate strongly, while sodium, magnesium, sulphate, and potassium did not influence the reaction rate much. For an actual process, it is important to use a buffer that is appropriate for the reaction-pH. In that way the amount of enzyme required in a process can be reduced considerably, in our case a factor of three was found.     

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.     

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.     

Modelling of the enzymatic kinetically controlled synthesis of cephalexin: influence of diffusion limitation

In this study the influence of diffusion limitation on enzymatic kinetically controlled cephalexin synthesis from phenylglycine amide and 7-aminodeacetoxycephalosporinic acid (7-ADCA) was investigated systematically. It was found that if diffusion limitation occurred, both the synthesis/hydrolysis ratio (S/H ratio) and the yield decreased, resulting in lower product and higher by-product concentrations. The effect of pH, enzyme loading, and temperature was investigated, their influence on the course of the reaction was evaluated, and eventually diffusion limitation was minimised. It was found that at pH >or=7 the effect of diffusion limitation was eminent; the difference in S/H ratio and yield between free and immobilised enzyme was considerable. At lower pH, the influence of diffusion limitation was minimal. At low temperature, high yields and S/H ratios were found for all enzymes tested because the hydrolysis reactions were suppressed and the synthesis reaction was hardly influenced by temperature. The enzyme loading influenced the S/H ratio and yield, as expected for diffusion-limited particles. For Assemblase 3750 (the number refers to the degree of enzyme loading), it was proven that both cephalexin synthesis and hydrolysis were diffusion limited. For Assemblase 7500, which carries double the enzyme load of Assemblase 3750, these reactions were also proven to be diffusion limited, together with the binding-step of the substrate phenylglycine amide to the enzyme. For an actual process, the effects of diffusion limitation should preferably be minimised. This can be achieved at low temperature, low pH, and high substrate concentrations. An optimum in S/H ratio and yield was found at pH 7.5 and low temperature, where a relatively low reaction pH can be combined with a relatively high solubility of 7-ADCA. When comparing the different enzymes at these conditions, the free enzyme gave slightly better results than both immobilised biocatalysts, but the effect of diffusion limitation was minimal.     

Use of aqueous two-phase systems for in situ extraction of water soluble antibiotics during their synthesis by enzymes immobilized on porous supports

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.     

Cephalexin synthesis by partially purified and immobilized enzymes

An enzyme which catalyzes the synthesis of cephalexin fromD -α phenylglycinemethylester (PGM) and 7-amino-3-desacetoxy-cephalosporanic acid (7-ADCA) was prepared from Xanthomonas citri (IFO 3835) and partially purified 30-fold by ammonium sulfate fractionation, DEAE-cellulose, and Sepharose-4B column chromatography. The K_m values for 7-ADCA, PGM, and cephalexin were determined as 11.1, 2.1, and 1.61 mM, respectively. The enzymatic cephalexin synthesis follows the reversible bi-uni reaction kinetics. The equilibrium constant is influenced by the initial mole ratios of 7-ADCA and PGM. The cephalexin hydrolysis is catalyzed by the same cephalexin synthesizing enzyme, but methanol does not participate in the hydrolytic reaction. The amount of enzyme in the reaction mixture affects the initial rate but does not influence the equilibrium product concentration. This cephalexin-synthesizing enzyme was immobilized onto several adsorbents. Among these, Kaolin and bentonite showed a higher retention of enzyme activity and stability for reuse. The immobilized-enzyme reaction kinetics were investigated and compared with those of the soluble enzyme. A rate expression for the enzymatic synthesis of cephalexin was derived. The results of computer simulation showed good agreement with the experimental results.     

Reaction kinetics of cephalexin synthesizing enzyme from Xanthomonas citri

In enzymatic synthesis of cephalexin from D-alpha-phenylglycine methyl ester (PGM) and 7-amino-3-deacetoxy-cephalosporanic acid (7-ADCA) using alpha-acylamino-beta-lactam acylhydrolase from Xanthomonas citri, it was found that this enzyme catalyzes all three reactions including PGM hydrolysis, cephalexin synthesis, and cephalexin hydrolysis. Based on our experimental results, a mechanistic kinetic model for cephalexin synthesizing enzyme system having acyl-enzyme intermediate was proposed. From this kinetic model, the reaction rate equations for three reactions were derived, and the kinetic parameters were evaluated. A good agreement between the simulation results and the experimental results was found.     

