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Showing papers on "Penicillin amidase published in 1993"


Journal ArticleDOI
TL;DR: An amino acid sequence similar to that of the active site of thioesterases was found in the isopenicillin-N acyltransferase, suggesting that this site is involved in the transfer of phenylacetyl residues from phenyl acetyl thioesters.
Abstract: The isopenicillin-N acyltransferase of Penicillium chrysogenum catalyzes the conversion of the biosynthetic intermediate isopenicillin N to the hydrophobic penicillins. The isopenicillin-N acyltransferase copurified with the acyl-CoA:6-aminopenicillanic acid (6-APA) acyltransferase activity which transfers an acyl residue from acyl-CoA derivatives (e.g. phenylacetyl-CoA, phenoxyacetyl-CoA) to 6-APA. Other thioesters of phenylacetic acid were also used as substrates. An amino acid sequence similar to that of the active site of thioesterases was found in the isopenicillin-N acyltransferase, suggesting that this site is involved in the transfer of phenylacetyl residues from phenylacetyl thioesters. Purified isopenicillin-N acyltransferase also showed isopenicillin-N amidohydrolase, penicillin transacylase and penicillin amidase activities. The isopenicillin-N amidohydrolase (releasing 6-APA) showed a much lower specific activity than the isopenicillin-N acyltransferase of the same enzyme preparation, suggesting that in the isopenicillin-N acyltransferase reaction the 6-APA is not released and is directly converted into benzylpenicillin. Penicillin transacylase exchanged side chains between two hydrophobic penicillin molecules; or between one penicillin molecule and 6-APA. The penicillin amidase activity is probably the reverse of the biosynthetic acyl-CoA:6-APA acyltransferase. Four P. chrysogenum mutants deficient in acyl-CoA:6-APA acyltransferase lacked the other four related activities. Transformation of these mutants with the penDE gene restored all five enzyme activities.

88 citations


Journal ArticleDOI
TL;DR: In this article, the authors developed a strategy for immobilization-stabilization of penicillin G acylase (PGA) from Kluyvera citrophila by controlled multipoint covalent attachment to agarose-aldehyde gels.
Abstract: We have developed a strategy for immobilization-stabilization of penicillin G acylase (PGA) from Kluyvera citrophila by controlled multipoint covalent attachment to agarose-aldehyde gels. This enzyme is composed by two dissimilar subunits noncovalently bound. Thus, in this article we establish clear correlations between enzyme stabilization and the multipoint immobilization and/or between enzyme stabilization and the involvement of the two subunits in the attachment of them to the support. We have demonstrated that important thermal stabilizations of derivatives were only obtained through a very intense enzyme-support multipoint attachment involving the whole enzyme molecule. In this way, we have prepared derivatives preserving more than 90% of catalytic activity and being more than 1000-fold more stable than soluble and one-point attached enzyme. In addition, the involvement of the two subunits in the covalent attachment to the support has proved to be essential to develop interesting strategies for reactivation of inactivated enzyme molecules [e.g., by refolding of immobilized PGA after previous unfolding with urea and sodium dodecyl sulfate (SDS)].

76 citations


Journal ArticleDOI
TL;DR: The results support the hypothesis that hpaB is a component of the 4-hydroxyphenylacetic acid degradative pathway of E. coli W.
Abstract: The hpaB gene encoding an aromatic hydroxylase of Escherichia coli ATCC 11105, a penicillin G acylase-producing strain, has been cloned and expressed in E. coli K-12. This gene was located near the pacA gene coding for penicillin G acylase. The hydroxylase has a molecular mass of 59,000 Da, uses NADH as a cosubstrate, and was tentatively classified as a 4-hydroxyphenylacetic acid hydroxylase, albeit it exhibited a rather broad substrate specificity acting on different monohydric and dihydric phenols. E. coli W, C, and B as well as Klebsiella pneumoniae M5a1 and Kluyvera citrophila ATCC 21285 (a penicillin G acylase-producing strain) but not E. coli K-12 contained sequences homologous to hpaB. Our results support the hypothesis that hpaB is a component of the 4-hydroxyphenylacetic acid degradative pathway of E. coli W.

