Utilization of gluconate by Aspergillus niger.

Zentralbl. ~Iikrobiol. 141 (1986), 461-469 VEB Gustav Fischer Verlag Jena [Institute of Animal Nutrition, University of Hohenheim, Stuttgart, FRG]

Utilization of Gluconate by Aspergillus niger. II. Enzymes of Degradation Pathways and Main End Products H.-M. MULLER With 1 Figure

Summary Aspergillus niger was grown from conidia on a medium with glucose as the source of carbon and potassium nitrate in non-limiting concentration as the source of nitrogen. After the exhaustion of glucose gluconate was added, this compound representing the almost only carbon source in the culture fluid at this time. Gluconate was used rapidly by the preformed mycelium, the main end-products of its metabolization being mycelial substance (including protein), CO 2 , and oxalate. In cell-free extracts from gluconate utilizing mycelia 8 enzymes of the EmbdenMeyerhof (EM) pathway, 5 enzymes of the tricarboxylic acid (TCA) cycle, and an oxalate forming enzyme, oxaloacetate hydrolase (EC 3.7.1.1) were identified. The addition offluoroacetate together with gluconate resulted in the accumulation of citrate, and in the inhibition of mycelial growth and of accumulation of oxalate. It is concluded that the EM pathway and the TCA cycle are involved in the formation of mycelial substance, CO 2 and oxalate from gluconate, There is good correspondence between the rates of gluconate utilization and of oxalate accumulation which were observed immediately after the addition of gluconate and the in vitro activities of glUCOllOkinase and oxaloacetate hydrolase, respectively, at this time.

Zusammenfassung Aspergillus niger wurde ausgehend von Konidien auf einem Medium mit Glucose als C-Quelle und Kaliumnitrat in nichtlimitierender Konzentration als N-Quelle kultiviert. Nach dem Verbrauch der Glucose wurde Gluconat zugesetzt, wobei diese Substanz zu diesem Zeitpunkt die fast einzige C-Quelle in del' Kulturfhissigkeit darstellte. Gluconat wurde durch das vorkultivierte ~Iycel schnell verbraucht, die hauptsitchlichen Endprodukte seines Stoffwechsels waren 1Iycelsubstanz (einschliel3lich Protein), CO 2 und Oxalat. In zellfreien Extrakten aus Gluconat verbrauchenden Mycelien wlirden 8 Enzyme des Embden-Meyerhof-Weges, 5 Enzyme rles Tricarbonsiture. zyklus und ein Oxalat bildendes Enzym (Oxalacetat-Hydrolase, EC 3.7. I.I) identifiziert. Der ZlIsatz von Fluoracetat zllsammen mit Gluconat fiihrte zur Anhiillfung von Citrat, und zur Hemmung des Mycelwachstums und del' Anhaufung von Oxalat. Es wird geschlossen, da13 del' Embdell~Ieyerhof-Weg und del' Tricarbonsiiurezyklus an del' Bildung von Mycelsllbstanz, CO 2 und Oxalat aus Gluconat beteiligt sind. Es besteht gute t'bereinstimmung zwischen den Geschwindigkeiten des Verbrauchs von Gluconat und del' Anhitufung von Oxalat, wie sie lInmittelbar nach dem Zusatz des Gluconats beobachtet wllrden, und del' in vitro Aktivitiit del' Gluconokinase hzw. del' Oxalacetat-Hydrolase zu diesem Zeitpllnkt.

In a preceding study (MULLER 1985) evidence was presented that the formation of 6-phosphogluconate and its metabolization via the pentose phosphate pathway are the first steps of gluconate utilization by a strain of A8pergillu8 niger. In the present investigation cell-free extracts from gluconate degrading mycelia were examined for enzymes of the Embden-Meyerhof (EM) pathway and the tri-

