JOURNAL
OF BIOSCIENCE
Vol. 87, No. 5, 61148.
AND BIOENGINEERING
1999
Enzymatic Synthesis of Fructose 1,6-Diphosphate with ATP
Regeneration in a Batch Reactor and a Semibatch Reactor
Using Purified Enzymes of Bacillus stearothermophilus
ARIEF WIDJAJA,’
MASAHIRO
HIROSHI
SHIROSHIMA,’ MASAHIRO YASUDA,’
NAKAJIMA,2 AND HARUO ISHIKAWA’*
HIROYASU
OGINO,’
Department of Chemical Engineering, Osaka Prefecture University, I-I Gakuen-cho, Sakai, Osaka 599-8531’ and
Basic Technology Department, Research and Development Center, Unitika Ltd., 23
Kozakura, Uji, Kyoto 611-0021,2 Japan
Received 7 December 1998/Accepted 26 January 1999
The enzymatic synthesis of fructose 1,Cdiphosphate (FDP), an important glycolytic intermediate whose
applications in the field of medicine have generated a great deal of interest, was performed in a batch reactor
and a semibatch reactor. Using the batch reactor, FDP was first synthesized from glucose by three enzymatic
reactions and the ATP consumed was regenerated simultaneously using conjugated enzymes, all of which were
purified from crude cell extract of thermophilic Bacillus sfearothermophilus. The results of the experiments
performed using several enzyme concentrations suggest the existence of an optimum concentration for each
enzyme at which the maximum FDP yield can be attained. Since the thermal decomposition of acetyl phosphate
reduced the yield of FDP in the batch reactor, the use of a semibatch reactor ln which acetyl phosphate was fed
continuously was examined. The yield of FDP was improved but the time required to complete the reaction was
longer, resulting in a lower productivity of FDP. The yields observed in the two reactors using various enzyme
and substrate concentrations were in good agreement with the theoretical predictions calculated based on
differential equations derived for the system using the rate equations and the kinetic parameters determined
previously. This means that these equations can be used for the analysis of the experimental results as well as
for determining the optimum experimental conditions.
[Key words: acetyl phosphate, glucose, glucose 6-phosphate, fructose 6-phosphate, FDP, yield, productivity]
Fructose 1,6-diphosphate (FDP), which is an important intermediate in the glycolytic pathway, has attracted the attention of many researchers due to its important applications in the field of medicine (1, 2). There
are several methods used for the production of FDP.
Enzymatic phosphorylation
of glucose with inorganic
phosphate using Saccharomyces carlsbergensis cells is one
of the methods commonly used for producing FDP (3).
Compagno et al. (4) studied the production of FDP
by bioconversion of molasses, a sucrose-rich substrate,
using S. cerevisiae cells. However, these methods have
several disadvantages. For example, in the former
method, cells are treated with organic solvents such as
toluene and diethyl ether to permeabilize the cell membrane for glucose, phosphate and FDP. This treatment
causes unwanted leakage of enzymes from the cells due
to the increased membrane permeability (5). This makes
the permeabilized cells rapidly lose their phosphorylating
ability. In the latter fermentation method, obtaining a
high level of conversion is difficult because the physiological conditions of the culture also affect the process
efficiency (6). Furthermore, isolation of the product
from the medium requires laborious and tedious use of
separation columns.
Nakajima et al. (7) proposed an enzymatic method of
FDP synthesis from glucose. The process consists of
three synthetic reactions catalyzed by glucokinase (GK),
phosphoglucose isomerase (PGI) and phosphofructokinase (PFK), and the ATP regeneration reaction catalyzed by acetate kinase (AK). The reactions are expressed as follows:
* Corresponding
Glucose+ATP
2
Glucose 6-phosphate (G6P)
+ADP
PC1
G6P ti
(1)
Fructose 6-phosphate (F6P)
F6P + ATP E% FDP + ADP
ADP + Acetyl phosphate 5
(2)
(3)
ATP + Acetic acid (4)
Using enzymes of B. stearothermophilus, they succeeded
in synthesizing FDP. However, they did not perform a
theoretical analysis of the process.
We believe that the enzymatic method of FDP synthesis from glucose is one of the most promising methods.
Therefore, in the present study, the enzymatic synthesis
of FDP from glucose and the enzymatic ATP regeneration reaction were performed simultaneously in a batch
reactor and a semibatch reactor at pH 8.0 and 30°C
using various enzyme and substrate concentrations.
Purified enzymes of B. stearothermophilus were used in
the present study. The experimental results of the yield
of the product FDP and the space-time yield were compared with the theoretical predictions calculated based
on the rate equations determined previously (8-10 and
Shiroshima, M. E. thesis, Osaka Prefecture University,
Osaka, 1994).
MATERIALS
AND METHODS
Materials
Purified enzymes of B. stearothermophilus, AK, GK, PGI and PFK, were produced by Unitika
Ltd. (Osaka). Aldolase was purchased from United
author.
611
612
WIDJAJA
ET AL.
