Preparative high-performance liquid chromatography of minor products of Streptomyces cinnamonensis.

Analytical and preparative high-performance liquid chromatography of 3 phenazines and furonaphthoquinone derivative on reversed-phase column are described. The mobile phase was methanol and water. The injected amount of the mixture was about 30 mg for a preparative chromatographic run requiring 80 min. Substances were detected directly in the column effluent by UV detection.

Effect of clomiphene on fatty acids, sterols and membrane fluidity in clavine producing Claviceps purpurea strains.

Clomiphene depressed the growth and enhanced clavine production of Claviceps purpurea strains 129,35 and 59. Mycelial content of 18:2 and 16:0 fatty acids decreased, whereas that of 18:1 and 18:0 acids increased. In the mutant strain 59 clomiphene, triadimefon and ergosterol stimulated the impaired function of chanoclavine cyclase. Their effect was counteracted by plant oil. Clomiphene decreased the content of total lipids (44%), triglycerides (32%), sterols (22%) and sterol/phospholipid molar ratio. The PC/PE ratio was 9X increased. Clomiphene and triadimefon enhanced membrane fluidity of protoplasts, ergosterol and oil reverted their effect.

Formation of conidia in a saprophytic strain Claviceps paspali producing simple lysergic acid derivatives.

A non-mutant saprophytic strain C. paspali which forms conidia both on a solid medium and during submerged fermentation is described. Conidiation proceeded in parallel with culture growth and production of alkaloids. The effect of composition of culture media on the intensity of conidiation is described.

Substrate regulation of elymoclavine formation by some saccharides.

Regulation of the production of clavine alkaloids, especially elymoclavine, by sucrose, maltose, and mixtures of these saccharides was studied in submerged cultures of strains Claviceps purpurea 129/35 and Claviceps sp. SD-58. The data were statistically processed on an EC 1040 computer. Fermentation medium containing sucrose (80 g/l) in combination with glucose (20 g/l) was the best for elymoclavine formation. Retarded release of glucose from maltose increased the formation of elymoclavine and suppressed the synthesis of undesirable extracellular glucans. Carbon source can affect both the total amount of produced alkaloids and the relative proportion of individual clavines in the alkaloid mixture.

Extracellular metabolism of sucrose in a submerged culture of Claviceps purpurea: formation of monosaccharides and clavine alkaloids.

Transformation of extracellular sucrose during cultivation of Claviceps purpurea led to the formation of mono- and oligosaccharides. Maltose was a suitable substrate for submerged fermentation of alkaloids. Fermentation in a medium with maltose was characterized by an insignificant formation of glucans, intensive sporulation, suspension growth of mycelium, and a higher formation of elymoclavine. Glucose alone yielded low levels of total alkaloids and high glucan formation; on the other hand, glucose promoted the formation of elymoclavine.

Srinivasan's coagulation test in taxonomically classified streptomycetes.

Srimivasan's coagulation test was performed in 18 strains of the genus Streptomyces and one strain of the genus Actinoplanes. The highest coagulation activity was detected in strains systematically classified in a series of streptomycetes with pink or red aerial mycelium: S. erythreus, Streptomyces sp. AJ/22, S. roseo-luteus and S. griseofuscus. With the exception of S. griscofuscus these three cultures also exhibited the highest inhibitory activity B. subtilis. When using hemoglobin as substrate it was possible to detect acid, neutral and alkaline proteinases with the highest poroteolytic activity at pH 3.0 to 4.0 in the most active strain of S. erythreus.

Electron-cytochemical localization of phospho(enol)pyruvate carboxylase (EC 4.1.1.31) in fungal cells.

New cytochemical method, based on biochemical experiments, was elaborated for the ultrastructural localization of phospho(enol)pyruvate carboxylase (EC 4.1.1.31). The procedure was used to study the saprophytic submerged mycelium of the ascomycetous fungus Claviceps purpurea Tul. producing clavine alkaloids. The pelleted mycelium was fixed in ice cold 3% glutaraldehyde in 50 mM cacodylate buffer pH 7.2 and washed repeatedly in the same cold buffer The reaction mixture contained 100 mM Tris-HCl buffer pH 9.0, 10 mM phospho(enol)pyruvate, 30 mM sodium potassium tartrate, 3 mM Pb(NO3)2, 60 mM MgCl2 and 30 mM NaHCO3. Enzyme activity was localized in vacuoles, particularly inside lipid globules (spherosomes) and less frequently in membranous vesicles. Acetyl-CoA activated PEP-carboxylase both in cell free extracts and in the cytochemical staining. Aspartate inhibited the enzyme in the biochemical assay with coupled malate dehydrogenase system; the cytochemical reaction was not influenced, probably due to the interference of asparagine synthase (EC 6.3.1.1).

The cellular role of nitrogen in the biosynthesis of alkaloids by submerged culture of Claviceps purpurea (Fr.) Tul.

Asparagine was a superior nitrogen source for clavine-alkaloid production in Claviceps purpurea. Its transport into the cell excedded the cell's biosynthetic need for this amino acid. Asparagine entered the cell without degradation. This disturbed the relative pool sizes of various amino acids resulting in a change in the genetically determined ratio at which amino acids were utilized for protein synthesis. Overproduction of alkaloids (4500 mug.ml-1) may be associated with increased availability of tryptophan because of the enhanced assimilation of asparagine-derived ammonia via glutamine synthetase (EC 6.3.1.2). However, ammonium salts in the fermentation broth led to a depression of the alkaloid yield. Partial replacement of the ammonium salt by aspartic acid elevated alkaloid production.

Correlation of certain alterations in metabolic activity with alkaloid production by submerged Claviceps.

Alkaloid biosynthesis in Claviceps paspali MG-6 was favored by unbalanced growth. A positive correlation between the rate of protein turnover and alkaloid formation was noted. The pattern of the orthophosphate content in the mycelium resembled that in the ripening sclerotia of the parasitic strains. Alkaloids were revealed as potentially effective in energy metabolism. Reduced adenosine triphosphate (ATP) utilization and an increase in the ATP pool were found to be favorable for alkaloid production. Acetyl-coenzyme A carboxylase activity and the level of cell lipids were directly related to the intensity of alkaloid biosynthesis. An inverse relationship was observed between the activities of the tricarboxylic acid and glycoxylate cycles and the rate of alkaloid formation.