Showing papers on "Penicillin amidase published in 2000"
TL;DR: Penicillin G acylase CLEAs had the same activity in the synthesis of ampicillin as cross-linked crystals of the same enzyme, but the accompanying hydrolysis of the side-chain donor was much less.
450 citations
TL;DR: Progressive establishment of new enzyme-support attachments was showed by the progressive irreversible covalent immobilization of several subunits of multi-subunits proteins, and the final stabilization factor become 100-fold comparing soluble penicillin G acylase and optimal derivative.
337 citations
TL;DR: Assay results show that mesoporous MCM-41 is a more effective support for the immobilization of Penicillin Acylase (PA) than many of other supports due to its structural and surface characteristics.
Abstract: Mesoporous MCM-41 having well ordered long-range structure, large pore diameters, narrow pore-size distribution, high pore volume and specific surface area has been synthesized. The surface of MCM-41 has an abundance of weakly acidic hydroxyl groups. Assay results show that MCM-41 is a more effective support for the immobilization of Penicillin Acylase (PA) than many of other supports due to its structural and surface characteristics. PA can be immobilized on MCM-41 through either direct immobilization or covalent coupling. The former gives higher activity of IME than the later. In the direct immobilization, PA molecules are immobilized on MCM-41 through the hydrogen-bonded interaction between hydroxyl groups of MCM-41 and carbonyl or amino groups in the PA molecule.
129 citations
TL;DR: The first crystal structure of a cephalosporin acylase from Pseudomonas diminuta is solved and provides structural evidence that four of the five different classes of cep Halospor in acylases can be grouped into one family of the Ntn hydrolase superfamily.
102 citations
TL;DR: The combined results of the structural and kinetic studies show the importance of alphaF146 in the beta-lactam binding site and provide leads for engineering mutants with improved synthetic properties.
Abstract: The binding of penicillin to penicillin acylase was studied by X-ray crystallography. The structure of the enzyme-substrate complex was determined after soaking crystals of an inactive betaN241A penicillin acylase mutant with penicillin G. Binding of the substrate induces a conformational change, in which the side chains of alphaF146 and alphaR145 move away from the active site, which allows the enzyme to accommodate penicillin G. In the resulting structure, the beta-lactam binding site is formed by the side chains of alphaF146 and betaF71, which have van der Waals interactions with the thiazolidine ring of penicillin G and the side chain of alphaR145 that is connected to the carboxylate group of the ligand by means of hydrogen bonding via two water molecules. The backbone oxygen of betaQ23 forms a hydrogen bond with the carbonyl oxygen of the phenylacetic acid moiety through a bridging water molecule. Kinetic studies revealed that the site-directed mutants alphaF146Y, alphaF146A and alphaF146L all show significant changes in their interaction with the beta-lactam substrates as compared with the wild type. The alphaF146Y mutant had the same affinity for 6-aminopenicillanic acid as the wild-type enzyme, but was not able to synthesize penicillin G from phenylacetamide and 6-aminopenicillanic acid. The alphaF146L and alphaF146A enzymes had a 3-5-fold decreased affinity for 6-aminopenicillanic acid, but synthesized penicillin G more efficiently than the wild type. The combined results of the structural and kinetic studies show the importance of alphaF146 in the beta-lactam binding site and provide leads for engineering mutants with improved synthetic properties.
86 citations
TL;DR: In this article, a penicillin G acylase (PA) was activated with glutaraldehyde and finally, PA was immobilized to these activated particles, both the conditions of the aminoalkylation-and the immobilization process were optimized.
Abstract: Nylon particles, grafted with diethylene glycol dimethacrylate (DGDA) using potassium persulphate as initiator, were treated with hexamethylene diamine (HMDA). The aminoalkylated particles were activated with glutaraldehyde and finally, penicillin G acylase (PA) was immobilized to these activated particles. Both the conditions of the aminoalkylation- and the immobilization process were optimized. The hydrolysis of cephalexin was used as model conversion. The retention of activity of the immobilized enzyme was 12%. This value improved to 30% by adding phenyl acetic acid (PAA), as active-site protecting agent, to the enzyme solution. The results suggest formation of multi-point attachment between the enzyme and the matrix.
58 citations
TL;DR: In this “aqueous solution–precipitate” system, accumulation of both products, ampicillin and d -(−)-phenylglycine, proceeds through the formation of their supersaturated solutions, which positively influences the efficiency of the biocatalytic process.
