Characterization of succinic semialdehyde dehydrogenase from Aspergillus niger.

The catabolism of fungal 4-aminobutyrate (GABA) occurs via succinic semialdehyde (SSA). Succinic semialdehyde dehydrogenase (SSADH) from the acidogenic fungus Aspergillus niger was purified from GABA grown mycelia to the highest specific activity of 277 nmol min-1 mg-1, using phenyl Sepharose and DEAE Sephacel chromatography. The purified enzyme was specific for its substrates SSA and NAD+. The substrate inhibition observed with SSA was uncompetitive with respect to NAD+. While product inhibition by succinate was not observed, NADH inhibited the enzyme competitively with respect to NAD+ and noncompetitively with respect to SSA. Dead-end inhibition by AMP and p-hydroxybenzaldehyde (pHB) was analyzed. The pHB inhibition was competitive with SSA and uncompetitive with NAD+; AMP competed with NAD+. Consistent with the kinetic data, a sequential, ordered Bi Bi mechanism is proposed for this enzyme.

Effect of addition of proton carriers in culture medium on growth and secretion of hybridoma cell line OKT3.

Monoclonal antibodies (MAb) constitute the centre of all in-vitro diagnostic measures and almost all in-vivo therapeutic manoeuvres now. Production emphasis for these antibodies is having a current shift from animal-based large-scale culture to in-vitro bioreactor-based high-density culture. One of the major difficulties in high-density culture is end-metabolite accumulation in batch and fed-batch cultures in the forms of H+, NH4+ etc.. thereby reducing cellular growth and secretions. In the present study, effects of added proton carries--NAD and NADP--over and above the metabolic pools of the molecules, were examined on the cellular growth and secretion kinetics. Although NADP fortification showed a remarkable improvement in cellular growth (time dependent 200-300% improvements compared to controls) and size, cumulative MAb titre was better with NAD fortification. Combined additional loads of the proton carriers would be interesting to study in high density culture conditions.

Kinetic analyses of the pyruvate dehydrogenase complex from Candida 107 (NCYC 911).

Candida 107 (NCYC 911) accumulates up to 45% of the biomass as triglycerides under conditions of nitrogenous substrate limitation in the medium. In oilseeds and adipocytes, lipid accumulation is preceded and accompanied by increased activity of key enzymes such as pyruvate dehydrogenase. However, in Candida 107, the activity of this complex was greatly reduced during lipogenesis. The initial velocity patterns were in accordance with a Hexa Uni Ping Pong mechanism. The Km values for the various substrates were similar to those found for the yeast Saccharomyces cerevisiae, but much higher than those reported for the mammalian enzyme. Product inhibition studies indicated that the Ki for acetyl coenzyme A and NADH were higher than those reported for other yeasts. The values for Ki were similar to those found for the liver enzyme, whereas the enzyme complex from heart had much lower Ki values for products. It has been suggested that in the heart and kidney, pyruvate dehydrogenase is regulated by product inhibition whereas in the liver this does not appear to be the mechanism. Therefore, it is probable, that like the liver enzyme, pyruvate dehydrogenase from Candida 107 may not be regulated by product inhibition.