Citric Acid Accumulation Research Articles

Presence and regulation of the alpha-ketoglutarate dehydrogenase multienzyme complex in the filamentous fungus Aspergillus niger.

alpha-Ketoglutarate dehydrogenase has been demonstrated for the first time in cell extracts from the filamentous fungus Aspergillus niger. A minimum protein concentration of 5 mg/ml is necessary for detecting enzyme activity, but a maximum of ca. 0.060 mumol/min per mg of protein is observed only when the protein concentration is above 9 mg/ml. alpha-Ketoglutarate can partly stabilize the enzyme against dilution in the assay system. Neither bovine serum albumin nor a variety of substrates or effectors of the enzyme could stabilize the enzyme against inactivation by dilution. A kinetic analysis of the enzyme revealed Michaelis-Menten kinetics with respect to alpha-ketoglutarate, coenzyme A, and NAD. Thiamine PPi was required for maximal activity. NADH, oxaloacetate, succinate, and cis-aconitate were found to inhibit the enzyme; AMP was without effect. Monovalent cations including NH4+ were inhibitory at high concentrations (greater than 20 mM). The highest enzyme activity was found in rapidly growing mycelia (glucose-NH4+ or glucose-peptone medium). We discuss the possibility that citric acid accumulation is caused by oxaloacetate and NADH inhibition of the alpha-ketoglutarate dehydrogenase of A. niger.

Influence of Nitrogen Metabolism on Lipid Accumulation by Rhodosporidium toruloides CBS 14

Rhodosporidium toruloides CBS 14 was grown in batch culture with urea as principal nitrogen source. The lipid content increased from 18% (w/w) with NH+ 4-grown cells to 52% (w/w) after 90 h growth. Urea was rapidly taken up and catabolized to release intracellular NH+ 4, which accumulated between 10 and 30 h growth. The increase in pool NH+ 4 content was mirrored by an increase in citric acid accumulation and excretion from the cells. The production of intracellular NH+ 4, sufficient to permit lipid accumulation, could be attributed to the increase in activity of urease over this period. Similarly, other catabolic enzymes, such as arginase, threonine dehydratase and NAD+: glutamate dehydrogenase, were also induced (or derepressed) when the respective amino acids were used as medium nitrogen source. Growth with mixed organic and inorganic nitrogen compounds considerably decreased the lipid content and was accompanied by a reduction in activity of the various catabolic enzymes concerned. The significance of nitrogen catabolism during lipid accumulation in this yeast is discussed.

The effect of the sugar source on citric acid production by Aspergillus niger

Under otherwise identical fermentation conditions, the sugar source has been shown to have a marked effect on citric acid production by Aspergillus niger. Sucrose was the most favourable source, followed by glucose and fructose and then lactose. No citric acid was produced from galactose. Strong relationships were observed between citric acid production and the activities of certain enzymes in myccelial cell-free extracts prepared from fermentation samples. When sucrose, glucose, or fructose was the sugar source pyruvate carboxylase activity was high, but 2-oxoglutarate dehydrogenase activity was not detected. When galactose was the sugar source pyruvate carboxylase activity was low, but 2-oxoglutarate dehydrogenase activity was high. It is suggested that whereas glucose and fructose repress 2-oxoglutarate dehydrogenase, thereby causing accumulation of citric acid, galactose does not. The activity of aconitase showed a direct relationship to the citric acid production rate. Thus, the activity was highest when sucrose was the sugar source, and lowest when galactose was the source. It is suggested that when large amounts of citric acid are lost from the cell the activity of aconitase increases as a response to the diminished intracellular supply of its substrate.

Pyruvate kinase from Aspergillus niger: a regulatory enzyme in glycolysis?

