THE ACTION OF TRANSGLUCOSIDASE OF ASPERGILLUS

ORYZAE ON MALTOSE*
BY JOHN H. PAZUR AND DEXTER FRENCH
(From the Department of Biological Chemistry, University of Illinois College of
Medicine, Chicago, Illinois, and the Department of Chemistry, Iowa State
College, Ames, Iowa)

(Received for publication, December 17, 1951)

In 1950 Pan and his associates (1) reported the conversion of maltose

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into non-fermentable sugars by an enzyme solution obtained from Asper-
gillus niger. They isolated a crystalline, non-fermentable, reducing trisac-
charide from the reaction mixture (2). Structural studies by French (3)
and by Wolfrom et al. (4) have shown this trisaccharide to be a 4-a-iso-
maltosyl-n-glucose (panose).
In a preliminary note (5) we described an enzyme of Aspergillus oryxae
which also synthesizes the trisaccharide panose from maltose. Other prod-
ucts of enzyme action on maltose were the disaccharide isomaltose, the
trisaccharide 6-ar-isomaltosyl-D-glucose (dextrantriose), and the tetrasac-
charide 4-or-dextrantriosyl-n-glucose.1 Since all the synthesized com-
pounds are formed by a transglucosidation reaction, it is probable that
only one enzyme is involved in their formation. In the present paper we
present evidence for the mechanism of action of this enzyme on maltose
and record the identification of the products of its action.

EXPERIMENTAL

Enzyme Xolution-The enzyme concentrate2 as obtained was a brown,

fluffy powder prepared from A. oryxae by alcohol precipitation procedures.
Due to the method of preparation, this concentrate contained a number of
carbohydrases, e.g., amylase, maltase, dextrinase, etc. A partial separa-
tion of these enzymes from the transglucosidase was accomplished by com-
plex formation of the enzymes with starch at low temperatures.
5 gm. of the enzyme material were stirred in 500 ml. of cold (2’) acetone-
water solution (1: 3). After the removal of a small amount of undissolved
material by filtration, the cold filtrate was poured into a beaker containing
* Journal Paper No. J-2020 of the Iowa Agricultural Experiment Station, Project
No. 1116, Ames, Iowa.
1 This system of nomenclature used by Wolfrom et al. (4) for panose supersedes
the one we previously used (5).
2 The first sample of enzyme material was kindly furnished by Dr. L. A. Under-
kofler and Mr. R. Miller, Department of Chemistry, Iowa State College, Ames, Iowa.
Later samples were made available by Takamine Laboratories, Clifton, New Jersey.
265
266 A. ORYZAE AND MALTOSE

50 gm. of defatted corn-starch. The resulting suspension was stirred con-

tinuously for 0.5 hour. Next, the starch was removed by filtration and a
clear enzyme solution obtained. The acetone in the solution was removed
by vacuum distillation in the cold. The enzyme solution was then layered
with toluene and stored in the refrigerator until it was to be used.
Maltose Digest-5 ml. of 2 per cent maltose3 solution were mixed with 5
ml. of enzyme solution. 1 ml. of the solution was removed as soon as
possible after the introduction of the enzyme to the maltose, heated to
boiling, and refrigerated until analyzed. Subsequent samples were inacti-
vated at 5, 10, 25, and 75 hour periods. These samples were analyzed for
reducing sugars and for phosphorylated sugars by paper chromatography
procedures (6, 7).

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Glucose and Glucose-l-phosphate Digests2 ml. of 5 per cent glucose solu-
tion were shaken with 2 ml. of enzyme solution. As in the maltose digest,
samples were removed and inactivated at 0, 5, 10,25, and 75 hour intefvals.
A parallel experiment was conducted in which 0.1 gm. of glucose-l-phos-
phate and 0.1 gm. of glucose in 2 ml. of phosphate buffer, pH 7.0, were
mixed with 2 ml. of enzyme solution. As before, samples were removed at
regular time intervals and examined for reaction products.
Maltose and C’4-Glucose Dige&--15 mg. of pure maltose and 0.1 mg. of
U4-glucose were dissolved in 0.6 ml. of enzyme solution. At 0, 6, 12, 24,
and 96 hour intervats 0.1 ml. aliquots were removed, placed in tightly
stoppered tubes, and heated in a boiling water bath for 3 minutes. Next,
0.01 ml. aliquots of these samples were introduced on squares of filter paper
at 1 inch intervals near the bottom of the paper. The reducing sugars in
the digest were resolved by paper chromatography procedures already
described (6). The finished chromatograms were placed in contact with
Kodak No-Screen x-ray film for 18 days. After development of the film,
a radioautograph showing the radioactive compounds in the digest was
obtained. Finally, the reducing sugars were located on the paper by spray-
ing with copper and phosphomolybdic acid reagent (6). The paper chro-
matogram and the radioautograph were photographed side by side and are
reproduced in Fig. 1.
3 As commercial c.p. maltose was found to contain glucose, amylotriose, and higher
molecular weight dextrins, we prepared the maltose by the action of @-amylase on a
starch paste. The limit dextrins in the digest were removed by precipitation with
methanol (50 per cent). The filtrate was concentrated under a vacuum to a syrup.
The carbohydrates in the syrup were adsorbed on carbon and the maltose preferen-
t,ially eluted with 15 per cent ethanol. The filtrate was concentrated to a syrup and
the maltose crystallized from hot ethyl alcohol solution. Physical properties,
microscopic examination of crystals, and paper chromatography of a solution of the
crystals showed our maltose to be free of other reducing sugars.
1 Carried out in cooperation with S. Aronoff and associates, Botany Division,
Institute for Atomic Research, Ames, Iowa.
 J. H. PAZUR AND D. FRENCH 267

