JOURNAL oF BACTERIOLOGY, Sept. 1972, p. 771-777 Vol. 111, No.

3
Copyright i 1972 American Society for Microbiology Printed in U.S.A.

Effect of Maltose on Glucoamylase Formation

by Aspergillus niger
LARRY L. BARTON,I CARL E. GEORGI, AND DAVID R. LINEBACK2
Department of Microbiology and Department of Biochemistry and Nutrition, University of Nebraska,
Lincoln, Nebraska 68508
Received for publication 23 May 1972

Low levels of glucoamylase are produced when Aspergillus niger is grown on

sorbitol, but substitution of the latter by glucose, maltose, or starch results in
greater formation of glucoamylase as measured by enzymatic activity. Both
glucoamylase I and glucoamylase II are formed in a yeast extract medium;
however, glucoamylase I appears to be the only form produced when ammo-
nium chloride is the nitrogen source. Maltose or isomaltose (1.4 x 10' M), but
no other disaccharides or monosaccharides, dextrins, dextrans, or starches,
stimulated glucoamylase formation when added to mycelia pregrown on sor-
bitol-ammonium salts. The induction of glucoamylase by maltose was inde-
pendent of sulfate concentration but showed a dependency on low pH and the
absence of utilizable carbon sources.

Starch hydrolysis in cultures of Aspergillus the carbohydrate moiety of the enzyme is well
niger may result from activity of two hydro- documented (24, 26), and many studies per-
lytic enzymes, namely, a-amvlase [a-D-(1-4)- taining to the effect of carbon and nitrogen
glucan glucanohydrolase, EC 3.2.1.1] or gluco- sources on the amount of glucoamylase pro-

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amylase [a-D-(1-4)-glucan glucohydrolase, EC
3.2.1.3]. Two molecular forms of glucoamylase
are produced by A. niger (19) and both liberate
glucose from starch by hydrolyzing a-D-(1-4)
and a-D-(1-6) glucosidic linkages (10, 20).
Additional similarities between the two glu-
coamylase forms include identical pH optima
(pH 4.5 to 5.0) with starch as the substrate,
temperature stability, action pattem on malto-
oligosaccharides, and N-terminal amino acid
(L-alanine) (10). G-lucoamylase I was not disso-
ciated with urea or acid under the conditions
investigated (10). Both forms are glycoproteins
containing D-glucose, D-mannose, and D-galac-
tose with one form, glucoamylase I, containing
18% carbohydrate and the other 10% carbohy-
drate (23). Although both forms of enzyme
have the same Km and Vm.x values with malt-
ose or small oligosaccharides, glucoamylase I
hydrolyzes starch about three times faster than
glucoamylase II (28).
Considerable attention has been given also
to the synthesis of glucoamylase by A. niger.
The incorporation of glucose and mannose into
'Present address: Department of Biology, University of
New Mexico, Albuquerque, N.Mex. 87106.
'Present address: Department of Grain Science and In-
dustry, Kansas State University, Manhattan, Kans. 66502.
771
duced have been reported (1, 9, 16). There is,
however, little information available con-
ceming the regulation of glucoamylase aside
from the apparent stimulation of enzyme pro-
duction when high levels (10%) of glucose, malt-
ose, starch, or glycogen were added to growing
cultures of A. niger (17). Evaluation of these
results in terms of regulation of glucoamylase
synthesis is difficult because high concentra-
tions of carbohydrates were used and because
it is unlikely that starch or glycogen is able
to enter the cell.
To clarify the question of regulation of glu-
coamylase in A. niger and examine the produc-
tion of the two enzyme forms, the following
investigation was initiated. We present evi-
dence for the induction of glucoamylase by
maltose or isomaltose.