J. BIOSCI. BIOENG.,
States Biochemical Co. (Cleveland, Ohio, USA). aGlycerophosphate dehydrogenase (a-GDH), NADH and
disodium salt of ATP were obtained from Oriental
Yeast Co. Ltd. (Osaka). Lithium potassium salt of acetyl
phosphate with enzymatic assay of approximately 90%
and triosephosphate isomerase (TPI) were purchased
from Sigma (St. Louis, MO, USA). Glucose, MgC12,
triethanolamine (TEA), and other chemicals were purchased from Wako Pure Chemical Ind. Ltd. (Osaka).
Determination of the FDP concentration
The FDP
concentration was determined by an enzymatic assay
using aldolase, TPI and a-GDH. Every one mole of FDP
in the sample was enzymatically converted to two moles
of NAD+ as shown below:
FDP aldolc D-Glyceraldehyde 3-phosphate (GAP)
+ Dihydroxyacetone phosphate (DAP)
(5)
GAP JpA DAP
(6)
DAP+NADH+H+
3
a-Glycerol-3-phosphate + NAD+
(7)
Therefore, the concentration of FDP in the reaction
mixture was monitored indirectly by measuring the
change in absorbance at 340nm due to the decrease in
NADH concentration (~~~=6220M-‘.cm-l)
using a
Shimadzu UV-2100 spectrophotometer (Shimadzu Co.
Ltd., Kyoto). The assays were carried out at 30°C in
a total volume of 3.0 ml of 0.1 M TEA buffer (pH 8.0)
containing 0.2 mM NADH, 0.21 kU.l-I aldolase, 8.84
kU.I-’ TPI and 0.56 kU.l-’ a-GDH.
Experiments in a batch reactor
Almost all the experiments were performed in a batch reactor consisting
of a lOO-ml-volume vial equipped with a jacket in which
temperature-regulated water was circulated to maintain
the temperature at 30°C. A Teflon-coated magnetic stirrer bar was placed inside the reactor to stir the reaction
solution. The procedure of the experiments using the
batch reactor was as follows: 27-28.5 ml of the buffer
solution containing the starting raw material glucose, the
substrate acetyl phosphate for the regeneration of ATP,
and the purified enzymes except GK were placed in the
reactor. The mixture was stirred using the Teflon-coated
magnetic stirrer bar until the liquid temperature attained
30°C. A Fluke 52K/J thermometer (John Fluke MFG.
Co. Inc., Everett, Wash., USA) was used to measure the
temperature of the reaction solution. Then, 1.5-3 ml
of the buffer solution containing 3.31 X lo-‘M GK was
added to the reactor to start the reaction. At appropriate
time intervals, an aliquot of 0.1 ml was removed from
the reactor and was added promptly to 0.9ml 0.54%
H3P04 solution (pH 1.6) to stop the reaction. The concentrations of FDP produced were then analyzed by the
method described above and the time courses of FDP
concentration were determined.
At the beginning of the experiments, the total liquid
volume was 30ml and the reaction mixture consisted
of 0.1 M TEA buffer (pH 8.0), 10 mM glucose, 20 mM
acetyl phosphate, 0.01 mM ATP, 1 mM MgClz, (1.663.31) x lo-* M GK, (2.63-10.5) x 1O-9 M PGI, (2.9311.7)x10-*M
PFK and (1.44-5.77)x10-*M
AK. The
molar concentrations of the enzymes GK, PGI, PFK and
AK were calculated based on the molecular masses of
67 kDa (ll), 189 kDa (9), 130 kDa (12), and 170 kDa
(13), respectively.
Experiments in a semibatch reactor
Two series of
experiments were performed in a lOO-ml-volume semibatch reactor equipped with a jacket. The reaction mixture was also stirred using a Teflon-coated magnetic stirrer bar. The semibatch reactor was equipped with a feed
line for acetyl phosphate. A peristaltic pump was used
to feed acetyl phosphate solution from a reservoir which
was maintained in an iced-water bath. The first series of
experiments was conducted to study the effect of acetyl
phosphate concentration in the feed solution on productivity. The second was conducted to study the feasibility
of the process using high-substrate concentrations. The
procedure in the first series of experiments was as follows: 27 ml of 0.1 M TEA buffer (pH 8.0) containing the
substrate glucose and all the enzymes except GK was
first placed in the reactor. The mixture was stirred until
the temperature reached 3O”C, and then 3 ml of the
buffer solution containing 3.31 x 1O-7 M GK was added
and the feeding of 0.1 M TEA buffer (pH 8.0) containing
15, 30 or 60mM acetyl phosphate into the reactor was
initiated. Acetyl phosphate was fed continuously at a
constant liquid flow rate of 13.5 ml-h-l until the predetermined amount of acetyl phosphate was fed, which was
equivalent to twice the initial molar amount of glucose.
This means that this semibatch process used the same
total molar amount of acetyl phosphate as that in the
batch reactor. At appropriate time intervals, 0.1 ml
aliquots of the reaction mixture were removed from
the reactor and were promptly added to 0.9 ml of H3P04
solution (pH 1.6) to stop the reaction, and the concentrations of FDP produced were analyzed.