Abstract: Penicillin acylase-catalyzed ampicillin synthesis via acyl group transfer in aqueous solution is highly dependent on the initial substrate concentration. The solubility of one substrate, 6-aminopenicillanic acid (6-APA), can be advantageously enhanced by the presence of acyl donor, the second substrate. Furthermore, a comparison of enzymatic synthesis in homogeneous solution with synthesis in a heterogeneous system having partially undissolved reactants, reveals major advantages for the latter approach. In this “aqueous solution–precipitate” system, accumulation of both products, ampicillin and d -(−)-phenylglycine, proceeds through the formation of their supersaturated solutions. Subsequent precipitation of the product ampicillin positively influences the efficiency of the biocatalytic process. As a result, ampicillin synthesis proceeds in 93% conversion on 6-APA and in 60% conversion on d -(−)-phenylglycine methyl ester.
54 citations
TL;DR: In this article, the thermal stability of penicillin V acylase from Streptomyces lavendulae in water-organic cosolvent monophasic systems was investigated.
41 citations
TL;DR: A new biocatalyst with enhanced activity in the hydrolysis of penicillin V respect to its soluble counterpart is presented, which can be recycled for at least 50 consecutive batch reactions without loss of catalytic activity.
Abstract: Penicillin acylase from Streptomyces lavendulae has been covalently immobilized to epoxy-activated acrylic beads (Eupergit C). Consecutive modification of the matrix with bovine serum albumin leads to a new biocatalyst (ECPVA) with enhanced activity (1.5 fold) in the hydrolysis of penicillin V respect to its soluble counterpart. This biocatalyst had a Km value of 7.6 mM, slightly higher than Km for native acylase (3 mM). In addition, ECPVA can be recycled for at least 50 consecutive batch reactions without loss of catalytic activity.
38 citations
TL;DR: By marker exchange mutagenesis, Bacillus megaterium strain UN‐1 was used to prepare a mutant strain B. megateria UN‐cat (Bm‐UNcat) lacking the penicillin G acylase gene (pac), and the resultant plasmid, pBA402, was introduced into Bm‐ UNcat and Bacillus subtilis.
Abstract: By marker exchange mutagenesis, Bacillus megaterium strain UN-1 (Bm-UN1) was used to prepare a mutant strain B. megaterium UN-cat (Bm-UNcat) lacking the penicillin G acylase gene (pac). The pac gene from Bm-UN1 was subcloned into pTF6 and the resultant plasmid, pBA402, was introduced into Bm-UNcat and Bacillus subtilis. Bm-UNcat harbouring pBA402 produced high penicillin G acylase (PAC) activity of 13·7, 19·5 and 20·4 U ml−1 at 24, 36 and 48 h of culture, respectively. This was two- to fivefold higher than PAC produced by B. subtilis harbouring pBA402 and about 20-fold higher than PAC produced by the parent strain, Bm-UN1.
30 citations
TL;DR: In this paper, a new catalytic membrane has been prepared using a nylon membrane grafted by γ-radiation with methylmethacrylate (MMA) and using hexamethylenediamine (HMDA) as spacer.
Abstract: A new catalytic membrane has been prepared using a nylon membrane grafted by γ-radiation with methylmethacrylate (MMA) and using hexamethylenediamine (HMDA) as spacer. Penicillin G acylase (PGA) and cephalexin were employed as catalyst and substrate, respectively. Cephalexin hydrolysis was studied in bioreactors operated under isothermal and non-isothermal conditions. A hydrolysis increase was found when the temperature of the warm membrane surface was kept constant and the temperature of the other membrane surface was kept at a lower value. The hydrolysis increase was linearly proportional to the applied temperature difference. Cephalexin hydrolysis increased to about 10% when a temperature difference of 1°C was applied across the catalytic membrane. These results have been attributed to the non-isothermal cephalexin transport across the membrane, i.e., to the process of thermodialysis. In this way, the enzyme immobilized on and into the membrane reacts with a substrate concentration higher than that produced by simple diffusion under isothermal conditions.
TL;DR: A new immobilized ECPVA was obtained by covalent binding of penicillin acylase from Streptomyces lavendulae on Eupergit C by utilizing a 23 factorial design of experiments, and the selected parameters for this study were pH, temperature and substrate concentration.
Abstract: A new immobilized penicillin acylase (ECPVA) was obtained by covalent binding of penicillin acylase from Streptomyces lavendulae on Eupergit C. Enzymic hydrolysis of penicillin V catalysed by ECPVA was optimized using a 2(3) factorial design of experiments, and the selected parameters for this study were pH, temperature and substrate concentration. The immobilized enzyme showed an optimal pH value of 9.5-10.5, and an optimal temperature of 60 degrees C, whereas its soluble counterpart showed the same optimal pH value and a lower optimal temperature of 50 degrees C.
