Penicillin G Acylase Research Articles

Characterization of the penicillin G acylase from Bacillus megaterium ATCC 14945

The purpose of this work was to characterize the enzyme penicillin G acylase (PGA) produced by Bacillus megaterium. Purification of the enzyme by ultra/diafiltration did not allow the detection of the PGA band by SDS-PAGE electrophoresis due to the high content of remaining proteins. However, using the DNA of the microorganism, it was possible to replicate the genes of the two B. megaterium PGA reported in literature, showing that the enzyme consisted of two sub-units, having 245 and 537 amino acids each and an average molecular mass of 26950 and 59070 Da, respectively. The parameters studied were: 1) the influence of temperature in the 25-60(0)C range, 2) pH in the 5-10 range and 3) substrate concentration, this was tested to obtain results on the Penicillin G hydrolysis reaction rate, using the initial velocities approach. The maximum hydrolysis rate was obtained at 37ºC and pH 8.0. The Michaelis-Menten model fitted well, resulting in estimated Km and Vmax parameters values of 1.83 mM and 0.165*10-3 mmol/min/UI, respectively.

Enzyme distribution and matrix characteristics in biocatalytic particles

In a study of Assemblase ®, an industrial immobilized penicillin-G acylase, various electron microscopic techniques were used to relate intra-particle enzyme heterogeneity with the morphological heterogeneity of the support material at various levels of detail. Transmission electron microscopy was used for the study of intra-particle penicillin-G acylase distribution in Assemblase ® particles of various sizes; it revealed an abrupt increase in enzyme loading at the particle surface (1.4-fold) and in the areas (designated halo's) surrounding internal macro-voids (7.7-fold). Cryogenic field-emission scanning electron microscopy related these abrupt local enzyme heterogeneities to local heterogeneity of the support material by revealing the presence of dense top layers surrounding both the particle exterior and the internal macro-voids. Furthermore, it showed a very distinct morphological appearance of the halo. Most probably, all these regions contained relatively more chitosan than gelatin (the polymers Assemblase ® was constructed of), which suggested local polymer demixing during particle production. A basic thermodynamic line of reasoning suggested that a difference in hydrophilicity between the two polymers induced local demixing. In the future, thermodynamic knowledge on such polymer interactions resulting in matrix heterogeneity may be used as a tool for biocatalyst design.

Selectivity of the enzymatic synthesis of ampicillin by E. coli PGA in the presence of high concentrations of substrates

Penicillin G acylase (PGA) catalyzes the synthesis/hydrolysis of acyl derivatives of phenylacetic acid through the formation of a covalent intermediate (the acyl–enzyme complex). When used for the kinetically controlled synthesis of β-lactam antibiotics, this enzyme promotes two undesired side reactions: the hydrolysis of the acyl side-chain precursor and of the antibiotic. Therefore, a high selectivity (synthesis/hydrolysis, S/H ratio) is very important for the process economics. Here, the enzymatic synthesis of ampicillin from d-phenylglycine methyl ester (PGME) and 6-aminopenicillic acid (6-APA), using PGA from Escherichia coli (EC 3.5.1.11) is studied. Kinetic assays provided S/H for high concentrations of substrates (up to 200 mM of 6-APA and 500 mM of PGME), using soluble PGA, at 25 °C, pH 6.5. S/H increased with 6-APA concentration, in accordance with the literature. However, when the concentration of 6-APA approached saturation, the rate of enzymatic hydrolysis tended towards zero (i.e., S/H tended to infinity). On the other hand, when the concentration of ester was augmented, S/H consistently decreased. This behavior, to the best of our knowledge still not reported, indicates that the acylation step may occur with 6-APA already positioned for the nucleophilic attack.