The reactions were carried out at 30°C in an initial
total volume of 30 ml of 0.1 M TEA buffer (pH 8.0) containing 10 mM glucose, 0.01 mM ATP, 1 mM MgC&,
3.31 x lo-* M GK, 1.05 x lo-* M PGI, 5.85 x lo-* M
PFK and 2.88 x lo-* M AK.
In order to investigate the feasibility of the process
using high substrate concentrations, experiments were
performed using lo-200 mM glucose. The experimental
procedure was the same as that described above, except
that the initial glucose concentrations were 10-200 mM
and that the 200 mM acetyl phosphate was fed continuously into the reactor. The ratio of the molar amounts
of glucose, acetyl phosphate and ATP was always constant at 1 : 2 : 0.001. The concentration of FDP produced
was determined by the method described above. In these
experiments, the pH of the solution was maintained at
around 8.0 by manual addition of 1 N or 6 N NaOH
solution.
THEORY
In the enzymatic method of FDP synthesis from glucose, the adenosine phosphates ATP and ADP are
present mainly in the forms of the complexes MgATP2and MgADP-,
respectively, in the presence of Mg*+
ions in the reaction mixture. Since these complexes are
ligands of the enzymes GK and AK, the concentrations
of the adenosine phosphates should be based on the complex ions. However, in the present study, the concentrations of ATP and ADP were used instead of the concentrations of the complexes MgATP*- and MgADP- for
simplicity. Therefore, the kinetic parameters of the rate
equations of the reactions catalyzed by GK and AK were
redetermined on the basis of the ATP and ADP concentrations (see Appendix).
VOL.
SYNTHESIS
87, 1999
OF FDP IN BATCH REACTOR
In the present work, a batch reactor and a semibatch
reactor were used to synthesize FDP. When the decomposition of acetyl phosphate is taken into account, the
basic differential equations for the changes in concentration with time of all the components involved in the
FDP synthesis in a semibatch reactor are given by:
A!%=
- v,,---e--c,
dt
Vo+Qt
@I
==
dt
Vo’GK-VpcI-
(9)
==
VpGI- VPFK--cj
dt
(10)
Vo+Qt
(11)
dc’_=-,K-dt
v
%=
vm--cb+kDc5
-==
dt
- v,,-
dt
+Qt
(CS
-
-
cS,f)
kDc5
0
(13)
Vo+Qt
vp,+
(12)
r&--C7
Vo+Qt
(14)
AND SEMIBATCH
REACTOR
613
basic differential equations, Eqs. 8-15, the rate equations, Eqs. Al-A4, and Eq. 16 are solved numerically
under the initial conditions of Eq. 17, the theoretical
concentrations of all the components involved in both a
batch reactor and a semibatch reactor can be calculated.
Based on the differential equations described above,
the theoretical time courses of the concentrations of
glucose, G6P, F6P, FDP and acetyl phosphate in a batch
reactor were calculated. Figure 1 shows the results of the
calculation using 10 mM glucose, 2OmM acetyl phosphate, 0.01 mM ATP, 1 mM MgC12, 3.31 x lo-*M GK,
1.05 x 1O-8 M PGI, 5.85 x 1O-8 M PFK and 2.88 x 1OV
M AK. Using these concentrations, the increase in the
concentration of the final product, FDP, is almost inversely proportional to the decrease in the concentrations
of the substrates, glucose and acetyl phosphate, and
the concentrations of the intermediates, G6P and F6P,
are maintained at low values. Glucose and acetyl phosphate are almost completely consumed at around t=87
min. However, since a certain amount of G6P remains,
the concentration of FDP produced is less than that of
the initial glucose concentration. This was attributed to
the thermal decomposition of acetyl phosphate. The
figure also shows that the G6P concentration decreases
slightly at around the time when almost all of the acetyl
phosphate is consumed. This is due to the isomerization
reaction catalyzed by PGI (G6P+F6P) because this reaction proceeds without acetyl phosphate.
(1%
where, C,, C,, C3, C4, C,, C,, C, and Cs represent the
concentrations of glucose, G6P, F6P, FDP, acetyl phosphate, acetic acid, ATP and ADP, respectively. kD is the
first-order rate constant of the thermal decomposition
of acetyl phosphate. VGK, VpG1, VP, and Vm express the
rates of the reactions catalyzed by the enzymes GK,
PGI, PFK and AK, respectively, and are given by Eqs.
Al-A4 in the Appendix. Their kinetic parameters are
listed in Tables Al-A4. Vo, Q and Cs,r express the initial
volume of the reaction mixture, the volumetric feeding
rate of acetyl phosphate and the concentration of acetyl
phosphate in the feed solution, respectively.