462

H.-M. MULLER

carboxylic acid (TCA) cycle. It is known from other organisms that these pathways can be involved in the utilization of intermediates of the pentose phosphate (PP) cycle (COCHRANE 1976). As yet, almost all enzymes of the EM pathway and the TCA cycle and part of the PP cycle enzymes have been identified in cell-free extracts from Aspergillus niger after growth from conidia with glucose or sucrose as carbon source (KUBICEK and ROHR 1977; see also BLUMENTHAL 1965,1968; NIEDl.;RPRUEM 1965 for reviews of literature). However, little is known on the enzyme make-up of gluconate utilizing cultures of this mold. LAKSHMINARAYANA et al. (1969a) adapted a strain of A. niger to rapid growth from conidia with gluconate as the sole carbon source. Cell-free extracts from this adapted mycelium were examined only for two enzymes of the EM pathway and one enzyme of the TCA cycle (LAKSHMINARAYANA et al. 1969b). Therefore, it was of interest to test gluconate utilizing mycelia also for other enzymes of these pathways. In order to investigate whether the TCA cycle is operating in vivo fluoroacetate was added to gluconate utilizing cultures. It was also studied which are the main end-products of gluconate utilization. Because it was found that oxalate was one of these products, cell-free extracts were also examined for the activity of an oxalate forming enzyme, oxaloacetate hydrolase (EC 3.7.1.1). Rates of disappearance of gluconate from the culture fluid and of production of oxalate were compared with in vitro activity of enzymes.

Materials and Methods I. Cultural conditions The strain used, the culture medium (containing 5 % glucose and 1 % KNO a as the sources of carbon and nitrogen), the inoculation with conidia and other experimental conditions were the same as described previously (MULLER 1965, 1966). The incubation temperature (43°C) allowed rapid growth but no formation of conidia.

II. Analytical methods For the determination of its dry weight the mycelium was washed with cold water and dried by lyophilization. The dry weight was corrected for the oxalate content of the mycelium. Total carbon in the lyophilized mycelium, the production of volatile metabolites (C0 2 , ethanol), the content of the culture fluid in glucose, and the content of the culture fluid and the mycelium in citrate and oxalate were determined as previously described (MULLER 1965, 1966, 1967). The content of the culture fluid in gluconate was determined according to MOELLERING and BERGMEYER (1974). For determination of the protein content of the mycelium a cell-free extract was prepared (extraction with 0.4 M potassium phosphate buffer, pH 6.5), and the protein content was determined according to BRADFORD (1976). Nitrate in the culture fluid was determined by the micro-Kjeldahl method after reduction with zinc and correction for the non-nitrate N.

III. Preparation of cell-free extracts Mycelial mats were removed from the culture fluid, washed with distilled water and gently pressed to remove as much water as possible. The whole mat was ground in a mortar with a pestle together with 5-6 g of quartz. After the mat was broken into small fragments extraction buffer was added in portions of 2-3 ml, and the homogenization was continued for 5-8 min until a homogeneous paste was obtained. This paste was brought to 25 ml with extraction buffer, and an aliquot (10 ml) was centrifuged at 34,000 g for 10 min. The residue was re-extracted twice with 5 ml of buffer, the supernatants were combined and brought to 20 ml with buffer. All these operations were carried out at I-3°C. The cell-free extract was kept in an ice bucket and was used for enzyme assays as soon as possible.

Utilization of Gluconate by Aspergillus niger. II.