Stabilization of enzymes by multipoint immobilization of thiolated proteins on new epoxy‐thiol supports

The controlled and partial modification of epoxy groups of Eupergit C and EP-Sepabeads with sodium sulfide has permitted the preparation of thiol-epoxy supports. Their use allowed not only the specific immobilization of enzymes through their thiol groups via thiol-disulfide interchange, but also enzyme stabilization via multipoint covalent attachment. Penicillin G acylase (PGA) from Escherichia coli and lipase from Rhizomucor miehei were used as model enzymes. Both enzymes lacked exposed cysteine residues, but were introduced via chemical modification under very mild conditions. In the first moments of the immobilization, a certain percentage of immobilized protein could be released from the support by incubation with DTT; this confirms that the first step was via a thiol-disulfide interchange. Moreover, the promotion of some further epoxy-enzyme bonds was confirmed because no enzyme release was detected after some immobilization time by incubation with DTT. In the case of the heterodimeric PGA, it was possible to demonstrate the formation of at least one epoxy bond per enzyme subunit by analyzing with SDS-PAGE the supernatants obtained after boiling the enzyme derivatives in the presence of mercaptoethanol and SDS. Thermal inactivation studies showed that these multipoint enzyme-support attachments promoted an increase in the stability of the immobilized enzymes. In both cases, the stabilization factor was around 12-15-fold comparing optimal derivatives with their just-thiol immobilized counterparts.

Penicillin G acylase catalyzed acylation of 7-ACA in aqueous two-phase systems using kinetically and thermodynamically controlled strategies: improved enzymatic synthesis of 7-[(1-hydroxy-1-phenyl)-acetamido]-3-acetoxymethyl-Δ 3-cephem-4-carboxylic acid

Penicillin G acylase (PGA) catalyzed acylation of 7-aminocephalosporanic acid (7-ACA) with R-mandelic acid and its derivatives gives 7-[(1-hydroxy-1-phenyl)-acetamido]-3-acetoxymethyl-Δ 3-cephem-4-carboxylic acid. This compound is a useful intermediate for the synthesis of some 3′-functionalized cephalosporins. However, acylations catalyzed by PGA isolated from Escherichia coli give poor results both considering a kinetical or a thermodynamical approach. In order to improve this enzymatic acylation, polyethylene glycol (PEG 600)-ammonium sulphate aqueous two-phase systems have been studied with the aim to have, during the reaction, a continuous extraction of the acylation product outside of the enzyme environment (the ammonium sulphate phase). This strategy shifts the equilibrium in the thermodynamically controlled synthesis and prevents the hydrolysis of the synthesized antibiotic in the kinetically controlled synthesis. The best results were achieved using PEG 600 (80% in water) equilibrated with 4 M ammonium sulphate. In these conditions, the acylation product was completely partitioned in the PEG phase ( K > 200), whereas the substrates maintained a suitable concentration in the enzyme environment. Both in the kinetic (88% yield) and the thermodynamic (75% yield) processes, the results obtained were sensitively improved in comparison with those achieved working in homogeneous solution (phosphate buffer). Using R-mandelic acid methyl ester, the yield increased from 65% (monophasic system) to 88%. The PEG solution, without isolation of the acylation product, was successfully used for the synthesis of Cefamandole and Cefonicid.

Kinetic study on substrate and product inhibitions for the formation of 7-amino-3-deacetoxy cephalosporanic acid from cephalosporin G by immobilized penicillin G acylase

Penicillin G acylase (PGA) is an important enzyme in β-lactam antibiotics pharmaceutical industry for the production of 7-amino-3-deacetoxy cephalosporanic acid (7-ADCA) from cephalosporin G (Ceph-G) as well as 6-aminopenicillanic acid (6-APA) from penicillin G (Pen-G). In this work, immobilized PGA was utilized to catalyze the conversion of Ceph-G to 7-ADCA. Higher concentration of Ceph-G was found to show inhibition effect on the reaction. Both 7-ADCA and PAA (phenylacetic acid) were found to inhibit the activity of PGA, however, via different mechanisms. In this work, with low concentrations of Ceph-G substrate, the inhibited kinetic models were obtained by adding different concentrations of PAA or 7-ADCA. The influence from both products was discussed and compared. Both inhibition kinetic mechanisms were different in that PAA showed noncompetitive inhibition to PGA whereas 7-ADCA appeared to be of competitive inhibition.

