A fedbatch strategy was developed coupling the feeding of the two inhibitory substrates glycerol and ammonium to alkali consumption. A continuous, automated substrate addition was achieved responding directly to the needs of the culture. Thus substrate concentrations were kept on a constant low, but non limiting level. The feeding was applied for the cultivation of Clostridium butyricum DSM 5431 and mutants with increased product tolerance. Compared to fedbatch cultivations with intermittent feeding cultivation times were considerably shortened.
Subtilisin Carlsberg adsorbed on silica particles has been used to catalyze the transesterification of CBZ-Ala-ONp and CBZ-Leu-ONp with 1-butanol in organic systems preequilibrated to water activity of 0.93. Initial reaction rates are conveniently followed by extraction of the released nitrophenol into an alkaline aqueous phase. Kinetic parameters were determined for varied ester concentrations in toluene, isopropyl ether, and hexane. The effect of solvent on substrate solvation was determined by solubility measurements. Much of the observed effect of solvent on V(m)/K(m) may be accounted for by solvation differences. The residual effect of solvent on K(m), after discounting solvation differences, is completely opposite to the apparent trend. (c) 1994 John Wiley & Sons, Inc.
Dense cell cultivation of the recombinant cell line BHK 21 pSVIL2 was performed in a fluidized bed bioreactor system containing porous borosilicate glass carriers. Experiments were carried out with different medium formulations for a period of 48 days. Due to an effective immobilization of the cells in the reactor, continuous operation was easy to perform. Maximal cell densities and product yields could be maintained, even when protein-free medium was perfused exceeding 2 reactor volumes per day. Final cell densities of magnitude 7.1 x 10(7) mL-1 intrasphere volume were reached, while the interleukin-2 production rate was 0.70 mg day-1. The cell specific productivity reached a value of 1.3 x 10(-10) mg day-1. The first results were presented with a cell line that grows under glutamine-free medium conditions. The use of a glutamine-free medium for the cultivation of the cells resulted in a drastic decrease in cell metabolism. Furthermore, the amino acids lysine and histidine were produced and secreted into the culture supernatant, although these metabolites normally are considered to be essential for animal cells grown in vitro. However, no lethal effect on the cells has been detected, and the total number of cells in the reactor remained constant. The metabolism of threonine has been detected to be directly dependent on the presence of glutamine. Cells grown in glutamine-free culture medium produced glycine yields 6 times higher than those grown in glutamine-containing medium. A bead-to-bead transfer of the cells has also been detected when the cells immobilized within the intrasphere volume of the borosilicate carriers reached the stationary phase.
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