463

IV. Enzyme assay procedures The reaction mixture for the assay of hexokinase (EC 2.7.1.1) contained Tris-HCl buffer, pH 7.7, 100,uM, MgS0 4 5,uM, ATP 5,uM, NADP 0.7 pM, glucose 6-phosphate dehydrogenase (5,ug ~ 1. 7 units), glucose 5,uM, cell·free extract (extraction with 0.4 M potassium phosphate buffer, pH 6.5), H 2 0 ad 1.5 m!. The reaction mixture for the assay of phosphoglucose isomerase (EC 5.3.1.9) contained Tris-HCl buffer, pH 7.7, 50It:\f, MgS0 4 5,uM, NADP 0.7,uM, glucose 6- phosphate dehydrogenase (5,ug ~ 1.7 units), fructose 6- phosphate 5 pM, cell-free extract (extraction with 0.8 M Tris-HCl buffer, pH 7.5). The activity of both enzymes was measured at 334 nm against a blank without substrate. Fructose-biphosphate aldolase (EC 4.1.2.13), glyceraldehyde-phosphate dehydrogenase (EC 1.2.1.12), phosphoglyceromutase (EC 2.7.5.3), enolase (EC 4.2.1.11), pyruvate kinase (EC 2.7.1.40) malate dehydrogenase (EC 1.1.1.37) were assayed according to BERGMEYER et a!. (1974), phosphofructokinase (EC 2.7.1.11) according to GANCEDO and GANCEDO (1971), citrate synthase (EC 4.1.3.7) according to RAMAKRISHNAN and MARTIN (1954), fumarate hydratase (EC 4.2.1.2) according to RACKER (1950), and the following extraction buffers were used: 0.02 M potassium phosphate, pH 7.5 (phosphofructokinase, enolase), 0.4 M potassium phosphate, pH 6.5 (other enzymes). Aconitate hydratase (EC 4.2.1.3), NADP-isocitrate dehydrogenase (NADP) (EC 1.1.1.42), and oxaloacetate hydrolase (EC 3.7.1.1) were extracted and assayed as described previously (1\H'LLER 1975; 1\hiLLER and FROSCH 1975). Gluconokinase (EC 2.7.1.12) and 6-phosphogluconate dehydrogenase (EC 1.1.1.43) were extracted with 0.4 M potassium phosphate buffer, pH 6.5, and assayed as described previously (MULLER 1985). All assays were done at 25 DC. The amount of mycelial extract added to the reaction systems was such as to provide substrate saturation. Activities were calculated from the initial rate of reactions, with the exception of gluconokinase whose activity was calculated once it had mounted to a constant value.

V. Chemicals D,L-Isocitrate (alIo.free), sodium salt was from Sigma, St. Louis, monofluoroacetate (sodium salt) and oxaloacetic acid were from Schuchardt, Hohenbrunn, other special chemicals and the auxiliary enzymes used in the enzyme assays were obtained from Boehringer, Mannheim, all other chemicals (reagent grade) from Merck, Darmstadt.

Results 1. Effect of gluconate on mycelial growth and the production of CO 2 and oxalate The effect of the addition of gluconate to preformed cultures can be seen from Fig. 1. In cultures to which no gluconate was added the mycelial dry weight began to decrease 78 h after inoculation. At this time the glucose of the culture medium had been used up almost completely and there were almost no other carbon sources available in the culture fluid (Table I). After the addition of a large amount of gluconate the mycelial dry weight continued to increase for about 70 h, i.e. up to the consumption of the greatest part of gluconate, then autolysis led to a significant loss of mycelial substance also in these cultures. During the first 50 h of gluconate consumption there was also an increase of the protein content of the mycelium per flask, this indicating true mycelial growth during this time. Net biosynthesis of protein was possible because at the time of gluconate addition only 2/ 3of the nitrogen source of the original medium (KNO a) had been consumed. The addition of gluconate resulted also in a marked stimulation of the production of CO 2 and oxalate as compared to control cultures which started to autolyze after the consumption of glucose.

464

H.-l\I. ~IuLLER

mg

BOO

A

600

400 200

of - - - - - - - - - J - - - - - - - L - - - - - I B

mg

30

20 10

O~------'---------'---_l

mM

C

8

DD, D\ D

'b

6

\"

4

2

':-

o

"\---" D

'-_,..._IVLa_....L....__ a_

100 200 HOURS AFTER INOCULATION

Fig. 1. Mycelial growth of Aspergillus niger with gluconate as carbon source and effect of fluoro. acetate on the utilization of gluconate. The mycelium was grown from conidia with glucose as carbon source, after exhaustion of glucose (78 h after inoculation) either sodium gluconate (8.5 mMI flask, in 5 ml H 2 0), or sodium gluconate (8.5 mM/flask, in 5 ml H 2 0) and monofluoroacetate (2.5 mM/flask, in 2.5 ml H 2 0), or H 2 0 (7.5 ml) were added and the incubation continued. All values are calculated per flask. A: )'Iycelial dry weight; B Protein in the mycelium; C: Gluconate in the culture fluid. 0: Gluconate added; 0: Gluconate and fluoroacetate added; \7: H 2 0 added.

Utilization of Gluconate by Aspergillus niger. II.

