Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
PROCESS FOR PRODWCIN~ PHYSIOLOGICALLY
ACTIVE SUBSTANCE BY MULTIENZYME PROCESS
FIELD OF THE INVENTION
The present invention relates to a process for
converting adenosine-5~-monophosphate (hereina-fter refer-
red to as AMP) into adenosine-5'-triphosphate (herein-
after referred to as ATP) and ~ process for producing aphysiologically active substance by an enzymic reaction
using ATP as an au~iliary factor.
BACKGROUN~ OF TI~E INVENTION --
In recent years, chemical reactions of living
bodies have been closely examined in order to attempt to
reproduce the chemical reactions of living bodies in a
reactor. In living bodies~ man-y biosynthetic reactions
are naturally performed in ~he presence of enzymes as a
catalyst in order to support life. Accordingly, living
bodies easily produce compounds which are difficult to
synthesize by chemical reactions. Knowledge of such
reaction is becoming important for satisfying social
requirements such as conserving energy and eliminating
public nuisances. Reproducing such reactions will
likely become an essential technique in chemical
industries. The practical use of such reactions has
already been found useful in the technical fields of
hydrolysis and isomerization.
- 1 -
When carrying out a synthetic reaction (which
is a particularly important reaction in biosynthetic
reactions), ATP is required as an energy source or an
auxiliary factor. In such reactions, ATP is consumed by
decomposing into adenosine-5'-diphosphate (hereinafter
re~erred to as ADP) or adenosine-5'-monophosphate after
it serves as an energy source-or an auxiliary factor.
Accordingly, in order to industrially reproduce the
synthetic reaction, it is necessary to supply ATP at a
moderate price. However, ATP is a very expensive
substance. Accordingly, it is important to convert ADP
and AMP which are in a state after consumption and,
particularly, AMP which is in a consumed state of ~he
lowest energy level, into ATP.
Many studies concerning reproduction and
conversion into ATP have been done. For example, since
production of ATP is carried out by a glycolysis reac~ion
in the living bodies, an attempt utilizing such a reac-
tion is knonw in T. Tochikura, M. Kuwahara, S. Yagi,
H. Okamoto, Y. Tominaga, T. Kano and K. Ogata, J. Ferment.
Tech., 45, 511 (1967); H. Samejima, K. Kimura, Y. Ado,
Y. Suzuki and T. Tadokoro, Enzyme Eng., 4, 2~7 ~1978)
and ~I. Asada, K. Yanamoto, K. Nakanis~hi, R. Matsuno and
- T. Kamikubo, Fur. J. Appl. Microbial. ~iotechnol., 12~
198 (1981). The concept of the reaction is that reproduc-
~P9~
tion and supply of consumed ATP are carried out using
microorganisms, wherein A~1P or adenosine is used as a
raw material for ATP. However, since the AMP or
adenosine is not a product after consumption o-f ATP,
it is additionally added as an ATP source. As a result
of this attempt, the conversion efficiency of AMP or
adenosine into ATP is very inferior and side reactions
are caused. Specifically, when utilizing glvcolysis of
microorganisms, negative result is only obtained
concerning effective reproduction of ATP from AMP which
is a product after consumption of ATP.
The u,s~ of an ATP conversion enzyme which is
c~ bl~,
not a heat-~4~ ~ enzyme has been attempted. Langer
et al. have reported process in converting A~IP into ATP
1~ by means of adenylate kinase in rabbit muscles and
acetate kinase in Escherichia coli in R.S. Langer,
B.K. Hamilton, C.~. Gardner, M.C. Archer and C.K. Colton,
AlchE J., 22, 1079 (1976); R.S. Langer, C.R. Gardner,
B.K. Hamilton and C.K. Colton, AlchE J., 23, 1 (1977),
and U.S. Patent 4,164,444. Further, reports have been
made with respect to converting adenosine into ATP using
adenosine kinase in addition to the above described two
kinds o~ conversion enzyme in R.L. Baughn, O. Adalsteinsson
and G.~I. Whitesides, J. Am. Chem. Soc., 1_ , 304 (1978).
