Showing papers on "Penicillin amidase published in 1996"
TL;DR: The analysis shows that the environment of the catalytically active N-terminal serine of the B chain contains no adjacent histidine equivalent to that found in the serine proteases, indicating that this must be an important recognition site for cleavage.
Abstract: PENICILLIN acylase (penicillin amidohydrolase, EC 3.5.1.11) is widely distributed among microorganisms, including bacteria, yeast and filamentous fungi. It is used on an industrial scale for the production of 6-aminopenicillanic acid, the starting material for the synthesis of semi-synthetic penicillins. Its in vivo role remains unclear, however, and the observation that expression of the Escherichia coli enzyme in vivo is regulated by both temperature and phenylacetic acid has prompted speculation that the enzyme could be involved in the assimilation of aromatic compounds as carbon sources in the organism's free-living mode1. The mature E. coli enzyme is a periplasmic 80K heterodimer of A and B chains (209 and 566 amino acids, respectively2,3) synthesized as a single cytoplasmic precursor containing a 26-amino-acid signal sequence to direct export to the cytoplasm4 and a 54-amino-acid spacer between the A and B chains which may influence the final folding of the chains5. The N-terminal serine of the B chain reacts with phenylmethylsulphonyl fluoride, which is consistent with a catalytic role for the serine hydroxyl group. Modifying this serine to a cysteine6'7 inactivates the enzyme, whereas threonine, arginine or glycine substitution prevents in vivo processing of the enzyme7, indicating that this must be an important recognition site for cleavage. Here we report the crystal structure of penicillin acylase at 1.9 A resolution. Our analysis shows that the environment of the catalytically active N-terminal serine of the B chain contains no adjacent histidine equivalent to that found in the serine proteases. The nearest base to the hydroxyl of this serine is its own α-amino group, which may act by a new mechanism to endow the enzyme with its catalytic properties.
434 citations
TL;DR: It is shown that, by controlling the pH, it is possible to inhibit the lateral undesirable reactions increasing the yield of the main reaction, in the first report where the specific effect of pH was studied in detail.
84 citations
TL;DR: Simultaneous utilization of these strategies has significantly improved this synthetic process with very high yields, reaction rates, and enzyme stability.
77 citations
TL;DR: Parameters relevant to the thermodynamically controlled synthesis of cephalothin utilizing highly active stabilized penicillin G acylase derivatives were studied and dynamic reaction design was implemented.
Abstract: Parameters relevant to the thermodynamically controlled synthesis of cephalothin utilizing highly active stabilized penicillin G acylase derivatives were studied These included solubility/stability of substrates, enzyme derivative activity/stability, reaction course and synthetic yields These parameters were altered by varying the pH, dimethylformamide concentration and temperature Simultaneous optimization of the selected parameters could not be achieved with a single set of conditions However, continuous adjustment of conditions throughout the reaction course allowed each parameter to be optimized (dynamic reaction design) This strategy works by optimizing those parameters that are critical to the overall reaction at a given point, whilst leaving others sub-optimal when their contribution to the total is minimal This strategy has achieved a 90% transformation of antibiotic nucleus to cephalothin at a final concentration of 20 g/l, high enzyme and reactant stability, with a reaction period of 3 h (using 1 ml of derivative/40 ml of reaction solution)
50 citations
48 citations
TL;DR: The rates of penicillin acylase-catalyzed reactions were found to be significantly reduced by the presence of a small amount of organic solvent, which indicates that interactions between the solvents and the enzyme are specific and reversible.
Abstract: The effects of various organic solvents on penicillin acylase-catalyzed synthesis of β-lactam antibiotics (pivampicillin and ampicillin) have been investigated in water-solvent mixtures. The rates of penicillin acylase-catalyzed reactions were found to be significantly reduced by the presence of a small amount of organic solvent. In particular, the rate of enzyme catalysis was extremely low in the presence of ring-structured solvents and acids while enzyme activities were fully restored after removing the solvents. This indicates that interactions between the solvents and the enzyme are specific and reversible. To correlate the inhibitory effects of organic solvents with solvent properties the influence of solvent hydrophobicities and solvent activity on the rate of pivampicillin synthesis was examined. The reaction rate was found to decrease with increasing solvent hydrophobicities, and a better correlation was observed between the reaction rate and solvent activity. The effects of ionic strength on the synthesis of pivampicillin and ampicillin were also examined. The ionic strength dependence indicates that electrostatic interactions are involved in the binding of ionic compounds to the enzyme. On the basis of the active site structure of penicillin acylase, a possible mechanism for molecular interactions between the enzyme and organic solvents is suggested.