Pyruvate kinase from the filamentous, citric acid producing fungus Aspergillus niger was purified about 100-fold by ammonium sulfate precipitation, DEAE-cellulose chromatography, and gel filtration. The addition of fructose-1,6-diphosphate was necessary to prevent loss of activity during purification. The enzyme purified in the presence of fructose-1,6-diphosphate (FDP) exhibits hyperbolic kinetics with respect to phosphoenolpyruvate (PEP) and ADP. Monovalent cations activated the enzyme (K+, NH4+). FDP neither activated nor inhibited the enzymatic activity from extracts freshly prepared in the absence of exogenous FDP; ATP showed a weak activation. In contrast the enzyme from crude extracts which had been stored in the presence of glycerol for 3 days showed activation by FDP or a metabolite thereof and inhibition by ATP. In the absence of FDP sigmoidal kinetics were obtained with respect to PEP, which became hyperbolic kinetics after addition of FDP. ATP inhibition turned into slight ATP activation in the presence of FDP. However, it was possible to reactivate inactivated pyruvate kinase (after dialysis in the absence of FDP) by adding FDP to the enzyme assay. From these results and because of the very high affinity of pyruvate kinase for FDP (Ka less than 0.1 microM), it is concluded that the enzyme probably has FDP bound to the protein in vivo. The significance of this hypothesis to the regulation of glycolysis in A. niger, with special reference to the mechanism of citric acid accumulation, is discussed.

Effect of calcium on synthesis of dipicolinic acid inPenicillium citreoviride and its feedback resistant mutant

Dipicolinic acid synthesis inPenicillium citreoviride strain 3114 was inhibited by Ca2+ ions, but not by Ba2+, Cu2+or Fe2+. Among the metals tested, only Zn2+ inhibited the synthesis of dipicolinic acid and promoted sporulation. None of these metals reversed the inhibition by Ca2+ or Zn2+. A mutant 27133-dpa-ca selected for resistance to feedback inhibition by dipicolinic acid: Ca2+ complex showed cross-resistance to inhibition by dipicolinic acid: Zn2+. Both 3114 and271 33-dpa-ca excreted a number of aliphatic and amino acids during secondary metabolism of dipicolinic acid. In the presence of 1000 ppm of Ca2+, accumulation of citric acid and α-aminoadipic acid was completely inhibited under conditions of inhibition of dipicolinic acid in parent strain 3114 but not in the mutant. Citric acid with or without Ca2+ did not inhibit thede novo synthesis of dipicolinic acid in the strain 3114. In fact, citric acid in the presence of Ca2+ improved significantly rate of dipicolinic acid synthesis. Apart from resistance to feed back inhibition by dipicolinic acid: Ca2+ complex, mutant differed from the parent in three other aspectsviz. (i) dipicolinic acid synthesis was not subject to catabolite repression by glucose, (ii) sporulation as well as dipicolinic acid synthesis was dependent on the presence of Ca2+ ions in the medium and (iii) Mg2+ requirement for the mutant increased three fold. Higher requirement of the Mg2+ could be partially relieved by Ca2+ during secondary metabolism. The results support the inference thatde novo synthesis of dipicolinic acid is regulated through feedback inhibition by dipicolinic acid: Ca2+complex.

Studies on some enzymes relevant to citric acid accumulation by Aspergillus niger

Enzymatic studies have been performed on a local strain of Aspergillus niger to find a correlation with citric acid accumulation. The activity of aconitase [aconitate hydratase, citrate(isocitrate) hydrolyase, EC 4.2.1.3] and isocitrate dehydrogenase (NADP +) [threo- d s-isocitrate:NADP + oxidoreductase (decarboxylating) EC 1.1.1.42] decreased after 4 days whereas that of citrate synthase [citrate oxaloacetate-lyase ( pro-3 S- CH 2 COO −→ acetyl CoA), EC 4.1.3.7] did so after 8 days, when citric acid accumulation in the medium reached a maximum (45.9 mg ml −1). In vitro studies with mycelial cell-free extracts demonstrated inhibition of citrate synthase activity by sodium azide and potassium ferricyanide on both the 4th and 8th days. Aconitase was inhibited by sodium arsenate, sodium fluoride, iodoacetic acid and potassium ferricyanide only on the 4th day. Isocitrate dehydrogenase (NADP +) activity on the 4th and 8th days was inhibited by iodoacetic acid but was stimulated by potassium ferricyanide. The possible existence of isozyme species of these enzymes is discussed.

Partial purification and regulatory properties of phosphofructokinase from Aspergillus niger.