465

Table 1. Production of mycelial substance and of metabolites during growth of Aspergillus niger with glucose and gluconate as sources of carbon in the culture fluid. The mycelium was grown from conidia with glucose as carbon source (total carbon in the noninoculated medium: 545 mg/flask). After the exhaustion of glucose (78 h after inoculation) 8.5 mM sodium gluconate were added per flask and the incubation continued for 72 h. During this time 8.1 mM gluconate were consumed per flask. Time of incubation

78 h (0-78 h after inoculation)

72 h (78-150 h after inoculation)

mg total carbon % per flask consumed

mg total carbon % ofgluconate1 ) per flask consumed

223.5 265.2 4.2 0.8 23.4 7.0 531.1

138 164

23.7 28.1

223 ND 525

38.2 ND 90.0

of glucose1 )

Mycelial dry substance Carbon dioxide Gluconate Citrate Oxalate Ethanol Sum

41.5 49.3 0.8 0.1 4.3 1.:~

97.3

1) Calculated on the basis of total carbon. XD: Not determined

2. Main end-products of gluconate utilization At the time of addition of gluconate to the preformed cultures this compound represented the almost only carbon source in the culture fluid. As can be seen from Table 1, there was very little glucose left at the time of addition of gluconate and only minor amonts of this, of oxalate and of (volatile) ethanol, and traces of citrate had been formed up to this time besides the main products mycelial substance and carbon dioxide. During the period of mycelial growth from gluconate (78-150 h after inoculation) mycelial substance, CO 2 and oxalate were the main end-products of gluconate utilization. The yields of total carbon in these three fractions, related to total carbon of gluconate consumed, were 24, 28 and 38 % (Table 1). The calculation of these yields did not take into account the changes in mycelial dry weight, CO 2 and oxalate observed during the same period in cultures without added gluconate because these cultures were subjected to autolysis. 3. Identification of enzymes

III

cell-free extracts

In cell-free extracts from mycelia which were able to grow with gluconate as the source of carbon after preculture with glucose (time of extraction: 40 h after addition of gluconate) the following enzymes of the EM pathway and the TCA cycle were identified: Hexokinase, phosphoglucose isomerase, phosphofructokinase, fructosebiphosphate aldolase, glyceraldehyde-phosphate dehydrogenase, phosphoglyceromutase, enolase, pyruvate kinase, citrate synthase, aconitate hydratase, isocitrate dehydrogenase (NADP), fumarate hydratase, and malate dehydrogenase. In addition, an oxalate forming enzyme, oxaloacetate hydrolase, was found. All these enzymes were also detected at the time of gluconate addition. 4. Effect of fluoroacetate on the utilization of gluconate Fluoroacetate is an inhibitor of the TeA cycle, its action involving prior enzymic conversion to fluorocitrate, an inhibitor of aconitate hydratase (PETERS 1957). Its addition together with gluconate inhibited mycelial growth completely, the mycelial

466

H.·)I.

l\WLLER

Table 2. Effect of fluoroacetate on the accumulation of citrate and oxalate from gluconate by Aspergillus niger. The mycelium was grown from conidia with glucose as carbon source, after exhaustion of glucose (78 h after inoculation) either sodium gluconate (8.5 mM/flask, in 5 ml H 20), or sodium gluconate (8.5 mM/flask, in 5 ml H 20) and monofluoroacetate (2.5 mM/flask, in 2.5 ml H 20), or H 20 (7.5 ml) were added and the incubation continued. Time after inoculation (h)

78

126

Citrate (mM/f1ask)

a b c

0.09 0.09 0.09

0.04 0.46 0.06

Oxalate (mM/flask)

a b c

2.0 2.0 2.0

7.3 2.9 3.1

a: gluconate; b: gluconate

+ f1uoroacetate;