48 citations
TL;DR: The kinetics of immobilized penicillin acylase inactivation was a multistage process, decay constants being evaluated for the free‐enzyme and enzyme complexes, from whose values modulation factors were determined for the effectors in each enzyme complex at each stage.
Abstract: Inactivation of immobilized penicillin acylase has been studied in the presence of substrate (penicillin G) and products (phenylacetic acid and 6-aminopenicillanic acid), under the hypothesis that substances which interact with the enzyme molecule during catalysis will have an effect on enzyme stability. The kinetics of immobilized penicillin acylase inactivation was a multistage process, decay constants being evaluated for the free-enzyme and enzyme complexes, from whose values modulation factors were determined for the effectors in each enzyme complex at each stage. 6-Aminopenicillanic acid and penicillin G stabilized the enzyme in the first stage of decay. Modulation factors in that stage were 0.96 for penicillin G and 0.98 for 6-aminopenicillanic acid. Phenylacetic acid increased the rate of inactivation in both stages, modulating factors being -2.31 and -2.23, respectively. Modulation factors influence enzyme performance in a reactor and are useful parameters for a proper evaluation.
47 citations
TL;DR: In this paper, the synthesis of four β-lactam antibiotics (penicillin G, ampicillin and pivampicillin) catalyzed by Escherichia coli penicillin acylase has been investigated in water-methanol mixtures.
Abstract: The synthesis of four β-lactam antibiotics (penicillin G, pivaloyloxymethyl ester of penicillin G, ampicillin and pivampicillin) catalyzed byEscherichia coli penicillin acylase has been investigated in water-methanol mixtures. The enzyme reactions were either thermodynamically or kinetically controlled at the same conditions using phenylacetic acid and d -α-phenylglycine methyl ester as acyl donors and 6-aminopenicillanic acid and pivaloyloxymethyl 6-aminopenicillanic acid as acyl acceptors. It has been found that the influences of the cosolvent content on the reaction rates and synthetic yields are significantly different depending on the substrates used in the experiments. On the other hand, within certain ranges of the methanol content (up to ca. 40% (v/v) the residual activities of the enzymes in water-methanol mixtures were only slightly lower than those in aqueous media. To analyze the factors that determine the reaction rate in water-cosolvent mixtures, the effect of methanol on the apparent pK values of the substrates has been investigated, and a mathematical model has been developed on the basis of the assumption that the enzyme binds non-ionized substrates. Model simulation results indicate that the solvent effect on reaction rates is mainly attributed to the kinetic effects of changes in apparent pK values.
46 citations
TL;DR: Reduction with sodium borohydride of the Schiff's bases formed between the amino groups of the protein and glutaraldehyde results in a dramatic improvement of the operational stability of the immobilized enzyme without affecting the catalytic activity.
46 citations
TL;DR: The high-expression system developed using the high PGA-producing strain Escherichia coli RE3 as a host and a recombinant plasmid pKA18 which was constructed by cloning the chromosomal pga gene coding for PGA in the strain RE3 on multicopy vector pK19 led to an increase in the specific activity of PGA.
37 citations
TL;DR: In this paper, the effects of reaction variables such as enzyme concentration, pH, temperature, and molar ratio of the substrates on PVM synthesis were investigated using an immobilized Escherichia coli penicillin acylase.
