Regulation of isopenicillin N synthetase and deacetoxycephalosporin C synthetase by carbon source during the fermentation of Cephalosporium acremonium

1984 ◽  
Vol 19 (4) ◽  
pp. 232-236 ◽  
Author(s):  
Jutta Heim ◽  
Yong-Qiang Shen ◽  
Saul Wolfe ◽  
Arnold L. Demain
Keyword(s):  
Carbon Source ◽  
Cephalosporium Acremonium ◽  
Deacetoxycephalosporin C Synthetase ◽  
Isopenicillin N Synthetase ◽  
Isopenicillin N

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

1983 ◽  
Vol 29 (5) ◽  
pp. 488-496 ◽  
Author(s):  
Jutta Kupka ◽  
Yong-Qiang Shen ◽  
Saul Wolfe ◽  
Arnold L. Demain
Keyword(s):  
Micrococcus Luteus ◽  
Gel Filtration ◽  
Ring Expansion ◽  
Exchange Chromatography ◽  
Cephalosporium Acremonium ◽  
Competitive Inhibitors ◽  
Isopenicillin N Synthetase ◽  
Soluble Enzymes ◽  
Temperature Optima ◽  
Isopenicillin N

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 δ-(L-α-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-δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine required reduction to δ-(L-α-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 α-ketoglutarate, were inactive. In addition to δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine, the enzyme converted adipyl-L-cysteinyl-D-valine, N-acetyl-δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine, and glycyl-δ-(L-α-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, deactoxycephalosporin 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.

Ammonium repression of cephalosporin production by Streptomyces clavuligerus

1985 ◽  
Vol 31 (8) ◽  
pp. 736-743 ◽  
Author(s):  
Alfredo F. Braña ◽  
Saul Wolfe ◽  
Arnold L. Demain
Keyword(s):  
Specific Activity ◽  
Relative Proportion ◽  
Streptomyces Clavuligerus ◽  
Defined Medium ◽  
Inhibitory Effects ◽  
Rate Limiting Step ◽  
Deacetoxycephalosporin C Synthetase ◽  
Isopenicillin N Synthetase ◽  
Rate Limiting ◽  
Isopenicillin N

Production of β-lactam antibiotics took place during growth of Streptomyces clavuligerus 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∙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.

Isolation, sequence determination and expression in Escherichia coli of the isopenicillin N synthetase gene from Cephalosporium acremonium

Nature ◽  
1985 ◽  
Vol 318 (6042) ◽  
pp. 191-194 ◽  
Author(s):  
S. M. Samson ◽  
R. Belagaje ◽  
D. T. Blankenship ◽  
J. L. Chapman ◽  
D. Perry ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cephalosporium Acremonium ◽  
Sequence Determination ◽  
Expression In Escherichia Coli ◽  
Isopenicillin N Synthetase ◽  
Isopenicillin N

scholarly journals Purification of isopenicillin N synthetase

1984 ◽  
Vol 222 (3) ◽  
pp. 789-795 ◽  
Author(s):  
C P Pang ◽  
B Chakravarti ◽  
R M Adlington ◽  
H H Ting ◽  
R L White ◽  
...  
Keyword(s):  
Streptomyces Clavuligerus ◽  
Protein Band ◽  
Maximum Activity ◽  
Polyacrylamide Gel ◽  
Major Protein ◽  
Synthetase Activity ◽  
Cephalosporium Acremonium ◽  
Major Protein Band ◽  
Isopenicillin N Synthetase ◽  
Isopenicillin N

Isopenicillin N synthetase was extracted from Cephalosporium acremonium and purified about 200-fold. The product showed one major protein band, coinciding with synthetase activity, when subjected to electrophoresis in polyacrylamide gel. An isopenicillin N synthetase from Penicillium chrysogenum was purified about 70-fold by similar procedures. The two enzymes resemble each other closely in their Mr, in their mobility on electrophoresis in polyacrylamide gel and in their requirement for Fe2+ and ascorbate for maximum activity. Preliminary experiments have shown that a similar isopenicillin N synthetase can be extracted from Streptomyces clavuligerus.

scholarly journals Incorporation of 3H from δ-(l-α-amino (4,5-3H)adipyl)-l-cysteinyl-d-(4,4-3H)valine into isopenicillin N

1979 ◽  
Vol 184 (2) ◽  
pp. 421-426 ◽  
Author(s):  
J O'Sullivan ◽  
R C Bleaney ◽  
J A Huddleston ◽  
E P Abraham
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Adipic Acid ◽  
Experimental Error ◽  
Acid Oxidase ◽  
Cephalosporium Acremonium ◽  
Amino Acid Oxidase ◽  
Free System ◽  
A Cell ◽  
Isopenicillin N

1. delta-(L-alpha-Amino[4,5-3H]adipyl)-L-cysteinyl-D-[4,4-3H]valine has been synthesized from its constituent amino acids, the L-alpha-amino[4,5-3H]adipic acid being obtained by reduction with 3H2 of methyl 5-acetamido-5,5-diethoxycarbonylpent-2-enoate and subsequent decarboxylation and hydrolysis. 2. In a cell-free system prepared by lysis of protoplasts of Cephalosporium acremonium 3H was incorporated from the doubly labelled tripeptide into a compound that behaved like penicillin N or isopenicillin N. The relative specific radioactivities of the alpha-aminoadipyl and penicillamine moieties of the penicillin were the same (within experimental error) as those of the alpha-aminoadipic acid and valine residues respectively of the tripeptide. 3. The behaviour of the labelled alpha-aminoadipic acid from the penicillin to the L-amino acid oxidase of Crotalus adamanteus venom showed that it was mainly L-alpha-aminoadipic acid. 4. The results are consistent with the hypothesis that the carbon skeleton of the LLD-tripeptide is incorporated intact into the penicillin molecule and that the first product is isopenicillin N.

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