Enzymatic Production of High Fructose Corn
Syrup (HFCS) Containing 55% Fructose in
Aqueous Ethanol
Kalevi Visuri and Alexander M . Klibanov
Department of Applied Biological Sciences, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02 139
Accepted for publication November 26, 1986
Production of high fructose corn syrup (HFCS) catalyzed
by immobilized glucose isomerase is by far the greatest
industrial success of enzyme technology to date. I The gist of
this process is the enzymatic isomerization of glucose (usu-
ally derived, also enzymatically, from corn starch) into a
mixture typically containing 42% fructose, 5 1% glucose,
and 7% oligosaccharides (remaining from incomplete
amylase-catalyzed hydrolysis of starch).*-' Although this
mixture is isosweet with sugar and suitable for some com-
mercial applications, it is insufficiently sweet for HFCS's
major use -in more acidic soft drinks (such as Coca-Cola
and Pepsi-Cola).' The latter market requires a fructose con-
tent of 55% of the total solids to give the same sweetness
level as sucrose (at the same concentration of solids).' Con-
sequently, glucose isomerase-catalyzed isomerization of
glucose syrup in industry is usually followed by chro-
matographic enrichment of the HFCS formed.'-'
Since the chromatographic step is a considerable expense,
it would be highly desirable to omit it. Conceptually, one
way to accomplish this is to conduct the isomerization reac-
tion not at the currently employed temperature of
60-65°C,3-5 but at 1oO-l10°C.6 Due to the fact that a tem-
perature increase favors fructose in its equilibrium with glu-
cose, HFCS containing 55% fructose can be enzymatically
formed directly above 100°C.' Unfortunately, glucose
isomerases are far too labile to work at such high tem-
peratures for even hours, let alone days and weeks required
for a commercial operation.6 Furthermore, recently eluci-
dated mechanisms of irreversible thermoinactivation of
enzymes',' strongly suggest that the upper limit of enzyme
thermostability is a half-life of several hours at loO°C, thus
making the foregoing stability levels unattainable.
In a search for an alternative approach, we turned to the
emerging area of enzyme catalysis in nonaqueous media. lo
It occurred to us that the equilibrium between glucose and
fructose might be shifted towards the latter not only at high
temperatures but also in aqueous-organic mixtures even at
the ambient temperature. For the reasons of safety for a food
application, cost and reactant solubility, ethanol was the
only choice for the organic solvent. In the present study, we
have discovered that glucose isomerase can directly produce
Biotechnology and Bioengineering, Vol. 30, Pp. 917-920 (1987)
0 1987 John Wiley & Sons, Inc.
HFCS containing 55% of fructose when the process is con-
ducted in 85-90% ethanol.
EXPERIMENTAL
Materials
Four different preparations of glucose isomerase
(D-xylose ketol isomerase, EC 5.3.1.5) were employed in
our work. Free glucose isomerase from mycelium of Strep-
tomyces rubiqinosus" was a generous gift of Finnish Sugar
Co. (Kantvik, Finland). The enzyme was purified by them
using lysozyme-assisted cell lysis, ammonium sulfate frac-
tionation, and subsequent recrystallization with ammonium
sulfate eight times. The resultant preparation (wet crystals
containing 41% protein, 6% ammonium sulfate, and 53%
water) was pure by SDS-polyacrylamidegel electrophoresis
and FPLC and had a specific activity of 0.32 international
units (IU) per mg of protein (fructose was used as a substrate
in this and subsequent determinations). The following three
immobilized glucose isomerases have been used: 1) The
first was the described above Streptomyces rubiqinosus en-
zyme adsorbed on DEAE-cellulose. The sample, also ob-
tained from Finnish Sugar and called Spezyme IGI, had an
activity of 11.8 IU/g dry wt. 2) The second was the enzyme
from Bacillus coagulans immobilized by covalent cross-
linking with gl~taraldehyde.~ This sample, obtained from
Novo Industri (Bagsvaerd, Denmark) and called Sweetzyme
S, had an activity of 2.9 IU/g dry wt. 3) Finally, the en-
zyme from Actinoplanes missouriensis was immobilized by
entrapment in a gelatin gel, followed by covalent cross-
'
linking with glutaraldehyde. This sample, obtained from
Gist-Brocades (Delft, The Netherlands) and called Max-
azyme GI, had an activity of 3.0 IU/g dry wt.
Crystalline a-D( +)-glucose and P-D(-)-fructose were
purchased from Sigma Chemical Co. (St. Louis, MO). High
fructose corn syrup, containing 71% (w/w) solids con-
sisting of 42% fructose, 52% glucose, and 6% oli-
gosaccharides, was a kind gift from Cargill Co. (Dayton,
OH); the sample (trade name Isoclear 42) was a viscous,
clear aqueous solution. The enzymes used for assays, yeast
CCC 0006-3592/87/070917- 04$04.00
hexokinase and phosphoglucose isomerase and Leuconostoc
mesenteroides glucose-6-phosphatedehydrogenase, as well
as NAD' and ATP, were purchased from Sigma. All other
chemicals and solvents used in this work were obtained
commercially and were of the highest purity availabIe.
Assays
Glucose isomerase (both free and immobilized) was as-
sayed as follows. An enzyme solution (1 mL) or 2 g
presoaked immobilized enzyme was added to 10 mL of
6% aqueous solution of fructose ( 0 . M Tris-HCl, pH 7.0,
