Role of glutamine synthetase in citric acid fermentation by Aspergillus niger

J . Biosci., Vol 7, Numbers 3 & 4 June 1985 , pp 269–287. © Printed in India.





Role of glutamine synthetase in citric acid fermentation by
Aspergillus niger



N. S. PUNEKAR*, C. S. VAIDYANATHAN and N. APPAJI RAO †

Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India
* Present address: Institute of Enzyme Research, 1710, University Avenue, Madison,
Wisconsin 53705, USA

MS received 5 October 1984; revised 25 February 1985

Abstract. The activity of glutamine synthetase from Aspergillus niger was significantly
lowered under conditions of citric acid fermentation. The intracellular pH of the organism as
determined by bromophenol blue dye distribution and fluorescein diacetate uptake methods
was relatively constant between 6·0-6·5, when the pH of the external medium was varied
between 2·3-7·0. Aspergillus niger glutamine synthetase was rapidly inactivated under acidic
pH conditions and Mn2+ ions partially protected the enzyme against this inactivation. Mn2+-
dependent glutamine synthetase activity was higher at acidic pH (6·0) compared to Mg2+-
supported activity. While the concentration of Mg2+ required to optimally activate glutamine
synthetase at pH 6·0 was very high (? 50 mM), Mn2+ was effective at 4 mM. Higher
concentrations of Mn2+ were inhibitory. The inhibition of both Mn2+ and Mg2+-dependent
reactions by citrate, 2-oxoglutarate and ATP were probably due to their ability to chelate
divalent ions rather than as regulatory molecules. This suggestion was supported by the
observation that a metal ion chelator, EDTA also produced similar effects. Of the end-
products of the pathway, only histidine, carbamyl phosphate, AMP and ADP inhibited
Aspergillus niger glutamine synthetase. The inhibitions were more pronounced when Mn2+
was the metal ion activator and greater inhibition was observed at lower pH values. These
results permit us to postulate that glutamine synthesis may be markedly inhibited when the
fungus is grown under conditions suitable for citric acid production and this block may result
in delinking carbon and nitrogen metabolism leading to acidogenesis.

Keywords. Citric acid fermentation; glutamine synthetase; regulation by metal ions.


Introduction

Overflow of fungal metabolism forms the basis of citric acid industry (Maill, 1978). The
mechanisms of citric acid fermentation are interesting not only because of their
relevance to fungal physiology, but also in understanding metabolic regulation of cell
growth and acidogenesis. Towards this objective, tricarboxylic acid cycle (TCA-cycle)
(Mattey, 1977; Kubicek and Rohr, 1978), glycolysis (Kubicek and Rohr, 1978) and the
enzymes therein (Kubicek and Rohr, 1977; Bowes and Mattey, 1980; Habison et al.,
1983) have received considerable attention. Inhibition by citric acid and the role of
Mn2+ ions in citric acid production (Bowes and Mattey, 1979, 1980) have led to the



† To whom correspondence should be addressed.
Abbreviations used: ?-GHA: ?-Glutamylhydroxamate; His, L-histidine; Glu, L-glutamic acid; TCA-cycle,
tricarboxylic acid cycle


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implication of mitochondrial NADP+-specific isocitrate dehydrogenase as an import-
ant enzyme for fermentation. Citrate, malate and 2-oxoglutarate levels were elevated
during acidogenesis. Also, under conditions of Mn2+ deficiency, significant increase in
concentrations of pyruvate and oxaloacetate was obtained. These studies led to the
suggestion that 2-oxoglutarate dehydrogenase may have a role in acidogenesis by
Aspergillus niger and that involvement of isocitrate dehydrogenase may be important
only during later stages of citric acid production (Kubicek and Rohr, 1978). Regulatory
aspects of citric acid fermentation have been recently reviewed by Rohr and Kubicek
(1981).
Although it is generally accepted that citric acid fermentation is a nitrogen limited
condition of the growth for the fungus (Berry 1975; Berry et al., 1977), very little is
known about the role of nitrogen metabolism during this process. The following
observations: (a) increase in the intracellular NH +
4 pool (Habison et al., 1979) and
elevated levels of TCA-cycle intermediates preceeding 2-oxoglutarate dehydro-
genase step as well as (b) occurrence of large amounts of glutamic acid and
metabolites derived from it (Kubicek et al., 1979) under fermentation conditions,
especially during Mn2+ deficiency, suggested to us that a block at glutamine synthetase
step could be one of the important factors responsible for excessive production of citric
acid. The results presented here provide some experimental support for this hypothesis.