MATERIALS AND METHODS
Culture and media. A. niger NRRL 330 was
maintained by periodic transfer on agar slants com-
posed of either ammonium salts or yeast extract
media with sorbitol as the carbon source. The basal
medium contained, per liter of distilled water:
KH2P04, 1.0 g; NH4C1, 2.0 g; MgSO4-7H,O, 0.5 g;
CuSO4*5H,0, 0.16 mg; FeSO4-7H,O, 1.0 mg;
ZnSO4 7H,O, 0.8 mg; NaMoO4 - 2H20, 0.06 mg; and
MnCl2 4H20, 0.07 mg. The yeast extract medium
772 BARTON, GEORGI, AND LINEBACK
contained 5 g of yeast extract (BBL) per liter and
the salts of the basal medium with the exception
that NH4Cl was omitted. Solutions of carbohydrates
were autoclaved separately and added aseptically to
the sterile basal medium to give a final concentra-
tion of 1.0%. The initial pH of both the basal salts
and yeast extract media was 4.5.
Preparation of spore inoculum. Conidia were
dislodged from freshly sporulated cultures and, after
suspension in sterile water, were washed once in
sterile distilled water. The spore suspension was fil-
tered through glass wool to remove mycelial frag-
ments, and the inoculum was standardized by enu-
merating conidia in a Petroff-Hauser chamber. The
inoculum, 0.5 ml, contained 5 x 101 spores that had
a viability of 90%.
Conditions of growth. After inoculating 50 ml of
media in 250-ml Erlenmeyer flasks, the flasks were
incubated at 28 C on a New Brunswick gyratory
shaker adjusted to 170 rev/min. Growth was followed
by measuring dry weight of submerged mycelia as
previously described (1).
Glucoamylase formation. Production of gluco-
amylase accompanying growtb of A. niger in various
media was determined by periodically assaying the
culture fluid. Induction of glucoamylase was deter-
mined by adding individual carbohydrates to a 48-hr
culture pregrown on ammonium salts-sorbitol (2.56
x 10-' M) medium and measuring the resulting spe-
cific activities after 24 hr. Induction is considered to
result from the carbohydrate added when the spe-
cific activity increases at least 15-fold. Monosaccha-
rides and disaccharides, including maltose, were ster-
J. BACTERIOL.
Comparison was made to a glucose standard. The
amount of glucose liberated from a starch substrate
was determined with glucose oxidase, a procedure
previously described (1). A unit of glucoamylase ac-
tivity is the quantity of enzyme which liberates 1
smole of glucose per min from starch under the reac-
tion conditions defined. Specific activity is ex-
pressed as units per milligram of dry mycelia.
Separation and identification of the two forms of
the glucoamylase were accomplished by column
chromatography on diethylaminoethyl (DEAE)-cel-
lulose, as previously described (19). Culture fluid,
200 ml, was either added directly to the column, or
enzymes were concentrated by precipitation with
two volumes of ethanol and charged to a column
after redissolution.
Sources of carbohydrates. Carbohydrates were
obtained from the following distributors: sorbitol,
Matheson Scientific; glucose, Fisher Scientific;
Lintner soluble starch, Merck and Co.; Schardinger
dextrins and dextrins, Mann Research; bacteriolog-
ical dextrin, technical maltose, glycogen, and meli-
biose, Pfanstiehl Chemical Co.; isomaltose, Pierce
Chemical Co.; sodium dextran sulfate 2000, Phar-
macia Fine Chemicals; com starch, Com Products
Co.; maltose and other carbohydrates, Nutritional
Biochemical Corp. Crystalline amylodextrin with a
molecular weight of approximately 3,600 (18) was
provided by J. H. Pazur.