Separation of Reaction Products in Maltose Digest-To obtain working

quantities of the synthesized saccharides, a large scale digest was prepared.
4 gm. of maltose in 80 ml. of water were treated with 5 ml. of enzyme solu-
tion. The mixture was allowed to stand at room temperature for 21 days.
Analyses of the digest showed that the concentrations of the synthesized
compounds were at a maximum in this reaction time. The enzyme was

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FIG. 1. A multiple ascent paper chromatogram (A) and a radioautograph (B) of

Cl4-glucose and maltose digest at several stages of enzymolysis. G, glucose; PI/L,
maltose; I, isomaltose; P, panose; D, dextrantriose; and T, tetrasaccharide (dextran-
triosylglucose).

destroyed by heat and the compounds were separated by large scale paper
chromatography.
A 10 ml. sample of the digest was placed in a continuous streak near one
edge of a rectangle of filter paper (35 X 40 cm.). The resolution of the
saccharides was accomplished by t,he multiple ascent t.echnique in t.he
solvent system (3 parts water, 4 part’s pyridine, and 6 part.s n-but.anol).
Two vert’ical strips were cut from the developed chromatogram, sprayed,
and used as markers for sectioning the remaining portions of the chro-
matogram. The individual saccharides were extracted from the paper with
boiling water. The extracts from five chromatograms were combined and
concentrated under a vacuum. The syrupy material was dried with ace-
tone and washed with a small amount of n-butanol. Attempts to crystal-
268 A. ORYZAE AND MALTOSE

lize the isomaltose and the panose have not yet been successful. Howe\-er,
crystalline flavazoles of these compounds mere prepared and identified by
x-ray diffraction methods as described later.
Isomaltose-The yield of isomaltose obtained by the above isolation pro-
cedure was 0.43 gm. The specific rotation of the isomaltose is [a], =
+ 119” (c 2, water); the published value is +120” (8).
200 mg. of isomaltose, 60 mg. of o-phenylenediamine, 360 mg. of phenyl-
hydrazine hydrochloride, 0.12 ml. of glacial acetic acid, and 5 ml. of water
were placed in a 25 ml. Erlenmeyer flask having a ground glass joint. The
mixture was refluxed in a boiling bath for 4 hours. The crude isomaltose
flavazole which formed was separated from the reaction mixture by filtra-
tion. It was dissolved in 2.5 ml. of hot n-propanol, filtered while hot into

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a 10 ml. beaker, and allowed to crystallize. After a few hours cryst’als of
the isomaltose flavazole appeared. These were collected on a filter paper
and air-dried. A powder x-ray diffraction photograph5 was obt#ained on
the air-dried material. Comparison of this photograph with t’hat ob-
tained for the flavazole of pure isomaltose” showed the two compounds to
be identical. The structure of the compound synthesized is therefore es-
tablished as 6-a-glucosyl-D-glucose (isomaltose).
4-wIsomaltosyl-D-glucose (Panose)-The weight of vacuum-dried panose
obtained by the separation scheme was 0.25 gm. The specific rotation
was [a], = + 150” (c 2, water) ; the published value is + 154” (2, 3).
100 mg. of panose were used for preparing the flavazole derivative. The
procedure was ident’ical with that for preparing the flavazole of isomaltose.
The panose flavazole was purified by recrystallization from n-propanol. As
before, x-ray diffraction photographs mere obtained on the air-dried crystals
and on panose flavazole prepared from the pure compound.7 Visual com-
parison of the photographs showed that our compound was 4-cu-isomaltosyl-
n-glucose (panose).
6-oc-Isomaltosyl-D-glucose (Dextrantriose)-The yield of this trisaccharide
(0.058 gm.) was markedly less than for the two previous compounds. The
specific rotation of the compound is f134” (c 0.5, water) and compares
well with the value of +136” calculated for such a compound by Freuden-
berg’s equation (9). On graded acid hydrolysis the trisaccharide was con-
verted first into glucose and isomaltose and finally completely to glucose.
Sufficient material was not available for derivatives of this compound.
4-wDextrantriosyl-D-glucose-The tentative structure suggested for the
5 The assistance of W. James, Department of Chemistry, Iowa State College,
Ames, Iowa, is acknowledged.
6 Pure isomaltose was obtained from M. L. Wolfrom, Department of Chemistry,
The Ohio State University, Columbus, Ohio.
7 Pure panose was obtained from S. C. Pan, Research Laboratories, Joseph l3.
Seagram and Sons, Inc., Louisville, Kentucky.
 J. H. PAZUR AND D. FRENCH 269

