Production of Thermostable alpha-Amylase, Pullulanase, and alpha-Glucosidase in Continuous Culture by a New Clostridium Isolate

The production of alpha-amylase, pullulanase, and alpha-glucosidase and the formation of fermentation products by the newly isolated thermophilic Clostridium sp. strain EM1 were investigated in continuous culture with a defined medium and an incubation temperature of 60 degrees C. Enzyme production and excretion were greatly influenced by the dilution rate and the pH of the medium. The optimal values for the formation of starch-hydrolyzing enzymes were a pH of 5.9 and a dilution rate of 0.075 to 0.10 per h. Increase of the dilution rate from 0.1 to 0.3 per h caused a drastic drop in enzyme production. The ethanol concentration and optical density of the culture, however, remained almost constant. Growth limitation in the chemostat with 1% (wt/vol) starch was found optimal for enzyme production. Under these conditions 2,800 U of pullulanase per liter and 1,450 U of alpha-amylase per liter were produced; the amounts excreted were 70 and 55%, respectively.     

How perchlorate improves excitation-contraction coupling in skeletal muscle fibers.

The effect of the "chaotropic" anion, perchlorate, on the activation of contraction has been studied in voltage clamped frog skeletal muscle fibers. It was found that the voltage dependence of either the contractile force or the intramembrane charge movement was shifted towards more negative membrane potentials. The maximum values of force or charge movement attained with large depolarizing pulses did not change significantly. It is concluded that a specific perchlorate effect on the movement of charged particles can explain the potentiating effect of perchlorate anions on contractile force, strengthening the view that these charged particles serve as voltage sensors regulating Ca2+ release from the sarcoplasmic reticulum.     

Coenzyme specificity of dehydrogenases and fermentation of pyruvate by clostridia

Summary Four clostridial species (C. pasteurianum, C. butylicum, C. butyricum and C. tetanomorphum) grow on pyruvate. Two other species (C. roseum and C. rubrum) only ferment this compound; this is probably due to their inability to synthesize hexose phosphates from pyruvate (fructose-1,6-diphosphatase and pyruvate carboxylase are absent).The fermentation of pyruvate by the above clostridia yields acetate, carbon dioxide, hydrogen and small amounts of compounds more reduced than acetate. Hydrogen pressure increases the amount of ethanol, butanol and butyrate formed during the fermentation of pyruvate. Since C. roseum and C. rubrum contain a ferredoxin: NADP reductase it seems likely that NADPH₂ is the coenzyme involved in ethanol formation. In accordance with this acetaldehyde and alcohol dehydrogenases exhibit activity with NADPH₂.The glyceraldehyde-3-phosphate dehydrogenase of the clostridia under investigation is NAD specific and so is the -hydroxy-butyryl-CoA dehydrogenase with the exception of C. kluyveri.The specific activity of hydrogenase and the coenzyme specificity of NAD(P) reductase vary among the clostridial species.     

Verwertung von Fructose durch Hydrogenomonas H 16

Zusammenfassung Hydrogenomonas H 16 wächst gut auf Fructose, kann aber Glucose nicht verwerten. Die Fructoseoxydation wird durch Glucose auch in höheren Konzentrationen nicht beeinflußt. Die im zellfreien Extrakt vorhandene Hexokinase phosphoryliert Glucose, Fructose und Mannose. Durch free space-Versuche wird nachgewiesen, daß Glucose nicht in das Zellinnere transportiert wird. Hydrogenomonas H 16 verhält sich also cryptisch gegenüber Glucose.

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Summary The only carbohydrate utilized by Hydrogenomonas strain H 16 is fructose. The oxidation of fructose is not influenced by glucose even at high concentrations. Cell-free preparations contain hexokinase which phosphorylated glucose, fructose, and mannose. By means of free-space experiments, it has been demonstrated, that glucose does not enter the cell. Strain H 16 behaves therefore as a cryptic with respect to glucose.

     

Parameters Affecting Solvent Production by Clostridium pasteurianum

The effect of pH, growth rate, phosphate and iron limitation, carbon monoxide, and carbon source on product formation by Clostridium pasteurianum was determined. Under phosphate limitation, glucose was fermented almost exclusively to acetate and butyrate independently of the pH and growth rate. Iron limitation caused lactate production (38 mol/100 mol) from glucose in batch and continuous culture. At 15% (vol/vol) carbon monoxide in the atmosphere, glucose was fermented to ethanol (24 mol/100 mol), lactate (32 mol/100 mol), and butanol (36 mol/100 mol) in addition to the usual products, acetate (38 mol/100 mol) and butyrate (17 mol/100 mol). During glycerol fermentation, a completely different product pattern was found. In continuous culture under phosphate limitation, acetate and butyrate were produced only in trace amounts, whereas ethanol (30 mol/100 mol), butanol (18 mol/100 mol), and 1,3-propanediol (18 mol/100 mol) were the major products. Under iron limitation, the ratio of these products could be changed in favor of 1,3-propanediol (34 mol/100 mol). In addition, lactate was produced in significant amounts (25 mol/100 mol). The tolerance of C. pasteurianum to glycerol was remarkably high; growth was not inhibited by glycerol concentrations up to 17% (wt/vol). Increasing glycerol concentrations favored the production of 1,3-propanediol.     

