Citrate regulation of NADP+-specific isocitrate dehydrogenase of Aspergillus niger [proceedings

Abstract

Many theories have been proposed to account for the accumulation of citrate in selected strains of Aspergillus niger (Berry et al., 1977). Citrate accumulation was attributed by Ramakrishnan et al. (1955) to loss of aconitase and isocitrate dehydrogenase activity, but the presence of these enzymes was clearly demonstrated by La Nauze (1966). The presence of tricarboxylic acid-cycle enzymes throughout the fermentation was further demonstrated by Ahmed et al. (1972), using intact mitochondria. Mattey (1977) reported the presence of an NADP+-specific isocitrate dehydrogenase in mitochondria of an A . niger strain during the phase of growth in which citrate was produced, which was inhibited by citrate, but was not affected by other tricarboxylic acid-cycle intermediates. He postulated that the regulation of isocitrate dehydrogenase activity by citrate could form the basis for the control of citrate production. If this is correct, then citrate inhibition of isocitrate dehydrogenase should be found in other strains of A. niger. In the present communication we report on the effect of citrate on NADP+-specific isocitrate dehydrogenase enzymes from two strains, one of which (72-4) can yield commercially useful quantities of citrate under suitable conditions, the other normally cultured for cellulolytic enzymes (50-565 ii). Stock cultures of A. niger strain 72-4 of Shu & Johnson (1947) and A . niger Van Tieghem strain C.M.I.50-565 ii (Commonwealth Mycological Institute, Kew, Surrey, U.K.) were maintained on Czapek-Dox agar. The organisms were grown in a highsucrose medium (Ahmed et al., 1972) in shake culture from an inoculum of 5 x lo7 conidia. Changes in mycelial weight, medium pH and citrate concentrations in the medium and mycelium were determined over an incubation period of 14 days. Citrate was determined by the method of Saffran & Denstedt (1948), modified by Spencer & Lowenstein (1967). Both strains showed similar batch culture growth kinetics. A . niger strain 72-4 showed a marked increase in citrate concentration in the mycelium from an initial value of 0.5mM to a maximum of 4 . 6 6 m ~ by the fifth day of cultivation, when citrate accumulation in the medium became significant. A. niger strain C.M.I. 50-565 ii showed no significant variation in citrate concentration in the mycelium, reaching 0.55 mM by the seventh day of cultivation. There is thus an 8-fold difference in internal citrate concentrations between the two strains. The pH of the medium fell from 3.1 to 1.9 during the first 4 says in both strains. The internal pH of the mycelium did not seem to be affected by the external pH under these conditions. Strain 72-4 produced 2.5g of citrate per g of dry mycelium by day 10 of cultivation, and strain C.M.I. 50-565 ii produced 0.609g of citrate per g of dry mycelium at this time. It would therefore appear that citrate concentrations in the medium and the mycelium are related. Mitochondria were isolated and disrupted as previously described (Mattey, 1977). Isocitrate dehydrogenase activity was assayed by the method of Ochoa (1948), with NADP+ and D,-threo-isocitrate as substrates. The NADP+-specific isocitrate dehydrogenase activity from mitochondria, isolated from day-6 mycelia, was inhibited by citrate, but was not affected by malate, fumarate, succinate, a-oxoglutarate or pyruvate. Fig. 1 shows a double-reciprocal (LineweaverBurk) plot of NADP+-specific isocitrate dehydrogenase activity versus substrate concentration, in the presence and absence of citrate, from both strains. It was previously reported that citrate inhibition of the NADP+-specific isocitrate dehydrogenase appeared to be competitive, but it is apparent that at higher isocitrate concentrations the enzyme does not obey conventional Michaelis-Menten kinetics. The kinetic data suggested that two similar enzymes were acting on the same substrate, but