Furthérmore, Whitesides et al. have reported that, when
;25
the above-described adenylate kinase and acetate kinase
are immobilized to Sepharose with cyanogen bromide to
continuously convert AMP into ATP, the residual activity
is only several percentages or less in the absence of a
stabilizer and stabili~y with the passage of time is
remarkably inferior as described in G.M. Whitesides,
A. Chmurny, P. Garrett and C.K. Colton, Enzyme Eng., 2,
217 (1974). Moreover, even if an immobilized enzyme is
used and a stabilizer is added, the reaction requires a
long period of time and conversion efficiency is not so
high, and it cannot be utilized for operating under a
level of chemical industry for a long period of time.
However, little is known with respect to the
production of useful substances by the above-described
synthetic reaction with reproducing ATP. There is a
process which comprises reproducing ATP which was
consumed when synthesizing glutathione by reacting
glutamic acid, cystein and glycine with y-glutamyl
cystein synthesis enzyme and glutathione synthesis . ,
enzyme, from ADP which is a product after consumption
by a function of acetate kinase originated from
Escherichia coli and using it again (as described in
K. Murata, K. Tani, J. Kato and I. Chibata, Eur. J.
Microbial Biotechnol., 10, 11 (1980i). However, this
process does not provide any information with respect to
converting the above-described A~IP consumed to the
lowest energy level into ATP, because it is only a
process for reproducing ATP from ADP.
A bioreactor for synthesizing a useful
substance by continuously consuming ATP into A~IP has
been considered, and it has been highly desired to
complete such a system as described in G.~. Whitesides 7
A. Chmurny, P. Garrett, L. La~otte and C.K. Colton,
Enzyme Eng., 2, 217 (1974).
_
SU~A~Y OF Tf-lE INVENTION
An object of the present invention is to
provide a process for converting A~1P ~which is a product
obtained by decomposing ATP to the lowest energy level)
into ATP in a high yield. Another object of the present
inven~ion is to provide a process for producing a
physiologically active substance by a multienzyme
process which comprises using AMP which is a product
obtained by decomposing to the lowest energy level as a
raw material.
As a result of earnest studies so as to attain
the above-described objects, the plesent inventors have
found that AMP can be converted into ATP in a high yield
in a short period of time, when conversion enzymes
produced from microorganisms having an optimum growth
temperature of 50C to 85C are used. As a result of
subsequent studies, we have found, on the basis of the
above-described knowledge, that a physiologically active
substance can be synthesized from A~!Y which is a product
obtained by decomposing to the lowest energy level as a
raw material, by combining (a) a reaction system of
conv~rting AMP into ATP with (b) a reaction system of
synthesizing a physiologically active substance from ATP.
The present invention relates to a process for
converting AMP into ATP which comprises using, as conver-
sion enzymes, an enzyme which converts AMP into ADP, theenzyme having been produced from microorganisms having
an optimum growth temperature of 50C to 85C, and an
enzyme which converts ADP into ATP, the enzyme having
been produced from microorganisms having an optimum
growtll temperature of 50C to ~5C. In addition, tne
invention relates to a process for producing a physio-
logically active substance by ~ multienzyme process
which comprises ~a) forming ATP from AMP using a combina-
tion of an enzyme which converts AMP into ADP and has
been produced from microorganisms having an optimum
growth temperature of 50C to ~5C and an enzyme which
converts A~P into ATP and has been produced from micro-
organisms having an op-timum growth temperature of 50C
to ~5C, ~b) synthesizing a physiologically active
substance with the resulting ATP, converting AMP result-
2S
in~ from the reaction in the step (b~ into ATP by the
reaction in the step (a), and repeatedly utili~ing the
converted ATP for synthesis of the physiologically
active substance in step (b).
S According to the present invention, it becomes
possible to stably carry out conversion of A~-~P into ATP
efficiently over a long period of time. Further, it is
possible to continuously and economically carry out an
enzymic reaction using ATP as an auxiliary factor with
very good eficiency, whereby it becomes possible to
realize operation of the so-called bioreactor wherein
synthetic reactions in the living body are carried out
as industrial chemical reactions outside the living body.