Phosphofructokinase (EC 2.7.1.11) from a citric acid-producing strain of Aspergillus niger was partially purified by the application of affinity chromatography on Blue Dextran--Sepharose and the use of fructose 6-phosphate and glycerol as stabilizers in the working buffer. The resulting preparation was still impure, but free of enzyme activities interfering with kinetic investigations. Kinetic studies showed that the enzyme exhibits high co-operativity with fructose 6-phosphate, but shows Michaelis--Menten kinetics with ATP, which inhibits at concentrations higher than those for maximal activity. Citrate and phosphoenolpyruvate inhibit the enzyme; citrate increases the substrate (fructose 6-phosphate) concentration for half-maximal velocity, [S]0.5, and the Hill coefficient, h. The inhibition by citrate is counteracted by NH4+, AMP and phosphate. Among univalent cations tested only NH4+ activates by decreasing the [S]0.5 for fructose 6-phosphate and h, but has no effect on Vmax. AMP and ADP activate at low and inhibit at high concentrations of fructose 6-phosphate, thereby decreasing the [S]0.5 for fructose 6-phosphate. Phosphate has no effect in the absence of citrate. The results indicate that phosphofructokinase from A. niger is a distinct species of this enzyme, with some properties similar to those of the yeast enzyme and in some other properties resembling the mammalian enzyme. The results of determinations of activity at substrate and effector concentrations resembling the conditions that occur in vivo support the hypothesis that the apparent insensitivity of the enzyme to citrate during the accumulation of citric acid in the fungus is due to counteraction of citrate inhibition by NH4+.

Malic and Citric Acids in Pickling Cucumbers

ABSTRACTThe content of major organic acids in pickling cucumbers has been determined. Malic acid was found to be the major organic acid in commercial size pickling cucumbers. The concentration among a group of six cultivars ranged from 0.2–0.3% on a fresh weight basis. Malic acid was present in all parts of the fruit. The highest concentration was in the outer 3 mm of the mesocarp, followed by the endocarp, the inner mesocarp, and the exocarp. During enlargement and maturation of cucumbers, citric acid became the principal organic acid, reaching levels in excess of 1% on a wet weight basis in the endocarp. This large accumulation of citric acid is the probable cause of the decline in endocarp pH during fruit maturation.

Effect of aeration and agitation on the biosynthetic activity of diffusely growing Aspergillus niger.

A considerable lowering of aeration demands occurs during diffuse growth of citric acid-producing Aspergillus niger in a submerged cultivation. However, the diffuse culture poses stricter demands on the type of aeration and agitation. The impeller frequency affects considerably the morphology of the producer fungus and the accumulation of citric acid. The effect of impeller frequency on the distribution of air in the medium and on the amount of air fed into the diffuse culture is less important.

Malate dehydrogenase isoenzymes inAspergillus niger

The accumulation and excretion of citric acid by Aspergillus niger is a notable example of fungal 'over-flow' metabolism, and for 30 years has been the world's primary source of citric acid. However, the biochemical information available on this process has to date not permitted its elucidation, but recently certain regulatory steps of A. niger energy metabolism have been investigated with respect to citric acid accumulation [ 1-4]. Malate dehydrogenase (EC 1.1.1.37) isoenzymes of A. niger were investigated because of the key role of oxaloacetate in the accumulation of citric acid [5]. They are involved in a shuttle mechanism which transports reduced NADH across the mitochondrial membrane and thereby generates mitochondrial oxaloacetate. On the other hand, there is a very active pyruvate carboxylase (EC 6.4.1.1), located intramitochondrially [6], whose activity in vivo has been shown to be strongly correlated with citric acid accumulation [7]. The present paper reports some molecular and regulatory properties of two malate dehydrogenase isoenzymes from A. niger. 2.1. Organism and conditions for growth

Pyridine nucleotide levels and ratios in Aspergillus niger.

A reliable method for the extraction and assay of pyridine nucleotides (NAD, NADH, NADP, and NADPH) in filamentous fungi is presented. The method is applied to a study of the physiology of citric acid accumulation by Aspergillus niger. The measurement of "catabolic reduction change" (NADH/(NAD + NADH)) and "anabolic reduction charge" (NADPH/(NADP + NADPH)) as a means of characterizing the metabolic state is discussed.

