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Biomedical subjects

M Flieger

Publications and source records attributed to M Flieger.

30 records · Page 2Linked to original sources
PubMed1992

Effect of growth rate on ethanol tolerance of Saccharomyces cerevisiae.

delta 5,7-Sterol-accumulating Saccharomyces cerevisiae cells growing in chemostat at a specific growth rate of 0.075/h exhibited higher ethanol tolerance measured as ethanol-induced death and anaerobic growth inhibition than the cells growing at 0.2/h, the difference being dependent on the carbon-to-nitrogen molar proportion in the medium. The observed difference in sensitivity to ethanol of anaerobic growth between the slowly and rapidly-growing cells was completely reversed as a result of a block in sterol synthesis causing a negligible synthesis of delta 5,7-sterols. Two physiological parameters, budding frequency and membrane composition, evidently affected ethanol tolerance. Differences between the delta 5,7-sterol-synthesizing and deficient strains documented a profound effect of the quality of the sterol present on the physiological state of the cell.

Anaerobiosis↗
PubMed1992

Effect of 5,7-unsaturated sterols on ethanol tolerance in Saccharomyces cerevisiae.

Structural membrane lipids are known to contribute to the high ethanol resistance of Saccharomyces cerevisiae (2, 4, 17). By manipulating the yeast cellular sterol level by changing the carbon-to-nitrogen source ratio in the chemostat growth medium, high delta 5,7-sterol levels were found to increase the resistance of yeast populations to ethanol-induced death. The resistance of the erg2 (delta 8----delta 7-sterol isomerase) mutant to ethanol-induced death was generally comparable with that of the delta 5,7-sterol-synthesizing strain. In contrast, the sensitivity of anaerobic growth to inhibition by ethanol was higher in the erg2 mutant in comparison with the delta 5,7-sterol-synthesizing strains but a high level of those sterols increased the vulnerability of anaerobic growth to ethanol inhibition.

Anaerobiosis↗
PubMed1989

Alanine dehydrogenase from Streptomyces fradiae. Purification and properties.

Alanine dehydrogenase was purified to homogeneity from a cell-free extract of Streptomyces fradiae, which produces tylosin. The enzyme was purified 1180-fold to give a 21% yield, using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The relative molecular mass of the native enzyme was determined to be 210,000 or 205,000 by equilibrium ultracentrifugation or gel filtration, respectively. The enzyme is composed of four subunits, each of Mr 51,000. Using analytical isoelectric focusing the isoelectric point of alanine dehydrogenase was found to be 6.1. The Km were 10.0 mM for L-alanine and 0.18 mM for NAD+. Km values for reductive amination were 0.23 mM for pyruvate, 11.6 mM for NH4+ and 0.05 mM for NADH. Oxidative deamination of L-alanine proceeds through a sequential-ordered binary-ternary mechanism in which NAD+ binds first to the enzyme, followed by alanine, and products are released in the order ammonia, pyruvate and NADH.

Alanine Dehydrogenase↗
PubMed1988

Isolation of pure anhydrotetracycline oxygenase from Streptomyces aureofaciens.

Anhydrotetracycline oxygenase was purified to homogeneity from Streptomyces aureofaciens, a producer of tetracycline. The enzyme was purified 60-fold in a 40% yield by a two-step procedure using a combination of hydrophobic chromatography and ion-exchange h.p.l.c. Purified anhydrotetracycline oxygenase was homogeneous according to SDS/polyacrylamide-gel electrophoresis, isoelectric focusing, ion-exchange h.p.l.c. on a Mono Q HR 5/5 column and size-exclusion h.p.l.c. on a TSK G 3000 SW column. The enzyme consists of two subunits of Mr 57,500, as determined by SDS/polyacrylamide-gel electrophoresis.

Chromatography, High Pressure Liquid↗
PubMed1988

Valine dehydrogenase from Streptomyces fradiae: purification and properties.

Valine dehydrogenase (VDH) was purified to homogeneity from cell-free extract of Streptomyces fradiae, which produces tylosin. The enzyme was purified 1508-fold in a 17.7% yield using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The Mr of the native enzyme was determined to be 218,000 and 215,000, by equilibrium ultracentrifugation and size-exclusion high-performance liquid chromatography, respectively. The enzyme is composed of 12 subunits of Mr 18,000. Using analytical isoelectric focusing the isoelectric point of VDH was found to be 4.7. Oxidative deamination of L-valine was optimal at pH 10.6. Reductive amination of 2-oxoisovalerate was optimal at pH 8.8. The Michaelis constants (Km) were 1 mM for L-valine and 0.029 mM for NAD+. Km values for reductive amination were 0.80 mM for 2-oxoisovalerate, 0.050 mM for NADH and 22 mM for NH4+.