containing 0.2mM CoC12) and shaken at 30°C. Periodi-
cally, aliquots were withdrawn and assayed for glucose as
outlined below.
Glucose was determined using the standard enzymatic
(hexokinase/glucose-6-phosphate dehydrogenase)method. l 3
Fructose (in the presence of glucose) was determined by the
phosphoglucose isomerase method. l4 When glucose isomer-
ization was catalyzed by free glucose isomerase, the latter
was irreversibly inactivated by 0.1M sulfuric acid prior to
the glucose/fructose assay to avoid interferences.
RESULTS AND DISCUSSION
The initial goal of this study was to ascertain whether the
equilibrium between glucose and fructose can be shifted
(hopefully in favor of fructose) when water is replaced with
aqueous-organicmixtures. To avoid working with emulsions
and due to the insolubility of the sugars in water-immiscible
solvents, we were limited to water-miscible organic sol-
vents. Since HFCS is a food ingredient, it was sensible to
select a FDA-approved solvent. These restrictions narrowed
the choice of the organic solvent down to just one -ethanol.
Since the isomerization of glucose to fructose is to be
enzymatically catalyzed, it was critical to establish whether
glucose isomerase can act as a catalyst in aqueous ethanol.
Table I depicts the dependence of the catalytic activity of
free Streptomyces rubiqinosus glucose isomerase on the
ethanol content of solution. One can see that in 80% (w/w)
ethanol the enzymatic activity is as high as 90% of that in
pure water and even in 90% (w/w) ethanol the activity is
almost one-quarter of that exhibited in a purely aqueous
solution. (At higher ethanol contents, the glucose isomerase
activity drops precipitously.) Hence, the enzyme can be
readily used to catalyze the isomerization reaction at least up
to 90% (w/w) of ethanol in the medium.
We then proceeded to determine whether the thermo-
dynamic equilibrium between glucose and fructose is indeed
different in aqueous ethanol from that in pure water. First,
the equilibrium was measured in the latter. To 100 mL of
aqueous buffer (50mM Tris-HC1, 1mM CoC12, 20mM
MgS04, pH 7.0), containing 3 g of either glucose or fruc-
tose, 1.33 g wet crystals of glucose isomerase was added,
and the solutions were stirred at 25°C. Periodically, the
reaction mixtures were assayed for glucose and fructose.
After three days, the reactant concentrations no longer
918 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 30, NOVEMBER 1987
Table I. Dependence of glucose isomerase activity on the concentration
of ethanol in the solvent."
Concentration of ethanol Enzymatic activity
1% (w/w)l (nmoI/s mg enzymeb)
0 2.80
40 2.65
80 2.51
90 0.68
92 0.44
94 0.10
96 0.044
98 0.044
a Glucose isomerase activity is expressed as the initial rate of enzymatic
isomerization of fructose to glucose in various mixtures of ethanol and
aqueous solution of sodium malate buffer (25mM, pH 7.0). The reaction
mixture (which contained 7pM CoC12 and 1 3 0 a MgSO,), consisting of
1.5% (w/w) fructose and 3.33 mg/mL Stregromyces rubiqinosus glucose
isomerase, was stirred at 25°C; periodically, aliquots were withdrawn and
analyzed for glucose as outlined in the Methods.
Wet enzyme crystals are as defined in the Materials section.
changed appreciably and were unaffected by addition of
more enzyme, thus indicating that the thermodynamic equi-
librium was approximately reached. At that point, the
concentration of fructose in the two solutions originally
containing only glucose or only fructose was 46.8% and
45.6%, respectively, which is in a reasonable agreement
with the literature data.7 When the same experiments were
conducted in 80% (w/w) ethanol (with the other 20% being
the aqueous buffer), then the equilibrium concentrations of
fructose obtained were 55.6 and 54.8%, respectively. That
is, replacement of water with 80% aqueous ethanol as the
reaction medium resulted in a 9% increase of the fructose
concentration in its equilibrium mixture with glucose.
The above results looked sufficiently promising to war-
rant a more detailed investigation. We decided to carry it out
with immobilized glucose isomerase, for this form of the
enzyme is currently used in i n d ~ s t r y . ~Initially,
-~ glucose
isomerase from Streptomyces rubiqinosus adsorbed on
DEAE-cellulose was employed to determine the position of
the thermodynamic equilibrium between glucose and fruc-
tose as a function of ethanol content. At each ethanol con-
centration, three independent experiments were run using
glucose, fructose or their equimolar mixture as substrates. In
all cases, a suspension of the immobilized enzyme in a
substrate solution was vigorously shaken at 30°C and the
concentrations of fructose and glucose monitored until they
nearly leveled off. The time courses of the enzymatic
isomerization in pure water and in 85% ethanol are depicted
in Figures 1(A) and 1(B), respectively. Similar patterns
were obtained at other ethanol concentrations, and the re-
sultant equilibrium fructose fractions are shown in Table 11.
It is seen that addition of ethanol to the reaction medium
substantially favors fructose in its equilibrium with glucose.
Also note that approximately the same equilibrium values
for fructose ensued regardless of whether the substrate was
glucose, fructose, or an equimolar mixture of the two