Abstract: Enzymatic synthesis of pivampicillin (PVM) from D-α-phenylglycine methyl ester (PGM) and pivaloyloxymethyl 6-aminopenicillanic acid (POM-6-APA) was investigated using an immobilized Escherichia coli penicillin acylase. The effects of reaction variables such as enzyme concentration, pH, temperature, and molar ratio of the substrates on PVM synthesis were investigated. The time-course profiles of the PVM synthesis reaction followed a typical pattern of the kinetically-controlled synthesis of β-lactam antibiotics: the concentration of PVM reached a maximum and then decreased gradually. By lowering the reaction temperature, the maximum yield was enhanced significantly. This was mainly attributed to the suppressed hydrolysis of PVM and PGM at low temperatures. A higher yield of PVM was also attained with increasing the molar ratio of PGM to POM-6-APA. When the molar ratio of PGM to POM-6-APA was 10, the maximum yield of 61.8% was obtained at 4°C. The addition of organic cosolvents, on the other hand, showed no improvement in the PVM synthesis yields due to inhibitory effect of the solvent; both synthetic and hydrolytic activities of penicillin acylase were reduced by organic solvents. The degree of inhibition was found to be more profound in the presence of less polar solvents.
TL;DR: In situ hydrated phosphates were employed for controlling the water activity in a benzene/water system (97:3 v/v), where the water is taken up by the salt with formation of the hydrated species.
TL;DR: With the mutant F360V, which differs from the wild-type enzyme in its ability to hydrolyse adipyl-L-leucine and has a kcat for 4-nitrophenyl acetate one-twentieth that of theWild- type enzyme, the corresponding values for the rates of formation and hydrolysis of the acetyl-enzyme were three times that for the Wild-Type enzyme.
Abstract: The kinetics of release of 4-nitrophenol were followed by stopped-flow spectrophotometry with two 4-nitrophenyl ester substrates of penicillin G acylase from Kluyvera citrophila. With the ester of acetic acid, but not of propionic acid, there was a pre-steady-state exponential phase, the kinetics of which were inhibited by phenylacetic acid (a product of hydrolysis of specific substrates) to the extent predicted from Ki values. This was interpreted as deriving from rapid formation (73 mM-1.s-1) and slow hydrolysis (0.76 s-1) of an acetyl derivative of the side chain of the catalytic-centre residue Ser-290. With the mutant F360V, which differs from the wild-type enzyme in its ability to hydrolyse adipyl-L-leucine and has a kcat for 4-nitrophenyl acetate one-twentieth that of the wild-type enzyme, the corresponding values for the rates of formation and hydrolysis of the acetyl-enzyme were 11.1 mM-1.s-1 and 0.051 s-1 respectively. The ratio of these rate constants was three times that for the wild-type enzyme, suggesting that the mutant is less impaired in the rate of formation of an acetyl-enzyme than in its subsequent hydrolysis.
TL;DR: Substrate specificity studies indicate that the Aeromonas sp.
Abstract: Aeromonas sp. ACY 95 produces constitutively and intracellularly a penicillin V acylase at an early stage of fermentation (12 h) and a cephalosporin C acylase at a later stage (36 h). Some penicillins, cephalosporin C and their side chain moieties/analogues, phenoxyacetic acid, penicillin V and penicillin G, enhanced penicillin V acylase production while none of the test compounds affected cephalosporin C acylase production. Supplementation of the medium with some sugars and sugar derivatives repressed enzyme production to varying degrees. The studies on enzyme formation, induction and repression, and substrate profile suggest that the cephalosporin C acylase and penicillin V acylase are two distinct enzymes. Substrate specificity studies indicate that the Aeromonas sp. ACY 95 produces a true cephalosporin C acylase which unlike the enzymes reported hitherto hydrolyses cephalosporin C specifically.
TL;DR: The proposed strategy, which involves maintaining the substrate concentration at the optimum value for a large part of the conversion, results in higher product concentrations than in batch operation, thereby reducing downstream procesing costs and the ease of implementation has been demonstrated.
Abstract: A general diffusion reaction model for immobilised biocatalysis has been developed. The model has been used to study the deacylation of penicillin G to 6-aminopenicillanic acid using two commercially available immobilised Penicillin acylases. The values of D e /R 2 for the enzyme pellets have been estimated using data on uptake of 6-aminopenicillanic acid and phenylacetic acid by the enzyme pellets. The kinetic parameters of the model were individually estimated from a suitably designed set of experiments. The values of the Thiele modulus from the kinetic parameters so calculated have been found to be in the range 1.67 to 9.8 for the two enzymes studied, implying that diffusional effects cannot be ignored. The effect of such diffusional limitations on the overall rates and hence on the utilisation of the intrinsic kinetic ability of the enzyme has been demonstrated. This paper also reports on the implementation of the fed-batch strategy for this system. The proposed strategy, which involves maintaining the substrate concentration at the optimum value for a large part of the conversion, results in higher product concentrations than in batch operation, thereby reducing downstream procesing costs. The productivity was also shown to be considerably higher than for batch operation. Further, the ease of implementation of this mode of operation has been demonstrated.
