THE JOURNAL OF BIO~QICAL CHEMISTRY

Vol. 234, No 8, August 1959

Printed in U.S.A.

The Action of an Amyloglucosidase of Aspergillus niger

on Starch and Malto-oligosaccharides*
JOHN H. PAZUR AND TADAHIKO ANDO

From the Department of Biochemistry and Nutrition, University of Nebraska, Lincoln, Nebraska

(Received for publication, March 23, 1959)

Earlier reports from a number of laboratories (l-3) have shown glucose-l-C4 and cyclohexaamylose by use of Bacillus macerans
that Aspergillus niger produces several enzymes with ability to amylase (10, 11).
hydrolyze the a-~-(1 ---f 4) glucosidic linkages in starch and malto- Enzyme Source-A sample of enzyme material, Diazyme, prc-
oligosaccharides. Important members of this group include pared from Aspergillus niger by an alcohol precipitation proee-
a-amylase, amyloglucosidase and maltase. Within recent years dure, was provided by Takamine Laboratory, Division of Miles
there has been considerable interest in the amyloglucosidase Laboratories Inc., Clifton, New Jersey. The enzyme material
group of enzymes since they are capable of converting starch was supplied as a yellow powder which was the starting material
completely to glucose (4-7). Evidence in the literature (1, 4) for the purification work. Preliminary experiments indicated
indicates that the enzymes of this group effect a hydrolysis of that this material contained enzymes with cY-amylase, amylo-
starch by a single chain mechanism in which glucose units are glucosidase and maltase activities.
removed from the nonreducing end of a linear chain until com- Other Materials-Commercial cornstarch extracted with hot
plete hydrolysis of the chain has been effected. Although high methanol was the starch substrate used in our experiments. The
yields of glucose are obtained from starch and amylopectin, re- cornstarch was fractionated into amylose and amylopectin by an
ports on the ability of the enzyme to hydrolyze the a-~-(1 ---f 6) amyl alcohol precipitation method (12) and the amylose and
linkages in the branched polysaccharides are conflicting (4, 6). amylopectin were also used as substrates for the enzyme. The
Little attention has been given to the elucidation of the action amylodextrin used in this series of experiments was prepared by
pattern of amyloglucosidase on malto-oligosaccharides or to the a method described previously (9).
possibility of transglucosylic reactions (8) occurring during amyl-
Methods and Results
oglucosidase action.
In our laboratories an amyloglucosidase of Aspergillus niger Assay Procedures-Two different procedures have been used
has been obtained in a highly purified state by a chromatographic for assaying for amyloglucosidasc activity. The first, suitable
procedure with the use of cellulose ion exchange materials. This for a small number of assays, involved the determination of glu-
enzyme has been used in studies reported herein on the mode of cose produced from starch under standardized conditions. In
action of a representative amyloglucosidase on starch and on the procedure adopted for use, 1.5 ml. of 4 per cent starch solu-
malto-oligosaccharides. The amyloglucosidase converted starch, tion buffered to pH 4.8 with 0.05 M citrate buffer was mixed with
amylose, amylopectin and amylodextrin to glucose in yields ap- 0.5 ml. of enzyme solution and incubated at 30” for 1 hour. The
proximating complete conversion. Results of experiments in glucose produced in this time in a 0.2-ml. sample of the digest
which malto-oligosaccharides labeled in position 1 with Cl4 were was separated by paper chromatography and determined quan-
used as substrates for the enzyme, showed that the hydrolysis of titatively with diphenylamine reagent (9). In some instances,
these oligosaccharides begins at the nonreducing end of the mole- an alkaline copper reagent (13) was used to obtain a reducing
cules and continues by a multi-chain mechanism. The purified value for the digest from which an apparent glucose value was
amyloglucosidase is capable of hydrolyzing maltose but is devoid calculated. A unit of enzyme activity is defined as that amount
of transglucosylic activity. of enzyme which produces 1 mg. of glucose from 60 mg. of starch
dissolved in 2 ml. of citrate buffer of pH 4.8 after incubation for
EXPERIMENTAL 1 hour at 30”.
The second assay procedure was a bioassay method based on
Materials
the measurement of growth zones of microorganisms on a starch-
Malto-oligosaccharides-Maltotriose and maltotetraose were agar medium and was particularly useful for assaying a large
isolated from an enzymatic hydrolysate of amylodextrin (9) and number of samples. The assay plates (30- x 20-cm. Pyrex
maltopentaose from an acid hydrolysate of amylose (10) by plates) used in this procedure contained a layer of agar and a
methods previously described. Maltose was obtained in crys- layer of agar-starch medium in which cells of the yeast, Sac-
talline form from a /3-amylase hydrolysate of starch (8). Malto- charomyces cerevisiae, were suspended. Details of the prepara-
oligosaccharides labeled in position 1 with Cl4 were prepared from tion of the media and the cells for this procedure are presented
elsewhere (3). Filter paper disks of 7.0 mm. diameter were
* Published with the approval of the Director as Paper No. 947
Journal Series, Nebraska Agricultural Experiment Station. Sup- dipped into the enzyme solution to be assayed, pressed on filter
ported in part by a grant from the Takamine Laboratory, Division paper to adsorb excess solution and placed on the agar-starch
of Miles Laboratories, Inc., Clifton, New Jersey. medium. After incubation for 1 hour at 30” the paper disks