DETAILED DESCRIPTION OF T~IE INVENTION
The present invention comprises conversion of
A~IP into ADP and conversion of the resulting ADP into
ATP. For example, adenylate kinase is used as an enzyme
for converting AMP into ADP, and ATP is used as a
phosphoric acid donator in this case. Examples of
enzymes for converting ADP into ATP include acetate
kinase, carbamate kinase, creatine kinase, 3-phospho-
glycerate kinase, pyrubate kinase and polyphosphate
kinase. Acetate kinase is preferably used considering
the price of phosphoric acid donator, activity for
converting into ATP and availability of enzymes, etc.
. , .
4~2~
In this case, acetylphosphate is used as a phosphoric
acid donator. As described above, though ATP and acetyl
phosphate are used as phosphoric acid donators when
using adenylate kinase and acetate kinase, it is suffi-
cient to supply only acetyl phosphate as the phosphoricacid donator, because ATP as the final conversion
product can be used as the phosphoric acid donator.
From the application of the above-described advantages
which are obtained when using the acetate kinase alone
and when using the combination of acetate kinase and
adenylate kinase, it becomes possible to plan the system
in which the ATP is effectively reproduced from the AMP.
As described above, it becomes possible to
convert AMP into ATP using two kinds of conversion
enzymes. However, these enzymes are those produced from
microorganisms having an optimum growth temperature of
50C to ~5C. Examples of such microorganisms include
microorganisms of the genus Bacillus, such as Bacillus
stearothermophillus, Bacillus brevis, Bacillus coagulans,
Baci.llus thermoproteolyticus or Bacillus acidocaldarius,
etc., microorganisms of the genus Clostridium, micro-
organisms of the genus Thermoactinomyces, microorganisms
of the genus Achromobacter, microorganisms of the genus
Streptomyces, microorganisms of the genus ~licropolyspora,
microorganisms of the genus Thermus such as Thermus
~ 9~
aquaticus, Thermus thermophilus or Thermus flavus, etc.,
microorganisms of the genus Thermomicrobium, etc.
Further, there are microor~anisms growing at a normal
temperature into which genes of the above-described
S microorganisms are introduced. Among these micro-
organisms, Bacillus stearothermophilus is particularly
suitable for producing both enzymes of adenylate kinase
and acetate kinase. Both enzymes obtained from this
microorganism can be easily purified and have a high
specific activity. In the present invention, it is
preferred to use the above-described enzymes in an
immobilized state. For this purpose, the enzymes are
bonded to, included in or absorbed in suitable carriers.
Examples of such carriers include polysaccharide deriva-
tives such as cellulose, dextran or agarose, etc., vinylpolymer derivatives such as polystyrene, ethylene-maleic
acid copolymer or cross-linked polyacrylamide, etc.,
polyaminoacids and polyamide derivatives such as L-
alanine-L-glutamic acid copolymer or polyaspartic acid,
etc., and inorganic derivatives such as glass, alumina
or hydroxyapatite, etc., preferably polysaccharide
derivatives, inorganic derivatives such as glass and
vinyl polymer derivatives such as polystyrene, which are
used by packing a reactor such as a column therewith.
The preferred amount of the above carriers used per the
~P~4~
enzyme is 1 ~g/enzyme unit to 100 g/enzyme unit
(indicated by enzyme activity Ullit), more preferably
10 ~g/enzyme unit to 10 g/enzyme unit.
In order to convert AMP into ATP according to
the present invention, it is preferred to ca~ry out
conversion of AMP -~ ADP ~ ATP in a packed bed reactor
by feeding 0.1 ~M -to 4 M, preferably 1 ~M to 2 M and,
more preferably 10 ~M to S00 mM of AMP, 0.1 ~1 to S00 mM,
preferably 1 ~q to 400 mM and, more preferably 10 ~M to
300 mM of acetyl phosphate, and ATP to an end of the
reactor. In this case, it is particularly preferred to
use ATP in an amount satisfying the formula (a).