Limitation of growth and lactic acid production in batch and continuous cultures of Lactobacillus helveticus

Summary Continuous and batch cultures of Lactobacillus helveticus operated under different conditions were studied with respect to the limitation of growth and lactic acid production by increasing undissociated lactic acid and hydrogen ion concentrations, respectively. In a single-stage continuous culture without pH control a final pH of 3.8 and 65 mm undissociated lactic acid was obtained. In two-stage continuous cultures provided with different growth media and run at different pH values, 65–70 mm free acid was obtained in the second stage. Further batch-culture experiments showed growth limitation at 60–70 mm lactic acid. After growth ceased, production of lactate continued until a lactic acid concentration of about 100 mm was reached; obviously an uncoupling of growth and acid production had occurred. Examining the effect of different concentrations of either lactic acid or hydrochloric acid, added to growing batch cultures of L. helveticus, it was shown that the undissociated lactic acid concentration was responsible for growth limitation and lactic acid production in this organism, whereas the pH value had only an indirect effect.     

H2: heterodisulfide oxidoreductase,a second energy-conserving system in the methanogenic strain Gö1

Washed everted vesicles of the methanogenic bacterium strain Gö1 catalyzed an H₂-dependent reduction of the heterodisulfide of HS-CoM (2-mercaptoethanesulfonate) and HS-HTP (7-mercaptoheptanoylthreonine phosphate) (CoM-S-S-HTP). This process was independent of coenzyme F₄₂₀ and was coupled to proton translocation across the cytoplasmic membrane into the lumen of the everted vesicles. The maximal H⁺/CoM-S-S-HTP ratio was 2. The tranmembrane electrochemical gradient thereby generated was shown to induce ATP synthesis from ADP+P_i, exhibiting a stoichiometry of 1 ATP synthesized per 2 CoM-S-S-HTP reduced (H⁺/ATP=4). ATP formation was inhibited by the uncoupler 3,5-di-tert-butyl-4-hydroxy-benzylidene-malononitrile (SF 6847) and by the ATP synthase inhibitor N,N-dicyclohexylcarbodiimide (DCCD). This energy-conserving system showed a stringent coupling. The addition of HS-CoM and HS-HTP at 1 mM each decreased the heterodisulfide reductase activity to 50% of the control. Membranes from Methanolobus tindarius showed F₄₂₀H₂-dependent but no H₂-dependent heterodisulfide oxidoreductase activity. Neither of these activities was detectable in membranes of Methanococcus thermolithotrophicus.Abbreviations _H+ transmembrane electrochemical gradient of H⁺ - CoM-SH 2-mercaptoethanesulfonate - F₄₂₀ (N-l-lactyl--l-glutamyl)-l-glutamic acid phosphodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin-5-phosphate - F₄₂₀H₂ reduced F₄₂₀ - HTP-SH 7-mercaptoheptanoylthreonine phosphate - DCCD N,N-dicyclohexylcarbodiimide - SF 6847 3,5-di-ert-butyl-4-hydroxybenzylidenemalononitrile - Mb. Methanobacterium - Ml. Methanolobus - Mc. Methanococcus - MV methylviologen - BV benzylviologen - MTZ metronidazole     

The pathway mediating insulin's effects on pyruvate dehydrogenase bypasses the insulin receptor tyrosine kinase

The effect of insulin on pyruvate dehydrogenase activity was examined in two different cell types that over expressed either normal or defective human insulin receptors, RAT 1 embryonic fibroblasts and Chinese hamster ovary (CHO) cells. Insulin stimulated pyruvate dehydrogenase activity in cells that expressed normal insulin receptors (RAT 1 HIRc, and CHO-WT and CHO-T cells), or receptors in which lysine 1018 in the ATP-binding site of the tyrosine kinase domain was exchanged for alanine (RAT 1 A/K1018 and CHO-mut cells). For both rat and hamster cell lines, the insulin dose-response curves from cells that expressed the mutant receptors were identical to those from the appropriate controls that over expressed the normal insulin receptors. Insulin failed to stimulate pyruvate dehydrogenase activity in CHO-delta cells, which expressed a mutant human insulin receptor that was truncated by 112 amino acids at the carboxyl terminal of the beta chain. Control studies verified that all the cells used in this study exhibited the expected phenotypes with respect to the number of insulin receptors which they expressed, insulin-stimulated tyrosine kinase activity, and the biological consequences of inactivating the insulin receptor tyrosine kinase. These findings show that the insulin receptor tyrosine kinase does not play an obligatory role in the insulin signaling pathway that stimulates pyruvate dehydrogenase activity.