1966

This is an Open Access article under the CC BY license.

August 1959 J. H. Pazur and T. Ando 1967

were removed and the plates were incubated for an additional the amylasc were located at 4 cm. and 0.5 cm. from the origin,
18 hours at 30”. Growth zones of microorganisms appeared at respectively.
the areas of the plate at which hydrolysis of starch had occurred. Temperature Stability-Some preliminary observations indi-
The diameters of the growth zones are a measure of the amount cated that the two amyloglucosidases of A. niger differed in their
of enzyme activity in the various samples. temperature stability. Storage of the enzyme solutions at pH
Chromatography on Cellulose Ion Exchange Columns-Cellulose 4.8 under toluene at 30” had little effect on the enzyme which
ion exchange materials used for the separation of proteins (14, migrated at the faster rate on paper clectrophoresis but resulted
15) were found to bc excellent materials for separating the car- in an inactivation of the other enzyme (18 per cent in 24 hours’
bohydrases of A. niger. A glass column (450 X 35 mm.) was storage and 52 per cent in 140 hours’ storage). Likewise, the
packed with 30 gm. of DEAE-cellulose ion cschange matcriall latter enzyme was inactivated more rapidly at clrvated tcmpera-
(r:lagcnt gradr, type 20) and washed thoroughly with citric acid- tures. In the inactivation studies aliquots of the enzyme solu-
disodium phosphate buffer of pH 8.0. A sample of 5 gm. of the tions were maintained at 50” and 60” for 30 minutes, cooled
enzyme powder was dissolved in 50 ml. of distilled water, filtered rapidly to 30” and assayed for activity by the m&hod drscribcd
and mised with 10 ml. of 0.04 M calcium acetatc. The small in “Methods and Results.” Gndcr thcsc conditions th(x ctstcnt
amount of prccipitatc which formed was removed by ccntrifuga- of inactivation for the latter amyloglucosidasc was 20 per cclnt at
tion. The supernatant contained 210 units of amyloglucosidase 50” and 58 per cent at 60” compared to 4 prr cent and 16 pclr cent
activity per mg. of nitrogen. The cnzymr solution was dialyzed for the rnzymc with the higher paper clectrophorrtic mobility.
against running water for 24 hours, diluted to 100 ml. with dis- Both enzymes were stable over a period of sclvcral months in the
tilled water and mixed with 100 ml. of 0.1 RI citric acid-disodium refrigerator.
phosphate buffer, pH 8.0. This solution was introduced on a pH Optimullz-The amvlogluc:osidasr acativity of the purified
column of DEAE-cellulose dcscribrd above. After absorption enzyme samplr used in subsequent csperiments was determined
of the enzyme was complctc, the column was washed with ap- on a starch substrate of various pH values. =\ typical pH curve
proximately 500 ml. of 0.05 M citric acid-disodium phosphate was obtained for the clnzymt: with masimal activity of pH 4.8
buffer of pH X.0, followed by 500 ml. of buffer, pH 6.0, and finally and with approsimatelp 50 per cent inactivation at pH values
by 500 ml. of buffer, pH 4.0. Fractions of 15 ml. were collected below 3.0 or above 6.5.
by means of a fraction collector during development of the Action of llmyloglucosidase on Starch, Amylopectin and Amylo-
column. Starch-hydrolyzing enzymes were present in fractions dextrin--ll 0.2 per cent starch solution buffered to pH 4.8 with
of pH 6.8 to 7.3 in Tubes 67 to 71, in fractions of pH 6.0 in Tubes citrate buffer was misrd with an equal volume of purified amylo-
79 to 82, and in fractions of pH 4.5 to 5.2 in Tubes 110 to 114. glucosidase (16 units per ml.) and incubated at room temperature
On the basis of the nature of hydrolytic products produced from for 18 hours. The enzyme was then inactivated by heat and