0.15 x 5 2 x AMP > ATP _ 0.04 x 5 2 x AMP (a)
~wherein AMP represents the concentration of AriP (~1),
ATP represents the concentra-tion of ATP (mM), and r repre-
sents a ratio of immobilized enzyme activity of the
enzyme which converts ADP into ATP to immobilized
enzyme activity of the enzyme which converts A~IP into
,, ~ ~f
ADP, which is a positive inte~e~ of 1 or more).
The reacting solution eluted from the reactor in this
case can be analyzed by a suitable analyzing system to
determine concentrations of AMP, ADP and ATP and a
conversion to ATP. In this case, the flow rate varies
- 1 0
~9~
according to the size of the reactor. For example, a
suitable flow rate can be selected from the linear
velocity range of 1 x 10-4 cm/hr to 1 x 106 m/hr. When
using the 5 liters reactor having inside diameter of
S 10 cm and leng~h of 63.5 cm, it is preferred to select
a suitable flow rate from the linear velocitv range of
6 x 10 2 cm/hr to 1 x 105 cm/hr. The apparatus for
supplying AMP, ATP and acetyl phospha-te to the reactor
are not particularly restricted, if they are capable of
varying the amount of sending flow by external control
signaIs. For example, metering pumps driven by a pulse
motor can be used (referred to as variable fluid sending
apparatus, hereinafter). Further, flow rates and concen-
trations of solutions of each substrate can be varied by
providing automatic controlling valves between vessels-
containing the solutions of each substrate and variable
fluid sending apparatus, and controlling opening and
closing o~ the automatic controlling valves by external
signals. The automatic controlling valves may be
electromagnetic valves. Further, the reactor may be
used, of course, at an ambient temperature. However,
it is preferable to add a means for maintaining any
given temperature. The analyzing system for analyzing
the reacting solution from the reactor is not particular-
ly restricted, if AMP, ADP and ATP are detected. An
-
~ ~9~2~
example of a preferred system is a high performance
liquid chromatographic apparatus.
In the present invention, in order to carry out
industrially, stably and economically substantial 100%
S conversion of AMP into ATP over a long period of time,
it is preferred to control the concentration of ATP so
as to satisfy ~he formula (a), and it is particularly
preferred to control the concentration of ATP so as to
be 0.08 x 5 2 x AMP or less in addition to the formula
(a). In order to satisfy the formula (a), the following
method can be used. Namely, control may be carried out
by a method which comprises previously feeding the
formula ~a) and data necessary for operation to an
arithmetical control uni-t, carrying out operation of
conversion of AMP into ATP from the above-described
formula and data and analyzed data from the analyzing
system, and sending signals from the arithmetical control
unit to at least one of the above-described variable
fluid sending apparatus and the automatic controlling
valve to vary the flow rate or the concentration so as
to satisfy the formula ~a). In this case, data necessary
for carrying out operation means the concentrations of
the AMP and ATP as raw materials and the ratio of the
immobilized enzyme activity of the enzyme for converting
- 12 -
ADP into ATP to the immobilized enzyme activity of the
enzyme for converting AMP into ADP, and analyzed data
means the concentrations of AMP, ADP and ATP in the
reacting solution from the reactor. Further, the
arithmetical control unit refers to an apparatus having
an arithmetical function and a function of sending
control signals to an external apparatus. For example,
a microcomputer can be used. Further, the immobilized
enzyme activity means activity of the immobilized enzyme.
For example, in case of adenylate kinase, activity in
the direction of AMP + ATP + 2-ADP is shown. With
respect to acetate kinase, activity in the direction of
ADP + acetyl phosphate -~ ATP + acetic acid is shol~n.
In order to measure the activity, a desired amount of
the immobilized enzyme, for example, 5 to 10 ~ according
to degree of activity, is added to a solution for
measuring activity, and activity ls measured by pursuing
it as a change in absorbance by means of a spectrophoto-
meter by the same manner as in case of free enzyme.
1 unit of enzyme activity means the amount of producing
1 micromole of ADP per minute at 30C in case of
adenylate kinase and the amoun-t of producing 1 micromole
of ATP per minute in case of acetate kinase.