Materials and methods

Chemicals

Imidazole, 2-mercaptoethanol, sodium arsenate, 2-oxoglutarate, carbamyl phosphate,
bromophenol blue, fluorescein, ATP, ADP, AMP, EDTA and all the amino acids were
obtained from Sigma Chemical Company, St. Louis, Missouri, USA. Glucose oxidase
reagent (GLOX) was obtained from Kabi Diagnostica, Stockholm, Sweden. All the
other chemicals were of analytical grade. Organic solvents were distilled before use.

Organism and growth conditions

A. niger (UBC 814) was maintained on potato dextrose-agar medium and an acid
producing strain of A. niger (ATCC 9142) was obtained from National Chemical
Laboratory, Poona, for comparative studies on citric acid fermentation.
A. niger (UBC 814 and ATCC 9142) were grown as a surface culture in one litre flasks
containing 100 ml of the culture medium. The composition of the medium for normal
growth of this fungus was essentially that described for A. nidulans by Pateman (1969),
with minor modifications. One per cent glucose was used as carbon source. NH4 NO3
(2·25 g/litre) was used as the nitrogen source. Addition of micronutrients (mg/litre):
FeCl3·6H2O, 20; ZnSO4·7H2O, 10; MnSO4 H2O, 3·0; NaMoO4·2H2O, 1·5 and
CuSO4 5H2O, 1·0; improved growth and yield of cells. The medium was buffered by
using Na2HPO4 ·2H2O (96 g/litre) and KH2PO4 (3 g/litre). The final pH of the
medium was adjusted to 5·5-6·0. For citric acid fermentation in the laboratory, the
following medium (1 litre) containing; glucose or sucrose, 140g; NH4NO3, 2·25g;
KH2PO4, 1·0 g; MgSO4·7H2O, 0·25g and Fe(NH4)(SO4)2·12H2O, 0·1 mg and

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A. niger glutamine synthetase and acidogenesis
271

sufficient HCl to decrease the pH to 2·3, was used (Perlman et al., 1946). Fermentation
being highly sensitive to trace metals (Kubicek and Rohr, 1977), especially Mn2+ ions,
the media were prepared in double distilled water and also using analytical grade
chemicals.
Uniform spore suspension (3 ml in double distilled water) obtained from 7-10 day
old cultures was used as inoculum. The cells were harvested during the maximal growth
phase (45 h after inoculation), washed and stored at — 20°C until further use.

Preparation of crude cell extracts and enzyme assays

Glutamine synthetase from A. niger (UBC 814) grown up to maximal growth phase
(45 h) on 66 mM glutamate as nitrogen source was purified by ammonium sulphate
fractionation, chromatography on DEAE-Sephacel and AMP affinity matrix and gel
filtration on Biogel A5M and its homogeneity was determined by gel filtration and
Polyacrylamide gel electrophoresis (Punekar et al., 1984).
Crude cell extracts were prepared and the enzyme was assayed by the colourimetric
determination of ?-glutamylhydroxamate (?-GHA) formed (Rowe et al., 1970).
Experimental details were same as those described before (Punekar et al., 1984). Specific
activity was expressed as units/mg protein. Protein was estimated according to Lowry
et al. (1951) using crystalline bovine serum albumin as the standard.

Inhibition studies

The enzyme was preincubated with appropriate concentration of the effectors (for
5 min) separately and the reaction was started by the addition of one of the substrates
(usually NH2OH). It was ensured that the pH of all the inhibitors used was adjusted to
corresponding assay pH values. 2-Oxoglutarate was found to interfere with the
colourimetric method for ?-GHA estimation (data not presented). In all these
experiments the amount of ?-GHA formed was read against an appropriate blank
containing the same amount of the interfering compound.

Citric acid estimation

Citric acid released into the medium during various stages of fermentation was
monitored colourimetrically (Spencer and Lowenstein, 1967) by pyridine-acetic
anhydride method.

Glucose estimation

Glucose remaining in the medium was estimated spectrophotometrically using glucose
oxidase (GLOX) reagent (Bergmeyer and Bernt, 1974).