The half-lives of the enzymes GK, PGI, PFK and AK
were 41 d (8), 164 d (9), 7.7 d (Shiroshima, M., M. E.
thesis, Osaka Prefecture University, Osaka, 1994) and
20d (Takase, S., M. E. thesis, Osaka Prefecture University, Osaka, 1987), respectively. As the longest experimental time required in the present work was 12 h, a
maximum of 3.2% enzyme inactivation is predicted to
occur. This means that the effect of enzyme inactivation
on the present experiments can be neglected. Therefore,
the change in the concentrations of the enzymes with
time is expressed by the following equation:
V&%
[Ed01 = ~V, +
Qt
(i=GK,
PGI, PFK and AK)
RESULTS
the batch reactor
The
effects of the concentrations of the four enzymes required for FDP synthesis in the batch reactor are shown
in Figs. 2a, d. The concentrations of glucose and acetyl
phosphate are 10 mM and 20mM, respectively, in all
these figures and the final yields are always less than the
maximum attainable yield (100%) due to the thermal
decomposition of acetyl phosphate. These results are in
agreement with the theoretical prediction shown in Fig.
1. At higher enzyme concentrations, the rates of FDP
synthesis were higher, and less acetyl phosphate was
decomposed. This resulted in higher final yields.
The lines in Fig. 2 represent the theoretical lines calculated based on the differential equations (Q=O) and the
rate equations described above. The agreement between
the theoretical lines and the experimental results is satisfactory, indicating that the rate equations of the reactions catalyzed by the enzymes, the kinetic parameters
Experimental
results in
20
(16)
The concentrations of the enzymes [EJO (i=GK, PGI,
PFK and AK) in the rate equations given by Eqs. Al-A4
must be replaced by [Ei (t)] given by Eq. 16. The initial
conditions for Eqs. 8-15 are given by:
t=o;
c,=c,,o,
c,=o,
c3=0,
c,=o,
(17)
When Q in Eqs. 8-16 is taken to be zero, the system is
reduced to the same as that in a batch reactor. When the
c5=c5,0,
c,=o,
c,=c7,0,
0
20
40
60
Time (mio)
80
100
c*=o
FIG. 1. Theoretical time courses of the concentrations of glucose,
G6P, F6P, FDP and acetyl phosphate in a batch reactor. The calculation was based on the differential equations described in Theory.
614
WIDJAJA ET AL.
J. BIOSCI.
and the rate constant of the
acetyl phosphate determined
thermore, the effect of FDP
neglected, although Nakajima
AK was activated by FDP.
z
0.8
i
0.6
z
0.4
c
-
0.2
3
0.8
%
0.6
5
0.4
6
0.2
so
0
The
z
100
150
100
150
(mln)
0.8
,o
0.6
.I
g
k
&I
0.4
0.2
0
0
so
Time (d)
FIG. 2. Effect of the concentration of the four enzymes required
for FDP production on FDP yield in a batch reactor. (a) The concentrations of PGI, PFK and AK were 1.05 x lo-* M, 5.85 X lo-* M
and 2.88 x lo-* M, respectively. The GK concentration was 1.66 x
lo-sM (o), 2.48x IO-sM (A) and 3.31 x lo-sM (0). (b) The concentrations of GK, PFK and AK were 3.31 x 10-s M, 5.85 x 10-s M and
2.88 x 10-s M, respectively. The PGI concentration was 2.63 x 1OV M
(o), 5.25 x 10-9 M(A) and 1.05 x lo-*M (0). (c)The concentrations
of GK, PGI and AK were 3.31 x 10m8M, 1.05 x 10m8M and 2.88 x
10-s M, respectively. The PFK concentrationwas2.93 x 1OVM ( q ),
5.85 x lo-*M
(0)
2.88 x lo-*M
(0)
and 1.17x 10m7M (A).
(d) The concentrations
and 5.77x 10-8M
The lines represent the
of GK, PGI and PFK were 3.31 x 10e8M, 1.05X lo-*M and 5.85 X
10-s M, respectively. The AK concentration was 1.44 x 1O-8 M ( 0 ),
theoretical predictions.
(A).
BIOENG.,
thermal decomposition of
previously are valid. Furactivation on AK can be
et al. (14) observed that
Experimental results in the semibatch reactor
The
reactor performance (the space-time yield) of a batch
reactor is generally affected by the decomposition of substrates and/or products. In FDP synthesis using a batch
reactor, the thermal decomposition of acetyl phosphate,
one of the substrates, was observed. Therefore, a semibatch operation with continuous feeding of acetyl phosphate was tested to improve the performance.
