Die Supervision auf dem Weg zur Profession? Professionalisierung im Spannungsfeld von Expansionsbestrebung und Selbstbescheidung

Kühl S. Die Supervision auf dem Weg zur Profession? Professionalisierung im Spannungsfeld von Expansionsbestrebung und Selbstbescheidung. Organisationsberatung - Supervision - Coaching. 2006;13(1):5-18

The utilization of 2-ketogluconate by Hydrogenomonas eutropha H 16.

During growth of Hydrogenomonas eutropha H 16 on 2-ketogluconate, 2-ketogluconate kinase and 2-keto-6-phosphogluconate reductase were formed. These enzymes were absent from cells grown on fructose, gluconate, acetate, succinate or autotrophically. There was no evidence for extracellular oxidation of glucose, fructose or gluconate with the formation of ketogluconic acids. The utilization of 2-ketogluconate is neither subject to catabolite inhibition by hydrogen nor is 2-keto-6-phosphogluconate reductase inhibited by ATP, ADP or phosphoenolpyruvate. The enzyme is characterized by a high affinity for its substrates

Localization and stability of hydrogenases from aerobic hydrogen bacteria.

Alcaligenes eutrophus strains H 16, B 19, G 27 and N9A contained two different hydrogenases. One enzyme catalyzed the reduction of NAD by hydrogen and was strictly localized in the soluble cell fraction, while the second enzyme was found to be particulate and unable to react with NAD. All other tested strains, Alcaligenes paradoxus SA 29, Pseudomonas facilis, P. palleronii RH 2, Pseudomonas sp. strain GA 3, Paracoccus denitrificans, Aquaspirillum autotrophicum SA 32, and Corynebacterium autotrophicum 14g and 7C contained only a single enzyme exclusively bound to membranes. This was established using fractional centrifugation, indicator enzyme systems, gentle methods of cell disintegration and discontinuous sucrose density gradient centrifugation. In cell-free extracts obtained by rough disruption (sonication) of cells, hydrogenase was associated to particles of different size and sedimentation velocity. A partial solubilization of hydrogenase caused by sonication was observed with P. facilis. Without exception, the particulate hydrogenases were found (1) to be unable to reduce pyridine nucleotides, and (2) to reduce methylene blue at an extremely high activity. The eminent reaction rate of 34 μmoles H2 oxidized per min and mg protein has been determined in particle suspensions of Pseudomonas sp. strain GA 3. All hydrogenases were stable during storage under hydrogen atmosphere, except the soluble enzyme from A. eutrophus H 16 which was shown to be more stable under aerobic conditions

Physiological characterization of the hydrogen bacterium <em>Aquaspirillum autotrophicum</em>.

Aquaspirillum autrotrophicum, an aerobic hydrogen bacterium recently isolated from an eutrophic freshwater lake, was characterized physiologically. It grew autotrophically in a fermenter with a doubling time of 4 h. Heterotrophic growth was faster. pH-Optimum ranged from 5.0-7.5, temperature optimum was about 28&deg; C. During autotrophic growth about 10 moles hydrogen were consumed per 1 mole carbon dioxide fixed. Hydrogenase activity is inducible. CO2 did not enhance the oxy-hydrogen reaction by intact cells. The hydrogenase activity was localized in the particulate fraction. The hydrogenase reduced methylene blue and phenazine methosulfate; pyridine nucleotides were not reduced. In cell-free extracts, hydrogenase was sensitive to oxygen. Ribulosebisphosphate carboxylase was present in autotrophically-grown cells and absent from heterotrophically grown cells. Hydrogenase induction in heterotrophically-grown cells followed parabolic kinetics. Oxygen and D-gluconate repressed hydrogenase synthesis, whereas citrate, DL-lactate and pyruvate stimulated its formation. The repressive effect was delayed. The results suggest that the control of hydrogenase synthesis occurred at the transcriptional level, and that mRNA coding for the hydrogenase had a relatively long life span. D-Gluconate was degraded via the Entner-Doudoroff pathway, the enzymes of which were constitutively formed. Enzymes of the pentosephosphate and Embden-Meyerhof pathways (except phosphofructokinase) were present, too. Hydrogen did not inhibit heterotrophic growth. The possible competitive advantage of the physiological properties described with regard to the natural habitat was discussed

Studies on a gram-positive hydrogen bacterium, <em>Nocardia opaca</em> 1 b : III. Purification, stability and some properties of the soluble hydrogen dehydrogenase.

Nocardia opaca strain 1 b has a NAD-dependent hydrogenase (hydrogen dehydrogenase). The enzyme has been purified from autotrophically grown cells and tested for optimal assay conditions and stability. The purification procedure involved protamine sulfate treatment, ammonium sulfate precipitation, and separation by DEAE-cellulose and Sephadex G-200 chromatography and resulted in a 63-fold increase of specific activity at a 11.7% enzyme recovery. The final specific activity was 103 &mu;moles H2/min&middot;mg protein. The purified enzyme was dependent on nickel and magnesium ions at 0.5 and 5.0 mM concentrations, respectively, as well as flavin mononucleotide at a 5-10 &mu;M concentration. Straight enzyme kinetics were achieved by preincubating the enzyme in the presence of NADH2. A high stability of the enzyme was observed in 0.1 M potassium phosphate buffer, pH 6.5, in the presence of 0.5 mM nickel and 5 mM magnesium ions under hydrogen atmosphere. Even under air the enzyme was remarkably stable, although less than under hydrogen. From double reciprocal plots of substrate saturation curves the Michaelis-Menten constants were calculated: For saturating NAD-concentration the Kmwas 0.063 mM H2 and for saturating hydrogen concentration the Kmwas 0.123 mM NAD

Excretion of metabolites by hydrogen bacteria - III. D(-)-3-hydroxybutanoate.