TL;DR: In this work, incubation under aerated conditions, of whole recombinant E. coli cells after glucose depletion and growth cessation, consistently resulted in 2- to 4-fold increases in penicillin acylase activity, suggesting that penicillins maturation occurs to a high extent even during the postfermentative stage.
Abstract: Active penicillin acylase is formed only after an in vivo post-translational processing of the polypeptide precursor. Such a maturation process is rare in procaryotes. In this work, incubation under aerated conditions, of whole recombinant E. coli cells after glucose depletion and growth cessation, i.e., during the postfermentative stage, consistently resulted in 2- to 4-fold increases in penicillin acylase activity. Such results suggest that penicillin acylase maturation occurs to a high extent even during the postfermentative stage. Accordingly, the effect of different incubation conditions, during the postfermentative stage, on penicillin acylase was determined. Incubation under anaerobic conditions resulted only in a 1.27-fold increase of enzyme activity, with respect to the end of the batch culture, whereas a 3- and 4- fold increase occurred during incubation under dissolved oxygen concentrations of 100 and 43% (with respect to air sat.), respectively. Only a small negative effect, on the maturation process, was observed during incubation with acetate concentrations above 0.6 g/L. No effect of pH, in the range of 6.0 to 8.0, was observed.
Journal Article•
TL;DR: Ampicillin was the affinity ligand that presented the best results for specific adsorption of penicillin acylase under affinity and pseudo-affinity adsorptive processes.
Abstract: Affinity ligand (6-Aminopenicillanic acid, Amoxycillin, Ampicillin, Benzylpenicillin and 4-Phenylbutylanzine) of penicillin acylase (EC 3.5.1.11) were attached to hydrophilic gels like Sepharose 4B-CNBr and Minileak 'medium'. Ampicillin and 4-Phenylbutylamine were the affinity ligands that presented the higher concentrations attached to both gels. Penicillin acylase adsorption on these affinity gels was mainly dependent on the activated group of the gel, the affinity ligand attached and the experimental conditions of enzyme adsorption. Under affinity conditions only the ligands Amoxycillin, Ampicillin and 4-Phenylbutylamine, immobilized on Minileak, adsorbed the enzyme from osmotic shock extracts at different pH values. These affinity ligand systems were characterized by low adsorption capacities of penicillin acylase activity (1.2-2.1 IU mL-1 gel) and specific activity (1.5-2.9 IU mg-1 prot). Under pseudo-affinity conditions all the ligands attached both activated to gels (Sepharose 4B-CNBr and Minileak) adsorbed the enzyme. The affinity gels were characterized by higher values of adsorption capacity (3.7 and 55.6 IU mL-1 gel) and adsorbed specific activity (2.0 and 6.1 IU mg-1 prot) than those observed under affinity conditions. The space arm of Minileak gel, shown to be fundamental to enzyme adsorption under affinity conditions, preferentially adsorbed proteins in relation to the enzyme under pseudo-affinity conditions. However, this effect was partially minimized when the gel was derivatized by the affinity ligands at concentrations higher than 6 mumol mL-1 gel. Ampicillin was the affinity ligand that presented the best results for specific adsorption of penicillin acylase under affinity and pseudo-affinity adsorption processes. The Sepharose 4B-CNBr derivatized gel also presented a good adsorption capacity of enzyme activity (26.8 IU mL-1 gel) under pseudo-affinity adsorption processes.
TL;DR: The introduction of a N-α-phenylacetyl moiety into glycine, methionine and aspartic acid derivatives using immobilized penicillin G acylase (PGA) as catalyst was studied and the synthesis of some phenyl acetyl derivatives on a preparative scale under the optimum conditions is described.