150 0.04 1.26 0.05 11.3 2.4 3.7

174 0.02 1.65 12.2 0.9

246 0.03 2.44 0.02 12.2 3.0 3.0

c: H 2O

dry weight and the protein of the mycelium decreasing with almost the same rate as in control cultures without gluconate (Fig. 1). The rate of gluconate disappearance at first was not affected by fluoroacetate but then was lowered to almost zero, this leading to the consumption of only 70 % of gluconate. In contrast, gluconate was used up completely in the absence of fluoroacetate (Fig. 1). The addition of this inhibitor also led to a strong decrease of the yield of oxalate but elicited the accumulation of citrate (Table 2). 5. Comparison of rates of gluconate utilization and of production of oxalate with enzyme activities in cell-free extracts Gluconate added to the culture fluid disappeared with a constant rate during almost the whole time of its utilization (Fig. 1). This rate is in good correspondence with the in vitro activity of gluconokinase, whereas the activities of 6-phosphogluconate Table 3. Rates of utilization of gluconate and of production of oxalate by Aspergillus niger and enzyme activities in cell·free extracts determined at the time of addition of gluconate. Rates of utilization of gluconate and of production of oxalate were determined graphically, all values are calculated per flask pM/min Rate of gluconate disappearance Rate of oxalate production Gluconokinase 6-Phosphogluconate dehydrogenase Phosphoglucose isomerase Phosphofructokinase Fructose-biphosphate aldolase Glyceraldehyde.phosphate dehydrogenase Phosphoglyceromutase Enolase Citrate synthase Aconitate hydratase Isocitrate dehydrogenase (NADP) Fumarate hydratase Oxaloacetate hydrolase

2.2 2.5 4.4 13.2 380 20 24 32 8 18 14.8 3.7 4.6 33 2.1

Utilization of G1uconate by Aspergillus niger. II.

467

dehydrogenase and of enzymes of the EM pathway were found to be significantly higher (Table 3). The correspondence is even better if gluconokinase was assayed at 43 0 0, the incubation temperature of the mycelium, because its activity determined at this temperature was 80% of that determined at 25 00 (MULLER 1986). These findings strongly suggest that the content of the mycelium in gluconokinase was the limiting factor of gluconate utilization. Likewise, the good correspondence between the activity of oxaloacetate hydrolase and the rate of oxalate accumulation (Table 3) suggest that the cleavage of oxaloacetate was the only oxalate yielding reaction.

Discussion From the identification of almost all of the enzymes of the EM pathway and the TOA cycle it can be concluded that these two pathways are involved in the metabolization of intermediates of the pentose phosphate (PP) pathway. According to a preceding study (MULLER 1985) the reactions of the PP pathway are the first steps of gluconate utilization after its phosphorylation to gluconate 6-phosphate. . In the presence of fluoroacetate, an inhibitor of the TOA cycle, the disappearance of gluconate was accompanied by the accumulation of citrate. However, in the absence of fluoroacetate no accumulation of this intermediate could be observed (Table 2). This and the identification of almost all of the TCA cycle enzymes provide strong evidence that the whole cycle was operating during the growth of our strain with gluconate as the sole source of carbon. This conclusion is also supported by the identification of (X-ketoglutarate dehydrogenase in extracts from Aspergillus niger cultivated on a glucose medium (MEIXNER-MoNORI et al. 1985). In fungi as well as in other organisms intermediates of the PP and EM pathways and of the TCA cycle can be used for the biosynthesis of components of the mycelium such as amino acids, nucleic acids, lipids and sterols, and the PP pathway can be considered to provide reducing power in the form of NADPH (COCHRANE 1976; WALKER and WOODBINE 1976; BERRY and BERRY 1976; MCCORKINDALE 1976). Our results provide evidence that these pathways are involved in the mycelial growth not only from glucose, sucrose and other sugars, but also from gluconate. Furthermore, in cell-free extracts from gluconate utilizing mycelia the enzyme phosphoglucomutase (EC 2.7.5.1) has been identified by us (unpublished results). This suggests that also cell wall polysaccharides and reserve carbohydrates such as R- and S-glucan and glycogen whose biosynthesis starts from glucose-I-phosphate (ROSENBERGER 1976; BLUMENTHAL 1976; COCHRAN}, 1976) can be formed from gluconate. As can· be seen from Table 1, oxalate is formed from gluconate with a yield of about 38 %, the sum of yields together with those of mycelial substance and CO 2 being about 90 %. If the hydrolytic splitting of oxalacetate represents the only biosynthetic origin of oxalate, the acetate formed via this reaction must be re-utilized at least partly, because otherwise the sum of yields would exceed 100%. The most probable pathway of this re-utilization is the formation of acetyl-CoA and its condensation with glyoxylate which is formed from isocitrate. All three key enzymes of this modified glyoxylate cycle (acetyl-CoA synthetase, EC 6.2.1.1, isocitrate lyase, EC 4.1.3.1, malate synthase, EC 4.1.3.2) have been identified in cell-free extracts of A. nigeT (SHAH and RAMAKRISHNAN 196:~), and its operation in gluconate utilizing cultures is also suggested by the decrease of the oxalate yield following the addition of fluoroacetate (Table 2). This effect can be explained by the inhibition of aeonitate hydratase which prevents the formation of isocitrate and thereby also of glyoxylate.