RESUTLTS
Glucoamylase production accompanying

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ilized by membrane filtration (Millipore Corp.) growth. The amount of glucoamylase
whereas polysaccharide suspensions, which could not (units/milliliter) accompanying growth on sor-
be filtered, or autoclaved without degradation, were bitol-ammonium salts is several times greater
added directly to the culture. The pH of the media than with sorbitol-yeast extract (Table 1). In
at the time of addition of the carbohydrates was near both cases, sorbitol was completely utilized by
2.0, and no bacterial contamination was observed day 3. Two different maximal levels of gluco-
throughout the induction period. amylase (4 and 6 days) occurred with yeast ex-
Assay and separation of glucoamylase. Produc- tract, in contrast to one maximal level (5 days)
tion of the extracellular enzyme elaborated by resulting with ammonium chloride. Although
growing cultures was determined by following the both glucoamylase forms show no loss of ac-
starch-hydrolyzing activity of the culture fluid. The
culture fluid, 0.2 ml, was mixed with an equal tivity at pH 2.5 for 24 hr (28) and only slight
volume of a 4.0% starch solution (Lintner) in 0.1 M
citrate-phosphate buffer at pH 4.8 and incubated at TABLE 1. Glucoamylase formation accompanying
30 C for 1 hr. Glucoamylase activity was stopped by growth on sorbitol
immersing the glass-stoppered reaction tubes in
boiling water for 2 min. Quantitation of glucose lib- Mycelia (mg dry Glucoamylase (10-2
erated could be accomplished with a colorimetric wt/50 ml) units/ml)
Day of in-
glucose oxidase-peroxidase system (2, 4). The oxi- cubation Ammo- Yeast Ammo- Yeast
dase-peroxidase reagent consisted of 2 ml of an al- nium t nium etat
cohol solution containing 3,3-dimethoxybenzidine chloride extract chloride.
extracta
(Eastman Organic Chemicals) added to 100 ml of 0.1
M citrate-phosphate buffer, pH 7.0, which contained 1 14 35 0.2 0.0
10 mg of purified glucose oxidase (Miles Chemical 2 80 135 0.9 0.4
Co.) and 5 gg of horseradish peroxidase type II 3 205 206 13.5 1.7
(Sigma). The hydrolyzed starch solution was diluted 4 241 296 39.0 8.2
to 1 ml and incubated with an equal volume of glu- 5 220 316 45.0 6.4
cose oxidase reagent for 5 min at 30 C. This reaction 6 215 297 28.5 10.8
was terminated by addition of 5 ml of 10 N H2SO4, 7 185 275 28.5 9.2
and the intensity of the resulting color was measured
at 525 nm with a Beckman DU spectrophotometer. a Nitrogen source.
VOL. 111, 1972 GLUCOAMYLASE FORMATION BY A. NIGER
loss of activity after 164 hr at pH near 5 (10),
some loss of enzyme activity was observed
with the ammonium salts and yeast extract
media. This may result from slow denaturation
of enzyme due to shaking. Loss of glucoamy-
lase activity as a result of proteolysis cannot
be discounted entirely, although it has been
found that both enzyme forms show marked
stability to proteolytic degradation (Lineback,
unpublished data).
Production of two different maximal levels
of glucoamylase has also been reported when
growth resulted from various organic nitrogen
sources (1). The production of a single level of
glucoamylase has also been previously ob-
served, but this response was associated with
cultures growing on cereal grains (3, 13, 24) or
on high concentrations of nutrient broth (1)
where the enzyme level was many times that
observed with sorbitol-ammonium salts. More
than one maximal level of enzyme production
in A. niger is not unique to glucoamylase, but
has been observed also in protease synthesis
(14).
Substitution of glucose, maltose, or starch
for sorbitol in the growth media results in
stimulation of glucoamylase synthesis as evi-
denced by specific activities (Table 2). The
pattern of glucoamylase formation does not
change with carbon sources since one period of
enzyme production occurs with ammonium
salts and two with yeast extract. The growth
(mycelial dry weight) changes slightly with the
various carbon sources but reflects the pattern
reported in Table 1 with maximum growth
occurring by day 3 or 4, followed by a gradual
decrease in mass. Glucoamylase activity de-
creases after day 5 or 6 with ammonium salts
or yeast extract media, respectively. The de-
crease in mycelial mass is greater than the loss
of enzyme activity, thereby accounting for the
increase in specific activity often observed at
TABLE 2. Comparison of glucoamylase levels
resulting from sorbitol, glucose, maltose, or starch
Nitrogen Carbon Specific activitya on day
source source 1 2 3 4 5 6
Ammonium Sorbitol 7 6 25 82 102 68
chloride Glucose 120 103 147 178 109
Maltose 101 169 234 292 243
Starch 101 186 315 314 272
Yeast ex- Sorbitol 0 2 4 14 10 18
tract Glucose 22 41 60 26 31
Maltose 5 39 18 24 48
Starch 35 139 105 128 171 149
aUnits of glucoamylase activity per gram of dry mycelia.
