Mathematical model for enzymatic

production of fructo-oligosaccharides
from sucrose
Kyung Hoon Jung, Jong Won Yun, Kyung Rae Kang, Jai Yun Lim
and Jae Heung Lee

R & D Centre, Cheil Sugar & Co., Ltd., Kyonggi-Do, South Korea

The production of fructo-oligosaccharides by the action of fructosyltransferase was investigated at

55C and pH 5.5. Enzyme kinetic studies with various substrates such as sucrose, 1-kestose, and
fructofuranosyl nystose revealed that the formation of fructo-oligosaccharides occurred from a
consecutive set of disproportionation reactions (viz. GF, + GF, ~ GF,_I + GF,+I). On the basis of
these experimental results, a mathematical model was proposed and computed. Although the data
points were scattered to some extent, good agreement was found between the model and experimental
results.

Keywords: Fructo-oligosaccharides; fructosyltransferase; disproportionation reactions; reaction mechanism; math-

ematical model

Introduction Materials and methods

Fructo-oligosaccharides, ~ in which one to three fruc-
tose units are bound to the beta-2,1 position of su-
crose, are mainly composed of 1-ketose (GFz), nystose
(GF3), and fructofuranosyl nystose (GF4). The fructo-
oligosaccharides which may be used in so-called
health food are produced commercially from sucrose
by the action of a fructosyltransferase system. This
enzyme has been found in fungi such as Aspergillus
sp.,2 Fusarium sp. ,3,4 and Aureobasidium sp. 5
In our previous work, 6 conditions for the produc-
tion of fructosyltransferase were studied. In the
present investigation, preliminary characterization of
this enzyme preparation was conducted. These in-
cluded optimum reaction conditions in terms of pH
and temperature, determination of kinetic parameters
such as Km and Vmax values, and enzyme kinetic
studies with various substrates. Based on these exper-
imental results, a possible reaction mechanism was
proposed and a mathematical model describing this
mechanism was compared with experimental data.
Address reprint requests to Dr. Lee at the R & D Centre, Cheil
Sugar & Co., Ltd., 522-1 Dokpyung-Ri, Majang-Myon, Ichon-Kun,
Kyonggi-Do, South Korea
Received 16 January 1988; revised 9 August 1988
Substrates
1-Kestose, nystose, and fructofuranosyl nystose were
purchased from Daichi Gakagu pharmaceutical com-
pany (Tokyo, Japan). Other chemicals used were
reagent grade.
Enzyme
The fructosyitransferase used in this study was pre-
pared in this laboratory by growing Aureobasidium
pullulans. The procedure for this enzyme preparation
was described previously. 6
Enzyme assay
Unless otherwise specified, fructosyltransferase ac-
tivity was determined by measuring the release of
glucose in the reaction mixture described below. One
fructosyltransferase unit is defined as the amount of
enzyme activity required to produce one micromole of
glucose per minute under the following conditionsS:
pH 5.5, temperature 55C, and reaction mixture con-
sisting of 7.5 ml of 80% (w/v) sucrose, 2.3 ml of 0.1 M
citrate buffer (pH 5.5), and 0.2 ml enzyme sample. The
enzyme reaction was stopped by heating at 100C for
10 min and the released glucose was measured.

@1989 Butterworth Publishers Enzyme Microb. Technol., 1989, vol. 11, August 491
Papers
Kinetic studies 100
Unless otherwise specified, enzyme reactions were
carried out for 1 h at 55C and pH 5.5 in test tubes
containing 10 ml of reaction mixture as described 80

above. When other substrates instead of sucrose were

used, only the sucrose was replaced. Throughout the I
m
course of this work, 5 units of enzyme per gram Ot 60
substrate were employed. The enzyme reaction was
m
stopped by heating and the reaction products were 0
analyzed.
40
ti
Analytical method I

Enzyme reaction products were analyzed by high-

20
pressure liquid chromatography (HPLC, Waters Asso-
ciates Model 244, equipped with a differential refrac-
tometer RI-401 detector), using the Ix Bondapak car-
bohydrate column (0.4 x 30 cm). A mixture of I I I I

acetonitrile/distilled water (75:25, v/v) was used as 0 2 4 6 8 10

the mobile phase at a flow rate of 1.5 ml min -]. PH

Results and discussion Figure 2 Effect of pH on fructosyltransferase activity: phos-