TL;DR: In this article, a penicillin G acylase (PGA) has been immobilized onto nylon membranes grafted with methylmethacrylate (MMA) or diethyleneglycoldimethacaryl (DGDA) monomers by means of γ-radiation.
Abstract: Penicillin G acylase (PGA) has been immobilized onto nylon membranes grafted with methylmethacrylate (MMA) or diethyleneglycoldimethacrylate (DGDA) monomers by means of γ-radiation. Hexamethylenediamine (HMDA) has been used as spacer between the grafted membranes and the enzyme. Glutaraldehyde (GA) was used as crosslinking to couple the enzyme to the HMDA. The catalytic membranes so prepared were studied as a function of pH and temperature of the solution containing the substrate. The membranes showing the best characteristics were the ones grafted with DGDA. The dependence of the behavior of these membranes on several experimental conditions was studied, i.e., the temperature and duration of the aminoalkylation process, spacer concentration, the glutaraldehyde concentration and the enzyme concentration. The experimental conditions giving the best performance of the catalytic membranes have been deduced. The time requested to obtain 50% of substrate conversion, i.e., hydrolysis of cephalexin, has been studied as a function of its initial concentration.
TL;DR: In this article, the effects of organic solvents on the penicillin acylase-catalyzed, kinetically controlled synthesis of cefazolin have been examined in various water-solvent mixtures.
Abstract: The effects of organic solvents on the penicillin acylase-catalyzed, kinetically controlled synthesis of cefazolin have been examined in various water–solvent mixtures. In the presence of water-miscible solvents, the initial rate and maximum yield of cefazolin (CEZ) synthesis reaction were found to be reduced. The extent of inhibition was increased with increasing hydrophobicity of the solvent in the reaction mixtures. Enzymatic synthesis of cefazolin was also carried out in the water–solvent biphasic systems. Among the water-immiscible solvents tested, ethyl acetate (EtOAc) and carbon tetrachloride (CCl 4 ) were found to markedly improve the yield of cefazolin in the two-phase reaction system. Our study showed that the enhancement effect of EtOAc and CCl 4 on the synthetic yield was mainly caused by a reduction of the hydrolysis of acyl donor and product in the two-phase system rather than extraction of the product into the solvent phase.
TL;DR: Penicillin V acylase (EC 3.5.1.11) from Streptomyces lavendulae showed both enhanced activity and stability in mixed water/ glycerol and water/glycols solvents, but further addition of the latter led to a gradual protein deactivation.
Abstract: Penicillin V acylase (EC 3.5.1.11) from Streptomyces lavendulae showed both enhanced activity and stability in mixed water/glycerol and water/glycols solvents. The catalytic activity was increased up to a critical concentration of these cosolvents, but further addition of the latter led to a gradual protein deactivation. The highest stabilizing effect was achieved in the presence of glycerol. Thermal stability was increased proportionally to the concentration of glycerol and glycols in the reaction mixture only if the amount added is below the threshold concentration. Reaction conditions that allow simultaneously enhanced activity and stability in the hydrolysis of penicillin V catalyzed by penicillin V acylase from S. lavendulae could be established.
TL;DR: Evidence is presented that the active enzyme is localized in the periplasmic space and maturation of pro-enzyme occurs during transport through the cytoplasmic membrane or rapidly after its entrance in theperiplasm.
TL;DR: A reasonable representation of this system was achieved under some operational conditions, but the model failed under others, Nevertheless, it will be useful whenever a simplified model is required, e.g., in model-based control algorithms for the enzymatic reactor.
Abstract: We present a kinetic model for the synthesis of amoxicillin from p-hydroxy-phenylglycine methyl ester and 6-aminopenicillanic acid, catalyzed by penicillin G acylase immobilized on agarose, at 25°C. Michaelis-Menten kinetic parameters (with and without inhibition) were obtained from initial velocity data (pH 7.5 and 6.5). Amoxicillin synthesis reactions were used to validate the kinetic model after checking mass transport effects. A reasonable representation of this system was achieved under some operational conditions, but the model failed under others. Nevertheless, it will be useful whenever a simplified model is required, e.g., in model-based control algorithms for the enzymatic reactor.
TL;DR: In this study, several fermentation media were tested for the production of penicillin G acylase (PGA) using Bacillus megaterium and showed a strong correlation between the nitrogen source and enzyme yield and the presence of glucose repression.