The ATP used in the present invention may be
the ATP which is the final conversion product of the
above-described reaction which is utilized by means of
- 13 -
~9~
circulation. In this case, it is sufficient to supplyonly acetyl phosphate as the phosphoric acid donator.
In the present invention, the enzymic reactions
for synthesizing physiologically active substances by
using ATP as an energy ~hereinafter referred to as reac-
tlon system for physiologically active substance) may
use one or more of the above-described synthetic reac-
tions as the main reactions. Examples of them include
a reaction for synthesizing peptide and peptide deriva-
tives from amino acids by means of aminoacyl t-RNA
synthetase, a reaction for synthesizing acetyl CoA or
acyl CoA from acetic acid or aliphatic acid and CoA by
means of acetyl CoA synthetase or acyl CoA synthetase,
a reaction for synthesizing L-pantothenic acid from
lS pantoic acid and ~-alanine by means of pantothenic acid-
synthetase, a reaction for synthesizing guanyIic acid
from xanthylic acid and ammonia or glutamine by means of
guanylic acid synthetase, a reaction for synthesizing
asparagine from aspartic acid and ammonia by means of
asparagine synthetase, a reaction for synthesizing acyl
CoA from carboxylic acid and CoA by means of bu~yryl CoA
synthetase, a reaction for synthesizing O-D-alanyl-poly-
(ribitol phosphate) from D-alanine and poly~ribitol
phosphate~ by means of D-alanyl-poly(ribitol phosphate)
synthetase and a reaction for synthesizing NAD from
- 14 -
. .
deamido NAD and L-glutamine by means of NAD synthetase,
etc.
In the present invention, ATP is consumed in
the above-described reaction system for making a physio-
logically active substance resulting the formation of
AMP. Ihis resulting AMP is converted into ATP using a
combination of an enzyme which converts into ADP and an
enzyme which converts into ATP, as described above (here-
inafter referred to as the reaction system for reproduc-
tion of ATP).
In order to synthesize a physiologically activesubstance from A~IP as a raw material in the above-
described reaction system for physiologically active
subs-tance, a reactor is first prepared. The reactor may
be a membrane type reactor or a column type reactor.
The membrane type reactor is particularly effective to
use when the physiologically active substance is a low
molecular material. In this case, since the enzymes are
high molecular materials, each enzyme can be used by
staying the enzyme in the reactor. The resulting A~IP is
eluted from the reactor because it is a low molecular
material. After it is separated from the physiologically
active substance by a simple operation such as ion-
exchange chromatography, etc., it is sent back to the
reactor, by which it is possible to reproduce ATP.
, .
~3~2~i
Further, if the so-called wa-ter-soluble high molecular
ATP whicll is obtained by previously introducing a
suitable spacer into ATP and bonding to a water-soluble
high molecular substance is used, the above-described
opera-tion for separation is not required. Various
materials having a molecular weight o~ 1,000 to 500,000
may be used as the water-soluble high molecular
substances. For example, it is possible to use poly-
saccharides such as a soluble dextran, vinyl polymer
derivatives such as polyacrylamide derivatives or
polyacrylic acid derivatives, and polyether derivatives
such as polyethylene glycol derivatives, etc.
The column type reactor can be used wi-thout
regard to the kind of physiologically active substance.
When a column reactor is used, each enzyme is packed in
the column in a form of the so-called immobilized enzyme
which is prepared by bonding to, including in or absorb-
ing in a suitable carrier as described above. In this
reactor, ~he resulting AMP flows out of the reactor
whether it is a high polymer or not, but it can be sent
back tG the reactor after being separated from the
physiologically active substance in the same manner as
described above. Further, in case of water-soluble high
molecular ATP, the operation for separation can be easily
carried out, because it can be separated by membrane
separation.
- 16 -
~4~
The above-described reactor has been explained
on the assumption that the operation is carried out
continuously, and other reactors may be designed on the
basis of such an idea. If necessary, a ba~ch type
reactor may be used in order to carry out a batchwise
operation.
