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Penicillin amidase activity

About: Penicillin amidase activity is a(n) research topic. Over the lifetime, 10 publication(s) have been published within this topic receiving 371 citation(s).

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TL;DR: A detailed study of the phase transition effect on thermal stability of the enzymes and protein-protein interactions has been carried out and found that change of the catalytic activity and Thermal stability of N-PEC-bound penicillin amidase is fully reversible and reproducible.
Abstract: Penicillin amidase, α-chymotrypsin and urease have been immobilized in water-soluble nonstoichiometric polyelectrolyte complexes (N-PEC). N-PEC are formed by modified poly(N-ethyl-4-vinyl-pyridinium bromide) (polycation) and excess poly(methylacrylic acid) (polyanion). N-PEC are a new class of polymers capable, characteristically, of phase transitions solution ⇄ precipitate induced by slight change in pH or ionic strength. Neither the chemical structure of the carrier nor the number of cross-linkages between an enzyme and a carrier change on phase transition. That gives an unique opportunity to elucidate the difference between enzymes immobilized on water-soluble and water-insoluble supports. A detailed study of the phase transition effect on thermal stability of the enzymes and protein-protein interactions has been carried out. The following effects were found. 1 Pronounced thermal stabilization of penicillin amidase and urease may be achieved on two conditions: (a) the enzyme is in the precipitate; (b) the-enzyme is linked to the N-PEC nucleus. Then the thermal stability of N-PEC-bound penicillin amidase increases 7-fold at pH 5.7, 60°C, and 300-fold at pH 3.1, 25°C, compared to the native enzyme. For urease, the thermal stabilization increases 20-fold at pH 5.0, 70°C. 2 The localization of enzyme on N-PEC has been established by titration of α-chymotrypsin bound to a polycation or polyanion with basic pancreatic trypsin inhibitor. Both in solution (pH 6.1) and in N-PEC precipitate (pH 5.7), an α-chymotrypsin molecule bound to a polyanion is fully exposed to the solution. If the enzyme is bound to a polycation, only 20% of α-chymotrypsin molecules in the precipitate and 40% in solution retain their ability for protein-protein interactions. This means that a polycation-bound enzyme is localized in the hydrophobic nucleus of the complex, whereas the polyanion-bound enzyme sits on the hydrophilic shell of the complex. 3 On pH-induced phase transition (pH decreases from 6.1 to 5.7), there occurs a stepwise decrease in penicillin amidase activity which is due to a 9.8-fold increase in the Km for 2-nitro-4-phenylacetamidobenzoic acid. 4 Change of the catalytic activity and thermal stability of N-PEC-bound penicillin amidase is fully reversible and reproducible. Such soluble-insoluble immobilized enzymes with controllable thermal stability and activity may be used for simulating events in vivo and in biotechnology.

97 citations

Journal ArticleDOI

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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.

82 citations

Journal ArticleDOI

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TL;DR: Penicillin amidase activity, resulting in the hydrolysis of phenoxymethylpenicillin to give 6-aminopenicillanic acid, was found to be widespread among micro-organisms and the pH optimum for enzyme activity in the direction of synthesis was 5.0 to 5.5.
Abstract: Penicillin amidase activity, resulting in the hydrolysis of phenoxymethylpenicillin to give 6-aminopenicillanic acid, was found to be widespread among micro-organisms. Partial purification of the enzyme, which was essentially exocellular, was obtained by precipitation with ammonium sulphate, or acetone, followed by dialysis. The pH optimum with phenoxymethylpenicillin as substrate was found to be approximately 9$\cdot $0 with maximum enzyme stability at approximately pH 10$\cdot $0. Over a relatively short period of reaction time the optimum temperature was approximately 50 degrees C. The K$_{m}$ value with phenoxymethylpenicillin was found to be 4$\cdot $0 mg/ml. Using as substrate a concentration of 1% phenoxymethylpenicillin, a conversion to 6-aminopenicillanic acid of 56% of theoretical was obtained in the enzyme reaction mixture. Isolation of pure 6-aminopenicillanic acid from this reaction mixture was obtained in 50% yield. 6-aminopenicillanic acid markedly inhibited the enzyme activity at a concentration of 4$\cdot $0 mg/ml. Phenoxyacetic acid at this concentration was without effect. At higher concentrations, incubation of 6-aminopenicillanic acid and phenoxyacetic acid with the enzyme resulted in the synthesis of phenoxymethylpenicillin. The pH optimum for enzyme activity in the direction of synthesis was 5$\cdot $0 to 5$\cdot $5 compared with approximately 9$\cdot $0 for hydrolysis.

54 citations

Journal ArticleDOI

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TL;DR: Isotopic exchange of (35)S between penicillins and 6-amino-penicillanic acid (6-APA) was observed in cell-free extracts of Penicillium chrysogenum, and penicillin V, G, K, X, and dihydro F showed isotopic exchange.
Abstract: Isotopic exchange of 35S between penicillins and 6-amino-penicillanic acid (6-APA) was observed in cell-free extracts of Penicillium chrysogenum. Sulfhydryl-containing compounds were required for activity. Dithiothreitol, dithioerythritol, mercaptoethanol, and glutathione served as activators. The acyltransferase was purified threefold by adsorption on calcium phosphate gel at pH 6 and elution at pH 8. The partially purified enzyme showed maximal activity at pH 8. The enzyme was stable at 25 C for at least 30 min at pH 8. Dissociable inhibitors or activators, other than the sulfhydryl-containing compounds, could not be demonstrated in the enzyme preparation. An apparent Michaelis constant of 1.5 ± 0.5 mm was determined for penicillin G at a 6-APA concentration of 5 mm. The enzyme did not appear to possess penicillin amidase activity. The exchange mechanism probably involves an acyl-enzyme intermediate. Penicillins V, G, K, X, and dihydro F showed isotopic exchange with 35S-6-APA. Penicillin N, methylpenicillin, and phenyl-penicillin did not show exchange. The level of acyltransferase in P. chrysogenum 51-20F3 was measured at times during the fermentation. The level of activity increased threefold between 40 and 55 hr, remaining high until about 90 hr.

49 citations

Journal ArticleDOI

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TL;DR: In submerged culture, Emericellopsis minima (Stolk) I. M. 69015 produced a substance resembling 6-aminopenicillanic acid in its behaviour on paper chromatograms and in its properties after reaction with phenylacetyl chloride, which was destroyed by penicillinase but was acid-stable.
Abstract: In submerged culture, Emericellopsis minima (Stolk) I.M.I. 69015 produced a substance resembling 6-aminopenicillanic acid in its behaviour on paper chromatograms and in its properties after reaction with phenylacetyl chloride. The substance was destroyed by penicillinase but was acid-stable. The culture also produced cephalosporin N and two unidentified antibiotics which have been named emericellopsin A and B. When other fungi of the genera Emericellopsis and Cephalosporium were examined in a similar manner, C. salmosynnematum and Cephalosporium I.M.I. 49137 were also found to produce small amounts of a substance resembling 6-aminopenicillanic acid. Examination of Emericellopsis minima (Stolk) also showed the presence of penicillin amidase activity in this culture.

36 citations

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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
19931
19911
19871
19851
19811
19801