Figure 3a shows the experimental results of the FDP
synthesis performed in the semibatch reactor as a plot of
the yield of FDP obtained against the reaction time with
the acetyl phosphate concentration in the feed solution
as a parameter. The solid lines show the theoretical
predictions calculated based on the above differential
equations for the semibatch reactor. For comparison,
the experimental data and the theoretical prediction
(broken line) for the batch reactor are also shown for
the case when the reaction mixture contained 1OmM
glucose and 20mM acetyl phosphate at the start of the
reaction using the same enzyme concentrations as those
used in this semibatch experiment. In the experiments
using the semibatch reactor, acetyl phosphate was fed at
a liquid flow rate of 13.5 ml.h-l and the feeding was
stopped when the total molar amount of acetyl phosphate fed into the reactor was equal to 600 pmol, which
was the same amount of acetyl phosphate used in the
batch reactor. As shown in the figure, the higher the concentration of acetyl phosphate in the feed, the higher
was the production rate of FDP. However, a lower
acetyl phosphate concentration resulted in a higher yield
of FDP. This is because of the efficient consumption of
acetyl phosphate at lower feed concentrations by the
ATP regeneration reaction so that the effect of thermal
decomposition was reduced. For example, when 60mM
acetyl phosphate was fed, about 94% of the acetyl phosphate was utilized to synthesize FDP. On the one hand,
when 15 mM acetyl phosphate was fed, almost all
(98.7%) was utilized. These results show that the yield
of FDP in the semibatch reactor is higher than that in
the batch reactor since the effect of thermal decomposition of acetyl phosphate was reduced by the gradual
feeding of acetyl phosphate.
Figure 3a also shows that it required a longer period
of time to attain the maximal yield and for the reaction
to reach completion, although feeding acetyl phosphate
at lower concentrations resulted in higher yields of FDP.
Therefore, it is necessary that productivity or the spacetime yield, which is defined by the amount of FDP
produced per reactor volume per unit time, should also
be considered in the evaluation of a reactor. In Fig. 3b,
the productivity
calculated from Fig. 3a is plotted
against the reaction time, and is compared with the theoretical predictions. All the processes shown in Fig. 3
were based on the total amount of 600 pmol acetyl phosphate in the reactor, and the productivity was based
on a 100-ml-volume reactor. It is clear from this figure
that reduced acetyl phosphate concentration resulted in
lower productivity. The maximal productivity in the
batch reactor was 1.77 mol.rnT3. h-l, which was higher
than that in the semibatch process. The highest productivity in the semibatch process was 1.63 mo10m-3. h-’
SYNTHESIS
VOL. 87, 1999
0
50
loo
150
200
I
*
I
loo
150
Time (min)
I
200
OF FDP IN BATCH REACTOR
0
I
50
,
,
FIG. 3. Effect of the acetyl phosphate concentration in the feed
solution on (a) the amount of FDP produced and (b) the productivity
in a semi batch reactor. (a) Acetyl phosphate concentration in feed
solution was 15mM (o), 30mM (A) and 6OmM (0). Solid lines
represent the theoretical predictions calculated based on Eqs. 8-15.
The experimental data (0) and the theoretical predictions (----) in the
batch reactor using 20mM acetyl phosphate and the same enzyme
concentrations as those used in this semibatch process are also shown.
(b) The productivity was calculated from the data in Fig. 3a on the
basis of a 100-ml-volume reactor.
obtained using an acetyl phosphate concentration of
60 mM.
Figure 4 shows the experimental results of the FDP
synthesis performed using various glucose concentrations
in a semibatch reactor. In these experiments, 200 mM
acetyl phosphate was fed into the reactor at a constant
feed rate of 13.5 ml.h-l and the enzyme concentrations
were the same as those used to obtain the results in Fig.
600o I
0
0
200
400
Time
REACTOR
615
3. In a preliminary experiment on FDP synthesis using a
high glucose concentration in the semibatch reactor, a
decrease in the pH value from 8 was encountered resulting in a very low yield of FDP. The pH value could be
maintained at around 8 by the manual addition of 1 N
or 6 N NaOH to the reaction mixture, and then the reactions proceeded as expected. As shown in Fig. 4, higher
initial glucose concentrations resulted in the production
of higher molar amounts of FDP. However, the experimental yield was lower when the initial glucose concentration
was higher (when the initial glucose concentrations were 10 mM, 20 mM, 40 mM, 60 mM, 100 mM and
200 mM, the yields of FDP were 0.92, 0.91, 0.89, 0.88,
0.85 and 0.78, respectively). This is because when the
glucose concentration was high, the time required for
the reaction to reach completion was longer so that the
amount of acetyl phosphate decomposed was greater.
The highest productivity
of 4.18 mol.m-3. h-l was
obtained using the highest glucose concentration of
200mM. The experimental yields of FDP were in good
agreement with the theoretical predictions as indicated
by the solid lines in the figure.
Time (min)
0
AND SEMIBATCH
600
(min)
FIG. 4. FDP synthesis in a semi batch reactor using various
glucose concentrations. The enzyme concentrations were 3.31 x lo-* M
GK, 1.05 x lo-* M PGI, 5.85 x lo-* M PFK and 2.88 x lo-* M AK.
Glucose concentration was 10 mM (0), 20mM (A), 40mM ( q ),
60 mM (V), 100 mM (0) and 200 mM (0). The molar ratio of glucose,
acetyl phosphate and ATP was always constant at 1 : 2 : 0.001.
DISCUSSION
The synthesis of FDP and simultaneous ATP regeneration using the purified enzymes of B. stearothermophilus
were performed in a batch reactor and a semibatch reactor. The experimental results of the FDP synthesis in the
batch reactor using various enzyme concentrations
showed that the yield of FDP was higher at higher enzyme concentrations. This was because when the enzyme
concentration was high, the rate of FDP synthesis was
also high, resulting in a reduction in the amount of
decomposition of acetyl phosphate. The decomposition
of acetyl phosphate was the reason why the yield of
FDP in the batch reactor was always below 100%.