773
day 7. With all media, the pH dropped rapidly
from the starting pH of 4.5 to between 2.5 and
2.1 and remained at this level throughout incu-
bation.
Incorporation of starch into the medium
with either ammonium salts or yeast extract
resulted in specific activities of glucoamylase
which were higher than when maltose, glucose,
or sorbitol was the carbon source. However,
glucoamylase production was not always found
to be greatest with polysaccharides as the
carbon source. In a yeast extract media, the
level of enzyme accompanying utilization of
maltose was greater than with dextrin, and
with Trypticase (0.12%) higher levels were ob-
tained with maltose than with starch.
Carbohydrases other than glucoamylase were
observed in many cultures. Glucosyltransferase
(transglucosylase, reference 21) and a-amylase
were present in all yeast extract media inde-
pendent of the carbon source. However, gluco-
syltransferase and a-amylase were detected in
ammonium salts media only when sta,rch was
the carbon source. These observations are con-
sistent with earlier reports which stated that
a-amylase and glucosyltransferase were pro-
duced in mannose-yeast extract medium but
not when ammonium salts were the nitrogen
source (9).
Enzyme forms. In two independent experi-
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ments, both forms of glucoamylase were found
in the culture fluid following growth in the
glucose-yeast extract medium. Resolution of
the two enzyme forms was readily accom-
plished by elution of enzymes from DEAE-
cellulose with buffers of decreasing pH values
(19). We found that glucoamylase II was eluted
at pH 6.8 to 6.0 in tubes 105 to 115, and glu-
coamylase I was removed at pH 4.5 to 4.0 in
tubes 148 to 158. Glucoamylase I and II were
found at an approximate ratio of 3:2, respec-
tively, at both day 2 and day 6 of incubation.
The observation that glucoamylase I is the
most abundant form of the two enzymes pro-
duced by A. niger agrees with other reports
(19, 24, 26).
Only one form of enzyme, glucoamylase I,
7 could be recovered from culture fluid when the
glucose-ammonium salts growth medium was
79 used. Repeated attempts to detect glucoamy-
155
221 lase II in the culture fluid on day 4 and 5 of
341 incubation were unsuccessful regardless of
17 whether the ethanol precipitate of the culture
40 fluid or the untreated culture fluid was exam-
68 ined. Similarly, only glucoamylase I was de-
tected when maltose was substituted for glu-
cose in the ammonium salts medium.
 rsMycelia
774 BARTON, GEORGI, AND LINEBACK J. BACTERIOL.
The effect of the nitrogen source on enzyme Induction by technical maltose could be due to
production was observed not only with glucoa- the presence of low-molecular-weight malto-
mylase II formation, but as indicated pre- dextrins (31). These results contradict the re-
viously, also with glucosyltransferase and a- port of Okazaki and Terui (17), who found that
amylase production. The role of the non-ni- glucose, glycogen, or starch stimulated gluco-
trogenous compounds in the yeast extract on amylase synthesis. Although these differences
the formation of a-amylase, glucosyl- may be explained by genetic variations within
transferase, and glucoamylase forms is not un- the cultures, it is most likely that a-amylase
derstood at this time. was being produced by the culture at the time
Addition of carbohydrates to pregrown of carbohydrate addition and that the products
mycelia. The effect of various branched, resulting from a-amylase action on these
cyclic, or linear polysaccharides on glucoamy- carbon sources were responsible for the stimu-
lase formation was examined, but none were lation of enzyme formation. Accordingly, the
found to induce glucoamylase (Table 3). Of the increase of glucoamylase production after the
mono- and disaccharides tested, only maltose transfer of growing cultures into glucose (17) or
or isomaltose stimulated enzyme formation. resulting from incorporation of glucose into the
growth medium (Table 2) results from the en-
TABLE 3. Effect of various carbohydrates on zymatic conversion of glucose into a suitable
glucoamylase formation inducer.