Abstract: In this study, several fermentation media were tested for the production of penicillin G acylase (PGA) using Bacillus megaterium. The carbon sources studied were glucose and lactose. The nitrogen sources studied were enzymatic casein hydrolysates produced with proteases of different specificities. The replacement of glucose with cheese whey and the addition of free amino acids in the PGA production were also tested. The results showed a strong correlation between the nitrogen source and enzyme yield and the presence of glucose repression. The highest enzyme concentration achieved was 138 IU/L using casein hydrolyzed with 0.6 L of Alcalase® and cheese whey.
TL;DR: A one-step method was developed to purify cephalothin from the enzymatic reaction mixture with the purity of 91% and the recovery yield of 96%.
TL;DR: In toluene, Celite R-640 is able to maintain the water activity constant within broad ranges of water concentrations, and the a w values are strongly related to the original hydration of the Celite batches, but prolonged drying confers comparable and reproduced properties to the different batches.
Abstract: The study describes how Celite R-640 adsorbs liquid water in toluene and vapour water from a gas phase. In toluene, Celite R-640 is able to maintain the water activity ( a w ) constant within broad ranges of water concentrations. The a w values are strongly related to the original hydration of the Celite batches, but prolonged drying confers comparable and reproducible properties to the different batches. The use of Celite R-640 in controlling the hydration and activity of covalently immobilised PGA in toluene is reported.
TL;DR: It was shown that the processing rates of wild-type and mutant precursor PAs depended on the pKa values of their side chain R group, and it was demonstrated that the lysine residue (K299) was involved in the precursor processing of PA together with N-terminal serine residue of the large subunit.
TL;DR: Structural data is presented that pertain to the unanswered questions that arose from the original strucutre of Penicillin Gacylase from Escherichia coli, and mutations that allow processing but yield inactive protein provide the structure of an ES complex with a true substrate.
Abstract: Penicillin acylases are used in the pharmaceutical industry for the preparation of antibiotics. The 3-D structure of Penicillin G acylase from Escherichia coli has been solved. Here, we present structural data that pertain to the unanswered questions that arose from the original strucutre. Specificity for the amide portion of substrate was probed by the structure determination of a range of complexes with substitutions around the phenylacetyl ring of the ligand. Altered substrate specificity mutations derived from an in vivo positive selection process have also been studied, revealing the structural consequences of mutation at position B71. Protein processing has been analyzed by the construction of site-directed mutants, which affect this reaction with two distinct phenotypes. Mutations that allow processing but yield inactive protein provide the structure of an ES complex with a true substrate, with implications for the enzymatic mechanism and stereospecificity of the reaction. Mutations that preclude processing have allowed the structure of the precursor, which includes the 54 amino acid linker region normally removed from between the A and B chains, to be visualized. Index Entries: Penicillin acylase; 3-D structures; site-directed mutagenesis; substrate binding; autocatalytic processing; precursor.
TL;DR: To the authors' knowledge, this is the first evidence demonstrating the limiting step for the production of P. rettgeri PAC and the existence of the P.Rettgeru PAC precursor.
Abstract: Heterologous production of Providencia rettgeri penicillin acylase (PAC) was optimized in Escherichia coli. Several factors, including carbon, temperature, and host effects, were identified to be critical for the enzyme overproduction. The optimum culture conditions for the enzyme production vary for different host/vector systems. With the optimization, both volumetric and specific PAC activities could be significantly improved by more than 50-fold compared to the native expression in P. rettgeri. The heterologous production could be possibly limited by translation or posttranslational steps, depending on the culture temperature and host/vector system. To our knowledge, this is the first evidence demonstrating the limiting step for the production of P. rettgeri PAC and the existence of the P. rettgeri PAC precursor.
TL;DR: New pseudo-affinity chromatographic supports for penicillin acylase were prepared and evaluated with three different samples: pure penicillins, industrial clarified feedstock and crude extract and may therefore be used on an industrial scale.
TL;DR: These buffers exhibited excellent performance in a variety of enzyme reactions in the MIER, such as in the case of penicillin G acylase and histidine decarboxylase, and were found to greatly stabilize enzyme activity, permitting operation of the MIERS over extended periods of time.
