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Glycerol as an initiator of citric acid accumulation in Aspergillus niger
Glycerol as an initiator of citric acid accumulation in
Aspergillus niger M. Legi~a* a n d M. M a t t e y Division o f Biochemistry, University o f Strathclyde, 31 Taylor Street, Glasgow G4 0NR UK *Kemijski Institut 'Boris Kidric', Hajdrihova 19, 61115 Ljubljana, Yugoslavia Glycerol accumulation has been shown to be an early event in the sequence of reaction leading to citric acid accumulation by Aspergillus niger. The concentration of glycerol found in the medium between 16 and 24 h of growth was shown to be capable of inhibiting mitochondrial NADP ÷dependent isocitrate dehydrogenase, which would lead to a self-perpetuating citrate accumulation.
Key words: Glycerol; citric acid; Aspergillus niger Introduction It is well known that Aspergillus niger, when grown in a manganese deficient medium, excretes large amounts of citric acid. ~ There have been several reviews o f theories proposed to explain the metabolism of A.niger during citric acid accumulation. 1-3 It is generally believed that there must be an inhibition of the TCA cycle, probably on the level o f isocitric dehydrogenase 4,5 or a-ketoglutarate dehydrogenase 6 while at the same time there should be an undisturbed metabolic flow through glycolysis. 7'8 Three different isocitric dehydrogenases (ICDH) have been found: NADP ÷ and NAD÷-specific mitochondrial enzymes and an NADP*-specific ICDH in the cytosol - the first may be responsible for citric acid accumulation since it was demonstrated that this enzyme could be inhibited b y physiological concentrations o f citrate in the absence o f Mn 2 ÷ ions. Indeed, there may be several isoenzymes o f the mitochondrial NADP*-dependent enzyme since the inhibition by citrate showed complex kinetics, s There still remains the question of what leads to the initial inhibition o f the mitochondrial NADP÷-specific ICDH: either an inhibitory level of citrate is present from the beginning or another regulator induces the initial inhibition of this enzyme. This paper describes studies on the initial stages o f A.niger development in a high citric acid yielding medium where, after germination of the spores, unusual growth in the form of enlarged bulbous ceils is seen and this is followed by a change to filamentous growth. 9
and Spencer. 12 The amount of biomass was detected gravimetrically after drying the cells at 105°C to constant weight. F o r the enzyme assay mycelial samples were disrupted in a Braun disintegrator model MKS with 0.5 mm glass beads. A ratio of 5 g mycelium to 15 m10.05 M T r i s - H C l b u f f e r to 10 ml beads was used and this mixture was shaken at maximum speed for 1 rain. The homogenate was centrifuged at 3000g for 30 s to remove the beads and unbroken mycelium. F r o m the supernatant the mitochondria were isolated by centrifugation at 16000g for 30 rain. The mitochondrial pellet was resuspended in four times its volume of 0.02 M T r i s - a c e t a t e buffer and sonicated for 2 min using an MSE ultrasonicator. The sonicate was centrifuged at 20000g for 20 min and the supernatant utilized in the assays. Isocitrate dehydrogenase activity was assayed by measuring the absorbance change at 340 nm due to the reduction o f NADP ÷ to NADPH. The reaction system contained in a final volume of 3.0 ml: 20 mN bicarbonate buffer, pH 7,6; 1 m u MgSO 4 ; 1 //M NADP ÷ and isocitrate. Specific enzyme activity was expressed in mU mg-1 after the amount o f proteins had been determined in the mitochondrial fraction by the method of Bradford la with crystalline serum albumin as a standard.