Novel approach to quantify immobilized‐enzyme distributions

The quantitative intraparticle enzyme distribution of Assemblase, an industrially employed polydisperse immobilized penicillin-G acylase, was measured. Because of strong autofluorescence of the carrier, the generally applied technique of confocal scanning microscopy could not be used; light microscopy was our method of choice. To do so, Assemblase particles of various sizes were sectioned, labeled with antibodies specifically against the enzyme, and analyzed light microscopically. Image analysis software was developed and used to determine the intraparticle enzyme distribution, which was found to be heterogeneous, with most enzyme located in the outer regions of the particles. Larger particles showed steeper gradients than smaller ones. A mathematical representation of the intraparticle profiles, based on in-stationary enzyme diffusion into the particles, was validated successfully for a broad range of particle sizes using data for volume-averaged particle size and enzyme loading. The enzyme gradients determined in this work will be used as input for a physical model that quantitatively describes the complex behavior of Assemblase. Such a physical model will lead to identification of the current bottlenecks in Assemblase and can serve as a starting point for the design of improved biocatalysts that also may be based on intelligent use of enzyme gradients.

Influence of Substrate Structure on PGA-Catalyzed Acylations. Evaluation of Different Approaches for the Enzymatic Synthesis of Cefonicid

The influence of the substrate structure on the catalytic properties of penicillin G acylase (PGA) from Escherichia coli in kinetically controlled acylations has been studied. In particular, the affinity of different β-lactam nuclei towards the active site has been evaluated considering the ratio between the rate of synthesis (v s ) and the rate of hydrolysis of the acylating ester (v h 1 ). 7-Aminocephalosporanic acid (7-ACA) and 7-amino-3-(1-sulfomethyl-1,2,3,4-tetrazol-5-yl)thiomethyl-3-cephem-4-carboxylic acid (7-SACA) showed a good affinity for the active centre of PGA. The enzymatic acylation of these nuclei with R-methyl mandelate has been studied in order to evaluate different approaches for the enzymatic synthesis of cefonicid. The best results have been obtained in the acylation of 7-SACA. Cefonicid (8) was recovered from the reaction mixture as the disodium salt in 65% yield and about 95% of purity. Furthermore, through acylation of 7-ACA, a one-pot chemo-enzymatic synthesis was carried out starting from cephalosporin C using three enzymes in sequence: D-amino acid oxidase (DAO), glutaryl acylase (GA) and PGA. Cefonicid disodium salt was obtained in three steps, avoiding any intermediate purification, in 35% overall yield and about 94% purity. This approach presents several advantages compared with the classical chemical processes.

Effects of spacer arm on penicillin G acylase purification using immobilized metal affinity membranes

For studying penicillin G acylase (PGA) purification efficiency, various chain lengths of diamine ( n = 2–10) were applied as spacer arm to prepare the regenerated cellulose-based immobilized metal affinity membrane (IMAM). The effects of spacer arm length on water content of the IMAM, immobilized metal ion capacity, and batch PGA adsorption were investigated. The use of 1,10-diaminodecane led to the smallest water content of membrane and the maximum capacity of Cu 2+ (62.06 μmol/disc), whereas the optimal PGA adsorption capacity (2.6 IU/disc) was achieved with 1,8-diaminooctane. When applying the IMAMs with the spacer arm of 1,8-diaminooctane to the flow chromatographic experiments, a performance of 2.73-fold purification and 70.27% recovery was obtained. The employment of diamine spacer arm on the preparation of IMAMs resulted in higher PGA adsorption amount but less purification efficiency.