Preparation of fluorescein diacetate

Fluorescein diacetate was prepared by reacting fluorescein with acetic anhydride in
pyridine. The product was recrystallized from 95% ethanol. The procedure was
essentially similar to that described for fluorescein dibutyrate (Kramer and Guilbault,
1963).

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Determination of intracellular pH

Internal pH of A. niger (UBC 814) mycelium was determined by two different methods.
In both cases, fine mycelial suspension of the fungus grown up to 36 h as suspension
culture in normal growth medium having NH4NO3 (28 mM) as nitrogen source was
used. Buffers (0·1 M) of different pH values were prepared using citrate and potassium
phosphate as buffer components, because such a buffer probably represents the
environment the organism experiences during fermentation.

Bromophenol blue method: The dye, bromophenol blue, distributes across the plasma
membrane in a pH dependent fashion (Kotyk and Janacek, 1975). The following
equation was used to calculate the internal pH (pHi):


where, pK value of the dye (bromophenol blue) was assumed to be 40; pHe (external
pH) was measured in the cell suspension; Ce (external concentration of the dye) was
determined in the centrifugate; and Ci (intracellular concentration of the dye) was
calculated from the difference between the initially added concentration and Ce. To
calculate Ci , the intracellular water volume was approximated by haematocrit method
(Kloppel and Hofer, 1976).
To a 3 ml cell suspension (about 4·5 mg dry wt/ml) maintained at predetermined pH
values using 0·1 ? citrate-phosphate buffer (at 25±3°C), 1 ml of bromophenol blue
solution (0·1 mM) was added and the suspension was constantly stirred. Samples (1 ml)
were withdrawn after 30 min at which time interval., the equilibration between the dye
inside and outside was reached. The samples were centrifuged at 2,000 g for 10 min in a
clinical table top centrifuge and the decrease in absorbance of the supernatant at
589 nm and at pH 7·5- was determined. The external concentration of the dye was
obtained from a standard curve.

Fluorescein diacetate as ? pH probe: By monitoring the monoanion-dianion transition
of intracellular fluorescein (Slavik, 1982), the values of internal pH were calculated from
a calibration curve prepared as follows: Fluorescein was dissolved in a series of buffers
(citrate-phosphate, 0·1 M) from pH 2·0-7·5. At each pH the fluorescence intensity at
520 nm of the sample was recorded (in a Perkin-Elmer, model 203 spectrofluorometer
with 150X xenon lamp source and a R212 photomultiplier tube) after excitation
at 535 nm and 490 nm and the ratio of these two intensities (I490/I435)was plotted
against pH.
The cells grown as fine suspension in the growth medium in shake cultures at 28°C
for 36 h were harvested, washed twice and resuspended in buffers of required pH. Prior
to fluorescence measurements, the cell suspension (4·5 mg dry wt/ml) was incubated at
28°C with fluorescein diacetate (10 ??) for about 20 min. The cells were washed
thoroughly and resuspended in the original volume of buffer before fluorescence
measurements.

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A. niger glutamine synthetase and acidogenesis
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Results

Citric acid production, mycelial growth and morphology of A. niger on surface cultures

Before correlating changes in glutamine synthetase activity with acidogenesis, it was
necessary to establish the fermentation characteristics of A. niger (UBC 841) strain. For
this reason, the organism was grown on citric acid fermentation medium and modified
Pateman medium (Materials and methods) separately and its growth characteristics
were compared. Sugar uptake, citric acid released and the pH of the medium were
monitored at different time intervals after inoculation and the results are shown
in figure 1. Citric acid was not produced when the organism was grown on
normal growth medium (figure 1a). However, when it was grown on fermentation
medium, two distinct phases of growth and citric acid production were apparent. In the
first growth phase (trophophase) there was an increase in the biomass (not shown), but
release of citric acid into the medium was not observed. In the second i.e., fermentation
phase (idiophase) increasing amounts of citric acid was excreted into the medium.
Maximal amount of citric acid (about 22 mg/ml) was formed after about 10–13 days.
These values are not as high as those encountered during industrial production, as
traces of metal ions were not rigorously excluded by adding a chelator to the
fermentation medium. Similar results were obtained when an industrially used acid-
production strain of A. niger (ATCC 9142) (data not shown) was grown under the same
conditions. Both the strains of A. niger (UBC 814 and ATCC 9142) sporulated within
72 h after inoculation into the normal growth medium, whereas no sporulation was
apparent even after 9–10 days of growth on the fermentation medium. Both the strains
produced non-sporulating, slimy, folded mats on the surface of the fermentation
medium. As both the organisms had similar properties, we used A. niger (UBC 814) in
these studies.

