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S Wolfe

Publications and source records attributed to S Wolfe.

At least 73 records · Page 4Linked to original sources
PubMed1986

Synthesis of benzylpenicillin by cell-free extracts from Streptomyces clavuligerus.

Cell-free enzyme concentrates from Streptomyces clavuligerus were found to convert phenylacetyl-L-cysteinyl-D-valine (PCV) directly into benzylpenicillin when incubated under reaction conditions which support the activity of isopenicillin N synthetase. The formation of benzylpenicillin was detected both by biological assay and by high performance liquid chromatography. Supplementation of PCV-containing reaction mixtures with cofactors required for ring expansion activity did not result in the production of cephalosporins. Incubation of phenoxyacetyl-L-cysteinyl-D-valine (PoCV) under the same reaction conditions did not result in the formation of penicillin V or any cephalosporin product.

Biological Assay↗
PubMed1985

Repression and inhibition of cephalosporin synthetases in Streptomyces clavuligerus by inorganic phosphate.

Cephalosporin production by growing cells of Streptomyces clavuligerus was reduced by 100 mM inorganic phosphate. Resting cell production was repressed by prior growth in high phosphate and inhibited by phosphate. The cell-free activity of desacetoxycephalosporin C synthetase (ring expansion activity) was repressed by prior growth in high phosphate and inhibited by phosphate. Isopenicillin N synthetase (cyclase) was inhibited but not repressed. Penicillin epimerase was neither inhibited nor repressed by phosphate.

Cephalosporins↗
PubMed1985

Ammonium repression of cephalosporin production by Streptomyces clavuligerus.

Production of beta-lactam antibiotics took place during growth of Streptomyces clavulgerus in chemically defined medium. The specific activities of isopenicillin N synthetase ("cyclase"), isopenicillin N epimerase, and deacetoxycephalosporin C synthetase ("expandase") increased during the exponential phase of growth. Specific cephalosporin productivity during fermentation followed a similar pattern, reaching a maximum near the end of the growth phase and decaying rapidly in the stationary phase. Ammonium chloride depressed cephalosporin production, presumably as a result of repression of cyclase and expandase formation, but not of epimerase. No inhibitory effects on enzyme activity by ammonium were found. Addition of tribasic magnesium phosphate [Mg3(PO4)2 X 8H2O] prevented the repression of cyclase and markedly stimulated cephalosporin production. Cephamycin C and, in smaller amounts, O-carbamoyldeacetylcephalosporin C were the only cephalosporins detected. Growth with ammonium resulted in lower titers of both compounds, and did not change the relative proportion of each. The correlation found between cephalosporin productivity and cyclase specific activity in different media suggests that formation of this enzyme may be the rate-limiting step in the pathway.

Amino Acid Isomerases↗
PubMed1985

Effect of side-chain substitution of a CH2 group by sulfur on the antimicrobial activity of natural penicillins and cephalosporins.

The S-carboxymethyl-D-cysteine side-chain analogs of cephalosporin C and deacetoxycephalosporin C were found to have markedly increased in vitro activity against Gram-positive and Gram-negative bacteria compared to the natural antibiotics. The S-carboxymethyl-L-cysteine analogs were less active than the S-carboxymethyl-D-cysteine analogs but against most organisms tested, still more active than the natural compounds. The effect of replacement of CH2 with S was less dramatic in the case of penicillin N and isopenicillin N. The S-carboxymethyl-D-cysteine analog of deacetoxycephalosporin C was found to be orally available in rats.

Administration, Oral↗
PubMed1984

Enzymatic approach to syntheses of unnatural beta-lactams.

Four enzymes associated with the transformation of the peptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) into the beta-lactam antibiotic desacetylcephalosporin C have been isolated from the prokaryotic organism Streptomyces clavuligerus and immobilized. Appropriate choice of the cofactors allows continuous and quantitative conversion of the peptide into either penicillins or cephalosporins at room temperature. The overall process includes four oxidations, two ring closures, and one epimerization. In contrast, cell-free transformations with the eukaryotic organism Cephalosporium acremonium do not proceed beyond the oxidation level of penicillin. The amino acids of the natural peptide ACV can be altered by chemical means; several of the resulting peptides are converted into novel antibiotics by the enzymes of Streptomyces clavuligerus.