starch and maltose the enzyme clutcd at pH 6.8 to 7.3 seems to the concentration of glucose in the digest was dctcrmined by the
br an ol-amylase whereas those cluted at pH 6.0 and 4.5 to 5.2 Shaffer and Somogyi m&hod (13). Similar rsperiments were
seem to bc amyloglucosidases. The two amyloglucosidases prrformrd with amylose, amylopectin and amylodrstrin as sub-
possess different mobilities on paper clcctrophoresis and diffrrcnt strates. From the concentration of thr glucose produced and
stabilities at clcvated temperatures. Since the amgloglucosidase the amount of original polysaccharide in the solution, the per
vvhic*hwas cluted at pH 4.5 to 5.2 was present in larger amounts cent conversion of the polysaccharide to glucose was calculated.
in the original sample it has been used in our esprriments as the The values obtained wcrc as follows: 95 per csc,ntfor starch, 93
cnzymc representative of the amyloglucosidase group. The per cent for amylosc, 98 per cent for amyloprctin, and 92 per cent
fractions containing this amyloglucosidasc were combined and for amylodestrin.
further purified by chromatography on a DEAE-ccllulosc column. The course of amyloglucosidasc action on starch was followed
The adsorption of the enzyme was effected from a solution of by identification of the low molecular weight hydrolytic products
pH 6.0 and the clution by buffer of pH 4.0. The samples of pH at various stagrs of hydrolysis. A 2 per cent starch solution of
4.6 to 5.0 in Tubes 44, 45, and 46 contained most of the amylo- pH 4.8 was treated with an equal volume of the purified amylo-
glucosidase activity. These wcrc combined and used as the glucosidasc (40 units per ml.) and samples taken for analysis
purified amyloglucosidase in the subsequent csperimcnts. Am- after reaction periods of 0, 1, 2, 4, 8, and 24 hours. Paper chro-
yloglucosidasc assays showed that the solution contained 2900 matographic examination of thrsr samplrs showed that glucose
units of activity per mg. of nitrogen. was the only low molrcular weight product at all stages of hy-
Paper ElectrophoresisPLk Spinco model R rlcctrophorcsis ccl1 drolysis. A parallrl experiment, in which an 0.8 per cent solu-
was used for determining the electromigration characteristics of tion of the nonpurified cnzymc material (approsimately equiva-
the original and purified enzyme samples. Samples of 0.01 to lent in activity to the rnzymr solution containing 40 units per
0.03 ml. of the enzyme solutions wcrc used in the clcctrophoretic ml.) was used, showed that maltose and some maltotriosc, in
separations in 0.1 M phosphate buffer, pH 7.6. Electrophoresis addition to glucose, wcrc present in the digest after hydrolysis
was carried out for 7 hours at a potential of 150 volts, direct cur- periods of 1, 2, and 4 hours. The maltose and maltotriosc were
rent and a currrnt of IO ma. Starch-hydrolyzing enzymes on slowly hydrolyzed to glucose and wrrr not tlrtrctctl in thr S- and
the paper clrctrophorctic strips were located by the starch- 24-hour samples. The appearance of maltose and maltotriosc
iodine color reaction (3). The amyloglucosidasc which was in the c,arly stages of hydrolysis of starch by the nonpurified
elutrd at pH 4.6 to 5.0 from the chromatographit: column mi- material confirms the presence of an cl-amyla,se typr enzyme in
grated on the paper as one homogeneous band and was located the original enzyme sample.
at, 6.5 cm. from the origin. The other amyloglucosidase and Action of Amyloglucosidase on P-labeled Malto-oligosacchar-
ides-Samplrs of 0.1 ml. of 0.1 M solution of maltotriosc-l-
1 A product of Brown Company, Berlin, New Hampshire. 04, maltotctraosc-l-C14 or maltopentaosr-1-P of pH 4.8 wrre
1968 Amyloglucosidczseof A. niger Vol. 234, No. 8