In the present invention, the reaction system
for physiologically active substance and the reaction
system for reproduction of ATP may be operated by combin-
ing them using different reactors, respectively. Further,the reaction system for physiologically active substance
and the reaction system for reproduction of ATP may be
operated in the same reactor. However, in order to
synthesize the physiologically active substance effi-
i5 ciently, it is desirable to supply A~IP produced in thereaction system for physiolGgically active substance to
the reaction system for reproduction of ATP together
with ATP, in both cases. In such cases, it is preferred
that .he ratio of A~IP to ATP is in the range shown by
the above-described formula Ca).
In order to operate the reaction system for
producing a physiologically active substance and the
reaction system for reproduction of ATP by combining
them using different reactors, it is possible to use,
for example, the following method. First, to a reactor
- 17 -
:,
of the reaction system for reproduction of ATP, O.l
to 4 M, preferably l ~M to 2 M and, more preferably
lO ~l to 500 mM of AMP, 0.] ~M to S00 mM, preferably
l ~M to 400 m~1 and, more preferably lO ~M to 300 mM of
acetyl phosphate and ATP in an amount of, preferably, 4~
or more based on AMP as shown in the formula ~a~, though
it varies according to the ratio in the reaction system
for reproduction of ATP, are fed to an end of the
reactor together with AMP to carry out conversion of
AMP ~ ADP ~ ATP in the reactor. The reacting solution
eluted from the reactor is analyzed by a suitable
analyzing system, by which concentrations of AMP, ADP
and ATP and conversion to ATP can be determined. In this
case, the flow rate varles according to the size of the
i5 reactor, and, for example, a suitable flow rate can be
selected from the linear velocity range of l x lO 4 cm/hr
to l x 106 m/hr. A part of the reacting solution (the
concentration of ATP is desired to be 4~ or more based
on the concentration of AMP? is circulated to send back
to the inlet of the reactor and feeding of the ATP
solution fed in the initial stage is stopped. The larger
part of the reacting solution is fed immediately to the
reactor of the reaction system for the physiologically
active substance while controlling the concentration
thereof together with substrates for the reaction system
L~
for the physiologically active substance in case that
substances excepting ATP in the reacting solution, for
example, acetic acid, do not inhibit the reaction system
for the physiologically active substance, or it may be
fed to an end of the reactor of the reaction system for
the physiologically active substance together with a
solution obtained by dissolving the substrates for the
reaction system for the physiologically active substance
after inhibiting substances are removed by a suitable
separation means such as ion-exchange resin, etc., in
case that they inhibit. In this case, the flow rate
varies according to the size of the reactor, and a
suitable flow rate may be selected1 for example, from
the linear velocity range of 1 x 10 4 cm/hr to
I5 1 x lQ6 m/hr. roncentrations of the substrates vary
according to the physiologically active substance.
When the solubility of the substrate is lower than the
concentration of ATP fed from the reaction system for
reproduction of ATP, the solubility of the substrate is
the upper limit of concentration. Further, when the
solubility of the substrate is higher than the concentra-
tion of ATP fed, the concentration similar to the concen-
tration of ATP is the highest concentration of the
substrate. Further, in the latter case, if the ATP fed
is concentrated, the synthetic reaction can be carried
- 19 -
Ollt at a higher concentration. In this case, operation
becomes discon~inuous because of the operation for
concentration. The reaction product (physiologically
active substance) and AMP are separated from the eluted
solution in the reactor by a suitable separation means
such as ion exchange resln, and AMP is sent back to an
end of the reactor of the reaction system for reproduc-
tion of ATP, by which ATP is reproduced again.
