Expression of the penDE gene of Penicillium chrysogenum encoding isopenicillin N acyltransferase in Cephalosporium acremonium: production of benzylpenicillin by the transformants

1991 ◽  
Vol 225 (1) ◽  
pp. 56-64 ◽  
Author(s):  
Santiago Gutiérrez ◽  
Bruno Díez ◽  
Emilio Alvarez ◽  
Jose L. Barredo ◽  
Juan F. Martín
Keyword(s):  
Penicillium Chrysogenum ◽  
Cephalosporium Acremonium ◽  
Pende Gene ◽  
Isopenicillin N

Replacement of Tyrosine-197 and the Corresponding Tyrosine-195 to Isoleucine in Cephalosporium acremonium and Streptomyces clavuligerus Isopenicillin N Synthase

2001 ◽  
Vol 56 (9-10) ◽  
pp. 806-809
Author(s):  
Paxton Loke ◽  
Tiow-Suan Sim
Keyword(s):  
Penicillium Chrysogenum ◽  
Biosynthetic Pathway ◽  
Specific Activity ◽  
High Specific Activity ◽  
Streptomyces Clavuligerus ◽  
Amino Acid Difference ◽  
Tyrosine Residue ◽  
Cephalosporium Acremonium ◽  
Isoleucine Residue ◽  
Isopenicillin N

AbstractIsopenicillin N synthase (IPNS) is one of the key enzymes in the penicillin and cephalosporin biosynthetic pathway which catalyses the conversion of δ-(ʟ-α-aminoadipyl)-ʟ-cysteinyl-ᴅ-valine to isopenicillin N. The IPNS from Penicillium chrysogenum 23X-80-269-37-2, a high penicillin V-producer, was found to possess an isoleucine residue instead of tyrosine at position 195. An attempt to increase the specific activity of IPNS from Cephalosporium acremonium and Streptomyces clavuligerus was undertaken by altering the corresponding tyrosine residue to an isoleucine at the corresponding location. Unfortunately, no apparent increase in specific activity was encountered when the purified mutant enzymes were analysed and thus, this amino acid difference is likely not responsible for high specific activity in IPNS.

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.

The isopenicillin N acyltransferases of Aspergillus nidulans and Penicillium chrysogenum differ in their ability to maintain the 40-kDa alphabeta heterodimer in an undissociated form

2003 ◽  
Vol 270 (9) ◽  
pp. 1958-1968 ◽  
Author(s):  
Francisco J. Fernandez ◽  
Rosa E. Cardoza ◽  
Eduardo Montenegro ◽  
Javier Velasco ◽  
Santiago Gutierrez ◽  
...  
Keyword(s):  
Aspergillus Nidulans ◽  
Penicillium Chrysogenum ◽  
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.

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