73 citations


Journal ArticleDOI
TL;DR: Transformation of E. coli G133 with a pBR322 derivative containing the Hindlll fragment of the PGA gene showed that the performance of E.'s coli G271 depends both upon the host strain properties and the plasmid structure.
Abstract: Penicillin G amidase (PGA) is a key enzyme for the industrial production of penicillin G derivatives used in therapeutics. Escherichia coli ATCC 11105 is the more commonly used strain for PGA production. To improve enzyme yield, the authors constructed various recombinant E. coli HB 101 and ATCC 11105 strains. For each strain, PGA production was determined for various concentrations of glucose and phenylacetic acid (PAA) in the medium. The E. coli strain, G271, was identified as the best performer (800 U NIPAB/L). This strain was obtained as follows: an E. coli ATCC 11105 mutant (E. coli G133) was first selected based on a low negative effect of glucose on PGA production. This mutant was then transformed with a pBR322 derivative containing the PGA gene. Various experiments were made to try to understand the reason for the high productivity of E. coli G271. The host strain, E. coli G133, was found to be mutated in one (or more) gene(s) whose product(s) act(s) in trans on the PGA gene expression. Its growth is not inhibited by high glucose concentration in the medium. Interestingly, whereas glucose still exerts some negative effect on the PGA production by E. coli G133, PGA production by itsmore » transformant (E. coli G271) is stimulated by glucose. The reason for this stimulation is discussed. Transformation of E. coli G133 with a pBR322 derivative containing the HindIII fragment of the PGA gene, showed that the performance of E. coli G271 depends both upon the host strain properties and the plasmid structure. Study of the production by the less efficient E. coli HB101 derivatives brought some light on the mechanism of regulation of the PGA gene.« less

50 citations



Journal ArticleDOI
01 Jan 1993-Polymer
TL;DR: Macroporous beaded polymers of varying pore sizes were synthesized coated with polyethyleneimine and derivatized with glutaraldehyde to generate aldehyde pendant groups and immobilization enhanced the thermal stability of the enzyme.

34 citations



Journal ArticleDOI
TL;DR: In this paper, the same enzyme was used to prepare both L- and D-enantiomers of these phosphorus analogues of alanine by stepwise enzymatic hydrolysis of their racemic N-phenylacetyl derivatives.
Abstract: D-Enantiomers of N-acylated 1-aminoethylphosphonic and 1-aminoethylphosphonous acids were able to be hydrolyzed with high concentrations of penicillin acylase in a reasonable time period. This finding was used to prepare both L- and D-enantiomers of these phosphorus analogues of alanine by stepwise enzymatic hydrolysis of their racemic N-phenylacetyl derivatives using the same enzyme - penicillin acylase - by simply changing the enzyme/ substrate ratio.

32 citations



Journal ArticleDOI
TL;DR: Data suggest that transport and translation of the penicillin acylase precursor are coupled and that the short Shine-Dalgarno-AUG distance interferes with a competent interaction between the translation initiation complex and the export system at high temperature.
Abstract: The enzyme penicillin acylase is synthesized by Escherichia coli only at growth temperatures below 30 degrees C. The biochemical basis of this strict temperature-dependent formation of an enzyme was investigated. When the gene (pac) was under the control of the lacUV5 promoter it showed the same temperature-dependent expression as the chromosomally encoded gene transcribed from its own promoter. This indicates that translation of the pac mRNA rather than transcription of the gene is temperature-dependent. This conclusion could be further confirmed by Northern hybridisation and by analysis of pac-lacZ transcriptional fusions. TnphoA insertion mutagenesis and experiments in which the promoter and 5' sequence encoding the signal peptide of the pac gene was exchanged with those of the cyclodextrin glycosyltransferase gene from Klebsiella oxytoca localised the region of pac mRNA responsible for the temperature-sensitive translation to the 5'-untranslated region and/or the signal peptide. Extension of the 5 nucleotide long spacer separating the Shine-Dalgarno motif from the AUG initiation codon by one or three nucleotides lead to partial or full synthesis of penicillin acylase precursor at 40 degrees C, respectively. The precursor of penicillin acylase formed at 40 degrees C by the mutant variants or when placed under the control of a heterologous upstream region was associated with the membrane but could not be translocated. Taken together these data suggest that transport and translation of the penicillin acylase precursor are coupled and that the short Shine-Dalgarno-AUG distance interferes with a competent interaction between the translation initiation complex and the export system at high temperature. Moreover, evidence was also provided which indicates a direct effect of temperature on the conformation of the precursor and it is proposed that the lack of translation at high temperatures has been selected to prevent the accumulation of transport-incompetent protein locked in the membrane.