When the reaction system for producing a
physiologically active substance and the reaction system
for reproduction of ATP are operated in the same reactor,
the operation can be carried out in only when substrates
in both enzymic systems do not inhibit the enzymic
reaction systems of each other. Concentrations of
substrates in both enzymic reaction systems ara desired
~o be selected according to a relation between the
concentration of ATP produced in the reac~ion system for
reproduction of ATP and the solubility of the substrate
in the reaction system for physiologically active
substance. For example, when the solubility of the
substrate in the reaction system for physiologically
active substance is lower than the concentration of ATP,
concentrations of AMP, ATP and acetyl phosphate may be
selected such that the concentration of ATP produced in
the reaction system for reproduction of ATP agrees with
- 20 -
~9~
the concentration of the substrate. In contrast with
this, when the solubili-ty of the substrate in the reac-
tion system for physiologically active substance is
higher than the concentration of ATP, it is desired that
the concentration of the substrate is allowed to agree
with the concentration of ATP produced in the reaction
system for reproduction of ATP. It is preferred that
the amount of ATP fed to the reaction system for reproduc-
tion of ATP together with AMP is more than 2 times,
preferably more than 4 times and, more preferably, more
than 5 times of the above-described case of using
different reactors respectively. The flow rate varies
according to the size of the reactor as well as the type
of reactor, and a suitable flow rate can be selected from
lS the linear velocity range, for example, 1 x 10 4 cm/hr
to 1 x 106 m/hr. Further, A~IP and the physiologically
active substance are separated from the reacting solution
eluted from the reactor by means of a suitable separation
means such as ion-exchange resin, etc., as described
above, and AMP can be used again by sending back as a
substrate to the inlet of the reactor.
In the present invention, the pH during the
reaction varies according to enzyme used ln case of the
reaction system for physiologically active sùbstance,
but a pH in a nearly neutral range, namely, 6 to 11 and,
- 21 -
~5
preferably, 6.5 to 9.0 is generally used. In case of the
reaction system for reproduction of ATP, a pH in the
range of 6.5 to 11, preferably 6.5 to 9.0 and, more
preferably 7 to ~ is used. As a buffer solution, it is
possible to use conventional solutions fit for these pH
ranges. For example, phosphates, imidazole salts, tris-
hydrochloride, collidine salts and barbital hydrochloride9
etc., can be used near pH 7. Further, the temperature
for treatment can be selected from the range of room
temperature to 50C. In case of the reaction system for
reproduction of ATP, though the reaction fGr reproduction
of ATP may be carried out at a higher temperature, it is
preferred to set at 5C or less which is the maximum
growth temperature of enzyme producing microorganisms.
i5 Moreover, in order for the reaction of adenylate kinase
and acetate kinase to effectively proceed in the reaction
sys~em for reproduction of ATP, various divalent metal
ions can be used. As the divalent metal ions, magnesium
ion and manganese ion are particularly recommended.
According to the present invention, change in
conversion shown in the prior conversion of A~P in~o ATP
can be overcome, and AMP can be converted effectively,
continuously and economically into ATP at a conversion
of substantial 1~0~ over a long period of time. In addi-
tion, ATP conversion can be kept stably for a long period
- 22 -
~94F~'5 -
of time because of having good operation properties.
Purther, it is an advantage of the present invention
that ATP converted from A~IP in the packed bed type
reactor can be used as a phosphoric acid donator.
Moreover, as a starting material, pure AMP is not
required, and mixlures of AMP wi-th ADP and ATP may be
used if they are controlled so as to sa~isfy the formula
~a). Accordingly, the process is very advantageous for
industrial use. Furthermore, the reaction system for
reproduction of ATP can be advantageously applied to
reproduction and utilization of ATP, and from a differ-
ent point of view, it-can be thought of as a process
for production of ATP using AMP as a raw material.
ATP is an important material as a medicine and
has been produced industrially. However, the ferment~-
tion process in the prior art has problems that by-
products are easily formed and productivi~y is inferior.
Consequently, the price of ATP has been high. Ho~ever,
according to the present invention, such problems can be
eliminated and ATP having high purity can be supplied
with good productivity.
According to the present invention, the en~ymic
reaction using ATP as an auxiliary factor can be carried
out continuously, effectively and economically by using
the above-described reactors. Accordingly, it becomes
- 23 -
~9~325
possible to realize the operation of the so-called bio-
reactor wherein coupling reactions carried out in the
living body are carried out as industrial chemical
reactions outside the living body. Particularly, it is
of great industrial value that protein synthesis reac-
tions and peptide synthesis reactions by means of amino
acid activating enzymes which are the most important
reactions in the living body can be utilized for practi-
cal application.
In the following, -the present invention is
illustrated in greater detail in examples.