The experimental results showed that the batch reactor
has the disadvantage of thermal decomposition of acetyl
phosphate and that the yield was reduced by more than
10% compared to the case without thermal decomposition. To overcome this problem, a semibatch process
was tested to reduce thermal decomposition by continuously feeding acetyl phosphate. As shown in Fig. 3a, the
process was improved resulting in almost 100% conversion of glucose to FDP and utilization of almost all
acetyl phosphate.
The FDP synthesis in the semibatch reactor was also
performed using various glucose concentrations. When
the initial glucose concentration was higher, a higher
molar amount of FDP was obtained, but the yield of
FDP was lower. This was because the amount of thermally decomposed acetyl phosphate increased due to the
longer reaction time at higher initial glucose concentrations. However, because the solubility of acetyl phosphate is limited (approximately 200 mM), it is impossible
to synthesize FDP in a batch reactor using glucose at
concentrations higher than 100 mM. Therefore, the semibatch reactor is superior to the batch reactor from the
viewpoint of the industrial production of FDP.
Irrespective of the type of operation, that is, batch or
semibatch, the experimental results of the yield of FDP,
the productivity or the total amount of FDP produced
were in good agreement with the theoretical predictions.
This indicates the validity of the rate equations of the
enzyme reactions as well as that of the kinetic parameters
616
WIDJAJA
ET AL.
and the rate constant of the thermal decomposition of
acetyl phosphate determined previously. Nakajima et al.
(14) studied the allosteric nature of B. stearothermophilus AK, and reported that FDP activated the reaction
catalyzed by AK. However, the good agreement between
the theoretical predictions and the experimental results
of the FDP synthesis demonstrated in the present study
indicates that the activation of the reaction catalyzed by
AK can be neglected under the present experimental conditions.
When we attempted to determine the optimum conditions for FDP synthesis in a batch reactor, both the
yield and the time required for the reaction to reach completion should be taken into consideration. Unfortunately, these two factors could not be optimized at the same
time, that is, a high yield of FDP was obtained with a
longer reaction time which finally resulted in lower
productivity. Therefore, a compromise between high
yield and high productivity should be made depending
on the criteria or the policy of the company who industrializes the process.
According to recent findings concerning the important
role of FDP as an effective agent for recovering some
physiological defects or metabolic disorders (2), the use
of high-quality FDP as an intravenous therapeutic agent
is promising. From this viewpoint, the requirement for
an efficient method to produce FDP with high purity
makes the enzymatic method of FDP synthesis more
promising than the traditional fermentation methods (3,
4).
Based on the satisfactory results obtained in the
present work, we further investigated the enzymatic
process of FDP synthesis using crude cell extract of B.
stearothermophilus. The results will be presented in our
following paper (15).
NOMENCLATURE
AK
: concentration of acetate kinase
: concentration of component j, M
cj
E
: enzyme
: initial concentration of enzyme, M
PI0
FDP
: fructose 1,6-diphosphate
GK
: glucokinase
Glucose: glucose
: first-order rate constant of decomposition of
kd
acetyl phosphate, s-l
Kp
: intrinsic equilibrium constant of the steps in
which the product binds or dissociates, M-l
: intrinsic equilibrium constant of the steps in
KS
which the substrate binds or dissociates, M-’
: intrinsic rate constant of the forward reaction
kr
of the slow steps, s-l
: intrinsic rate constant of the reverse reaction of
k
the slow steps, s-l
PFK
: phosphofructokinase
PGI
: phosphoglucose isomerase
: volumetric feeding rate of acetyl phosphate,
Q
ml.s-l or ml.h-1
t
: time, s or min
: rate of reaction catalyzed by acetate kinase,
vAK
Mess’
: rate of reaction catalyzed by glucokinase, M. s1
VGK
: rate of reaction catalyzed by phosphoglucose
VPGI
isomerase, M. s-l
: rate of reaction catalyzed by phosphofructoVPFK
J.
BIOSCI.
BIOENG.,
kinase, M. s-l
: initial volume of reaction mixture, ml
vo
Y
: yield of FDP, = [FDP]/[Glucoselo, <Subscript >
0
: value under initial condition
f
: value in the feed solution
1
* glucose
2
I G6P
3
. F6P
4
; FDP
5
: acetyl phosphate
6
* acetic acid
; ATP
ii
: ADP
<Greek letters >
: kinetic parameter in Eqs. Al, A3 and A4
;;
: kinetic parameter in Eqs. Al, A3 and A4
APPENDIX
Kate equations of the reactions catalyzed by B.
steurothermophiltrs GK, PGI, PFK and AK
Glucokinase
Ishikawa et al. (8) measured the initial rates of the phosphorylation of glucose catalyzed by
B. stearothermophilus GK over a wide range of substrate
concentrations at pH 7.5 and 37°C. They found that the
reaction mechanism was essentially the ordered Bi Bi
mechanism of the monomeric enzyme in which glucose
adds to the enzyme before MgATP2- and G6P is released
from the enzyme after the dissociation of MgADP-.