tetrasaccharide is 4-cr-dextrantriosyl-n-glucose. The compound was syn-

thesized by the addition of a glucosyl unit to the panose molecule. Evi-
dence for this structure for the compound rests on (1) its migration velocity
on paper in comparison with that of similar compounds, (2) the mechanism
of synthesis of this series of compounds, and (3) the products of partial
acid hydrolysis of the compound. In an acid hydrolysate, glucose, maltose,
isomaltose, panose, and dextrantriose were identified.
DISCUSSION

We have observed two types of enzymes with carbohydrate-synthesizing

ability in the filtrates of A. oryxae. One is the transglucosidase which is
described in this paper; the other, to be reported at a later date, is a trans-

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fructosidase. At present it can be stated that in transfructosidase reactions
fructosyl units of sucrose are transferred to cosubstrate saccharides, result-
ing in the formation of tri- and tetrasaccharides composed of glucosyl and
fructosyl moieties.
The transglucosidase of A. oryxae synthesizes from maltose the disac-
charide isomaltose, the trisaccharides 4-cr-isomaltosyl-n-glucose (panose),
and 6-or-isomaltosyl-D-glucose (dextrantriose), and the tetrasaccharide 4-(Y-
dextrantriosyl-n-glucose. Initially, these reducing sugars were identified
by paper chromatography. Later, sufficient quantities of the compounds
were isolated for determination of physical and chemical properties. The
specific rotations determined for our compounds agree with the literature
values. On complete acid hydrolysis of the saccharides glucose was the
sole monosaccharide produced. On graded acid hydrolysis all the theoreti-
cally expected hydrolytic products were identified in the hydrolysates.
This technique proved especially useful for characterizing the saccharides
of higher molecular weight of which the supply was limited. Finally,
crystalline flavazole derivatives were prepared from the isomaltose and the
panose isolated from the digest. The x-ray diffraction patterns of the
flavazoles matched the patterns obtained for the flavazoles prepared from
isomaltose and panose of established purity.
In most of the studies reported on the synthesis of carbohydrates, it has
been found that such synthesis proceeds by a phosphorylation mechanism
in which phosphorylated sugars function as intermediates. The possibility
that transglucosidase action is a type of phosphorolysis was first considered.
In studies on the r61e of phosphorus in sucrose phosphorylase reactions,
Doudoroff et al. (10) point out the difficulties encountered in attempting
to remove phosphorus from enzyme preparations. Consequently, we did
not attempt to prepare phosphorus-free enzyme solutions but tested the
action of our enzyme on phosphorylated sugars which would be expected
as intermediates in the reactions. We also examined the digest aliquots
for phosphorylated intermediates at various stages of reaction. The en-
270 A. ORYZAE AND MALTOSE

zyme did not synthesize oligosaccharides from glucose-l-phosphate and

glucose or from glucose alone. Furthermore, there was no evidence of the
presence of phosphorylated intermediates in the maltose digest. The phos-
phorolytic route for the action of transglucosidase would seem to be elimi-
nated.
All of the oligosaccharides that are synthesized from the maltose by
transglucosidase contain 1,6 linkages. Apparently the enzyme is capable
of transferring glucose units linked by 1,4 linkages to the 6 position of
appropriate cosubstrates in the system. The net result is the conversion
of 1,4-linked glucosyl compounds into 1,6-glucosyl compounds. The re-
action can be conveniently represented as occurring in two steps. In the
first step the enzyme removes a glucosyl unit from the maltose and forms a

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glucosyl-enzyme intermediate. The energy state of the glucosyl-enzyme
complex is such that the free energy change for the reaction in the direction
of synthesis of 1,6 linkages is negative, while the free energy change for the
reaction in the direction of synthesis of 1,4 linkages is positive. Evidence
for this interpretation was obtained from experiments with radioactive
glucose and maltose. The C14-glucose in the reaction mixture was in-
corporated into the isomaltose and dextrantriose but not in the maltose
and panose.
In Step 2 of the reaction, the glucosyl portion of the intermediate complex
is transferred to a cosubstrate saccharide. In a maltose digest, glucose,
maltose, isomaltose, and panose function as cosubstrates. The complete
action on maltose is shown in the accompanying equations. Horizontal
lines represent 1,4-glucosidic bonds, vertical lines 1,6-glucosidic bonds,
and G--, the glucose unit with the free aldehyde group.
A similar reaction mechanism has been suggested by Hehre (11) for the
Step 1. G-G- + E + G-E +G-
Step 2. (a) G-E + G- -+ G +E