Purification and characterization of inducible cephalexin synthesizing enzyme in Gluconobacter oxydans

Cephalexin synthesizing enzyme (CSE) of Gluconobacter oxydans ATCC 9324 was purified up to about 940-fold at a yield of 12%. CSE biosynthesis in G. oxydans was found inducible in the presence of D-phenylglycine but not its substrate phenylglycine methyl ester. The purified enzyme was shown homogeneous on SDS-PAGE and exhibited a specific activity of 440 U per mg protein. The apparent molecular mass of the native enzyme was estimated to be 70 kDa over a Superdex 200 gel filtration column and 68 kDa on SDS-PAGE, indicating that the native enzyme is a monomer. Its isoelectric focusing point is 7.1, indicating a neutral character. The enzyme had maximal activity around pH 6.0 to 6.5, and this activity was thermally stable up to 40 degrees C. Synthesis of cephalexin from D-phenylglycine methyl ester and 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) by the purified CSE was demonstrated. Its L-enantiomer was not accepted by CSE. Apart from cephalexin, ampicillin was also synthesized by the purified CSE from its acyl precursors and 6-aminopenicillanic acid (6-APA). Substrate specificity studies indicated that the enzyme required a free alpha amino group and an activated carboxyl group as a methyl ester of D-form phenylglycine. Interestingly, the purified enzyme did not catalyze hydrolysis of its products, e.g., cephalexin, cephradine, and ampicillin, in contrast to enzymes from other strains of Pseudomonadaceae.     

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.     

Evaluation of magnetic polymer micro-beads as carriers of immobilised biocatalysts for selective and stereoselective transformations

The kinetic, selective and stereoselective properties of enzyme immobilised on magnetic polymer beads with diameters in the range 1 microm was studied with penicillin amidase from E. coli. The enzyme was immobilised on epoxy and glutaraldehyde-activated poly(vinyl alcohol), poly(methylmetacrylate) and poly(vinyl acetate-divinylbenzene) magnetic beads. The amount of covalently bound active protein was dependent on the chemical modification of the matrix and increased at higher ionic strength of the immobilisation buffer. The small size of the magnetic beads, that reduces mass transfer limitations, and the decreased charge density in the electric double layer resulted in lower apparent Km values and higher efficiency for benzylpenicillin hydrolysis, higher stereoselectivity in condensation of R-phenylglycine amide with S- and R-Phe and in hydrolysis of racemic phenylacetyl-Phe and higher selectivity in kinetically controlled synthesis of cephalexin compared to the enzyme immobilised on larger and porous carriers.     

Cephalexin synthesis by immobilised penicillin G acylase under non-isothermal conditions: reduction of diffusion limitation

The effect of thermodialysis on the enzymatic kinetic synthesis of the antibiotic cephalexin was investigated. As reference points, two existing models for an immobilised enzyme (Assemblase^®) and for the free enzyme were used. For Assemblase^®, it is known that diffusion limitation occurs and that therefore considerably more of the undesired side-product phenylglycine is formed.

The enzyme was immobilised on a membrane, and under isothermal conditions (293 K) the course of the reaction resembled that of the Assemblase^® enzyme. However, if a temperature gradient was applied across the membrane, with an average temperature of 293 K for the enzyme, than the course of the reaction changed. For large temperature gradients (30° and more), the course of the reaction resembled that of free enzyme. Thermodialysis enhances mass transfer across the membrane and therewith reduces diffusion limitations in the immobilised enzyme on the membrane.

The stability of the immobilised enzyme is such that the reactor can be re-used repeatedly. This, together with the positive effect of the temperature gradient on the course of the reaction, makes thermodialysis an interesting new technique that has potential to be applied on a larger scale if the membrane surface area per volume of reactor can be improved.     