Results and discussion During the first 24 h in a high citric acid yielding medium, spore germination occurred yielding enlarged bulbous cells. 9 The subsequent growth of these cells resulted in a considerable drop in pH to about 1.7 (Figure 1). To determine
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1.0 ==
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Materials and methods
A. niger spores (strain N R R L 2270) were harvested from a 7 days old wort agar slant and suspended in 25 ml 0.1% Tween 80 solution. The suspension was filtered through Whatman 54 filter paper to remove the clumped spores. One ml o f this suspension (106 spores) was used to inoculate 50 ml of the medium 9 in 500 ml baffled flasks which were shaken on a rotary shaker (New Brunswick)at 100 rev rain-1 at 30°C. Glycerol in the medium was detected enzymatically using glycerol kinase. 1° Citric acid was determined according to Saffran and Denstedt 11 modified according to Lowenstein
258
E n z y m e Microb. T e c h n o l . , 1986, vol. 8, M a y
.it
~0.5
2.0
O I
10
15
20
25
30
35 40 Time (h)
Figure 1 Glycerol production during the early stages of growth of A. niger in a high citric acid yielding medium. Glycerol was measured enzymatically using glycerol kinase as described in Materials and methods. Citric acid was measured colorimetrically. H +, Glycerol; x, citric acid; e, pH; v, dry weight
0141 --0229/86/050258--02 $03.00 © 1986 Butterworth & Co. (Publishers) Ltd
Rapid communication
Glycerol (M) 0.06
~> 0.03
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I
I
I
0.01
0.02
0.03 I/Isocitmte (pM-=l
Figure 2 Double reciprocal plots of glycerol inhibition of mitochondrial NADP+-specific ICDH measured at different substrate concentrations
whether anything other than protons was excreted during this phase an ether extraction of the medium was performed. After evaporation of the ether a residue was found, which was shown to be glycerol by infrared spectroscopic analysis. Clearly this could not be responsible for the pH drop which appears to be due solely to proton excretion. Quantitative determination of glycerol revealed that significant concentrations were present in the medium, up to 0.1 M (Figure 1). Glycerol accumulated in the medium between 16 and 24 h and this phase was followed by a rapid disappearance of glycerol from the medium. The peak of glycerol concentration corresponded to the start of citric acid accumulation in the medium and to a change in morphology: on the surface of the enlarged cells, buds appeared which later developed into filamentous hyphae with an increased growth rate. The rate of citric acid accumulation was initially slow but it increased as glycerol was assimilated from the medium (Figure 1). During the growth of enlarged bulbous cells no citric acid could be detected in the medium. Because of the juxtaposition of the glycerol peak and the initiation of citrate accumulation in the medium and because of free permeability of glycerol across the lipid bilayers in the cell membranes, 14 indicating that a high concentration of glycerol should be present also in the mitochondria, the effect of glycerol on the activity of mitochondrial NADPLspecific ICDH was examined (Figure 2). Glycerol inhibited ICDH at physiological concentration: a concentration of 0.1 M reduced the activity by ~ 90%, which should lead to the accumulation of citric acid because of the equilibrium position of aconitase. Inhibition by glycerol of mitochondrial NADP÷-specific ICDH from the enlarged cells (22 h) and from the filamentous cells (72 h) was similar, with K i value for glycerol of 8 raM, suggesting that both types have the same isoenzyme. The main difference between them was in their Vmax value which was 270 mU mg-1 for enlarged cells and 110 mU mg-I for the filamentous stage. Double-reciprocal plots (Figure 2) of the activity of the mitochondrial NADP*-specific ICDH versus isocitrate concentration in the presence of different amounts of added glycerol, suggest a competitive nature for the inhibition. We could detect inhibition only in the presence of Mg2. ions (1 mM), while Mn 2. ions (1 mM) permitted normal activity, even in the presence of high concentrations of glycerol. The concentration of Mg 2. in a high citric acid yielding medium 9 was 1 mM. It has been shown that the concentra-
tion of divalent metal ions in the mycelium of Aspergillus niger does not differ significantly from that of the extracellular medium over a wide range, s while it has been shown that an Mn 2 * concentration as low as 1/.tM drastically deceases the yield of citric acid. is A similar effect was observed with the citrate inhibition s of this enzyme, indicating that both effectors may have a similar action. Glycerol3-phosphate did not show any inhibition at lower concentrations (0.01-0.05 M), while at higher concentrations ( 0 . 0 6 0.1 M) it gave only ~ 50% inhibition of ICDH either from enlarged or filamentous cells. Since such high cytosol concentrations of a phosphorylated molecule would not be expected this molecule probably does not cause significant inhibition in vivo. The present results indicate the importance of the morphological change from enlarged or filamentous cells during the early stages of A. niger growth in a high citric acid yielding medium. We propose a model for the initiation of citric acid accumulation based on these observations, namely that the accumulation of up to 1% glycerol by the enlarged cells is the cause o f the initial inhibition ofmitochondrialNADP*specific ICDH which then leads to a build up of citrate via aconitase. Once the concentration of citrate reaches a critical value it brings about the feed-forward inhibition of NADP +specific ICDH as previously demonstrated, s despite the eventual assimilation of glycerol from the medium. These results support the idea that mito chondrial NADP*specific ICDH is of primary importance in the citric acid fermentation. Acknowledgement We thank Research Council o f Slovenia and the British Council for financial support. (Received 31 October 1985 ; revised 31 January 1986)