Evidence for the involvement of arginyl residue at the active site of penicillin G acylase from Kluyvera citrophila

Penicillin G acylase (PGA) is used for the commercial production of semi-synthetic penicillins. It hydrolyses the amide bond in penicillin producing 6-aminopenicillanic acid and phenylacetate. 6-Aminopenicillanic acid, having the beta-lactam nucleus, is the parent compound for all semi-synthetic penicillins. Penicillin G acylase from Kluyvera citrophila was purified and chemically modified to identify the role of arginine in catalysis. Modification with 20 mM phenylglyoxal and 50 mM 2,3-butanedione resulted in 82% and 78% inactivation, respectively. Inactivation was prevented by protection with benzylpenicillin or phenylacetate at 50 mM. The reaction followed psuedo-first order kinetics and the inactivation kinetics (V(max), K(m), and k(cat)) of native and modified enzyme indicates the essentiality of arginyl residue in catalysis.

Design of large-pore mesoporous materials for immobilization of penicillin G acylase biocatalyst

In this study, functionalization of large-pore nanoporous silica materials was carried out by condensation of tetraethylorthosilicate (TEOS) and 3-aminopropyltriethoxysilane (APTES), 3-mercaptopropyltrimethoxysilane (MPTMS), phenyltrimethoxysilane (PTMS), vinyltriethoxysilane (VTES), and 4-(triethoxysilyl)butyronitrile (TSBN), respectively, in the presence of non-ionic surfactant under acidic conditions. The TSBN functionality was subsequently converted to carboxyl group while APTES was further functionalized with glutardialdehyde, a cross linker. The various functionalized materials were used as supports for immobilization of enzyme penicillin G acylase (PGA). Experimental data showed that the functionalized materials except for the material functionalized with MPTMS possess a faster loading kinetics and a higher loading amount of enzyme PGA than the pure-silica counterpart. The enzymatic catalytic activities of the immobilized biocatalysts varied from 52.2 to 167.5 U/g of solid. The glutardialdehyde-activated material displayed the highest initial immobilized enzyme activity and the most stable activity among all the support materials. PGA immobilized on VTES-functionalized nanoporous silica showed the highest initial enzymatic activity (67.7 U/mg of PGA, much higher than that of free PGA (300 U/mg of PGA). Experimental data along with theoretical analysis results indicate that glutardialdehyde is a good cross linker, offering covalent binding of PGA with the support materials while VTES-functionalized nanoporous silica is a very good potential support for physical entrapment of PGA enzyme.

Production of a fully functional, permuted single-chain penicillin G acylase.

Penicillin G acylase (PGA) is a heterodimeric enzyme synthesized as a single-polypeptide precursor that undergoes an autocatalytic processing to remove an internal spacer peptide to produce the active enzyme. We constructed a single-chain PGA not dependent on autoproteolytic processing. The mature sequence of the beta-domain was expressed as the N terminus of a new polypeptide, connected by a random tetra-peptide to the alpha-domain, to afford a permuted protein. We found several active enzymes among variants differing in their linker peptides. Protein expression analysis showed that the functional single-chain variants were produced when using a Sec-dependent leader peptide, or when expressed inside the bacterial cytoplasm. Active-site titration experiments showed that the single-chain proteins displayed similar k(cat) values to the ones obtained with the wild-type enzyme. Interestingly, the single-chain proteins also displayed close to 100% of functional active sites compared to 40% to 70% functional yield usually obtained with the heterodimeric protein.

Optimization of the host–plasmid interaction in the recombinant Escherichia coli strains overproducing penicillin G acylase

Segregational stability of the recombinant plasmid pKA18 bearing the pga gene encoding penicillin G acylase (PGA) and the production stability for PGA was studied in Escherichia coli strain W and its descendants. The hosts differ in the number of indigenous plasmids (pRK1–3) and the specific activity of PGA. The production of PGA by the recombinant strain RE3(pKA18) is the highest among the hosts and the increase of activity is proportional to the increase of copy number (CN) of the plasmid pKA18. The plasmid is segregationally stable in spite of the presence of the indigenous plasmid pRK2 that belong to the same ColE1 plasmid family. Both plasmids (total sum of CN equals 271) are maintained without selection pressure during 102 generations. Subculture-based selection experiments taking advantage of a very high segregational and production stability of plasmid pKA18 in the host RE3 were used to study the optimization of the host–plasmid interaction. The clones with increased synthesis of the enzyme average value of 833 U/g DM were isolated: the expression of PGA per plasmid molecule increased in these strains from 3 to 4 U. Once this expression is reached, the population of segregationally stable, high-producing strains becomes heterogeneous: segregationally stable, low-producing clones and segregationally unstable, high-producing clones appear in population.