Induction by maltose. After addition of
Carbohydratea Specific
activity" maltose to a 48-hr culture, a lag of about 12 hr
occurred before an increase of glucoamylase
Polysaccharides activity was observed (Table 4). This lag re-
Amylopectin 28 mained virtually unchanged regardless of the
Amylodextrin 24 amount of maltose added. Long lag periods
Dextran 9 accompanying production of fungal enzymes
Dextrin 23 have been observed with amylase (recognized
Glycogen 20
by the authors to be primarily glucoamylase)
Lintner starch 11
Corn starch 29 induction in Neurospora (5) and in formation
Schardinger a-dextrin 12 of glucoamylase by A. niger after transfer of

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Schardinger ,3-dextrin 17 culture into fresh media (17).
Disaccharides Maltose-induced glucoamylase over a range
Maltose (technical) 183 of 2.8 x 10-' to 1.4 x 10-1 M and amount of
Maltose 162 enzyme formed were proportional to the
Isomaltose 147 amount of maltose added. Although it would be
Trehalose 14
Sucrose 25 expected that maltose would be readily hydro-
Lactose 8 lyzed, only traces of glucose were found in the
Melobiose 22 media after addition of maltose, this could
Cellobiose 15 result from the low level of glucoamylase
Monosaccharides and polyols
Sorbitol 2 TABLE 4. Induction of glucoamylase by maltose
Inositol 10
Hours . Glucoamy-
Mannitol 18 nraeI Increase in
after lase Specific
Sorbose 2
Fructose 11
maltose
added
(mgWdiy
w)
(total activitya
units)acity
activityf
Mannose 5
Galactose 28 0 73 0.9 12
Glucose 13 4 106 2.3 21 9
Xylose 21 8 140 3.1 22 8
Ribose 19 12 170 4.8 28 11
Rhamnose 12 16 194 10.7 55 33
20 211 17.4 82 58
a Concentration of mono- and disaccharides was 24 234 34.4 147 117
1.4 x 10-4 M, and polysaccharide was 0.05%, equiva-
lent to 1.4 x 10-4 M maltose. Additions were made a Units of glucoamylase activity per gram of dry
to 48-hr cultures growing on sorbitol-basal salts me- mycelia.
dium, and enzyme production was followed as de- 'Determined by calculating difference between
scribed in Materials and Methods. specific activity resulting from addition of maltose
b Units of glucoamylase activity per gram of dry and specific activity occurring when no maltose is
mycelia. added.
V/OL. 111, 1972 GLUCOAMYLASE FORMATION BY A. NIGER
present in the culture before induction. This
stimulation could be shown also when maltose
(1.4 10-' M) was added to 1-, 3-, 4- or 5-day
x was
cultures; however, the resulting induction was
not as reproducible as that obtained with 2-
day cultures.
Glucoamylase formation by A. niger pre-
grown on sorbitol-yeast extract could be in-
duced also by maltose (1.4 x 10-I M). This
stimulation, unlike that obtained with the cul-
ture pregrown on sorbitol-ammonium salts,
resulted in only a two- to threefold increase in
glucoamylase synthesis.
Effect of pH on induction. Adjustment of
the pH of the culture fluid to several different
values at the time of induction produced a
greater effect on enzymatic activity than on
fungal growth (Table 5). Greatest induction
occurred at pH 2.0, with lower enzyme activi-
ties resulting with increases in pH. In compar-
ison, Okazaki and Terui (17) reported that op-
timal production of glucoamylase occurred at
pH 3.0. It is not known whether the transport
system for maltose is more efficient at low pH
values or if pH adjustments are injurious to
the cells.