Glutamine synthetase levels in A. niger grown on fermentation medium

To determine the effects of composition of the media, pH, Mn2+ ions, on the levels of
glutamine synthetase, the specific activities of the enzyme in crude extracts obtained
from cells grown on normal growth medium (modified Pateman) and fermentation
medium were determined. Both the strains (UBC 814 and ATCC 9142) showed
approximately a two fold lowered levels of glutamine synthetase (specific activity 0·10
and 0·11 ?mol/ min per mg of protein, respectively) when grown on citric acid
fermentation medium as compared to the level of the enzyme in the mycelia grown on
normal growth medium (0·20 and 0·21, respectively). The addition of MnSO4 H2O
(200 mg/litre) to the fermentation medium resulted in an increase of glutamine
synthetase levels in both the strains (0·10–0·18 ?mol/min per mg protein for UBC 814
and 0·11–0·20 ?mol/mm per mg protein for ATCC 9142). In addition to determining
the glutamine synthetase levels at a fixed time interval (the maximal growth phase,
45 h), the activity of the enzyme was also estimated at different time intervals during
growth and a reciprocal relationship with citric acid excretion was observed. The
enzyme levels were high (0·22 units) until about 48 h at which time no citric acid
accumulated and thereafter the levels decreased rapidly as citric acid started
accumulating in the medium. When the medium was supplemented with different

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Figure 1.
Fermentation characteristics of A. niger (UBC 814) strain. The organism was
grown on (a) normal growth medium and (b) fermentation medium, by inoculating uniform
spore suspension obtained from 6–8 day old cultures. Changes in the pH of the medium (O),
glucose utilization (?) and citric acid released into the medium (?) were monitored at time
intervals indicated.

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A. niger glutamine synthetase and acidogenesis
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amounts of Mn2+, the increase in glutamine synthetase activity paralleled the increasing
concentration of ?n2+ added. The ratio of the Mg2+ -dependent synthetase
activity to ?n2+-dependent ?-glutamyltransferase activity remained constant
0·15–0·17 under these conditions, suggesting that the activity was probably not being
modulated by covalent modification as in Escherichia coli.

Effect of pH of the medium on internal pH of A. niger mycelia

Two different methods were employed to evaluate the internal (cytoplasmic) pH in
UBC 814 strain of A. niger. Although the pH of the medium in which A. niger mycelia
were suspended was varied by about 5 units in the range 2·3–7·0, the internal pH
remained reasonably constant between 6·0 and 6·8, and infact was essentially
unchanged until the external pH values were increased beyond 5·0 (figure 2a). At an
external pH of 5·3, the intracellular pH was determined to be 6·4 using the fluorescent
probe, fluorescein diacetate (figure 2b). Similarly, when the pH of the medium outside
was 2·3, an internal pH of 5·9 was observed (figure 2b). In these studies, cells without the
probe had negligible fluorescence at the wavelengths used. The results obtained by both
the methods were in good agreement. Our data indicated that A. niger mycelia have an
efficient in vivo buffering capacity and maintain their internal pH acidic within narrow
limits.


Effect of pH on metal ion saturation

The pH optima for the Mg2+- and Mn2+-dependent synthetase reactions are 7·8 and
5·5, respectively (Punekar et al., 1984). Figure 3 shows the saturation of glutamine
synthetase by Mg2+ and Mn2+, at two different pH values in the presence of 10 mM
ATP. It is evident that at pH 6·0 the Mg2+-supported synthetase activity was very low
until the concentration of Mg2+ was increased beyond 20 mM. On the otherhand at
pH 8·0, saturation pattern was sigmoid with a lag phase until about 5 mM Mg2+, and
maximum activity was observed around 20 mM Mg2+. The saturation pattern of
glutamine synthetase by Mn2+ was qualitatively similar at both the pH values with a
maximum around 4 mM Mn2+ and concentrations beyond this value were inhibitory.
For the same amount of enzyme protein (9·9 µg/assay) the maximum value for Mn2+-
supported synthetase activity at pH 8·0 was about half of that at pH 6·0.

