SUMMARYThe reduction of tetrathionate to thiosulphate by anaerobically growing Citrobacter caused a significant rise in specific growth rate and molar growth yield. It enabled also anaerobic growth on non-fermentable carbon sources and resembled therefore the respiratory reduction of nitrate to nitrite. The efficiency of energy production by tetrathionate respiration amounted to only two-thirds that of nitrate respiration.
Escherichia coli HB101 harboring an expression plasmid that bears the calf prochymosin gene controlled by the tac promoter was cultivated under different conditions in order to find an optimal fermentation arrangement that would lead to maximal prochymosin yield. Our results indicate that it is advantageous to use lactose in the double role of inducer and carbon/energy source when foreign gene expression is controlled by the tac promoter and the gene product is only moderately toxic owing to its accumulation in the form of an intracellular body. Glucose, on the other hand, may be used when expression should be repressed. Growth temperature substantially influenced the specific rate of prochymosin and beta-lactamase gene expression and the plasmid copy number. Three phases were distinguished in the time course of the fermentation on lactose: exponential growth practically without prochymosin synthesis, linear growth with prochymosin synthesis, and prochymosin synthesis without growth of biomass. The synthesis of prochymosin in the form of intracellular inclusion body was accompanied by the loss of respiratory activity of the cell and the loss of its ability to multiply. Sixteen hours cultivation at 37 degrees C in a complex medium with lactose as inducer and carbon/energy source resulted in up to 30% of the volume and 48% of the total protein of biomass being accumulated for as prochymosin inclusion bodies. The concentration of extractable enzymatically active chymosin in the culture reached 12 mg/L.
SUMMARYDuring the submerged batch cultivation of a riboflavin-producing strain of Eremothecium ashbyi three phases were observed. The first phase was characterized by rapid growth of mycelium, rapid utilization and oxidation of glucose and a decrease of pH value caused by accumulation of pyruvic acid. Subsequently acetoin accumulated in the medium. Glucose was oxidized incompletely since only 1 -8 pmole oxygen were consumed/pmole glucose utilized. The end of this phase was marked by exhaustion of glucose and cessation of growth. The second phase began with sporulation and was characterized by rapid synthesis of cell-bound riboflavin. Simultaneously a rapid increase in catalase activity and decreases of pyruvate and acetoin were observed. This was accompanied by a marked decrease in Qo, on glucose while the Qo, on pyruvate, threonine or acetaldehyde increased to a maximum. Ammonia accumulated in the medium and alkaline pH values were reached. The third phase was characterized by autolysis of mycelium which led to the release of riboflavin and to a decrease of enzymic activities. A comparison of all important physiological parameters was made with two strains of E. ashbyi of different riboflavin productivity. On the basis of correlation between riboflavin formation, catalase activity and respiration on acetaldehyde, an hypothesis is proposed to explain overproduction of riboflavin by a shift from the initial cytochrome type of terminal respiration to the flavoprotein type which is, however, accompanied by over-production of the flavin prosthetic group.
S U M M A R YCyanide (I mM) strongly inhibited aerobic respiration, and 2,4 dinitrophenol (0.2 mM) apparently uncoupled oxidative phosphorylation, in nongrowing Citrobacter. At these inhibitor concentrations, anaerobic tetrathionate reductase activity was not much affected. Aeration inhibited tetrathionate reductase activity; 0.2 m~-2 , 4 DNP did not influence oxygen inhibition, but I mM-KCN restored the reductase activity quantitatively. The process of aerobic respiration rather than the oxygen molecule itself therefore inhibits tetrathionate reductase activity. Induced synthesis of reductase required anaerobic conditions. Cyanide and 2,4 DNP allowed anaerobic synthesis of reductase; aeration prevented it. This effect of oxygen was abolished neither by KCN nor by 2,4 DNP. Oxygen therefore represses the synthesis of tetrathionate reductase directly.
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