References 1 Lockwood, L.B. in The Filamentous Fungi (Smith, J.E. and Berry, D.R., eds), Edward Arnold, London, 1975, vol. 1, pp. 140-157 2 Perlman, D. and Sih, C.J. in Progress in Industiral Microbiology (Hockenhull, D.J.D., ed.) Heywood, London, 1960, vol, 2, pp. 169-200 3 Berry, D.R., Chmiel, A. and AI Obaidi, Z. in Genetics and Physiology of the Aspergilli (Smith, J.E. and Pateman, J.A., eds), Academic Press, London - New York - San Francisco, 1977, pp. 405-426 4 Mattey, M. FEMSMicrobiol. Lett. 1977, 2, 71-74 5 Bowes, I. and Mattey, M. FEMS Microbiol. Lett 1979, 6, 219-222 6 Kubicek, C.P. and Rtihr, M. J.Appl. Microbiol. Biotechnol. 1977, 4, 167-173 7 Habison, A., Kubicek, C.P. and R6hr, M. FEMS Microbiol. Lett. 1979, 5, 39-42 8 R6hr, M. and Kubicek, C.P. Process Biochem. 1981, 16 (4), 34-37 9 Legisa, M., Cimerman, A. and Sterl¢, M. FEMS Microbiol. Lett. 1981, 11,149-152 10 Eggstein, M. and Kuhlmann, E. in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.), Verlag Chemie, Weinheim/ Academic Press Inc., New York - London, 1974, vol. 4 pp. 1825-1831 11 Saffran, M. and Denstedt, O.F.J. Biol. Chem. 1948, 175, 849-855 12 Spencer, A.F. and Lowestein, J.M. Biochem. J. 1967, 103, 342-348 13 Bradford, M.M. Anal. Biochem. 1976, 72,248-254 14 Moore, T.J.J. Lipid Res. 1968, 9,642-646 15 Shu, P. and Johnson, M.J.J. Bacteriol. 1948, 56,577-585
Enzyme Microb. Technol., 1986, vol. 8, May
259
Aspergillus niger M. Legi~a* a n d M. M a t t e y Division o f Biochemistry, University o f Strathclyde, 31 Taylor Street, Glasgow G4 0NR UK *Kemijski Institut 'Boris Kidric', Hajdrihova 19, 61115 Ljubljana, Yugoslavia Glycerol accumulation has been shown to be an early event in the sequence of reaction leading to citric acid accumulation by Aspergillus niger. The concentration of glycerol found in the medium between 16 and 24 h of growth was shown to be capable of inhibiting mitochondrial NADP ÷dependent isocitrate dehydrogenase, which would lead to a self-perpetuating citrate accumulation.
Key words: Glycerol; citric acid; Aspergillus niger Introduction It is well known that Aspergillus niger, when grown in a manganese deficient medium, excretes large amounts of citric acid. ~ There have been several reviews o f theories proposed to explain the metabolism of A.niger during citric acid accumulation. 1-3 It is generally believed that there must be an inhibition of the TCA cycle, probably on the level o f isocitric dehydrogenase 4,5 or a-ketoglutarate dehydrogenase 6 while at the same time there should be an undisturbed metabolic flow through glycolysis. 7'8 Three different isocitric dehydrogenases (ICDH) have been found: NADP ÷ and NAD÷-specific mitochondrial enzymes and an NADP*-specific ICDH in the cytosol - the first may be responsible for citric acid accumulation since it was demonstrated that this enzyme could be inhibited b y physiological concentrations o f citrate in the absence o f Mn 2 ÷ ions. Indeed, there may be several isoenzymes o f the mitochondrial NADP*-dependent enzyme since the inhibition by citrate showed complex kinetics, s There still remains the question of what leads to the initial inhibition o f the mitochondrial NADP÷-specific ICDH: either an inhibitory level of citrate is present from the beginning or another regulator induces the initial inhibition of this enzyme. This paper describes studies on the initial stages o f A.niger development in a high citric acid yielding medium where, after germination of the spores, unusual growth in the form of enlarged bulbous ceils is seen and this is followed by a change to filamentous growth. 9
and Spencer. 12 The amount of biomass was detected gravimetrically after drying the cells at 105°C to constant weight. F o r the enzyme assay mycelial samples were disrupted in a Braun disintegrator model MKS with 0.5 mm glass beads. A ratio of 5 g mycelium to 15 m10.05 M T r i s - H C l b u f f e r to 10 ml beads was used and this mixture was shaken at maximum speed for 1 rain. The homogenate was centrifuged at 3000g for 30 s to remove the beads and unbroken mycelium. F r o m the supernatant the mitochondria were isolated by centrifugation at 16000g for 30 rain. The mitochondrial pellet was resuspended in four times its volume of 0.02 M T r i s - a c e t a t e buffer and sonicated for 2 min using an MSE ultrasonicator. The sonicate was centrifuged at 20000g for 20 min and the supernatant utilized in the assays. Isocitrate dehydrogenase activity was assayed by measuring the absorbance change at 340 nm due to the reduction o f NADP ÷ to NADPH. The reaction system contained in a final volume of 3.0 ml: 20 mN bicarbonate buffer, pH 7,6; 1 m u MgSO 4 ; 1 //M NADP ÷ and isocitrate. Specific enzyme activity was expressed in mU mg-1 after the amount o f proteins had been determined in the mitochondrial fraction by the method of Bradford la with crystalline serum albumin as a standard.