They derived the following rate equation:
-- VGK
_
[EGKIO
--
(AlI
where VGKis the reaction rate [M. S-I], [EG& the initial
GK concentration [Ml, Cr the glucose concentration
[Ml, C7 the MgATP2- concentration [M] and Cs the
MgADP- concentration [Ml. (rr and pj (j = 1-6) are the
kinetic parameters consisting of the rate and equilibrium
constants and have positive values.
Since the conditions used in this study for FDP synthesis
were slightly different from those used by Ishikawa et al.
(8), the initial reaction rates were measured (Shiroshima,
M., M. E. thesis, Osaka Prefecture University, Osaka,
1994) under the same conditions (pH 8.0 and 3O’C) that
were used for the FDP synthesis. All the experimental
reaction rates were fitted to Eq. Al by a nonlinear regression procedure and the kinetic parameters (Y~and /9j (j=
l-6) were determined. The correlation was carried out on
the basis of the concentrations of ATP and ADP, not
on the basis of those of the complexes MgATP2- and
MgADP-, respectively. The kinetic parameters thus determined are listed in Table Al.
Phosphoglucose isomerase
Widjaja et al. (9) clarified the kinetics and mechanism of the reaction catalyzed
by B. stearothermophilus PGI and proposed the followTABLE
a,=2.77x
/9,=2.23x
Al.
10’“M-2.
101 M-’
,82=5.&tX lo3 M-’
/3,=6.15x
IO’ M-2
Kinetic
’
‘-
parameters
,9,=2.65x
,&=2.68x
,&=1.24x
of Eq. Al
102 M-’
10’ M-2
10’“M--3
SYNTHESISOF FDP IN BATCH REACTOR AND SEMIBATCH REACTOR
81, 1999
VOL.
TABLE A4. Kinetic parametersof Eq. A4
TABLE A2. Kinetic parametersof Eq. A2
kr =8.46x
Ks=4.9Ox
k, =1.83x
102 SF]
10ZM-’
Kp=4.90X
l@s-’
l@ M-’
p6 =1.59x
1V4M-*
‘4’ =1.%x1@‘M-2
,98 =2.19x
10” M-2
j; =1.06x
1@4M-z
p,,=1.12~
1029~-2
/3,,=5.32xlO”M-*
/3,2=1.93~1077M--2
B,z=3.23
x 10” M-*
j3,4=4.28
x 1g9 M-*
&=3.30x
1036M-3
,c&=7.66X
1WMm3
/3,,=2.19x
lO=M-’
p,8=1.29x
1W’M-3
8,9=8.40x
KG’* M-3
j3i0=4.32
x 1029 M-3
81, =4.96x
10’9 M-3
,!~**=6.31 x lPOM-’
,!Iz3=3.20x
1039M-4
jIa=4.65
x lO=’ M-4
&=1.36x
102’ M-4
a, =4.11xlO”M-*.
ing rate equation:
-- VP,,
_
a8 =1.46x
ao --2.22x
1015M-*.
103 6 Mm
x[(1+KsC2)3+(1+KpC3)3t3KsKpC3C2(2tKPC3+KsC2)-1]
(1+KsC2)4+(1+KpC3)4+12KsKpC3C2
+ 12KsKp2Cs2C2+ 12Ks2KpC3Cs2
+ 6KS2Kp2C32C22
+ 4KsKp3Cs3Cs+ 4KS3KpC3Cz3- 1
(-42)
a10=3.49x
a,,-6.16xW
al*=1
58x
a’311:34x
1@‘Mm3.
Mm
1V4M-’
l@:M-:::I:
where [,?ZpoI]Orepresents the concentration of PGI [Ml,
C2 the concentration of G6P [Ml, and C3 the concentration of F6P [Ml. kf, k,, KS, and Kp are the intrinsic
kinetic parameters and their values determined at pH 8.0
and 30°C are listed in Table A2.
Phosphofructokinase
The initial rates of the reaction catalyzed by B. stearothermophilus PFK were measured at pH 8.0 and 30°C and using a wide range of F6P,
ATP, FDP and ADP concentrations (Shiroshima, M.,
M. E. thesis, Osaka Prefecture University, Osaka, 1994).
The experimental rate data were in good correlation with
the rate equation which was derived based on the assumptions that the reaction obeys the general reaction
scheme based on the random Bi Bi mechanism and that
the enzyme behaves like a dimeric enzyme even though it
is a tetrameric enzyme. The rate equation was given by:
j32=3.21X
[~PGIIO
W&G
617
- 4KpkrCd
3.
:-
1
1
’
.::I
,t?,=5.77
x 109 M-’
108 M-’
81=1.74xlWM-’
j84=4.74x
102’M-’
&=3.68x
103*M-*
I
._
I
.