(b) G-E +G-G--G 4-E

I
A-G-
(c)G.E +G +G +E
(j-
4
G-
(c2)G.E +G +G +E
I I
G-G- G
I
G--G-
 J. H. PAZUR AND D. FRENCH 271

enzyme dextran-dextrinase which converts a-l ,4-glucose polymers (dex-

trin) into 1,6-glucose polymers (dextran). Conclusive evidence for the
transglucosidation mechanism for our enzyme was forthcoming from the
tracer experiments. In these experiments a trace amount of C14-glucose
(1 per cent of total carbohydrate of the system) was included in the reac-
tion mixture. According to Step 2 (a), this marked glucose should act as
a cosubstrate in the synthesis of radioisomaltose. The radioisomaltose, in
turn, can function as a cosubstrate, Step 2 (c), in the formation of radio-
dextran trisaccharide.
The rate of formation of the reducing sugars and the radio compounds
in the digest was determined by analyzing aliquots of the mixture at vary-
ing time intervals. First, the sugars in the digest aliquots were resolved

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on paper chromatograms; second, radioautographs of these were prepared;
and, finally, the locations of the reducing sugars on the paper were found by
spraying with appropriate reagents. Photographs showing the reducing
sugars and the radioactive sugars in the digest at different stages of enzy-
molysis are reproduced in Fig. 1. Comparison of the paper chromatogram
and the radioautograph reveals the following facts: Initially, the digest
contained glucose and maltose as reducing components, and glucose as the
only radioactive component. After a 6 hour reaction period, isomaltose
and panose were synthesized at the expense of the maltose, with a corre-
sponding liberation of glucose. Of the synthesized compounds only the
isomaltose was radioactive. The next period of analysis (12 hours) showed
the presence of all the synthesized compounds we have listed. The iso-
maltose and the panose were present in higher concentrations than the
dextran trisaccharide and the tetrasaccharide. The compounds that were
radioactive at this point were isomaltose and dextrantriose. The same
reducing and radioactive products were present in the digest at the next
two periods of analysis. The results of the tracer experiment are in accord
with the reaction mechanism presented in Steps 1 and 2.

SUMMARY

The transglucosidase of Aspergillus oryxae synthesizes from maltose

the disaccharide isomaltose, the trisaccharides 4-a-isomaltosyl-n-glucose
(panose), and 6-oc-isomaltosyl-D-glucose (dextrantriose), and the tetrasac-
charide 4ol-dextrantriosyl-n-glucose. The identities of these compounds
were established from a comparison of the mobilities on paper of the syn-
thesized and reference compounds, from optical rotation values, from
identification of products of partial acid hydrolysis, and by preparation of
crystalline flavazole derivatives. By using CJ4-glucose in the reaction mix-
ture, it has been shown that the enzyme action involves the transfer of
glucosyl units from 1,4-glucosidic compounds to the 6 position of cosub-
strate saccharides resulting in the synthesis of new 1,6-oligosaccharides.
272 A. ORYZAE AND MALTOSE

BIBLIOGRAPHY

1. Pan, S. C., Andreasen, A. A., and Kolachov, P., Science, 112, 115 (1950).
2. Pan, S. C., Nicholson, L. W., and Kolachov, P., J. Am. Chem. Sot., 73,2547 (1951).
3. French, D., Science, 113, 352 (1951).
4. Wolfrom, M. L., Thompson, A., and Galkowski, T. T., J. Am. Chem. Sot., 73,
4093 (1951).
5. Pazur, J. H., and French, D., J. Am. Chem. Sot., ‘73, 3536 (1951).
6. French, D., Knapp, D. W., and Pazur, J. H., J. Am. Chem. Sot., 72, 5150 (1950).
7. Aronoff, S., and Vernon, L., Arch. Biochem., 28,424 (1950).
8. Montgomery, E. M., Weakley, F. B., and Hilbert, G. B., J. Am. Chem. Sot., 71,
1682 (1949).
9. Freudenberg, K., Friedrich, K., and Bremann, I., Ann. Chem., 494, 41 (1932).
10. Doudoroff, M., Barker, H. A., and Hassid, W. Z., J. Biol. Chem., 168,725 (1947).

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11. Hehre, E. J., J. Biol. Chem., 192, 161 (1951).