775
duced was less as the concentration of sulfate
was decreased. The amount of enzyme formed
generally related to cell mass, as evi-
denced by comparison of the resulting specific
activities.
Effect of carbon sources on induction.
Since the production of glucoamylase is less
when the carbon source is increased in the
growth media (1), it was suspected that gluco-
amylase formation could be decreased by cer-
tain carbon sources. Sorbitol, fructose, and
xylose, which produce little glucoamylase
when used as carbon sources (1), reduced the
amount of enzyme produced when added si-
multaneously with maltose (Table 7). Simi-
larly, the level of glucoamylase produced in
the presence of glycerol, a-ketoglutarate, and
pyruvate was lower than when only maltose is
added.
This effect of carbon sources on induction by
maltose could be explained by inhibition of
TABLE 6. Effect of sulfate concentration on cell
growth and glucoamylase productiona
Gluco-

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Mcla amylase
Previous growth studies indicated that initi- Sulfate concn Mycela
(mg dry
Specific
ating growth at pH values from 6.0 to 2.0 (1) (M) wt)
(10- 2 activity"
units/ml)
had little effect on the amount of glucoamylase
produced. Perhaps this is because the pH 1.25 x 10- 60 14 115
drops rapidly in the culture medium and is near 2.5 x 10-1 75 18 120
2.0 by the second day. 1.25 x 10-4 100 16 80
Effect of sulfate on induction. Since the 1.0 x 10-s 210 46 109
production of several extracellular enzymes by 2.0 x 10-s 210 52 123
bacteria and fungi has been found to be stimu- a Maltose (1.4 x 10-3 M) was added to a 48-hr cul-
lated by cultivation of the organism in sulfate ture grown on sorbitol, and measurements were
concentrations which were suboptimal for made 24 hr later. The growth medium contained
growth (12, 15, 27, 29, 30), the induction of magnesium as the chloride salt and sulfate as the
glucoamylase was examined when A. niger was sodium salt.
grown on varying concentrations of sulfate Units of glucoamylase activity per gram of dry
(Table 6). The amount of glucoamylase in- mycelia.

TABLE 5. Induction of glucoamylase at various pH TABLE 7. Effect of various carbon sources on

values induction
Glucoamylase Mycelia (mg Specific
Compound addeda Specific activity"
pHa activity (10-2I dry wt) activity"
units/ml) None 167
Sorbitol 79
2 51 205 125 Fructose 66
3 21 210 50 Xylose 56
4 15 200 35 Glycerol 31
5 13 195 33 a-Ketoglutarate 42
6 10 195 26 Pyruvate 38
a
At 48 hr, the culture was adjusted to the pH in- a
Carbon sources (5.6 x 10-i M) were added with
dicated with 0.5 N NaOH, and maltose (1.4 x 10-8 maltose (1.4 x 10-9 M) to a 48-hr culture, and spe-
M) was added. All measurements were made at 72 hr. cific activity was determined 24 hr later.
"
Units of glucoamylase activity per gram of dry Units of glucoamylase activity per gram of dry
mycelia. mycelia.
776 BARTON, GEORGI, AND LINEBACK /
maltose uptake as well as by catabolite repres-
sion. Since inhibition of glucoamylase produc-
tion could be shown when acetate or oxalace-
tate was incorporated into the media 16 hr
after the addition of maltose, it would appear
that factors other than inhibition of maltose
uptake must be responsible.
DISCUSSION
Glucoamylase synthesis in A. niger is in-
duced by maltose or isomaltose but not by
polymeric glucans, which apparently are too
large to enter the cells. The high level of en-
zyme accompanying growth on starch may be
due to the continual release of inducer mole-
cules due to the action of a-amylase produced
by A. niger. This would be in accord with a
previous report in which the supply of inducer
over an extended period has been found to be
important in the production of various carbo-
hydrases by fungi (25).