'"OF-
3 6
days days
Figure 1. Time courses of glucose isomerase-catalyzed isomerization of
(a) glucose, (b) fructose, or their (c) equimolar mixture in (A) water or (B,
C, and D) 85% ethanol: (A and B) Streptomyces rubiqinosus glucose
isomerase immobilized by adsorption on DEAE-cellulose; (C) Bacillus
coagulans glucose isomerase immobilized by crosslinking with glu-
taraldehyde; (D) Actinoplanes missouriensis glucose isomerase entrapped
in gelatin and crosslinked with glutaraldehyde. General conditions are 10%
(w/w) substrate, 3 0 T , and shaking at 250 rpm. Other conditions are
(A) 50mM Tris. HCl aqueous buffer (pH 8.2), 0.17 g/mL enzyme; (B, C,
and D) 85% aqueous ethanol (5OmM final Tris.HC1 concentration, pH
adjusted to 8.2 prior to mixing with ethanol), 0.25 g/mL enzyme.
(which is quite close to the ultimate equilibrium). This fur-
ther validates the reported results.
We then employed two other, unrelated immobilized glu-
cose isomerases (both commercial catalysts manufactured
by Novo and Gist-Brocades)-from Bacillus coagulans
crosslinked with glutaraldehyde and from Actinoplanes mis-
souriensis entrapped in gelatin and crosslinked with
glutaraldehyde- to equilibrate glucose and fructose in 85%
aqueous ethanol. As one can see in Figures 1(B), 1(C), and
1(D), the positions of the equilibrium obtained are virtually
identical for all three immobilized glucose isomerases and
much more favorable towards fructose than in water.
Operational stability is a practically important character-
istic of a bio~ata1yst.l~ With that in mind, we examined the
remaining activity of the three immobilized glucose isomer-
ases as a function of time of the catalyzed reaction [30"C,
shaking at 250 rpm, 10% (w/w) substrate; for other condi-
tions see Figures 1(B), 1(C), and l(D)]. After three days,
the residual activity was ca. 25, 35, and 57% of the initial
level for the FinnSugar, Gist-Brocades, and Novo enzymes,
respectively. It is difficult to directly compare these numbers
with their counterparts in water, because in 10% aqueous
solutions of sugars at 30°C microbial growth begins in just
a few hours (which presumably does not happen in 85%
ethanol) and leads to enzyme inactivation. However, it is
known from the that at 60-65°C (where the
microbial growth is essentially suppressed), the half-life of
immobilized glucose isomerase is of the order of a month.
Hence, it appears that addition of ethanol significantly labil-
izes the biocatalyst. The operational stability of the enzymes
in aqueous ethanol mixtures will have to be enhanced to
make them feasible as industrial catalysts.
The next step was to verify that our approach to enriching
glucoseifructose mixtures with the latter by adding ethanol
is directly applicable to commercial HFCS. To that end, we
obtained high fructose corn syrup from Cargill. The solu-
tion, which according to the manufacturer contained 42%
fructose, 52% glucose (according to our analysis 41.5 and
52.5%, respectively), and 6%oligosaccharides, was diluted
with ethanol to bring its ratio with water to 85: 15 (the
overall solution composition (w/w) was 10% dry substrates,
76.5% ethanol, and 13.5% H,O), and the pH was adjusted