Anti-Bacterial Agents↗
PubMed1984

A pure enzyme catalyzing penicillin biosynthesis.

Isopenicillin N synthetase (cyclase) has been purified to homogeneity from Cephalosporium acremonium strain C-10. The enzyme has a molecular weight of 40,000 to 42,000 and yields a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was purified in 10 percent yield by a combination of protamine sulfate and ammonium sulfate precipitations, gel filtration, and ion-exchange high-performance liquid chromatography. The purified enzyme can be stabilized with sucrose and stored at -20 degrees C for several weeks without any loss in activity.

Acremonium↗
PubMed1984

Enzymatic synthesis of the penicillin and cephalosporin nuclei from an acyclic peptide containing carboxymethylcysteine.

The tripeptide delta-(L- carboxymethylcysteinyl )-L-cysteinyl-D-valine (L-CMC-CV) is converted sequentially into the CMC analog of isopenicillin N, the CMC analog of penicillin N, and the CMC analog of desacetoxycephalosporin C by, respectively, isopenicillin N synthetase, isopenicillin N epimerase, and desacetoxycephalosporin C synthetase, all isolated from the beta-lactam producing prokaryote Streptomyces clavuligerus.

Amino Acid Isomerases↗
PubMed1984

Enzymatic conversion of the unnatural tripeptide delta-(D-alpha-aminoadipyl)-L-cysteinyl-D-valine to beta-lactam antibiotics.

Incubation of the unnatural tripeptide delta-(D-alpha-aminoadipyl)-L-cysteinyl-D-valine (DLD-ACV) with a partially purified extract of Cephalosporium acremonium resulted in the production of deacetoxycephalosporin C. The extract contained isopenicillin N synthetase (cyclase) and deacetoxycephalosporin C synthetase (expandase) but no penicillin epimerase activity, and was incubated aerobically in the presence of the components of the cyclase and expandase reaction mixtures (Fe++, ascorbate, dithiothreitol, alpha-ketoglutarate and ATP). The reaction was sensitive to penicillinase, indicating penicillin N to be an intermediate. However, when ring expansion was prevented by omission of alpha-ketoglutarate and ATP, no penicillin N was detected.

Acremonium↗
PubMed1984

Repression of beta-lactam production in Cephalosporium acremonium by nitrogen sources.

A variety of inorganic and organic nitrogen sources were added to fermentation media to determine their regulatory effects on the production of beta-lactam antibiotics by Cephalosporium acremonium. (NH4)2SO4 at concentrations higher than 100 mM (1.3%) strongly inhibited beta-lactam production. L-Asparagine and L-arginine proved to be the best nitrogen sources tested for beta-lactam production. The optimum concentration of asparagine was 1.2%. Higher concentrations led to NH3 accumulation, increase in pH, and lower growth rates. Addition of tribasic magnesium phosphate [Mg3(PO4)2 X 8H2O] to the (NH4)2SO4-containing medium stimulated beta-lactam production markedly and ammonium repression of the ring-expansion enzyme was reversed. It appears that the ring-expansion step is a very sensitive part of beta-lactam biosynthesis in C. acremonium with respect to nitrogen source repression. Other enzymes may also be sensitive in view of the fact that nitrogen source derepression not only led to increases in cephalosporin C but, to a lesser extent, penicillin N and total beta-lactam titers.

Acremonium↗
PubMed1983

Analysis of penicillin N ring expansion activity from Streptomyces clavuligerus by ion-pair high-pressure liquid chromatography.