Modifying the substrate specificity of penicillin G acylase to cephalosporin acylase by mutating active-site residues

The penicillin G acylase (PGA) and cephalosporin acylase (CA) families, which are members of the N-terminal (Ntn) hydrolases, are valuable for the production of backbone chemicals like 6-aminopenicillanic acid and 7-aminocephalosporanic acid (7-ACA), which can be used to synthesize semi-synthetic penicillins and cephalosporins, respectively. Regardless of the low sequence similarity between PGA and CA, the structural homologies at their active-sites are very high. However, despite this structural conservation, they catalyze very different substrates. PGA reacts with the hydrophobic aromatic side-chain (the phenylacetyl moiety) of penicillin G (PG), whereas CA targets the hydrophilic linear side-chain (the glutaryl moiety) of glutaryl-7-ACA (GL-7-ACA). These different substrate specificities are likely to be due to differences in the side-chains of the active-site residues. In this study, mutagenesis of active-site residues binding the side-chain moiety of PG changed the substrate specificity of PGA to that of CA. This mutant PGA may constitute an alternative source of engineered enzymes for the industrial production of 7-ACA.

A novel support of MCM-48 molecular sieve for immobilization of penicillin G acylase

As a novel support of immobilizing penicillin G acylase (PGA), MCM-48 and Co-MCM-48 molecular sieves were synthesized and characterized by XRD, N 2 adsorption, NH 3-TPD, FT-IR and so on. The studies show that MCM-48 and Co-MCM-48 has well ordered long-range structure, narrow pore size distribution, larger surface area and higher concentration of the weakly acidic silanol groups on their surface. Penicillin G acylase was immobilized on MCM-48 or Co-MCM-48 by interacting silanol groups on the surface. The presence of cobalt in the framework of MCM-48 increases the amount of the weak acid sites. For the hydrolysis of penicillin G catalyzed by PGA/Co-MCM-48 (Co/Si=0.01), its specific activity reaches 1682 U/g. After used for six cycles, PGA/MCM-48(0.01) can keep 1375 U/g of the specific activity. If MCM-41 was used as the support, the activity of immobilized PGA is only 402 U/g.

Modifying the substrate specificity of penicillin G acylase to cephalosporin acylase by mutating active-site residues

The penicillin G acylase (PGA) and cephalosporin acylase (CA) families, which are members of the N-terminal (Ntn) hydrolases, are valuable for the production of backbone chemicals like 6-aminopenicillanic acid and 7-aminocephalosporanic acid (7-ACA), which can be used to synthesize semi-synthetic penicillins and cephalosporins, respectively. Regardless of the low sequence similarity between PGA and CA, the structural homologies at their active-sites are very high. However, despite this structural conservation, they catalyze very different substrates. PGA reacts with the hydrophobic aromatic side-chain (the phenylacetyl moiety) of penicillin G (PG), whereas CA targets the hydrophilic linear side-chain (the glutaryl moiety) of glutaryl-7-ACA (GL-7-ACA). These different substrate specificities are likely to be due to differences in the side-chains of the active-site residues. In this study, mutagenesis of active-site residues binding the side-chain moiety of PG changed the substrate specificity of PGA to that of CA. This mutant PGA may constitute an alternative source of engineered enzymes for the industrial production of 7-ACA.

Cloning, overexpression, and characterization of a novel thermostable penicillin G acylase from Achromobacter xylosoxidans: probing the molecular basis for its high thermostability.