Abstract: The introduction of a N-α-phenylacetyl moiety into glycine, methionine and aspartic acid derivatives using immobilized penicillin G acylase (PGA) as catalyst was studied. High synthetic yields (86–97%) were obtained in biphasic systems under thermodynamic control using fully carboxy-protected derivatives of amino acids such as H-Gly-OMe, H-Gly-OBzl, H-Met-OEt, H-Asp(OBut)-OMe, H-Asp(OMe)-OMe and H-Asp(OBzl)-OBzl. Moderate yields (50–62%) were obtained for the N-α protection of β-carboxy free, α-carboxy esters of aspartic acid such as H-Asp-OMe and H-Asp-OBzl in methanol/buffer mixtures under kinetic control. The synthesis of some phenylacetyl derivatives on a preparative scale under the optimum conditions is also described.
TL;DR: Penicillin acylase from Bacillus megaterium was purified 6.22 fold using ultrafiltration followed by ammonium sulphate precipitation and gel filtration chromatography using Sephadex G-100 and was free from interfering proteases.
Abstract: Penicillin acylase from Bacillus megaterium was purified 6.22 fold using ultrafiltration followed by ammonium sulphate precipitation and gel filtration chromatography using Sephadex G-100. The final specific activity was 2.62 U/mg protein and was free from interfering proteases.
TL;DR: The stereoselectivity of penicillin amidase (PA, EC 3.5.1.11) from E coli and homologeous enzymes from other sources has been determined as a function of temperature and substrate for hydrolysis and kinetically controlled synthesis.
Abstract: The stereoselectivity of penicillin amidase (PA, EC 3.5.1.11) from E coli and homologeous enzymes from other sources has been determined as a function of temperature and substrate for hydrolysis and kinetically controlled synthesis. The stereoselectivity of these reactions decreased almost by one order of magnitude from 5 to 45°C. It increased with the substrate (kcat/Km) and nucleophile (kT/kH) specificity, and was found to differ in the S1- (R-specific) and S′1-(S-specific)-binding subsites of the active site. The S1-stereoselectivity was determined mainly by differences in the activation energy, i.e. the turnover number. The stereoselectivity of PA from different sources differed by almost an order of magnitude for the same substrate.
TL;DR: A high degree of nucleophile conversion into the corresponding cephem at biotechnologically relevant concentrations (50 mM) was achieved.
Abstract: Penicillin amidase (EC 3.5.1.11) catalysed the synthesis of cephem 7-(2-benzoxazolon-3-yl-acetamido)-desacetoxycephalosporanic acid by a kinetically controlled transfer of the non-specific 2-benzoxazolon-3-yl-acetyl moiety from its polyethyleneglycol ester (m.w. 400, nine monomeric units) to the nucleophile 7-aminodesacetoxycephalosporanic acid. Penicillin amidase from E.Coli immobilised in polyacrylamide gel was used as biocatalyst. A high degree of nucleophile conversion (98 %) into the corresponding cephem at biotechnologically relevant concentrations (50 mM) was achieved.
TL;DR: In this paper, N-protected P α-methyl nucleoside triphosphates were deblocked by treatment with penicillin amidase at pH 7.8, 25°C.
Abstract: 5′-O-(methylphosphonyl)-N-(phenylacetyl)-2 ′-deoxycytidine, deoxyadenosine and deoxyguanosine were pyrophosphorylated and the resulting N-protected P α-methyl nucleoside triphosphates were deblocked by treatment with penicillin amidase at pH 7.8, 25°C to give P α-methyl nucleoside triphosphates.
TL;DR: A reactor-separator combining immobilized penicillin acylase (IPA) and electrodialysis was developed for the production of 6-aminopenicillanicacid (6-APA), and strong concentration polarization of Pen-G was observed in the vicinity of anion exchange membrane.
Abstract: A reactor-separator combining immobilized penicillin acylase (IPA) and electrodialysis was developed for the production of 6-aminopenicillanicacid (6-APA)from the enzymatic hydrolysis of penicillin G (Pen-G) accompanying the continuous removal of by-product phenylacetic acid (PAA). The kinetics of Pen-G hydrolysis by IPA, the separation of PAA, 6-APA and Pen-G with electrodialysis, as well as the reaction-separation phenomena of Pen-G hydrolysis with IPA in the electrodialyzer were studied. The products inhibition mechanism and rate equation of Pen-G hydrolysis by IPA were obtained. The separation of PAA, 6-APA and Pen-G with electrodialysis was dependent on their molecular size and affinity toward the anion exchange membrane. The transport rates of three components decreased in the sequence PAA > 6-APA > Pen-G. Strong concentration polarization of Pen-G was observed in the vicinity of anion exchange membrane. An increase in Pen-G concentration enhanced the selectivity factor of PAA to 6-APA but ...