Results and discussion During the first 24 h in a high citric acid yielding medium, spore germination occurred yielding enlarged bulbous cells. 9 The subsequent growth of these cells resulted in a considerable drop in pH to about 1.7 (Figure 1). To determine
1.5
!
).1
2.5
_---,
5
1.0 ==
I=
Materials and methods
A. niger spores (strain N R R L 2270) were harvested from a 7 days old wort agar slant and suspended in 25 ml 0.1% Tween 80 solution. The suspension was filtered through Whatman 54 filter paper to remove the clumped spores. One ml o f this suspension (106 spores) was used to inoculate 50 ml of the medium 9 in 500 ml baffled flasks which were shaken on a rotary shaker (New Brunswick)at 100 rev rain-1 at 30°C. Glycerol in the medium was detected enzymatically using glycerol kinase. 1° Citric acid was determined according to Saffran and Denstedt 11 modified according to Lowenstein
258
E n z y m e Microb. T e c h n o l . , 1986, vol. 8, M a y
.it
~0.5
2.0
O I
10
15
20
25
30
35 40 Time (h)
Figure 1 Glycerol production during the early stages of growth of A. niger in a high citric acid yielding medium. Glycerol was measured enzymatically using glycerol kinase as described in Materials and methods. Citric acid was measured colorimetrically. H +, Glycerol; x, citric acid; e, pH; v, dry weight
0141 --0229/86/050258--02 $03.00 © 1986 Butterworth & Co. (Publishers) Ltd
Rapid communication
Glycerol (M) 0.06
~> 0.03
Q0/, 002
I
I
I
0.01
0.02
0.03 I/Isocitmte (pM-=l
Figure 2 Double reciprocal plots of glycerol inhibition of mitochondrial NADP+-specific ICDH measured at different substrate concentrations
whether anything other than protons was excreted during this phase an ether extraction of the medium was performed. After evaporation of the ether a residue was found, which was shown to be glycerol by infrared spectroscopic analysis. Clearly this could not be responsible for the pH drop which appears to be due solely to proton excretion. Quantitative determination of glycerol revealed that significant concentrations were present in the medium, up to 0.1 M (Figure 1). Glycerol accumulated in the medium between 16 and 24 h and this phase was followed by a rapid disappearance of glycerol from the medium. The peak of glycerol concentration corresponded to the start of citric acid accumulation in the medium and to a change in morphology: on the surface of the enlarged cells, buds appeared which later developed into filamentous hyphae with an increased growth rate. The rate of citric acid accumulation was initially slow but it increased as glycerol was assimilated from the medium (Figure 1). During the growth of enlarged bulbous cells no citric acid could be detected in the medium. Because of the juxtaposition of the glycerol peak and the initiation of citrate accumulation in the medium and because of free permeability of glycerol across the lipid bilayers in the cell membranes, 14 indicating that a high concentration of glycerol should be present also in the mitochondria, the effect of glycerol on the activity of mitochondrial NADPLspecific ICDH was examined (Figure 2). Glycerol inhibited ICDH at physiological concentration: a concentration of 0.1 M reduced the activity by ~ 90%, which should lead to the accumulation of citric acid because of the equilibrium position of aconitase. Inhibition by glycerol of mitochondrial NADP÷-specific ICDH from the enlarged cells (22 h) and from the filamentous cells (72 h) was similar, with K i value for glycerol of 8 raM, suggesting that both types have the same isoenzyme. The main difference between them was in their Vmax value which was 270 mU mg-1 for enlarged cells and 110 mU mg-I for the filamentous stage. Double-reciprocal plots (Figure 2) of the activity of the mitochondrial NADP*-specific ICDH versus isocitrate concentration in the presence of different amounts of added glycerol, suggest a competitive nature for the inhibition. We could detect inhibition only in the presence of Mg2. ions (1 mM), while Mn 2. ions (1 mM) permitted normal activity, even in the presence of high concentrations of glycerol. The concentration of Mg 2. in a high citric acid yielding medium 9 was 1 mM. It has been shown that the concentra-