Glucoamylase hydrolyzes a variety of a-D-
glucosides (22), but only certain substrates
stimulate enzyme formation. Levels of glucoa-
mylase resulting from maltose or isomaltose
induction are about the same even though the
a-D-(1-4) linkage is hydrolyzed 30 to 40 times
as fast as the a-D-(1-6) bond (10, 20). No in-
duction was observed with trehalose or sucrose,
even though both can be slowly hydrolyzed by
glucoamylase (22).
Induction of glucoamylase may also result
from malto-oligosaccharides. Maltotriose, mal-
totetraose, and maltopentaose, all isolated
from technical-grade maltose (32), have been
found to be superior to maltose as inducers of
a-amylase in Bacillus stearothermophilus (32).
Similarly, a component in dextran stimulates
production of amylases in Neurospora (6). The
greater stimulation by technical maltose as
compared to purified maltose (Table 3) sug-
gests that contaminating maltodextrins also
induce glucoamylase formation. Maltodextrins
could arise in starch-containing media by the
activities of a-amylase or glucosyltransferase
(19).
In addition to appropriate carbon and ni-
trogen sources, it is evident that with defined
media certain salts are necessary for produc-
tion of high levels of glucoamylase. Both sul-
fate (Table 6) and trace elements (9) are neces-
sary, not for stimulation of enzyme formation,
but rather for optimal growth. On the other
hand, the pH at the time of maltose addition
is more important for induction of glucoamy-
lase than for growth. When the pH is adjusted
to a level higher than that established by the
J. BACTERIOL.
growing culture, the amount of glucoamylase
resulting decreases appreciably. Whether this
response, after adjustment of pH, is due to
impairment of transport systems or metabolic
alterations in the cell cannot be determined at
this time.
Various carbon sources reduce the level of
glucoamylase induced in A. niger, and similar
results have been found in other carbohydrase-
producing systems. Repression of a-amylase in
B. stearothermophilus occurs with fructose (31);
a-amylase synthesis by Aspergillus is reduced
by glucose (5); invertase, maltase, and tre-
halase are repressed in Neurospora by man-
nose, glucose, fructose, or xylose (11); and
amylase synthesis by Neurospora is reduced by
glycerol (6).
Because deamination products of alanine
and glutamic acid, i.e., pyruvate and a-keto-
glutarate, have been shown to have a detri-
mental effect on glucoamylase production
(Table 7), media containing organic nitrogen
would be expected to produce low levels of
glucoamylase. Perhaps this explains the low
level of enzyme produced with yeast extract
(Table 2; references 1 and 3), with amino acids
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in chemically defined media (1), or with com-
plex nitrogen sources rich in amino acids (3).
Glucoamylase formation by A. niger in media
containing a mixture of carbon sources would
appear to be subject to catabolite repression or
inhibition of maltose uptake. The resolution
and importance of these phenomena await fur-
ther studies.
Media have been shown to influence the
formation of glucoamylase isoenzymes, as well
as the amount of enzyme produced, and it will
be important to reexamine isoenzyme forma-
tion accompanying growth on different ni-
trogen sources. This will not be easily accom-
plished because at the present time it is im-
possible to assay glucoamylase I and II indi-
vidually except after complete separation. The
varying ratio of glucoamylase forms observed
with growth in different media and the infor-
mation that enzyme forms do not arise from
artifacts in isolation (10, 19, 28) suggest that
glucoamylase isoenzymes are products of dif-
ferent genes. This would agree with previous
reports that the two enzymes produced by A.
niger (26) and the two forms of glucoamylase
produced by Candida pelliculosa (8) are under
separate genetic control.
The presence of carbohydrate moieties
firmly bound to glucoamylase is well estab-
lished (8, 23), and it is possible that carbohy-
drates could regulate the activity of the nu-
cleotide pathway involved in the addition of
VOL. 111, 1972 GLUCOAMYLASE FORMATION BY A. NIGER
carbohydrates into the carbohydrate portion of
the enzymes (24, 26), as well as regulate the
synthesis of enzyme protein. We suggest that
glucoamylase formation in A. niger could serve
as a useful model for studying the regulation of
carbohydrate-containing isoenzymes.
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