TL;DR: Experimental findings show that the periplasmic penicillin amidase does not show any variation by the chloroform treatment, and this analysis was also extended to the E. coli cells grown at various concentrations of phenylacetic acid plus glucose and lactic acid.
Abstract: Penicillin amidase is a periplasmic enzyme in Escherichia coli. Conventionally, the periplasmic enzymes are released into the medium by osmotic shock which is tedious involving a number of centrifugation steps. The present communication deals with a simple technique for the release of penicillin amidase by chloroform shock. Experimental findings show that the periplasmic penicillin amidase does not show any variation by the chloroform treatment. This analysis was also extended to the E. coli cells grown at various concentrations of phenylacetic acid, optimal concentration of phenylacetic acid plus glucose and lactic acid.
01 Jan 1996
TL;DR: Pressure activation and inactivation is investigated for several enzymes like penicillin amidase, glutamate dehydrogenase, lipase and lipase in the pressure range between 1 bar and 4000 bar and hence their enantioselectivity can be directed.
Abstract: Summary Pressure activation and inactivation is investigated for several enzymes like penicillin amidase ( E. coli ), glutamate dehydrogenase ( P. woesei ) and lipase ( Rhizopus arrhizus ) in the pressure range between 1 bar and 4000 bar. In dependance of pressure and temperature the enzymes are acivated or inactivated and hence their enantioselectivity can be directed. The activation of the enzymes corresponds to a decrease in the K M value which results in a higher substrate affinity.
TL;DR: According to this hypothesis the experimental phenomena taking place in biosynthesis of penicillin and cephalosporin, such as the inhibition by glucose and carbon dioxide and the reduction of the yield, can be satisfactorily explained.
TL;DR: Experimental findings show that the biosynthesis of penicillin amidase in E. coli show a little variation among batch reactor modes and significant variation on the continuous mode of operation, and analysis shows that the different reactor modes also influence periplasmic localization of the enzyme in the cell.
Abstract: Different operational mode of bioreactors influence the biosynthesis of the enzyme and related products as well as the growth of industrial microorganisms. This communication deals with the effect of mode of operation of various bioreactors with different geometric configurations, viz., batch (includes commercially available batch stirred tank, and custom-designed cylindrical and tapered reactors), batch-fed, continuous flow stirred tank reactors on the biosynthesis of penicillin amidase in Escherichia coli. Experimental findings show that the biosynthesis of penicillin amidase in E. coli show a little variation among batch reactor modes and significant variation on the continuous mode of operation. Further analysis show that the different reactor modes also influence periplasmic localization of the enzyme in the cell.
TL;DR: A recombinant immobilized E. coli cell biocatalyst was used to design an enzymatic process for the synthesis of ampicillin and inhibition of PGME hydrolysis by methanol and pH control allowed synthesis reactions with 75% yields.
Abstract: A recombinant immobilized E. coli cell biocatalyst was used to design an enzymatic process for the synthesis of ampicillin. Reduction of water activity did not increase conversion yield. Inhibition of PGME hydrolysis by methanol and pH control allowed synthesis reactions with 75% yields.
TL;DR: In this article, the authors used Hess' law to measure the heat involved in the 6-aminopenicillanic acid (6-APA) hydrolysis process using dynamic calorimetry.
TL;DR: The hollow tubes reactor revealed to be slightly superior to the hollow zones reactor, and the application to the conversion data obtained in both reactors of the α, β kinetic model derived by Cardoso and Costa gave evidence of the superior performance of the hollow tubes reactors.
Abstract: The comparative efficiencies of two special biocatalytic reactors designed to handle gel-like immobilized biocatalysts, the hollow tubes and the hollow zones reactors, have been assessed for the performance of the hydrolysis of benzylpenicillin to 6-aminopenicillanic acid and phenyl acetic acid by an immobilized penicillin amidase preparation.