phate buffer (ll) citrate buffer (O)
Effect of temperature and pH
The influence of temperature on fructosyltransferase Determination of kinetic parameters
activity was investigated by measuring activity in the The effects of sucrose, l-kestose, or nystose concen-
temperature range of 40 to 70C. As shown in Figure tration on fructosyltransferase activity were investi-
I, the curve of enzyme activity is fairly symmetrical gated at 55C and pH 5.5. The Km and Vmax values for
and maximum activity was measured at 55C. each substrate were determined using the
The effect of pH on enzyme activity was tested by Lineweaver--Burk plot. As illustrated in Table 1,
measuring the amount of 1-kestose produced in buffers increases in the number of fructose units in the sub-
a t p H ranging from 3 to 8 because sucrose may be strate resulted in decreased Vmax and increased Km
hydrolyzed at lower pH values. The enzyme was values. These kinetic parameters obtained from exper-
active above pH 5 but the optimum pH was found to iments were used in later computer simulation studies.
be 5.5 (Figure 2). In order to investigate any types of enzyme inhibi-
tion that may occur in the enzyme system, glucose in
the range 20--80 g 1-] was added to the reaction
system with sucrose as a substrate. In Figure 3, the
reaction rate with and without glucose is shown. The
competitive type of inhibition by glucose was evident
and the value of the inhibition constant K~ was
10o determined to be 30 g 1-1. Similar experiments were
A
N also carried out to determine whether the enzyme
E
reaction might be inhibited by one of the other com-
~e ponents such as sucrose, l-kestose, nystose, and
fructofuranosyl nystose. As a result, it was concluded
m
that the effect of inhibition caused by the other com-
ponents was negligibly small.
50
Proposed mechanism for enzyme reaction
E In order to investigate the enzyme reaction mechanism
N
C involved in the production of fructo-oligosaccharides,
i
Table 1 Km and Vmaxvalues for various substrates
}/jr I I I l I
40 50 60 70 80 Sub.rate Vmax(gl l h 1) Km (g 1-1 )

Temperature (C) GF 130 330

GFF 30 750
Figure 1 Influence of temperature on fructosyltransferase GFFF 16 850
activity

492 Enzyme Microb. Technol., 1989, vol. 11, August

 Mathematical model for production of fructo-oligosaccharides: K. H. Jung et al.
nystose were produced from 1-kestose, while 1-
kestose and fructofuranosyl nystose were formed from
nystose. Therefore, one can generally express the
enzyme reaction as follows:
GFn + GF, ~ GF,-1 + GF,+I
If n is equal to 1, GF,-1 becomes equal to GF0 which
J=
m
4 0 indicates glucose.
In Figure 4 an enzyme reaction mechanism network
0
ql= is shown together with the molecular weights of sub-
X 3 strates and products. The sucrose as a starting sub-
,'1> strate undergoes a number of enzyme reaction steps to
yield 1-kestose, nystose, and fructofuranosyl nystose.
As shown in Figure 4, the enzyme reactions are
eventually accompanied by the liberation of glucose. It
1
is worthwhile to note that further enzyme reaction
with fructofuranosyl nystose as a substrate did not
occur in up to 50 h in a batch enzyme reaction system
4 ~ i t / I I I I I at 55C and pH 5.5, probably due to its small Wmax
-2 2 4 6 8 10 value and its large Km value.
1 X103 ( i g _ l )
Development o f mathematical model
Figure 3 Lineweaver-Burk plots of enzyme reaction rates According to the proposed mechanism as shown in
using sucrose as a substrate with and without glucose or Figure 4, 8 moles of sucrose disappear to form 4 moles
1-kestose. Without glucose (), 40 g 1-1 glucose supplemented of glucose and 4 moles of 1-kestose. The rate of
(B), 40 g 1-1 1-kestose supplemented (e)
sucrose disappearance may be written:
dS Vms" S
enzyme reactions with various substrates were carried
out as illustrated in Table 2. When glucose plus dt [Kms + S + (Kms/Kig)G]
fructose were used as substrates, no enzyme reaction 2 342 Vmk"K
occurred. When sucrose was employed as a substrate,
however, only both glucose and l-kestose were pro- + 4 50-----~ " (Kmk + K) (1)
duced. The molar ratio of glucose to 1-kestose was where S indicates sucrose, G indicates glucose, K
found to be about 1 : l, indicating that a disproportio- indicates 1-kestose, Vms indicates Vma, for sucrose,
nation reaction mechanism was involved, i.e. sucrose Vmk indicates Vmax for 1-kestose, Kms indicates the
acts as either a donor or an acceptor so that 1 mole of Michaelis constant for sucrose, Kink indicates the
glucose and 1 mole of 1-kestose are formed simulta- Michaelis constant for 1-kestose, and Kig indicates a
neously from 2 moles of sucrose: competitive inhibition constant for glucose.
GF+GF~G + GFF The rate of glucose production can be expressed:

Similar reaction patterns were found with other sub- dG 4 180 Vms" S
strates such as 1-kestose and nystose. Sucrose and d---t = 8 342 [Kms + S + (Kms/Kig)G] (2)