468

H.-M. MULLER

As can be seen from Fig. 1 the utilization of gluconate started immediately after its addition to the culture fluid. This is in accordance with the finding that gluconokinase, 6-phosphogluconate dehydrogenase and enzymes of the EM pathway and the TeA cycle could be identified in the mycelial extracts at that time, and that there was a good correspondence between the activity of gluconokinase and the rate of disappearance of gluconate (Table 3). According to LAKSHMINARAYANA et al. (1969b) gluconokinase is not formed constitutively by A. niger but is induced by gluconate. The presence of this enzyme at the time of addition of gluconate can be explained by the accumulation of gluconate from glucose by which gluconokinase was induced. In contrast, the enzymes needed for the metabolization of gluconate-6-phosphate can be considered to be formed constitutively, with the exception perhaps of oxaloacetate hydrolase and of the key enzymes of the modified glyoxylate cycle which has been proposed above to be involved in the re-utilization of acetate. LAKSHMINARAYANA et al. (1969a) "adapted" a strain of A. niger to grow from conidia with gluconate as the sole source of carbon. The same ability was found by these authors also with the parent "non-adapted" strain, the only effect of the "adaptation" being the acceleration of growth on the medium containing gluconate as the sole carbon source. This justifies the assumption that the strain used by us is able to metabolize gluconate not only after precultivation on glucose but also during growth from conidia.

Refel'ences BERGMEYER, H. U., GAWEHN, K., und GRASSL, l\f.: Enzyme als biochemische Reagentien. In: Methoden der enzymatischen Analyse (H. U. BERGMEYER, Hg.), Weinheim 1974, 454-558. BERRY, D. R., and BERRY, E. A.: Nucleic acid and protein synthesis in filamentous fungi. In: The Filamentous Fungi, Vol. 2 (J. E. SMITH, D. R. BERRY, Eds.). London 1976, 238-291BLUMENTHAL, H. J.: Carbohydrate metabolism. 1. Glycolysis. In: The Fungi, Vol. 1 (G. C. AINSWORTH, A. S. SUSSMAN, Eds.). New York, London 1965, 229-268. - Glucose catabolism in fungi. Wallerstein Lab. Comm. 31 (1968),171-191- Reserve carbohydrates in fungi. In: The Filamentous Fungi, Vol. 2 (J. E. SMITH, D. R. BERRY, Eds.). London 1976, 292-307. BRADFORD, M. M.: A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72 (1976), 248-254. COCHRANE, V. W.: Glycolysis. In: The Filamentous Fungi (J. E. SMITH, D. R. BERRY, Eds.). London 1976, 65-92. GANCEDO, J. M., and GANCEDO, C.: Fructose-l,6-diphosphatase, phosphofructokinase, and glucosephosphate dehydrogenase from fermenting and nonfermenting yeasts. Arch. Microbiol. 76 (1971),132-138. KUBICEK, C. P., and ROHR, M.: Influence of manganese on enzyme synthesis and citric acid accu· mulation in Aspergillus niger. Europ. J. Appl. Microbiol. 4 (1977),167-175. LAKSH1IfINARAYANA, K., MODI, V. V., and SHAH, V. K.: Studies on gluconate metabolism in Aspergillus niger. I. Nutritional requirements of Aspergillus niger cultivated in gluconate medium. Arch. Mikrobiol. 66 (1969a), 389-395. - - - Studies on gluconate metabolism in Aspergillus niger. II. Comparative studies on the enzyme make-up of the adapted and parent strains of Aspergillus niger. Arch. Mikrobiol. 66 (1969b), 396-405. l\'[CCORKINDALE, N. J.: The biosynthesis of terpenes and steroids. In: The Filamentous Fungi, Vol. 2 (J. E. SMITH, D. R. BERRY, Eds.). London 1976, 369-422. MEIXXER-MoNORI, B., KUBICEK, C. P., HABISON, A., KUBICEK-PRANZ, E. M., and ROHR, M.: Presence and regulation of iX-ketoglutarate dehydrogenase multienzyme complex in the fila. mentous fungus Aspergillus niger. J. Bacteriol. 161 (1985), 265-271. MOLLERING, H., und BERGMEYER, H. U.: D-Gluconat. In: Methoden der enzymatischen Analyse. Be!. II (H. U. BERGMEYER, Hg.), Weinheim 1974, 1288-1292.