Abstract: A novel class of amphoteric, polymeric buffers, is described, consisting of grafting onto growing polyacrylamide chains weakly acidic and basic acrylamido-monomers (called Immobilines; protolytic groups as N-substituents on the nitrogen of the amido bond), for operating a membrane-immobilized enzyme reactor (MIER) in an electric field. With these soluble, polymeric buffers, it is possible to operate the membrane reactor at any optimum of pH activity, for any given enzyme, in the pH 3-10 scale. Such buffers, being amphoteric, are confined in the enzyme reaction chamber by the same isoelectric trapping mechanism. The best buffers were found to be those polymerized in presence of 9% neutral monomer (acrylamide) and containing 20 mM Immobiline as buffering ion. To decrease their viscosity in solution, the polymeric buffers are synthesized at high temperatures (70 degrees C) and in presence of a chain-transfer agent. The weight average molecular size in these conditions has been found to be ca. 200,000 Da. These buffers exhibited excellent performance in a variety of enzyme reactions in the MIER, such as in the case of penicillin G acylase and histidine decarboxylase and were found to greatly stabilize enzyme activity, permitting operation of the MIER over extended periods of time. As an example, in a penicillin G acylase reactor, >75% enzyme activity was maintained over a 10-d cycle of operation, while with conventional buffers more than 90% inactivation was experienced over the same period of time. This novel class of macromolecular, amphoteric buffers could also be exploited in other types of conventional bioreactors not based on an isoelectric trapping mechanism.
TL;DR: A novel activity of penicillin G acylase from E. coli, immobilized onto agarose gels by multipoint covalent attachment, was used for removing the benzyloxycarbonyl amino protecting group from selected amino acids and oligopeptides in different reaction systems.
Abstract: A novel activity of penicillin G acylase from E. coli is presented. This enzyme, immobilized onto agarose gels by multipoint covalent attachment, was used for removing the benzyloxycarbonyl (Z) amino protecting group from selected amino acids and oligopeptides in different reaction systems. Quantitative deprotection yields were obtained for simple amino acids, while the efficiency with oligopeptides varied depending on the sequence.
TL;DR: Investigation of the influence of the cultivation temperature, phenylacetic acid, and glucose on the proteolysis of the ppPA in the cytoplasm explains a new aspect of regulation of active PA production in E. coli.
Abstract: The yield of periplasmic enzyme, penicillin amidase (PA), from E. coli ATCC 11105 is regulated at the post-translational level by two competitive processes-intracellular proteolysis of newly synthesised pre-pro-PA (ppPA) in the cytoplasm and membrane transport and maturation of ppPA. Intracellular proteolysis results in a significant loss in the yield of active PA. Immunochemical analysis were used to study the influence of the cultivation temperature, phenylacetic acid, and glucose on the proteolysis of the ppPA in the cytoplasm. An increase in glucose concentrations or temperature during the cultivation resulted in a significant loss of PA activity due to the increased rate of intracellular proteolysis of ppPA. Addition of phenylacetic acid reduced the intracellular proteolysis of ppPA and, as a consequence, increased the PA production. Taken together, these data explain a new aspect of regulation of active PA production in E. coli.
TL;DR: The main purpose of this work was to study the stability of a dimeric enzyme, penicillin acylase, in reversed micelles and the sugar alcohol mannitol shown to provide an increase of more than 300% in the retention of activity.
Abstract: The main purpose of this work was to study the stability of a dimeric enzyme, penicillin acylase, in reversed micelles. Kinetic studies were carried out in order to understand the enzyme behaviour in reversed micelles. The enzyme activity profile as a function of the water content of the system exhibited three maxima at w0 equal to 14, 20 and 23, which were assigned to the light sub-unit, to the heavy sub-unit and to the enzyme itself. The effect of AOT concentration on the activity of penicillin acylase was also studied and an increase in the surfactant concentration, at a constant w0 was shown to produce an increase in enzyme activity. The influence of the water content (w0), AOT and enzyme concentration on the enzyme stability in reversed micelles was then investigated.Enzyme stabilisation by the addition of alcohols and sugars was examined and the sugar alcohol mannitol shown to provide an increase of more than 300% in the retention of activity.
TL;DR: A bipyramidal crystal form was obtained from a solution containing polyethylene glycol (MW 3350) and calcium chloride as discussed by the authors, which is tetragonal with the space group P4(1)2(1)/P4(3)2/1/2.
TL;DR: It was concluded that, when the ultrafiltration operation retained 100% of protein, the concentrates from reverse osmosis could be successfully directly fed to the enzyme reactor, giving high enzymatic conversion yield of benzylpenicillin to 6-aminopenicillanic acid.
Abstract: Benzylpenicillin filtered broths purified by ultrafiltration and fermented broths clarified by ultrafiltration and afterwards concentrated by reverse osmosis were used directly for enzymatic conversion of benzylpenicillin to 6-aminopenicillanic acid and phenylacetic acid by immobilised penicillin G acylase or amidase. It was concluded that, when the ultrafiltration operation retained 100% of protein, the concentrates from reverse osmosis could be successfully directly fed to the enzymatic reactor, giving high enzymatic conversion yield of benzylpenicillin to 6-aminopenicillanic acid.