Equilibrium position, kinetics, and reactor concepts for the adipyl-7-ADCA-hydrolysis process

One of the building blocks of cephalosporin antibiotics is 7-amino-deacetoxycephalosporanic acid (7-ADCA). It is currently produced from penicillin G using an elaborate chemical ring-expansion step followed by an enzyme-catalyzed hydrolysis. However, 7-ADCA-like components can also be produced by direct fermentation. This is of scientific and economic interest because the elaborate ring-expansion step is performed within the microorganism. In this article, the hydrolysis of the fermentation product adipyl-7-ADCA is studied. Adipyl-7-ADCA can be hydrolyzed in an equilibrium reaction to adipic acid and 7-ADCA using glutaryl-acylase. The equilibrium reaction yield is described as a function of pH, temperature, and initial adipyl-7-ADCA concentration. Reaction rate equations were derived for adipyl-7-ADCA-hydrolysis using three (pH-independent) reaction rate constants and the apparent equilibrium constant. The reaction rate constants were calculated from experimental data. Based on the equilibrium position and reaction rate equations the hydrolysis reaction was optimized and standard reactor configurations were evaluated. It was found that equilibrium yields are high at high pH, high temperature and low-initial adipyl-7-ADCA concentration. The course of the reaction could be described well as a function of pH (7-9), temperature (20-40 degrees C) and concentration using the reaction rate equations. It was shown that a series of CSTR's is the best alternative for the process.     

Enzymatic removal of a cephalosporin methyl ester blocking group

Over 7000 microorganisms were screened to find an enzyme source for the hydrolysis of a C₄ methyl ester blocking group on 7-aminodesacetoxycephalosporanic acid (7-ADCA). Only one culture, Streptomyces capillispira Mertz and Higgens nov. sp., produced an enzyme that catalysed the reaction. Enzyme synthesis in a defined mineral salts medium was repressed by NH₃ and amino acids. Under optimum fermentation conditions, the maximum rate of substrate hydrolysis was 6 × 10^?10 mol min^?1 mg^?1 cell. The enzyme was recovered from the mycelia and partially purified by gel filtration. Kinetic studies by pH-stat titration indicated that the pH optimum was 7.5–8.5, the temperature optimum was 25–30°C, and the substrate K_m value was 2.3 mg ml^?1. The reaction products, 7-ADCA and methanol, were weak competitive inhibitors of the enzyme with K₁ values of 6.63 and 0.188 mg ml^?1, respectively. The enzyme also hydrolysed cefaclor and cephalexin methyl esters but did not hydrolyse cephalosporin ethyl esters. With further improvements in enzyme yields and stability, enzymatic deblocking of cephalosporins could provide an alternative to chemical deblocking processes.     

Evaluation of the performance of immobilized penicillin G acylase using active-site titration

Penicillin G acylase from Escherichia coli was immobilized on Eupergit C with different enzyme loading. The activity of the immobilized preparations was assayed in the hydrolysis of penicillin G and was found to be much lower than would be expected on the basis of the residual enzyme activity in the immobilization supernatant. Active-site titration demonstrated that the immobilized enzyme molecules on average had turnover rates much lower than that of the dissolved enzyme. This was attributed to diffusion limitations of substrate and product inhibition. Indeed, when the immobilized preparations were crushed, the activity increased from 587 U g-1 to up to 974 U g-1. The immobilized preparations exhibited up to 15% lower turnover rates than the dissolved enzyme in cephalexin synthesis from 7-ADCA and D-(-)-phenylglycine amide. The synthesis over hydrolysis ratios of the immobilized preparations were also much lower than that of the dissolved enzyme. This was partly due to diffusion limitations but also to an intrinsic property of the immobilized enzyme because the synthesis over hydrolysis ratio of the crushed preparations was much lower than that of the dissolved enzyme.     