Regulation of citric acid production by oxygen: Effect of dissolved oxygen tension on adenylate levels and respiration in Aspergillus niger

The mechanism of the control of citric acid accumulation by oxygen was investigated by means of pilot plant fermentation using Aspergillus niger. The critical dissolved oxygen tension (DOT) for oxygen uptake of this fungus was about 18–21 and 23–26 mbar for trophophase and idiophase, respectively. Minimal DOT for citric acid production was about 25 mbar. Citric acid production increased steadily between 40–150 mbar. Short time changes in the DOT produced immediate, irreversible changes in the rate of product formation. Adenine nucleotides paralleled growth but showed no evidence for control function in the oxygen effect on citric acid fermentation. A branched respiratory system was identified by experiments using specific inhibitors (antimycin, cyanide, azide, rotenone, amytal and salicylhydroxamic acid). Growth was sensitive towards inhibitors of the standard respiratory chain, but only slightly sensitive towards salicylhydroxamic acid (SHAM). Citric acid synthesis was highly sensitive towards SHAM during trophophase, but sensitive towards antimycine during idiophase. Interruptions in aeration cause an impairment of the SHAM sensitive oxidase during trophophase, and of the antimycin sensitive oxidase during idiophase.

Metabolic Pathway of α-Ketoglutarate in Citrus Leaves as Affected by Phosphorus Nutrition

The uptake and metabolism of alpha-[5-(14)C]ketoglutarate by phosphorus-deficient and full nutrient (control) lemon (Citrus limon) leaves were studied over various time intervals. After 45 minutes in P-deficient leaves, the bulk of incorporated (14)C appeared in organic acids and much less in amino acids, while in the control leaves, the (14)C contents of organic and amino acids were equal. In P-deficient leaves, after longer incubation times the (14)C content of organic acids and amino acids increased, while that of CO(2) and residue fractions remained low. In full nutrient leaves the (14)C content of amino acids and organic acids decreased after longer incubation time and increased in the insoluble residue and CO(2). In full nutrient leaves the organic and amino acid metabolism were closely related and accompanied by protein synthesis and CO(2) release, while in P-deficient leaves an accelerating accumulation of arginine and citric acid was linked together with inhibition of protein synthesis and CO(2) liberation.

The role of the tricarboxylic acid cycle in citric acid accumulation by Aspergillus niger

Determinations of the momentary levels of various intermediates related to the activity of the tricarboxylic acid cycle have been made during citric acid production in high-accumulating (manganese deficient) and lowaccumulating (manganese supplemented) mycelia of Aspergillus niger. During the growth period the levels of almost all TCA cycle acids, with the exception of 2-oxo-acids, were unusually high; during the induction phase of citrate accumulation malate, fumarate, and isocitrate decreased, whereas pyruvate, oxalacetate, and citrate increased. The presence of succinate could not be demonstrated. The interrelations of the momentary concentrations of the intermediates mainly demonstrate a lack in activity of 2-oxoglutarate dehydrogenase, representing a block in the TCA cycle concomitant with a strongly operating glycolysis as a prerequisite for citrate accumulation. Inhibition studies with crude enzyme preparations suggest that an inhibition of malate dehydrogenase by citrate and also inhibition of isocitrate dehydrogenase by citrate and 2-oxoglutarate occur during the production phase as additional factors.

Influence of manganese on enzyme synthesis and citric acid accumulation inAspergillus niger

A comparison of citric acid fermentations in manganese-deficient and manganese-containing media showed that manganese strongly influences idiophase metabolism. In the presence of manganese, cell growth increases, sugar consumption is diminished and acidogenesis decreases drastically. An investigation of the key enzymes of glycolysis, the pentosephosphate pathway, TCA-cycle, nitrogen metabolism, and gluconeogenesis indicated that manganese deficiency was accompanied by a repression of anabolic and TCA-cycle-enzymes with the exception of citrate synthase. The activity of this enzyme and the enzymes of glycolysis paralleled the sugar consumption rate. In the presence of manganese, no repression of enzyme synthesis was observed. Activities of 2-oxoglutarate dehydrogenase and isocitrate lyase could not be detected in either case. The results support the hypothesis that manganese deficiency mainly affects the operation of biosynthetic reactions inAspergillus niger, thus leading to an overflow of citric acid as an end product of glycolysis.

Effect of Aminoethoxy Analog of Rhizobitoxine on Ripening of Pears

Ripening reactions in pears (Pyrus communis L.) were differentially affected by an aminoethoxy analog of rhizobitoxine (l-2-amino-4-[2-aminoethoxy]-trans-3-butenoic acid) (AAR). Ethylene production of both ;Anjou' and ;Bartlett' pears was inhibited by AAR. Decrease in firmness, increase in protein N and soluble pectin were delayed by AAR in ;Anjou' but not in ;Bartlett' pears. While loss in malic acid was retarded in ;Anjou' pears, rates of citric acid accumulation and malic acid reduction were not affected by AAR in ;Bartlett' pears.