FIG. 1. A paper chromatogram (A) and a radioautograph (B) of the products in digest of maltotriose-1-W and purified amylo-
glucosidase. G, glucose; Gs, maltose; Gs, maltotriose; R, reference malto-oligosaccharides; S, substrate.

TABLE I the same chromatogram after incubation of the digest for periods
Radioactivity (c.p.m.) of products in digest of maltotetraose-1-P of 5, 15, 30, 60, and 120 minutes. The products in the samples
and purified amyloglucosidase were separated by paper chromatography and located by the
Compound
permanganate-periodate spray reagent (16) and by radioautog-
Time raphy (8). A photograph of the paper chromatogram and
Maltotetraose Maltotriose Maltose Glucose radioautogram obtained for the maltotriose-l-Cl4 digest is repro-
duced in Fig. 1. The radioactivities of the products in the malt-
minutes
otetraose-l-Cl4 and maltopentaose-l-Cl4 were measured directly
0 1680 14 6 0
on the paper chromatograms with Geiger-Mtiller counting as-
5 1590 107 9 3
15 1230 470 16 1 sembly. Typical values for these experiments are recorded in
30 845 766 89 5 Tables I and II. For comparison purposes, results of a similar
60 326 1010 364 14 experiment with maltotetraose-1-W and nonpurificd enzyme ma-
120 53 282 1350 82 terial are presented in Table III.
Action of Amyloglucosidase on Maltose-The purified and non-
purified amyloglucosidase hydrolyzed maltose rapidly to glucose.
TABLE II In order to determine whether the amyloglucosidase possesses
Radioactivity (c.p.m.) of products in digest of maltopentaose-l-C~4 transfer activity, these experiments were performed with high
and purijied amyloglucosidase concentrations of maltose. Samples of 0.5 ml. of 60 per cent
Compound maltose solution were treated with 0.5 ml. of amyloglucosidase
Time
- (400 units per ml.) or 0.5 ml. of a 2.0 per cent solution of the
Mabto- Malto- Maltotriose Maltose Glucose
pentaose tetraose
TABLE III
minutes
Radioactivity (c.p.m.) of products in digest of maltotetraose-l-04
0 1920 38 11 16 5 and nonpurijied amyloglucosidase
5 1570 310 23 17 9
15 1390 540 95 21 12 Compound
30 880 720 310 52 18 Time
60 390 610 870 160 22 Maltotetraose M&hose Maltose Glucose
120 80 94 650 1210 85 minu1es
-
0 1690 21 11 2
treated with 0.1 ml. of purified amyloglucosidase (80 units per 15 1021 273 238 10
30 843 493 415 9
ml.) and incubated at 30”. A sample of 0.005 ml. of untreated
60 467 654 622 24
oligosaccharide solution was placed on a paper chromatogram 120 154 680 950 57
and subsequent samples of 0.01 ml. of the digest were placed on
August 1959 J. H. Pazur and T. Ando 1969

FIG. 2. Paper chromatograms of products in digests of maltose with nonpurified (A) and purified (B) amyloglucosidase. G, glu-
cose; Gz, maltose; G1, maltotriose; I, isomaltose; P, panose (4-or-isomaltosyl-n-glucose); R, reference malto-oligosaccharides.