30 citations


Journal ArticleDOI
TL;DR: In this paper, N 6 -Phenylacetyl-2′-deoxyadenosine and N 2 -phenyl-acetyl 2′deoxyguanosine are readily deprotected in reactions catalyzed by free and immobilized penicillin amidase at pH 7.8 and 25°C.

Journal ArticleDOI
TL;DR: In this paper, a simple and efficient strategy is developed for resolving racemic mixtures of chiral compounds of industrial interest using enzymatic derivatives, which avoid the prior handling of the racemic mixture.

Journal ArticleDOI
TL;DR: Whereas the cloned pac gene was found to produce higher levels of penicillin acylase constitutively, the process parameters remained about the same for both the parent and the recombinant.

Journal ArticleDOI
TL;DR: Direct activity determination by a flow-through microcalorimetry in the enzyme thermistor system was employed for a fast comparison of (poly)acrylamide gel-entrapped penicillin G acylase preparations and the validity of the results was corroborated by spectrophotometric measurements.
Abstract: Direct activity determination by a flow-through microcalorimetry in the enzyme thermistor system was employed for a fast comparison of (poly)acrylamide gel-entrapped penicillin G acylase preparations. Composition of the pre-polymerization cocktail and both the storage and operational stabilities of optimal gel-entrapped enzyme preparations isolated from the Escherichia coli industrial strain were optimized by this method. The validity of the results was corroborated by spectrophotometric measurements.

Journal ArticleDOI
D.E. Kerr1
TL;DR: Results show that this new compound, which uses 2-nitro-5-(phenoxyacetamido)-benzoic acid as a substrate, is useful in determining the presence and levels of penicillin-V amidase.

Journal ArticleDOI
TL;DR: It is proposed that the inactivation of PA by EEDQ proceeds through a two-step reaction, with the initial and rapid reversible binding followed by a slow, time-dependent, non-covalent, reversible inactivating step.
Abstract: Penicillin acylase (PA) from Kluyvera citrophila was inhibited by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), a specific carboxy-group-reactive reagent. Enzyme activity progressively decreased to a residual value depending on EEDQ concentration. Neither enzymic nor non-enzymic decomposition of EEDQ is concomitant with PA inactivation. Moreover, enzyme re-activation is achieved by chromatographic removal of EEDQ, pH increase or displacement of the reagent with penicillin G. It was then concluded that PA inactivation is due to an equilibrium reaction. The kinetics of enzyme inactivation was analysed by fitting data to theoretical equations derived in accordance with this mechanism. Corrections for re-activation during the enzyme assay were a necessary introduction. The pH-dependence of the rate constant for EEDQ hydrolysis either alone or in the presence of enzyme was studied by u.v. spectroscopy. It turned out to be coincident with the pH-dependence of the forward and reverse rate constants for the inactivation process. It is suggested that previous protonation of the EEDQ molecule is required for these reactions to occur. The thermodynamic values associated with the overall reaction showed little change. Finally it is proposed that the inactivation of PA by EEDQ proceeds through a two-step reaction. The initial and rapid reversible binding is followed by a slow, time-dependent, non-covalent, reversible inactivating step. The expected behaviour in the case of enzyme modification by covalent activation of carboxy residues is also reviewed.