Amino Acid Oxidoreductases↗
PubMed1988

Isolation and characterization of valine dehydrogenase from Streptomyces aureofaciens.

Valine dehydrogenase was purified to homogeneity from the crude extracts of Streptomyces aureofaciens. The molecular weight of the native enzyme was 116,000 by equilibrium ultracentrifugation and 118,000 by size exclusion high-performance liquid chromatography. The enzyme was composed of four subunits with molecular weights of 29,000. The isoelectric point was 5.1. The enzyme required NAD+ as a cofactor, which could not be replaced by NADP+. Sulfhydryl reagents inhibited the enzyme activity. The pH optimum was 10.7 for oxidative deamination of L-valine and 9.0 for reductive amination of alpha-ketoisovalerate. The Michaelis constants were 2.5 mM for L-valine and 0.10 mM for NAD+. For reductive amination the Km values were 1.25 mM for alpha-ketoisovalerate, 0.023 mM for NADH, and 18.2 mM for NH4Cl.

Amino Acid Oxidoreductases↗
PubMed1987

Partial purification and characterization of anhydrotetracycline oxygenase of Streptomyces aureofaciens.

Anhydrotetracycline oxygenase was purified both by affinity chromatography and by hydrophobic interaction chromatography. Molecular weight of anhydrotetracycline oxygenase was determined to be 115,000 by Sephadex G-200 gel filtration. Using preparative isoelectric focusing the isoelectric point of the enzyme was estimated to be 5.3. The enzyme showed a sensitivity to thiol-specific inhibitors. During the hydrophobic interaction purification step, the activity dropped considerably. Reactivation occurred when a heat treated crude extract was added to the reaction mixture.

Chromatography, Affinity↗
PubMed1987

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.

Carbohydrate Metabolism↗
PubMed1982

Quantitative changes of the alkaloid complex in a submerged culture of Claviceps paspali.

Claviceps paspali FA produced high concentrations of alkaloid under submerged conditions. Their production was found to depend on the developmental stage and treatment of the filamentous culture inoculum. A medium containing Bacto-peptone with a constant composition of amino acids was selected for the preparation of the inoculum. A two-week fermentation in a synthetic medium with mannitol at 24 +/- 1 degrees C resulted in an increased production of total alkaloids from the original value of 100-200 micrograms/mL to more than 2000 micrograms/mL. Addition of tryptophan did not further increase the production of alkaloids but resulted in changes of the spectrum of some metabolites, 2,3-Dihydroxybenzoic acid accompanied the alkaloids in the fermentation medium. alpha-Hydroxyethyllysergamide was the predominant component of extracellular alkaloids (80% in the first days of fermentation). During fermentation the level of this alkaloid continuously decreased while the concentration of the accompanying alkaloids, i.e. lysergamide and the corresponding minor isomers, increased.

Alkaloids↗
PubMedDate not supplied

Electrokinetic separation of enantiomers using a capillary coated with poly-terguride.

Enantioseparation of 2-arylpropionic acids (Flobufen) and dansyl-derivatives of amino acids can be achieved in 10-15 min by electrochromatography using a capillary coated with poly-terguride. It was found that the analytes, in the range of the buffer pH between 2.5-4.0, were driven by anodic electroosmotic flow originated by the positively charged moieties of the ergolinic skeleton, and, only partially, by their anodic electrophoretic mobility. The enantiomers eluted with the same sequence, e.g. L- or (S)-isomer more retained than D- or (R)-, which was observed in HPLC separations on terguride-silica based CSPs, thus indicating that racemates were resolved by a similar chiral recognition mechanism. The retention of the solutes was affected by the concentration and composition of the effluent. An other mobile phase variable, strongly affecting the selectivity, was the pH of the buffer.

Amino Acids↗
PubMedDate not supplied

Use of cyclodextrins for the enantioselective separation of ergot alkaloids by capillary zone electrophoresis.

Ergot alkaloid enantiomer derivatives were resolved using capillary zone electrophoresis. The effect of cyclodextrins, added to the background electrolyte, on the migration time and the resolution was studied. Good separation for epimeric ergot alkaloid derivatives was also obtained using phosphate buffer at pH 2.5. Separation was improved by supplementing the background electrolyte with 30 mM of gamma-cyclodextrin. Good resolution of racemic ergot alkaloid derivatives in their enantiomers was achieved in a background electrolyte containing either beta-cyclodextrin or its derivative, or gamma-cyclodextrin.

Cyclodextrins↗