D(-)-3-hydroxybutanoic acid was produced by a double mutant of Alcaligenes eutrophus, unable to synthesize poly-3-hydroxybutanoic acid and to utilize 3-hydroxybutanoate as a substrate. About 3.4 g of 3-hydroxybutanoate/l were produced under optimum conditions at pH 7.0 to 7.3, at a temperature of 30&deg;C after exhaustion of ammonia and at restricted aeration rates allowing 10-12% of the maximum respiratory rate of the cells to occur. D(-)-3-hydroxybutanoic acid was identified by means of gas and ion exchange chromatography, IR-spectrometry and specific rotation

Studies on a gram-positive hydrogen bacterium, <em>Nocardia opaca</em> strain 1b - I. Description and physiological characterization.

A new hydrogen bacterium has been isolated by enrichment culture on propane. It is a strictly aerobic, Gram-positive, non acid-fast bacterium, characterized by filamentous growth, and has been tentatively assigned to Nocardia opaca (strain 1 b). It grows heterotrophically, on many organic compounds (71 out of 138 tested substrates including organic acids and sugars), on hydrocarbons (C11-C18) as well as under autotrophic conditions (under an atmosphere of hydrogen, oxygen, and carbon dioxide=8:1:1) In the absence of a nitrogen source storage materials, mainly carbohydrates, are accumulated. Hydrogenase is an inducible enzyme. Under appropriate growth conditions the specific hydrogenase activity reaches high values: 2700 enzyme units/g cell protein. The formation of hydrogenase is repressed by fructose. With increasing oxygen concentrations during growth the specific hydrogenase activity decreases. In resting cell oxygen progressively inhibits the oxyhydrogen reaction. Cell-free extracts of autotrophically grown cells are able to reduce oxygen benzyl-and methyl viologen, dichlorphenolindophenol, methylene blue and nicotinamide adeninedinucleotide with hydrogen

Kinetics and properties of &beta;-ketothiolase from <em>Clostridium pasteurianum</em>.

1. Ketothiolase of Clostridium pasteurianum was purified 130-fold by ammonium sulphate fractionation and by column chromatography using DEAE-Sephadex A-50 and hydroxylapatite. Subjected to gel electrophoresis &beta;-ketothiolase revealed two distinct bands; by isoelectric focusing two enzymes with isoelectric points at pH 4.5 and 7.6 were separated. As established by sucrose density gradient centrifugation the molecular weight of both enzymes was found to be 158000. 2. The condensation reaction was measured by a coupled optical test using &beta;-hydroxybutyryl-CoA dehydrogenase as auxiliary enzyme and either acetyl-CoA or free coenzyme A plus acetyl-phosphate and phosphotransacetylase (regenerating system) or acetyl-CoA plus regenerating system as substrates. &beta;-Ketothiolase from C. pasteurianum used only 20% of the chemically synthesized acetyl-CoA; the enzyme from Alcaligenes eutrophus H 16 used 25%. When the regenerating system was added the condensation reaction continued. The enzyme from C. pasteurianum was inactivated by free coenzyme A, while the enzyme from A. eutrophus was inhibited. When acetyl-CoA was added as the substrate the initial velocity determination was impeded by the lack of linearity. With acetyl-CoA as the substrate the Km-value was found to be 2.5 mM acetyl-CoA. If free CoASH (or acetyl-CoA) plus regenerating system was added the Km was 0.44 mM (0.42 mM) acetyl-CoA. 3. The &beta;-ketothiolase activity was measured in the direction of acetoacetyl-CoA cleavage by an optical assay following the decrease of the enol and chelate form of acetoacetyl-CoA by absorption measurement at 305 nm. The activity was maximal at 24 mM MgCl2. The apparent Km values for acetoacetyl-CoA were 0.133 mM and 0.105 mM with 0.065 and 0.016 mM CoASH, respectively. The Km-values as calculated for only the keto form of acetoacetyl-CoA were 0.0471 and 0.0372 mM, respectively. The cleavage reaction was inhibited by high acetoacetyl-CoA concentrations; the inhibition was partially relieved by CoASH. In the range of low concentrations of acetoacetyl-CoA only a slight inhibition by CoASH was observed. The Km for CoASH was found to be 0.0288 and 0.0189 mM with 0.09 and 0.045 mM acetoacetyl-CoA, respectively. High concentrations of CoASH exerted an inhibitory effect on the cleavage reaction. With respect to enzyme kinetics and sensitivity to inhibitors and metabolites the &beta;-ketothiolases of C. pasteurianum and A. eutrophus were rather similar