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MULLER, H.-l\:L: Untersuchungen zum 8iiurestoffwechsel von Aspergillus niger. I. Der EinfluB des CjN-Verhiiltniss€s der Ausgangs-Nahrlosung auf den pH-Wert und die Oxalsaureanhiiufung. Arch. Mikrobiol. 52 (1965), 251-265. Untersuchungen zum 8aurestoffwechsel von Aspergillus niger. II. Der EinfluB des CjN-Verhiiltnisses in der Ausgangs-Niihrlosung auf die Glucon-, Citronen- und Oxalsiiureanhaufung bei kontrolliertem pH-Wert und mittlerer Temperatur (aO cC). Arch. Mikrobiol. 55 (1966), 77-9I. Untersuchungen zum Saurestoffwechsel von Aspergillus niger. VI. Zur Interpretation der Beziehung zwischen dem CJN-Verhiiltnis in der Ausgangs-Niihrl6sung und der Oxalsaure. anhiiufung. Zbl. Bakt. II 121 (1967), 617-6a5. Oxalate accumulation from citrate by Aspergillus niger. I. Biosynthesis of oxalate from its ultimate precursor. Arch. Microbiol. 103 (1975),185-189. and FROSCH, S.: Oxalate accumulation from citrate by Aspergillus niger. II. Involvement of the tricarboxylic acid cycle. Arch. Microbiol. 104 (1975), 159-162. - Utilization ofgluconate by Aspergillus niger. I. Enzymes of phosphorylating and nonphosphorylating pathways. Zbl. Mikrobiol. 140 (1985), 475-484. - Gluconate accumulation and enzyme activities with extremely nitrogen-limited surface cultures of Aspergillus niger. Arch. Microbiol. 144 (1986), 151-157. NIEDERPRUEM, D. J.: Carbohydrate metabolism. 2. Tricarboxylic acid cycle. In: The Fungi, Vol. I (G. C. AINSWORTH, A. S. SUSSMAN, eds.). New York-London 1965, 269-aOO. PETERS, R. A.: Mechanism of toxicity of the active constituent of Dichapetalum cymosum and related compounds. In: Adv. Enzymol. Vol. 18 (F. F. NORD, Ed.). New York-London 1957, 114-159. RACKER, E.: Spectrophotometric measurements of the enzymatic formation of fumaric and cisaconitic acids. Biochim. biophys. Acta (Arnst.) 4 (1950), 211-214. RAMAKRISHNAN, C. V., and MARTIN, S. M.: The enzymatic synthesis of citric acid by cell-free extracts of Aspergillus niger. Canad. J. Biochem. Physiol. 32 (1954), 434-439. ROSENBERGER, R. F.: The cell wall. In: The Filamentous Fungi, Vol. 2 (J. E. S~nTH, D. R. BERRY, Eds.). London 1976, 328-344. SHAH, V. K., and RAMAKRISHNAN, C. V.: Studies on acid metabolism in Aspergillus niger. Part II. Metabolic changes during citric acid utilization by Aspergillus niger. Enzymologia 26 (1963), 33-43. \VALKER, P., and \VOODBINE, M.: The biosynthesis of fatty acids. In: The Filamentous Fungi, Vol. 2 (J. E. SMITH, D. R. BERRY, eds.). London 1976, la7-158. Author's address: Prof. Dr. H.-M. MULLER, Institute of Animal Nutrition, University of Hohenheim, Emil-WolffStraJ3e 10, D - 7000 Stuttgart 70.

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Zentralbl. Mikrobiol., Bd. 141