tion of divalent metal ions in the mycelium of Aspergillus niger does not differ significantly from that of the extracellular medium over a wide range, s while it has been shown that an Mn 2 * concentration as low as 1/.tM drastically deceases the yield of citric acid. is A similar effect was observed with the citrate inhibition s of this enzyme, indicating that both effectors may have a similar action. Glycerol3-phosphate did not show any inhibition at lower concentrations (0.01-0.05 M), while at higher concentrations ( 0 . 0 6 0.1 M) it gave only ~ 50% inhibition of ICDH either from enlarged or filamentous cells. Since such high cytosol concentrations of a phosphorylated molecule would not be expected this molecule probably does not cause significant inhibition in vivo. The present results indicate the importance of the morphological change from enlarged or filamentous cells during the early stages of A. niger growth in a high citric acid yielding medium. We propose a model for the initiation of citric acid accumulation based on these observations, namely that the accumulation of up to 1% glycerol by the enlarged cells is the cause o f the initial inhibition ofmitochondrialNADP*specific ICDH which then leads to a build up of citrate via aconitase. Once the concentration of citrate reaches a critical value it brings about the feed-forward inhibition of NADP +specific ICDH as previously demonstrated, s despite the eventual assimilation of glycerol from the medium. These results support the idea that mito chondrial NADP*specific ICDH is of primary importance in the citric acid fermentation. Acknowledgement We thank Research Council o f Slovenia and the British Council for financial support. (Received 31 October 1985 ; revised 31 January 1986)
References 1 Lockwood, L.B. in The Filamentous Fungi (Smith, J.E. and Berry, D.R., eds), Edward Arnold, London, 1975, vol. 1, pp. 140-157 2 Perlman, D. and Sih, C.J. in Progress in Industiral Microbiology (Hockenhull, D.J.D., ed.) Heywood, London, 1960, vol, 2, pp. 169-200 3 Berry, D.R., Chmiel, A. and AI Obaidi, Z. in Genetics and Physiology of the Aspergilli (Smith, J.E. and Pateman, J.A., eds), Academic Press, London - New York - San Francisco, 1977, pp. 405-426 4 Mattey, M. FEMSMicrobiol. Lett. 1977, 2, 71-74 5 Bowes, I. and Mattey, M. FEMS Microbiol. Lett 1979, 6, 219-222 6 Kubicek, C.P. and Rtihr, M. J.Appl. Microbiol. Biotechnol. 1977, 4, 167-173 7 Habison, A., Kubicek, C.P. and R6hr, M. FEMS Microbiol. Lett. 1979, 5, 39-42 8 R6hr, M. and Kubicek, C.P. Process Biochem. 1981, 16 (4), 34-37 9 Legisa, M., Cimerman, A. and Sterl¢, M. FEMS Microbiol. Lett. 1981, 11,149-152 10 Eggstein, M. and Kuhlmann, E. in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.), Verlag Chemie, Weinheim/ Academic Press Inc., New York - London, 1974, vol. 4 pp. 1825-1831 11 Saffran, M. and Denstedt, O.F.J. Biol. Chem. 1948, 175, 849-855 12 Spencer, A.F. and Lowestein, J.M. Biochem. J. 1967, 103, 342-348 13 Bradford, M.M. Anal. Biochem. 1976, 72,248-254 14 Moore, T.J.J. Lipid Res. 1968, 9,642-646 15 Shu, P. and Johnson, M.J.J. Bacteriol. 1948, 56,577-585
Enzyme Microb. Technol., 1986, vol. 8, May
259