Showing papers by "Arnold L. Demain published in 1984"

A pure enzyme catalyzing penicillin biosynthesis

Abstract: 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.

Enzymatic approach to syntheses of unnatural beta-lactams.

Abstract: 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.

Probable involvement of sulfhydryl groups and a metal as essential components of the cellulase of Clostridium thermocellum

Abstract: The crude extracellular cellulase from Clostridium thermocellum was oxidatively inactivated by air and inhibited by sulfhydryl reagents. Activity-loss was prevented and reversed by the addition of a high concentration (10 mM) dithiothreitol (DDT) at zero time and up to 24 h respectively. In the presence of a low concentration (0.4 mM) of DTT, the enzyme was more rapidly inactivated than in air alone. This was probably due to autoxidation of the low DTT concentration to H2O2 as shown by its prevention by a high DTT concentration, exclusion of air, or catalase; and by the oxidative inactivation of the enzyme by H2O2. The inactivation by H2O2 could be prevented by a high concentration of DTT but not by air exclusion. EDTA protected the enzyme from inactivation in air by a low concentration of DTT or by H2O2. This is presumably due to the role of metals in oxidation of SH groups. Furthermore, copper (5 μM) also caused inactivation and this was prevented by the presence of a high DTT concentration. Even in the protective atmosphere of a high DTT concentration, cellulase was inactivated by certain apolar chelating agents such as o-phenanthroline and α-α1-dipyridyl, such inactivation being preventable by the prior incubation of the chelator with a mixture of Fe2+ and Fe3+. These data suggest that the clostridial cellulase, unlike the enzyme from aerobic fungi, contains essential sulfhydryl groups and is stimulated by iron. The endo-β-glucanase component of the cellulase complex was not susceptible to oxidative inactivation.

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

Abstract: In a chemically defined medium with glucose and sucrose as major carbon sources (standard medium), Cephalosporium acremonium excretes the intermediate of the β-lactam biosynthetic pathway, penicillin N, into the medium during growth; production of cephalosporins is delayed until glucose is completely utilized. Deacetoxycephalosporin C synthetase, the ring-expansion enzyme (“expandase”), does not appear as long as glucose is present. Afterwards, initiation of its formation is accompanied by the production of cephalosporins. Feeding additional glucose during the fermentation turns off expandase synthesis without affecting formation of isopenicillin N synthetase, the ring-cyclization enzyme (“cyclase”). The above results point to a strong glucose catabolite repression of the expandase as one of the main regulatory mechanisms in β-lactam biosynthesis by Cephalosporium acremonium and the reason for accumulation of penicillin N during the fermentation. Cyclase shows a biphasic pattern in activity, the first very high peak not being correlated with the excretion of any β-lactam antibiotic into the medium.

Prevention of phosphate inhibition of cephalosporin synthetases by ferrous ion

Abstract: Phosphate interference in the production of cephalosporins by Streptomyces clavuligerus had been associated with repression of expandase (desacetoxycephalosporin C synthetase) and inhibition of both expandase and cyclase (isopenicillin N synthetase). The present work shows that inhibition of enzyme action could be prevented by increasing the Fe2+ added to the cell-free reactions or to resting cells incubated with chloramphenicol. Since excess Fe2+ could not reverse phosphate interference of antibiotic synthesis in complete fermentations, it is clear that the major cause of the phosphate effect in fermentations is phosphate repression, rather than phosphate inhibition caused by Fe2+ deprivation.

Enzymatic Synthesis of a Sulfur-Analog of Penicillin Using the “cyclase” of Cephalosporium Acremonium

Abstract: Enzymatic Synthesis of a Sulfur-Analog of Penicillin Using the “cyclase” of Cephalosporium Acremonium

Dependence of betaine stimulation of vitamin b(12) overproduction on protein synthesis.

Abstract: The betaine-stimulated differential synthesis of vitamin B(12), i.e., the increase in B(12) per increase in dry cell weight, by Pseudomonas denitrificans was inhibited by rifampin and chloramphenicol but not by benzylpenicillin and carbenicillin at concentrations of antibiotic that inhibit growth. The level of the first enzyme of corrin (and porphyrin) biosynthesis, delta-aminolevulinic acid synthetase, was decreased to a much greater degree by rifampin and chloramphenicol than by the penicillins. These data support the concept that betaine stimulation of B(12) synthesis is a result of its stimulation of synthesis of delta-aminolevulinic acid synthetase, a labile and presumably rate-limiting enzyme of corrin formation requiring continuous induction. In further support of this hypothesis, it was found that chloramphenicol immediately interfered with both vitamin B(12) and delta-aminolevulinic acid synthetase formation, no matter when it was added to the system.

In vitro biological activities of 6-isosteric penicillins and 7-isosteric cephalosporins

Abstract: Antibiotic and penicillinase inhibitor activities of various penicillin and cephalosporin analogs are reported. The compounds include C-6 penicillin and C-7 cephalosporin carbon, oxygen and sulfur analogs obtained by replacing the NH of the amide side chains with CH2, O and S, respectively. In almost all cases, analogs were considerably less active than the standard compounds (benzylpenicillin and cephalothin), However, some of the analogs act as penicillinae inhibitors.

Gramicidin S synthetase stabilization in vivo.

Abstract: We recently proposed a novel way of approaching the problem of optimal microbial production of secondary metabolites, such as antibiotics, namely increasing the in vivo stability of enzymes (synthetases) that catalyze the biosynthesis of these important chemicals.’ This suggestion reflects our current awareness that in vivo inactivation of microbial enzymes appears to be a general regulatory phenomenon irreversibly affecting the catalytic activity of many enzymes in the ceL2 The rapid in vivo inactivation of antibiotic synthetases is widespread and occurs during the idiophase of many batch fermentations.’ As a model system, we have attempted to understand and control the in vivo inactivation of gramicidin S synthetase, the enzyme complex responsible for the formation of gramicidin S (GS), a cyclic decapeptide antibiotic, in Bacillus brevis ATCC 9999. The pattern of appearance and disappearance of the synthetase‘ is typical of a large number of antibiotic fermentations: after the appearance of the enzyme complex in late exponential growth phase and the attainment of a peak value in specific activity, it rapidly disappears (half-life = 1-2 hours) as the cell population moves into stationary phase.’ Previous work6 had shown that inactivation of GS synthetase in vivo is oxygen-dependent and irreversible.

Capabilities of microorganisms (and microbiologists)

Abstract: Up to this point in the conference, I have absorbed several general impressions from the previous speakers. They involve the difficulties of studying microbial ecology, the importance of microbial survival, and the promise of microbial genetics in solving some of the problems of environmental pollution. These bring to mind several quotations, which I feel are relevant to these topics: “Ecology is physiology under the worst possible conditions.” — Thomas D. Brock “We may rest assured that as green plants and animals disappear one by one from the face of the globe, some of the fungi will always be present to dispose of the last remains.” — B.O. Dodge “...when you have mutants, you are better off than when you don’t.” — Salvador E. Luria