The gene encoding a novel penicillin G acylase (PGA), designated pgaW, was cloned from Achromobacter xylosoxidans and overexpressed in Escherichia coli. The pgaW gene contains an open reading frame of 2586 nucleotides. The deduced protein sequence encoded by pgaW has about 50% amino acid identity to several well-characterized PGAs, including those of Providencia rettgeri, Kluyvera cryocrescens, and Escherichia coli. Biochemical studies showed that the optimal temperature for this novel PGA (PGA650) activity is greater than 60 degrees C and its half-life of inactivation at 55 degrees C is four times longer than that of another previously reported thermostable PGA from Alcaligenes faecalis (R. M. D. Verhaert, A. M. Riemens, J. V. R. Laan, J. V. Duin, and W. J. Quax, Appl. Environ. Microbiol. 63:3412-3418, 1997). To our knowledge, this is the most thermostable PGA ever characterized. To explore the molecular basis of the higher thermostability of PGA650, homology structural modeling and amino acid composition analyses were performed. The results suggested that the increased number of buried ion pair networks, lower N and Q contents, excessive arginine residues, and remarkably high content of proline residues in the structure of PGA650 could contribute to its high thermostability. The unique characteristic of higher thermostability of this novel PGA provides some advantages for its potential application in industry.

Optimization of the host?plasmid interaction in the recombinant Escherichia coli strains overproducing penicillin G acylase

Segregational stability of the recombinant plasmid pKA18 bearing the pga gene encoding penicillin G acylase (PGA) and the production stability for PGA was studied in Escherichia coli strain W and its descendants. The hosts differ in the number of indigenous plasmids (pRK1–3) and the specific activity of PGA. The production of PGA by the recombinant strain RE3(pKA18) is the highest among the hosts and the increase of activity is proportional to the increase of copy number (CN) of the plasmid pKA18. The plasmid is segregationally stable in spite of the presence of the indigenous plasmid pRK2 that belong to the same ColE1 plasmid family. Both plasmids (total sum of CN equals 271) are maintained without selection pressure during 102 generations. Subculture-based selection experiments taking advantage of a very high segregational and production stability of plasmid pKA18 in the host RE3 were used to study the optimization of the host–plasmid interaction. The clones with increased synthesis of the enzyme average value of 833 U/g DM were isolated: the expression of PGA per plasmid molecule increased in these strains from 3 to 4 U. Once this expression is reached, the population of segregationally stable, high-producing strains becomes heterogeneous: segregationally stable, low-producing clones and segregationally unstable, high-producing clones appear in population.

Evaluation of a monolithic epoxy silica support for penicillin G acylase immobilization

In this work, a new type of penicillin G acylase (PGA)-based monolithic silica support was developed and evaluated for the chiral separation in HPLC. The preparation procedure consisted of two steps: preparation of an epoxy derivatized monolithic silica column and chemical modification of the epoxide groups with the enzyme chiral selector. The epoxy Silica-Rod column for the immobilization of PGA was prepared with the in situ modification process by using epoxy-silanes and the identification of the species bound to the surface was achieved by solid-state nuclear magnetic resonance. The enzyme was covalently immobilized to the surface of the derivatized monolithic column. The enantioselectivity and the performance of the developed column are discussed and compared to the corresponding experimental data obtained with a PGA-based microparticulate (5 μm) silica column.

Effect of temperature on the enzymatic synthesis of cefaclor with in situ product removal

An integrated process of cefaclor synthesis from phenylglycine methyl ester (PGME) and 7-aminodesacetoxymethyl-3-chlorocephalosporanic acid (7-ACCA) catalyzed by penicillin G acylase (PGA) with in situ product removal (ISPR) was established. The integrated process was more significantly influenced by temperature than the separate synthesis process as without ISPR. The difference between the overall yields with and without ISPR was minified as reaction temperatures rose. For instance, the maximum 7-ACCA conversion was 86 and 68%, respectively, in the process with and without ISPR at 5 °C. Both the maximum conversions, however, decreased to around 45% at 40 °C. The effect of substrate concentration on the overall conversion was also obviously dependent on the reaction temperature. The product cefaclor inhibited PGME hydrolysis in the enzymatic synthesis. ISPR stimulated cefaclor synthesis at lower temperatures, but was not useful at higher temperatures which accelerated PGME hydrolysis.