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.

Ammonium ions repress δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase in Streptomyces clavuligerus NRRL 3585

1989 ◽  
Vol 35 (3) ◽  
pp. 399-402 ◽  
Author(s):  
Jinyou Zhang ◽  
Saul Wolfe ◽  
Arnold L. Demain
Keyword(s):  
Streptomyces Clavuligerus ◽  
Defined Medium ◽  
Antibiotic Biosynthesis ◽  
Chemically Defined Medium ◽  
Ammonium Ions ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Negative Effect ◽  
Major Factors ◽  
Rate Limiting

Production of cephems (predominantly cephamycin C) by Streptomyces clavuligerus grown in chemically defined medium supplemented with 120 mM NH4Cl was sharply reduced. This concentration of ammonium ions in the medium repressed δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase formation by about 75%. Of the other cephalosporin synthases, cyclase was repressed by 70%, expandase by 50%, and epimerase only to a very small extent. Inhibition of the action of ACV synthetase was only slight in the presence of 100 mM NH4Cl. Repression of ACV synthetase, cyclase, and expandase appears to be the major factors contributing to the negative effect of ammonium on S. clavuligerus NRRL 3585. ACV synthetase is probably the rate-limiting step of cephalosporin biosynthesis in this strain.Key words: β-lactam biosynthesis, antibiotic biosynthesis, cephalosporins, Streptomyces clavuligerus, ACV synthetase.

scholarly journals A multi-enzyme cascade for efficient production of d-p-hydroxyphenylglycine from l-tyrosine

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Xu Tan ◽  
Sheng Zhang ◽  
Wei Song ◽  
Jia Liu ◽  
Cong Gao ◽  
...  
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
Enantiomeric Excess ◽  
Efficient Production ◽  
Enzyme Cascade ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rotation Strategy ◽  
Rate Limiting

AbstractIn this study, a four-enzyme cascade pathway was developed and reconstructed in vivo for the production of d-p-hydroxyphenylglycine (D-HPG), a valuable intermediate used to produce β-lactam antibiotics and in fine-chemical synthesis, from l-tyrosine. In this pathway, catalytic conversion of the intermediate 4-hydroxyphenylglyoxalate by meso-diaminopimelate dehydrogenase from Corynebacterium glutamicum (CgDAPDH) was identified as the rate-limiting step, followed by application of a mechanism-guided “conformation rotation” strategy to decrease the hydride-transfer distance d(C6HDAP−C4NNADP) and increase CgDAPDH activity. Introduction of the best variant generated by protein engineering (CgDAPDHBC621/D120S/W144S/I169P with 5.32 ± 0.85 U·mg−1 specific activity) into the designed pathway resulted in a D-HPG titer of 42.69 g/L from 50-g/L l-tyrosine in 24 h, with 92.5% conversion, 71.5% isolated yield, and > 99% enantiomeric excess in a 3-L fermenter. This four-enzyme cascade provides an efficient enzymatic approach for the industrial production of D-HPG from cheap amino acids.

scholarly journals Mutation of Aspartate 238 in FAD Synthase Isoform 6 Increases the Specific Activity by Weakening the FAD Binding

2019 ◽  
Vol 20 (24) ◽  
pp. 6203 ◽  
Author(s):  
Piero Leone ◽  
Michele Galluccio ◽  
Stefano Quarta ◽  
Ernesto Anoz-Carbonell ◽  
Milagros Medina ◽  
...  
Keyword(s):  
Binding Sites ◽  
Specific Activity ◽  
Synthesis Rate ◽  
General Increase ◽  
Respiratory Chain Deficiency ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Acyl Coa Dehydrogenases ◽  
Rate Limiting ◽  
Fad Binding

FAD synthase (FADS, or FMN:ATP adenylyl transferase) coded by the FLAD1 gene is the last enzyme in the pathway of FAD synthesis. The mitochondrial isoform 1 and the cytosolic isoform 2 are characterized by the following two domains: the C-terminal PAPS domain (FADSy) performing FAD synthesis and pyrophosphorolysis; the N-terminal molybdopterin-binding domain (FADHy) performing a Co++/K+-dependent FAD hydrolysis. Mutations in FLAD1 gene are responsible for riboflavin responsive and non-responsive multiple acyl-CoA dehydrogenases and combined respiratory chain deficiency. In patients harboring frameshift mutations, a shorter isoform (hFADS6) containing the sole FADSy domain is produced representing an emergency protein. With the aim to ameliorate its function we planned to obtain an engineered more efficient hFADS6. Thus, the D238A mutant, resembling the D181A FMNAT “supermutant” of C. glabrata, was overproduced and purified. Kinetic analysis of this enzyme highlighted a general increase of Km, while the kcat was two-fold higher than that of WT. The data suggest that the FAD synthesis rate can be increased. Additional modifications could be performed to further improve the synthesis of FAD. These results correlate with previous data produced in our laboratory, and point towards the following proposals (i) FAD release is the rate limiting step of the catalytic cycle and (ii) ATP and FMN binding sites are synergistically connected.