Light stimulated 'shunt-metabolism' succinate-alpha-ketoglutarate-isocitrate cycle and accumulation of citric acid in Aspergillus niger.

Studies with light of the visible range had shown that light plays significant role in the biosynthesis and accumulation of citric acid in Aspergillus niger. Accumulation of 14C-labelled carbon atoms in alpha-ketoglutaric, isocitric, succinic and glycolic acids in the cultures grown under illumination suggest a probable 'shunt-metabolism' leading to the succinate-alpha-ketoglutarate-isocitrate (SKI) cycle. This shunt metabolism minimizes the accumulation of citric acid in cultures due to depletion of intermediates.

Regulation by zinc and adenosine 3′,5′-cyclic monophosphate of growth and citric acid accumulation in Aspergillus niger

The citric acid fermentation by Aspergillus niger is divided into two consecutive phases, growth phase when the cells proliferate but do not accumulate citrate, followed by an accumulating phase when they excrete citrate but do not proliferate. The phase alternative was controlled by the concentration of zinc: high zinc (2 muM) maintained growth phase, and a zinc 'deficiency' apparently signaled the ransition to the accumulating phase. Cyclic AMP affected the rates of growth and acidogenesis when added to cultures growing at low but not at high zinc: that is, cAMP did not induce the phase transition, zinc deficiency did, Cyclic AMP enhanced growth early in the fermentation, but at later stages the response of the mycelia to cAMP changed, and then cAMP inhibited growth. When citrate eventually began to accumulate cAMP augmented its sypthesis. The growth and acidogenic responses were quite specific to cAMP, and were sensitive to concentrations of about 1 muM. Cyclic AMP also either promoted or retarded the appearance of an unidentified yellow pigment. It is proposed that the growth and accumulating phases are distinct differentiated states, at least with respect to cAMP metabolism.

The citric acid fermentation by Aspergillus niger: regulation by zinc of growth and acidogenesis

The citric acid fermentation by Aspergillus niger is divided into two consecutive phases, a growth phase when the cells proliferate but do not accumulate citrate, followed by an accumulating phase when they produce citrate but do not proliferate, or else do so at a much reduced rate. When studied in a low sucrose (0.4-0.8%) mpinimal salts medium the growth-accumulation alternative was controlled by the concentration of zinc: high zinc (about 1-2 muM) maintained growth phase, while at low zinc (below 1 muM) growth became limited by zinc deficiency and the cultures passed into accumulating phase. Addition of zinc to accumulating cultures resulted in their reversion to growth phase. Iron, manganese, calcium at concentrations as high as 5-10 muM had no influence on either growth or citrate accumulation. These results suggest that zinc plays a role in the regulation of growth and citric acid accumulation.

A COMPARISON OF THE EFFECTS OF THE CALCITONINS, STEROID HORMONES AND THYROID HORMONES ON THE RESPONSE OF BONE TO PARATHYROID HORMONE IN TISSUE CULTURE

A bone culture system was used to compare the effects of several hormones on the response of 5-day-old mouse calvaria to parathyroid hormone (PTH). The results showed that salmon calcitonin was almost 10-5 times more active than any other hormone in preventing the PTH-induced release of calcium and caused a dose-related inhibition of calcium release over a range of 0-2-200 milli MRC units/culture. A high dose of calcitonin (200 milli MRC units) caused a net accretion of calcium in the absence of PTH. Progesterone and testosterone were more active than the naturally occurring oestrogens although a synthetic oestrogen (stillboestrol diphosphate) had approximately the same potency. High concentrations of these hormones caused a net accretion of calcium whether or not PTH was present. Cortisol was only effective at high doses, as was the steroid precursor cholesterol. In the present culture system the thyroid hormones (triiodothyronine and thyroxine) inhibited the action of PTH. It was concluded that these agents acted in a similar fashion to the oestrogens. That is, they prevented the accumulation of citric acid induced by PTH by reducing the rate of glycolysis. None of the hormones affected the inhibition of citrate oxidation caused by PTH. The results also showed that, whilst these hormones inhibited PTH-mediated bone resorption, they had an action on bone independent of PTH. Experiments with clomiphene citrate failed to demonstrate an oestrogen receptor in bone.