Enhancement effect of polyethylene glycol on enzymatic synthesis of cephalexin

In an enzymatic synthesis of cephalexin (CEX) using an acylase from Xanthomonas citri, the effect of polyethylene glycol (PEG) on the synthetic reaction of 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and D-alpha-phenyl-glycine methyl ester (PGM) to CEX was investigated. The addition of PEG (MW 300-20,000) increased the yield significantly. This yield enhancement effect tended to increase with the increasing molecular weight of PEG. Addition of PEG to the reaction system did not affect both the CEX and PGM hydrolytic reactions. The PEG added to the reaction medium used in these experiments did not depress the water activity significantly, and the product yield improvement could not be explained by the activity alone. The PEG stabilized the enzyme activity to some extent, but this stabilizing effect was only partially attributable to the yield enhancement of CEX. The enhancing effect of PEG on the synthetic yield increased with the increasing PEG molecular weight or the length of the poly(oxy-1,2-ethanediyl) chain, which increases the hydrophobicity of PEG. This finding consequently has led to the conclusion that the PEG structure renders the affinity between enzyme and 7-ADCA, which is a hydrophobic substrate. The microenvironmental hydrophobicity of PEG and its interaction with the hydrophobic substrate was found to be the main reason for the improvement of the CEX yield. In fact, the Michaelis-Menten kinetic constant for 7-ADCA, K(7-ADCA) in the presence of PEG was smaller than that in the control system (without PEG addition). (c) 1993 John Wiley & Sons, Inc.     

聚丙烯腈纤维固定化青霉素酰化酶合成头孢氨苄的研究

将巨大芽孢杆菌胞外青霉素酰化酶通过共价键结合到聚丙烯腈纤维的衍生物上。制成的丝状固定化青霉素酰化酶表现活力达 1 5 3U g(湿重 )。固定化酶合成头孢氨苄的最适pH为 6 5 ,最适温度为 40℃。 7 ADCA的投料浓度以 4%为好 ,7 ADCA与PGME的投料量比率为1∶2 ,最佳用酶量为 1 70U g 7 ADCA。在pH6 5、温度 3 0℃时 ,固定化酶对 7 ADCA的表观米氏常数K7 ADCA为 0 1 6 2mol L ,对PGME的表观米氏常数KPGME为 0 3 6 4mol L ,最大反应速度Vmax为0 0 4 6 2mol·L- 1·min- 1,用固定化酶合成头孢氨苄 ,使用 5 0次保留酶活力 83 9%     

Kinetically controlled synthesis of ampicillin and cephalexin in highly condensed systems in the absence of a liquid aqueous phase

Advantages of performing penicillin G amidase catalysed synthesis of ampicillin and cephalexin by enzymatic acyl transfer to the β-lactam antibiotic nuclei in a highly condensed system using mainly undissolved substrates, with no apparent aqueous liquid phase, were demonstrated. It was shown that synthesis can be performed in the absence of a liquid phase formed by water or an organic co-solvent. This highly condensed system is formed by a liquid phase given by one of the reactant, the phenylglycine methyl ester (PGM), that remains liquid in these operative conditions and the partially dissolved β-lactam nucleus. Operating in such highly condensed system, the water that causes the hydrolysis of PGM is limited to the water hydrating the support on which the enzyme is covalently immobilised. In this way the reaction system is maintained at a controlled degree of hydration.

In the present work the reaction system was modulated by eliminating the solvent (aqueous or aqueous/organic), reducing the amount of water to the minimum for the biocatalytic activity and using PGM as solvent and reagent at the same time. The synthesis was conducted with equimolar amounts of PGM and the β-lactam nucleus, with a reduced hydrolysis of the activated acyl donor. We have also studied a simple and efficient method for the workup of the reaction where the unreacted reagents can be recovered after selective filtration and precipitation.     

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.).     

氨基酸酯水解酶产生菌的筛选及其合成头孢立新的研究

From ten genera and 146 bacterial strains, 22 strains producing alpha-amino acid ester hydrolase were selected. Among them, AS 1.586 and 41-2 were the best. The optimal conditions for synthesis of cephalexin by pseudomonas aeruginosa 1.204 were investigated. The optimal pH and temperature for enzymatic synthesis reaction was pH 6.8 and 25 degrees C, respectively. By using 1% 7-ADCA, 3% PGME and 4% biomass, about 70% of 7-ADCA was converted to cephalexin under the mentioned conditions.     

Enzymatic biosynthesis of cephalexin

The reaction kinetics of the enzymatic of cephalexin from 7-aminodea-cetoxy cephalosporanic acid and phenylglycine methylester was studied using the synthesizing enzyme obtained from Xanthomonas citri. The activation energy, K_m value for 7-aminodeacetoxy cephalosporanic acid and phenylglycine methylester, and K_i value for phenylglycine methylester were determined as 8.63 kcal/mol, 3.7mM, 14.5mM, and 70mM, respectively. The enzyme was found to be constitutive and susceptible to deactivation.