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

Phosphorylation of Ca-ATPase of Sarcoplasmic Reticulum with Different Substrates

1983 ◽  
Vol 38 (9-10) ◽  
pp. 845-847 ◽  
Author(s):  
Hector Barrabin ◽  
Leopoldo de Meis
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Low Temperatures ◽  
Hplc Analysis ◽  
Calcium Concentration ◽  
Specific Activity ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
High Calcium ◽  
High Calcium Concentration ◽  
Rate Limiting

ATP and GTP as substrate for phosphorylation of sarcoplasmic reticulum ATPase are compared. Maximal levels of phosphoenzyme are between 4.5 and 4.8 nmol per mg of protein when either substrate is used provided that phosphoenzyme hydrolysis are strongly inhibited by high calcium concentration (20 mм) and low temperatures ( 0 ° C ) . The maximal values obtained with GTP are lower than those previously reported. It is shown that this difference is due to underestimation of the specific activity of labeled nucleotides used in previous studies, as revealed by UV absorption and HPLC analysis. The dependence of the phosphoenzyme levels on calcium concentration, pH and temperature confirm previous findings indicating that ATP, but no GTP, accelerates the rate limiting step of the catalytic cycle.

Isopenicillin N synthase and desacetoxycephalosporin C synthase activities during defined medium fermentations of Streptomyces clavuligerus: effect of oxygen and iron supplements

1989 ◽  
Vol 35 (12) ◽  
pp. 1111-1117 ◽  
Author(s):  
Malcolm J. Rollins ◽  
Susan E. Jensen ◽  
Donald W. S. Westlake
Keyword(s):  
Dissolved Oxygen ◽  
Total Duration ◽  
Fold Increase ◽  
Streptomyces Clavuligerus ◽  
Ring Expansion ◽  
Defined Medium ◽  
Cephamycin C ◽  
Defined Media ◽  
Isopenicillin N

When the level of dissolved oxygen was increased to saturation in defined media fermentations of Streptomyces clavuligerus, the total duration of activity of the penicillin ring cyclization enzyme, isopenicillin N synthase (IPNS), was extended by at least 20 h; however, no increase in the stability of the ring expansion enzyme, desacetoxycephalosporin C synthase (DAOCS), was observed. Consequently, the conversion of the excreted intermediate penicillin N to cephamycin C was 15–20% less efficient at this high oxygen concentration. The increased dissolved oxygen level also led to the complete loss of IPNS and DAOCS activities for 4 h during the period of fastest growth, and the rate of specific cephamycin C production fell to zero. A several hundred fold increase in the level of iron in the defined media resulted in a sixfold improvement in the rate of specific cephamycin C production after 60 h fermentation. This increased rate appeared to be due to an elevation in the in vivo activities of a number of the cephamycin biosynthetic enzymes, particularly those catalysing later pathway steps.Key words: Streptomyces clavuligerus, isopenicillin N synthase, desacetoxycephalosporin C synthase, oxygen, iron.

scholarly journals Exploiting two-dimensional morphology of molybdenum oxycarbide to enable efficient catalytic dry reforming of methane

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexey Kurlov ◽  
Evgeniya B. Deeva ◽  
Paula M. Abdala ◽  
Dmitry Lebedev ◽  
Athanasia Tsoukalou ◽  
...  
Keyword(s):  
Contact Time ◽  
Specific Activity ◽  
Catalytic Performance ◽  
Dry Reforming ◽  
Dry Reforming Of Methane ◽  
Two Dimensional ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Oxygen Coverage ◽  
Rate Limiting

Abstract The two-dimensional morphology of molybdenum oxycarbide (2D-Mo2COx) nanosheets dispersed on silica is found vital for imparting high stability and catalytic activity in the dry reforming of methane. Here we report that owing to the maximized metal utilization, the specific activity of 2D-Mo2COx/SiO2 exceeds that of other Mo2C catalysts by ca. 3 orders of magnitude. 2D-Mo2COx is activated by CO2, yielding a surface oxygen coverage that is optimal for its catalytic performance and a Mo oxidation state of ca. +4. According to ab initio calculations, the DRM proceeds on Mo sites of the oxycarbide nanosheet with an oxygen coverage of 0.67 monolayer. Methane activation is the rate-limiting step, while the activation of CO2 and the C–O coupling to form CO are low energy steps. The deactivation of 2D-Mo2COx/SiO2 under DRM conditions can be avoided by tuning the contact time, thereby preventing unfavourable oxygen surface coverages.

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