An ion-pair, reversed-phase, high-pressure liquid chromatographic method for the analysis of penicillin N ring expansion activity has been developed which allows simultaneous measurement of both substrate and product. The high-pressure liquid chromatography conditions were as follows: stationary phase, C18; flow rate, 2 ml/min; detection, 220 nm. The stationary phase was preconditioned with 4.5 mM tetrabutylammonium bromide in 0.05 M KH2PO4 (pH 4.0)-methanol (85:15, vol/vol) and then equilibrated with 0.06 mM tetrabutylammonium bromide in 0.05 M KH2PO4 (pH 4.0)-methanol (95:5, vol/vol) for analysis of reaction mixtures. These conditions separated authentic samples of penicillin N and desacetoxycephalosporin C and allowed cell-free studies of the ring expansion of penicillin N to desacetoxycephalosporin C by a partially purified enzyme from Streptomyces clavuligerus to be followed conveniently.

Cephalosporins↗
PubMed1983

Studies on the ring-cyclization and ring-expansion enzymes of beta-lactam biosynthesis in Cephalosporium acremonium.

Micrococcus luteus was found to be very sensitive to isopenicillin N and was used as assay organism for purification of the enzyme isopenicillin N synthetase, which cyclizes delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N. Purification of the enzyme from the crude extract obtained by sonication of mycelia of Cephalosporium acremonium CW-19 was carried out by ammonium sulfate precipitation, desalting with Sephadex G-25, gel filtration on LKB ultrogel AcA44 or ion-exchange chromatography on DEAE-Sepharose. The cyclization enzyme was separated from the ring-expansion enzyme and was purified considerably more than 50-fold by this procedure. Using the purified enzyme, we found that the disulfide bis-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine required reduction to delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine in order to behave as a substrate. The enzyme activity was stimulated by FeSO4 and ascorbate, but other cofactors, including alpha-ketoglutarate, were inactive. In addition to delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine, the enzyme converted adipyl-L-cysteinyl-D-valine, N-acetyl-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine, and glycyl-delta-(L-alpha-aminoadipyl)L-cysteinyl-D-valine to penicillins. All of these latter peptides were competitive inhibitors of the cyclization reaction. The Km of the cyclization enzyme is 10 times higher than that of the ring-expansion enzyme, deacetoxycephalosporin C synthetase. The pH and temperature optima of the two enzymes were rather similar. Phosphate inhibited ring expansion, but not cyclization. Both enzymes appear to be soluble enzymes of about 31 000 molecular weight.

Acremonium↗
PubMed1983

Partial purification and characterization of isopenicillin N epimerase activity from Streptomyces clavuligerus.

Epimerase activity, which converts isopenicillin N to penicillin N, has been partially purified from cell-free extracts of Streptomyces clavuligerus. No stimulating cofactors of this activity were found, and neither EDTA nor anaerobic incubation caused significant inhibition of activity. Although pyridoxal phosphate did not stimulate epimerase activity, the presence of this cofactor was necessary for the stabilization of enzymic activity during the purification process. Epimerase activity was purified 35.5-fold by a combination of salt precipitation, gel filtration, and ion exchange chromatography. Gel filtration indicated that the epimerase has a molecular weight of 60 000 and sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the 35.5-fold purified epimerase showed a major protein band running near that location. Pyridoxal phosphate antagonists did not uniformly inhibit epimerase activity, but the inhibitory effect of hydroxylamine could be partially reversed by pyridoxal phosphate.

Amino Acid Isomerases↗
PubMed1983

Experimental ridge augmentation with porous hydroxyapatite implants.

A new hydroxyapatite ceramic implant material for restoration of atrophic edentulous ridges was tested in a canine animal model. The structure of this material is unique, in that it is produced from the skeleton of a coral, and in that it is penetrated by a uniform network of interconnected pores. Results indicate that bone penetrates its structure to a greater degree than had been observed with similar implants constructed of different materials. Implant insertion through a simple surgical technique involving minimal morbidity is a viable method of placement.

Alveolar Ridge Augmentation↗