I
I.
where C3 is the F6P concentration [Ml, C4 the FDP concentration [Ml, C7 the ATP concentration [M] and Cs
the ADP concentration [Ml. aj (j=l-13)
and /9j (j=l25) are the kinetic parameters consisting of the rate and
equilibrium constants as given in Table A3.
Acetate kinase
Ishikawa et al. (10) measured the
initial rates of the ATP regeneration catalyzed by B.
stearothermophilus AK using a wide range of ADP,
acetyl phosphate, ATP and acetic acid concentrations.
The initial rate data were correlated well with the following rate equation:
a’C7C3+(Y2C72C3+LY3C7C32+(Y4C7C3C*
+ qc7c3c4
+
c&,2c,2+
a~C8C~+a2C82C~+(u3C8C52+(Y4C8C~C7
(Y,C,C3C8C4
+a
-a&*c4-a9c,c~c4-a&3c~c4
-- VPFK
WPFKIO
_
-- VAK
1+ihc7+~2c3+~3c8+~4c4+/%c72+t%c7c3
+ p7c7c8
+ bSc7c4+
b9c3’+
bK10
t%Oc3c8
+h~3~4+/%2~82+~13~8~4+~14~42
+ hc7c3-c8c4
+ pZ,c7’c6
+ ~25c82c42
TABLE A3. Kinetic parametersof
10”
a4
as
Lye
cq
10’ M-3.
109 M-3.:-’
=1.81x
=4.%x
=o
=1.91x
q,,=7.92x
a11=5.18x
alz=1.68x
a,,=7.71
M-2.
10’6M-4.
10”M-‘.sc’
10” M-3.s-’
10’ Mm3.sc’
x 109 Mm4.s-’
,8, =2.20 x 1O-2
p2=2.41
x 106
,93=1.69x
103
,&=6.50x
102
Bs= 1.67 x 109
M-’
M-’
M-’
M-’
M-2
’
-~~~c72c6-(Y,2c7c62-~,3c72c62
1+/h~8+~2~5+~3~7+~4~6+/%~82+~6~8~5
+~7c8c7+B8c8c6+89c*2+810csc7
+ /hSc8c5c6
+ ~23c72c32
t.43)
01, =9.50x
_
+~15~82~,+~16~8~52+~17~8~S~7
+/%8~7~3~4+~19~7~8~4+~20~3~8~4
+ b22c8c4’
-
_ _
~9C8C7C6-&OC&,C6
+p,,~,c,+‘~~2~,i+~~3~7c6+jg~~c62
+~~s~72~3+~16~7~32+P17~7~3~8
+ ~21c82c4
_ _
5c 8c c c 6 +,,c*2c~2+a,c*c~c,c6
--a8ti7i76--
-~~~C,2C4-(u~~C~C42-~,3C,2C,2
+ ,&9c8c7c6
+ j922c7c6’+
+ t%cSc5c7c6+
+ h&5~7~6
p23c82c52
b25c7’c6’
b44)
Eq. A3
p6 =8.58x
108 M-*
/3, =4.26x
lo3 M-2
BE =1.18x
l@ Mm2
p9 = 1.93 x 10’ M-*
,&=7.89x
10’ M-*
,‘3,,=1.02x
108 M-*
/3,2=3.72X106 M-Z
b13=2.47 x 10’ M-*
,~?,~=7.09 x lo3 M-*
&=2.88x
lOI M-3
,&=3.48x
10” M-3
,&=5.16x
10’2M-3
/?,8=1.43x10’2M-3
,&=3.50x
10”M-3
/3m=1.15x10’2M-3
&=5.74x
lo6 M-’
,&=2.0
x 10’“M-3
,Bz3= 1.69 x lOLs Mm4
t??4=0
&=3.81
X log M-4
This equation was derived based on an assumption that
the reaction obeys the random Bi Bi mechanism and on
the fact that the enzyme behaves like a dimeric enzyme
even though it is a tetrameric enzyme (13). In Eq. A4, C,
is the acetyl phosphate concentration [Ml, C6 the acetic
acid concentration [Ml, C7 the MgATP2- concentration
[Ml, and Cs the MgADP- concentration [Ml. /Xj (j = l13) and /3j (j = l-25) are the kinetic parameters consisting
of the rate and equilibrium constants. These kinetic
parameters were previously determined on the basis of
the fact that ATP and ADP exist as forms of MgATP2and MgADP-,
respectively, in the presence of Mg2+
ions. However, in the present paper, the values of aj and
Bj were redetermined on the basis of the concentrations
of ATP and ADP instead of those of MgATP2- and
MgADP-, respectively. The values are listed in Table A4.
Note that in Eq. A4, C7 and Cs are the concentrations
of ATP and ADP, respectively.
618
WIDJAJA
ET AL.
J. BIOSCI. BIOENG.,
ACKNOWLEDGMENTS
This work was partially supported by a Grant-in-Aid for Developmental Scientific Research (No. 06555250) from the Japanese Ministry of Education, Science, Sports and Culture. The authors express
their thanks for its support.
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