Table 11. Equilibrium fractions of fructose in its mixtures with glucose in different aqueous ethanol
solutions. a

Equilibrium fraction of fructose enzymatically

Concentration of formed when the substrateb was
ethanol in the
solvent 100% glucose 100% fructose 50% glucose + 50% fructose
(%) (%I (%) (%)

0 45.1 46.0 45.1

50 48.5 49.3 49.0
80 53.7 54.5 53.9
85 55.9 56.7 56.0
90 57.8 59.4 58.2

A substrate was dissolved in a reaction medium consisting of varying proportions of ethanol and
aqueous Tris . HCI buffer (25mM, pH 7.0, containing ImM CoCI2 and 20mM MgS04). Then
immobilized glucose isomerase was added (0.2 g/mL), and the suspension was shaken at 250 rpm
and 30°C for 2 days which is sufficient to reach the practical equilibrium between glucose and
fructose.
The substrate concentration was 1) 12% (w/w) in pure water, in 50% ethanol, and in 80% ethanol;
2) 6% (w/w) in 85% ethanol; and 3) 3% (w/w) in 90% ethanol. Lower concentrations of the sugars
at higher ethanol contents are due to a decreasing solubility upon addition of the organic solvent.

COMMUNICATIONS TO THE EDITOR 919

to 8.2 (where the enzyme is more stable than at pH 7.0)
-
following addition of Tris HC1 buffer (the final concen-
tration in the aqueous ethanol mixture of 50mM). A
2.5 x 106-cm column was packed with presoaked immo-
bilized glucose isomerase (Finnish Sugar, 200 g dry wt).
Then the above-mentioned substrate solution in aqueous
ethanol was passed through the column (ascending direc-
tion, using a peristaltic pump) at 70 mL/h for 62 h. As a
result, 4.3 L of the product solution which contained 51.6%
fructose, 42.4% glucose, and 6% oligosaccharides was ac-
cumulated.
The foregoing data suggest the following straightforward
prototype for an industrial setup. First, HFCS containing
42% fructose is enzymatically produced the same way as at
present: in water at 60-65"C.3-s Then, instead of enriching
it by ion-exchange chromatography, the syrup is diluted
with ethanol (ideally, also produced from starch hydrolysate
by fermentation at the same site) and passed through a
smaller glucose isomerase column (or columns) to enhance
the fructose content to a desired level. Ethanol is then recov-
ered by distillation and reused. We have demonstrated that
this approach can be used for the production of the syrup
containing 55% fructose needed for acidic soft drinks (see
the Introduction). The effluent from the aforedescribed col-
umn was supplemented with more ethanol [to bring its ratio
with water to 9: 1 (w/w)] and then passed through another
column half the size of the first one. The effluent of the
second column contained 55.3% fructose, 38.7% glucose,
and 6% oligosaccharides. It is hoped that the novel approach
reported in this communication will be useful for the manu-
facturing of HFCS.
920 BIOTECHNOLOGY A N D BIOENGINEERING, VOL. 30, NOVEMBER 1987
This work was supported, in part, by Miles Laboratories. Kalevi
Visuri is a Finnish Sugar Company visiting research fellow.
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