nonpurified enzyme material. Aliquots of the digest were ana- tetraose-l-C4 and maltopentaose-l-C4; i.e. a stepwise removal
lyzed by paper chromatographic methods as described above. of the glucose units from the nonreducing end of the oligosac-
Photographs of the finished chromatograms of the digests with charides. This sequence of production of radioactive products
the two enzyme solutions are reproduced in Fig. 2. from the oligosaccharides labeled at position 1 with Cl4 points to
the multi-chain mechanism as the predominate mechanism by
DISCUSSION which these linear oligosaccharides are hydrolyzed by amylo-
The chromatographic method with DEAE-cellulose is a simple glucosidase.
and rapid procedure for purifying the amyloglucosidase of A. The action of amyloglucosidase on labeled oligosaccharides
niger. The enzyme so purified has been used for investigating differs from that of an enzyme of the a-amylase class. In
the mode of action of an amyloglucosidase on starch and malto- general, cr-amylases attack the interior glucosidic bonds of malto-
oligosaccharides. As measured by a reducing sugar method (13), oligosaccharides at a rate many times greater than that for
the yield of glucose produced from starch and related compounds terminal bonds (10, 17, 18). As a result the major radioactive
by the amyloglucosidase is in agreement with that espected for hydrolytic product expected in digests of maltotetraose-l-Cl4
complete hydrolysis. Paper chromatographic examination of with cr-amylases is maltose-1-Ci4. That the A. niger produces
the digest at various stages of hydrolysis showed that glucose an enzyme of the ol-amylase type is indicated by a comparison
was the only low molecular weight sugar produced during the of the radioactivities of the hydrolytic products produced from
course of hydrolysis of starch. In order to convert starch com- maltotetraose-l-Cl4 by the purified (Table I) and nonpurified
pletely to glucose, the amyloglucosidase must be capable of hy- (Table III) enzyme preparations. These values and the nature
drolyzing the a-~-(1 ---f 6) linkage as well as the a-~-(1 -+ 4) of the hydrolytic products in digests of starch with the purified
linkage in the polysaccharide. Results of experiments in prog- and the nonpurified enzyme show that the amylase has been
ress on the hydrolysis of glucosyl oligosaccharides containing the separated from the amyloglucosidase by the chromatographic
a-~-(1 + 6) and the a-~-(1 + 4) linkages by the amylogluco- procedure.
sidase substantiate this suggestion. Fig. 2 shows that the purified amyloglucosidase is capable of
Use has been made of Ci4-labeled malto-oligosaccharides for hydrolyzing maltose to glucose but it does not effect the synthe-
demonstrating that amyloglucosidase action on linear glucose sis of transglucosylic products. In contrast, the original enzyme
polymers proceeds from the nonreducing end of the molecule. sample from A. niger was capable of synthesizing transglucosylic
Thus, the initial products of enzyme action on maltotriose-l-Cl4 products (isomaltose and panose) during the hydrolysis of malt-
(Fig. 1) were maltose-l-C4 and nonradioactive glucose. As the ose to glucose. These observations are interpreted as evidence
concentration of maltose-l-C4 in the digest increased, the disac- for the presence of a maltase with transfer activity in the non-
charide was itself hydrolyzed by the amyloglucosidase to yield purified enzyme preparation from A. niger. The maltase was
radioactive and nonradioactive glucose. Values in Tables I and separated from the amyloglucosidase by the chromatographic
II indicate a similar action pattern of amyloglucosidasc on malto- procedure. Although amyloglucosidase, itself, possesses maltase
1970 Amyloglucosidase of A. niger Vol. 234, No. 8

type activity, e.g. hydrolysis of maltose to glucose, it does not the use of cellulose ion exchange materials. The cnzymc con-
effect transglucosylation reactions. verts starch, amylose, amylopectin, and amylodcxtrin to glucose
in yields approximating complete conversion. Evidence from
SUMMARY experiments in which malto-oligosaccharides labeled in position
1 with Cl4 were used as substrates indicates that the amyloglu-
An amyloglucosidase of Aspergillus niger has been obtained cosidase hydrolyzes the oligosaccharides by a multi-chain mcch-
in a highly purified sta.te by a chromatographic procedure with anism beginning at the nonreducing end of thrl molcculcs.
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