Table 2 Data for enzyme reaction studies with various substrates

Products a
Concentration Molar ratio
Substrate (g 1-1) GF._I (g 1-1) GFn+I (g 1-1) (GF._I/GFn.I)

G 250

F 250
300 10.3 29.2 0.98
GF 400 11.8 34.7 0.96
600 13.4 29.5 1.26
300 1.7 4.3 0.78
GFF 400 2.5 6.6 0.84
600 4.0 9.0 0.86
300 1.0 1.7 1.23
GFFF 400 1.9 2.8 1.10
600 3.5 6.1 0.94

Enzyme reactions were carried out for 20 min instead of 1 h at 55C and pH 5.5

Enzyme Microb. Technol., 1989, vol. 11, August 49:3

Papers
8 x 342
600
8GF4 I
500 A

4 x 504 4 G F2 400
300

GF 2o0

2 x 666 2 100

0 10 20 30 40 50 10 20 30 40 50
O F2
Tlmelh)
4 x 828 1 GF 4
Figure 5 Comparison of experimental data with the mathemat-
! ical model at 55C and pH 5.5. (A) 50% sucrose, (B) 65% sucrose.
'. Glucose (O), sucrose (Q), 1-kestose (A), nystose (IlL fructofu-
ranosyl nystose (~)

Figure 4 Network of the proposed reaction mechanism the production of fructo-oligosaccharides by the action
of fructosyltransferase occurs from a consecutive set
of disproportionation reactions. It should be men-
tioned that small amounts of fructose (below 1%) were
The rate of 1-kestose production is complicated: 4
also accumulated slowly as the enzyme reaction pro-
moles of 1-kestose are produced from 8 moles of
gressed. The production of fructose is most likely due
sucrose and 1 mole of 1-kestose is formed from 2
to the action of another enzyme such as invertase as a
moles of nystose. Simultaneously 2 moles of nystose
contaminant in the fructosyltransferase enzyme prepa-
and 2 moles of sucrose are also formed from 4 moles of
ration.
1-kestose.
dK 4 x 504 Vr~s" S
dt 8 X 342 [Kms + S + (Kms/Kig)G] Nomenclature
Vmk " K 504 Vmn " N F fructose concentration (g 1-~)
+ - - (3) G glucose concentration (g l -J)
(Kink+K) 2 x666 (Km.+N) K 1-kestose concentration (g 1-~)
where N indicates nystose, Vm~ indicates Vmax for Kig competitive inhibition constant for glucose
nystose, and Kmn indicates the Michaelis constant for (g 1-1)
nystose. Kink Michaelis constant for 1-kestose (g 1-l)
With respect to the production of nystose, 2 moles Krnn Michaelis constant for nystose (g 1-l)
of nystose are produced from 4 moles of 1-kestose and Krns Michaelis constant for sucrose (g 1-l)
the nystose formed is removed to form 1 mole of N nystose concentration (g 1-l)
fructofuranosyl nystose and 1 mole of 1-kestose. P fructofuranosyl nystose concentration (g l-l)
S sucrose concentration (g 1-1)
dN 2 x 666 Vmk " K Vmn " N t time (h)
- - = - - (4)
dt 4 x 504 (Kmk+ K) (Km. + N) Vmk maximum velocity for 1-kestose (g 1-l h l)
maximum velocity for nystose (g 1-l h -l)
Finally, the rate of fructofuranosyl nystose produc-
Vms maximum velocity for sucrose (g 1-1 h -1)
tion is given by:
dP 828 Vmn " N

d--~- = 66------~
2 x " (Kin, + N)
where P indicates fructofuranosyl nystose.
The simultaneous integration of the differential
equations describing the proposed mechanism was
carried out using a digital computer with a fixed step
size of 0.1 h. As illustrated by Figure 5, which shows
the computer curve and the experimental data, good
agreement was found between the model and the
experimental results, although the data points were
scattered to some extent. Therefore, it appears that
(5) References
1 Hidaka, H., Eida, T., Adachi, T. and Saitoh, Y. Nippon
Nogeikagaku Kaishi 1987, 61, 915-923
2 Pazur, J. H. J. Biol. Chem. 1952, 199, 217-225
3 Gupta, A. K. and Bhatia, I. S. Phytochemistry 1980, 19,
2557-2563
4 Gupta, A. K. and Bhatia, I. S. Phytochemistry 1982, 21,
1249-1253
5 Smith, J. A., Grove, D., Luenser, S. J. and Park, L. G. US Pat.
4 309 505 (1982)
6 Jung, K. H., Lira, J. Y., Yoo, S. J., Lee, J. H. and Yoo, M. Y.
Biotechnol. Lett. 1987, 9, 703-708

494 Enzyme Microb. Technol., 1989, vol. 11, August