Journal ArticleDOI
TL;DR: The permeation rate of phenylacetic acid, in this electrodialysis system was found to be much faster than those of two other compounds, penicillin G and 6-aminopenicillanic acid, and therefore, the membranes selected here might be suitable for continuous removal ofphenylacefic acid from the reaction mixture for peniillin G hydrolysis.
Abstract: Phenylacefic acid, an inhibitory product, was formed through the hydrolysis of penicillin G by immobilized penicillin acylase. In this study, electrodialysis was applied to remove phenylacetic acid continuously from the reaction mixture and to enhance the efficiency of the reaction.The permeation rate of phenylacetic acid, in this electrodialysis system was found to be much faster than those of two other compounds, penicillin G and 6-aminopenicillanic acid. Therefore, the membranes selected here might be suitable for continuous removal of phenylacefic acid from the reaction mixture for penicillin G hydrolysis. However, the permeation rate of phenylacetic acid was significantly influenced by the penicillin G concentration; it decreased with increasing penicillin G concentration. A theoretical model was developed to describe this phenomenon and was applied to the hydrolysis of penicillin G by means of a combination of immobilized penicillin acylase and electro dialysis. The experimental data were compared to those predicted by a model using independently determined parameters such as the Michaelis constant, inhibition constants and permeation rate constants. Fairly good agreement was found between the theoretical and experimental data. When a 100 mol·m–3 penicillin G solution was used as the substrate, the time required to reach 98% conversion could be reduced to 24.7% by using the combination with electrodialysis.

Journal ArticleDOI
TL;DR: The results indicated that the induction of the plasmid cloned gene was the main factor which rendered it a target for inactivation by insertions of the host encoded IS2 elements.
Abstract: Structural instability of the cloned penicillin acylase gene (pac) from E.coli ATCC11105 was studied under various physiological conditions. Structural changes which adversely affect the expression of penicillin acylase gene were selected for only under conditions in which the gene was active and fully induced. In E.coli strain YMC the predominant mutations were the insertions of the IS2 element at various sites within the 700 bp proximal portion of the pac gene. The results indicated that the induction of the plasmid cloned gene was the main factor which rendered it a target for inactivation by insertions of the host encoded IS2 elements. The mutational events were host specific and they were not influenced by mutual positions and orientations of key marker genes on the plasmid.

Journal ArticleDOI
J Martín1, I Prieto1, J M Mancheño1, J L Barbero1, R. Arche1 
TL;DR: It is proposed that binding of substrate involves the formation of hydrogen bonds between the substrate and the essential ionizable groups in the enzyme which lie within the hydrophobic environment of the active site of penicillin acylase.
Abstract: The variation with pH of the kinetic parameters of penicillin acylase from Kluyvera citrophila has been used to gain information about the chemical mechanism of the reaction catalysed by the enzyme. The pH-dependence of log (V/Km) for penicillin G showed that a group with a pK value over 4.7 must be deprotonated and that a group with a pK value over 9.7 must be protonated in the free enzyme for activity. The solvent perturbation and temperature studies indicated that these groups are respectively of cationic and neutral acid type with ionization enthalpies of 29.7 and 111 kJ/mol. It was proved that penicillin G sulphoxide is a reversible linear competitive inhibitor with respect to the hydrolysis of penicillin G. The similarity of the pH profile and the magnitude of the pK values derived from the dissociation constant, Ki, suggest that both groups are concerned with the binding of penicillin G and its analogues to the enzyme. It is proposed that binding of substrate involves the formation of hydrogen bonds between the substrate and the essential ionizable groups in the enzyme which lie within the hydrophobic environment of the active site of penicillin acylase. This suggestion is supported by the finding that the profile of V (Vmax.) is similar to the V/Km profile, except that the low and high pK values are respectively shifted downward and upward due to the entry of substrate. Moreover, the bell shape of the V profile indicated that they are also essential in the catalytic steps subsequent to binding.