pH inactivation of the enzyme

To further examine the role of glutamine synthetase in citric acid fermentation which is
sensitive to changes in pH, the stability of the enzyme at various pH values was
monitored. The sigmoid pH-stability profile suggested that the enzyme was more stable
at neutral pH values than at acidic pH values with a mid point of inactivation around
5·5 (figure 4). Similar pH inactivation experiments were carried out in the presence of
either Mn2+ (0·5 mM) or ATP: Mg2+ (25:10 mM) also. It can be seen that Mg ATP
shifted the pH inactivation profile towards the acidic side with a mid point at 5·2,
whereas Mn2+ caused a more significant shift with a midpoint pH of 4·8. These results
demonstrated that Mn2+ and Mg· ATP protected the enzyme against inactivation at
acidic pH values, as indicated by higher activity under these conditions.

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Figure 2.
Correlation between extra- and intracellular pH of A. niger (UBC 814),
(a) Bromophenol blue-dye distribution method, (b) Fluorescein diacetate method. Expe-
rimental details are given in Materials and methods.

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A. niger glutamine synthetase and acidogenesis 277


Figure 3. pH dependence of metal ion saturation of A. niger glutamine synthetase. The
Mg2+-dependent (solid lines) and Mn2+ dependent (broken lines) synthetase activity was
assayed at pH 8·0 (?)and at pH 6·0 (O), in 100 mM imidazole hydrochloride buffer, using
10 mM ATP and the concentrations of Mg2+ (0-50 mM) or Mn2+ (0–20 mM) indicated in the
figure. ?-GHA formed was estimated.


Is A. niger glutamine synthetase regulated by feed back inhibition ?

pH-Dependence of inhibitions: The effect of a number of possible feedback inhibitors
(alanine, anthranilic acid, arginine, asparagine, ?-aminobutyric acid, glutamine, glycine,
histidine, methionine, serine, tryptophan, AMP, ADP, GTP, NAD+, NADP+,
carbamyl phosphate, DL-glucosamine-HCl, all at 20 mM) on the Mg2+ -dependent (at
pH 7·8 and 6·7) and Mn2+-dependent (at pH 5·5) synthetase activities was checked.
Among all the compounds tested, alanine, glycine, histidine, serine, ADP, AMP, GTP
and carbamyl phosphate caused some inhibition and hence the effect of these
compounds was probed further. The results of such a study are shown in table 1.
Alanine, glycine and serine had no effect on the Mg2+-dependent synthetase activity (at
both pH values), whereas Mn2+-dependent synthetase activity was inhibited. Histidine
significantly inhibited the Mg2+-dependent activity at acidic pH (6·7) and also the
Mn2+-supported synthetase activity. It is also evident that GTP (at 5 mM) was without
effect on the Mg2+-supported reaction. AMP and ADP inhibited both the activities,
while carbamyl phosphate inhibited the Mg2+-dependent synthetase reaction only.

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Figure 4. H inactivation of A. niger glutamine synthetase. The enzyme (14·3 µg) was
incubated at 28°C for 30 min in the presence of 0·5 mM Mn2+ (O), 10:2, 5 mM Mg2+: ATP
(?) or without any additionally added metal ion (?)in 50 ?l of buffer (100 mM) of different pH
values indicated sodium acetate/acetic acid between 3·7–5·62 and imidazole hydrochloride
between 6·0–7·7. At the end of this incubation, the residual activity was estimated by
readjusting the pH to 6·0 and assaying Mn2+-dependent ?-glutamyl transferase activity In
each ease the activity at pH 7·5 was normalized to 100 as the enzyme was stable throughout the
incubation period at this pH. The activity at other pH values were expressed as per cent of this
normalized value.


Kinetics of inhibition by histidine, carbamyl phosphate and adenine nucleotides: In view
of the inhibition of enzyme activity by these compounds, it was of interest to examine
kinetic mechanism of this process in some detail. Fractional inhibition analysis, Dixon
plots of inhibition and double reciprocal plots were drawn for histidine, AMP, ADP
and carbamyl phosphate. These plots and the derived plots were used to analyze the
nature of inhibition and obtain inhibition constants. The different inhibition constants
obtained from kinetic experiments and the pattern of inhibitions are summarized in
table 2.

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