Cephamycin production and isopenicillin N synthetase activity in cultures of Streptomyces clavuligerus

1987 ◽  
Vol 27 (3) ◽  
Author(s):  
L.C. Vining ◽  
S.E. Jensen ◽  
D.W.S. Westlake ◽  
Y. Aharonowitz ◽  
S. Wolfe
Keyword(s):  
Streptomyces Clavuligerus ◽  
Synthetase Activity ◽  
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 Biochemical studies on the activity of δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase from Streptomyces clavuligerus

1992 ◽  
Vol 283 (3) ◽  
pp. 691-698 ◽  
Author(s):  
J Zhang ◽  
S Wolfe ◽  
A L Demain
Keyword(s):  
Enzyme Activity ◽  
High Temperature ◽  
Bioactive Compounds ◽  
Streptomyces Clavuligerus ◽  
Synthetase Activity ◽  
Enzyme Specificity ◽  
Acidic Ph ◽  
Reaction Parameters ◽  
Biochemical Studies ◽  
Isopenicillin N

The enzyme activity of purified delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase from Streptomyces clavuligerus was studied biochemically. The dependence of ACV synthetase activity on reaction parameters, including substrates, cofactors, temperature and pH, were determined, resulting in a substantially increased enzyme activity. The activity is very labile to high temperature and is also unstable at acidic pH. The enzyme specificity is strict towards L-alpha-aminoadipate, but rather loose with respect to L-valine; certain modifications of L-cysteine can also be tolerated. Some unnatural tripeptides synthesized by ACV synthetase can be converted into bioactive compounds by isopenicillin N synthase. The only nutrient found to negatively affect ACV synthetase activity is phosphate, but various compounds such as thiol-blocking reagents and ATP-utilization products (AMP and pyrophosphate) are inhibitory to the enzyme.

Cloning and nucleotide sequence determination of the isopenicillin N synthetase gene from Streptomyces clavuligerus

Gene ◽  
1988 ◽  
Vol 62 (2) ◽  
pp. 187-196 ◽  
Author(s):  
Brenda K. Leskiw ◽  
Yair Aharonowitz ◽  
Moshe Mevarech ◽  
Saul Wolfe ◽  
Leo C. Vining ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Streptomyces Clavuligerus ◽  
Sequence Determination ◽  
Isopenicillin N Synthetase ◽  
Nucleotide Sequence Determination ◽  
Isopenicillin N

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.

Purification of isopenicillin N synthetase from Streptomyces clavuligerus

1986 ◽  
Vol 32 (12) ◽  
pp. 953-958 ◽  
Author(s):  
Susan E. Jensen ◽  
Brenda K. Leskiw ◽  
Leo. C. Vining ◽  
Yair Aharonowitz ◽  
Donald W. S. Westlake ◽  
...  
Keyword(s):  
High Performance ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Streptomyces Clavuligerus ◽  
High Performance Liquid Chromatographic ◽  
Inhibitory Effect ◽  
Isopenicillin N Synthetase ◽  
Isopenicillin N

Isopenicillin N synthetase was purified from Streptomyces clavuligerus by sequential salt precipitation, ion-exchange and gel-filtration chromatography using both conventional open column and high-performance liquid chromatographic techniques. Material from the final purification step had a specific activity of 204.1 × 10−3 units/mg of protein which represented a 130-fold purification over the cell-free extract. The purified isopenicillin N synthetase was determined to have a molecular weight of 33 000 by sodium dodecyl sulfate – polyacrylamide gel electrophoresis and to have a Km of 0.32 mM with respect to its substrate δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine. The enzyme showed a sensitivity to thiol-specific inhibitors with N-ethylmaleimide giving the strongest inhibitory effect.

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