EXA2~1PLES 1 SEp~Ro~
After 5 g of activated CH-S~p~arose 4B
(produced by Pharmacia Fine Chemicals) was washed to
swell, 2,000 units of acetate l~inase obtained from
Bacillus stearothermophilus NCA 1503 (optimum growth
temperature: ~0C) (sold by Seikagaku Kogyo Co.) were
added thereto and the reaction was carried out to obtain
1,000 units of immobilized acetate kinase. The same
operation as described above was carried out using 250
units of adenylate kinase (sold by Seikagaku Kogyo Co.)
instead of acetate kinase to obtain 100 units of immobil-
ized adenylate kinase. The ratio of immobilized enzyme
activity of acetate kinase to that of adenylate kinase
in this case was 10.
~rr~e ~ark - 24 -
A glass column having an inside diameter ot
1.6 cm and a length of 10 cm was packed with both of
these immobilized~enzymes, and each substrate dissolved
in a 25 m~l imidazole hydrochloride buffer solution
containing 10 m~ magnesium chloride having a pH of 7.5
was fed to the column at a flow rate of 150 mQ/hour.
A variable fluid sending apparatus, an electromagnetic
valve and a microcomputer were equipped on this column.
The temperature in the column was kept at 30C. Concen-
trations of AMP, ADP and ATP in the reacting solution
- eluted from the column were measured by a high perform-
ance liquid chromatographic apparatus. The concentration
of AMP was fixed at 1.5 mrl and the concentration of
acetyl phosphate was fixed at 5 m~l.
Conversion to ATP was then determined with
varying the condition so as to satisfy the formula (a)
by the microcomputer such that it was 0.063 mM ATP (ratio
by concentration o-f ATP to AklP was 0.042; Example 1),
0.07 m~l ATP (ratio by concentration of ATP to A~P was
0.047; Example 2), 0.13 mM ATP (ratio by concentration
of ATP to AMP was 0.087; Exam~le 3) and 0.19 mM ATP
(ratio by concentration of ATP to AMP was 0.127; Example
4).
As a result, af~er feeding to the column, no
AMP was detected after only 20 minutes and 98.5% of ATP
and 1.5% of ADP were detected.
- 25 -
Further, the same procedure was carried out
with varying the concent-ration o-f ATP to 0.03 m~l ATP
(ratio of,concentration of ATP to AMP was 0.02; Example
5), 0.024 ml~ ATP ~ratio by concentration o~ ATP to AMP
S was 0.016; Example 6) and 0.01~ m~I ATP (ratio by concen-
cration of ATP to hMP was 0.009; Example 7).
As a result, 95% of ATP, 3% of ADP and 2% of
AMP were detec-ted in Example S, 89~ of ATP, 8% of ADP
and 3% of AMP were detected in Example 6, and 72% of ATP,
20% of ADP and 8% of AMP were detected in Example 7.
E AMPLE 8
After the reaction was initiated under the same
condition as in Example 2, a solution eluted from the
column after 20 minutes was circulated to feed to the
column so ti.at the ratio of ATP fed to the column to AMP
was 0.047.
As a result, ATP was kept in the range of 98%
to 98.5% over 5 hours after 20 minutes after the eluent
from the reactor was used instead of ATP.
EXAMPLE 9
A glass column having an inside diameter of
2.0 cm and a length of 12 cm was packed with 2,000 units
of immobilized acetate kinase and 200 units of immobil-
ized adenylate kinase obtained by the same method as in
Example 1, and 3.0 mM of AMP, 0.13 mM of ATP (ratio by
- 2~ -
9~
concentration of ATP to AMP was 0.043~ and 10 mM of
acetyl phosphate which were dissolved in a 50 m~l
imidazole-hydrochloride buffer solution containing
25 mM magnesium chloride and 0.0~% sodium azide having
a p~I of 7.5 were fed to the column at a flow rate of
300 mQ/hour, and flol~ rates and concentrations of ArlP
and ATP were maintained so as to satisfy the formula (a).
As a result, the conversion to ATP was kept in
the range of 98.5% to 99.0% over 10 days after initiation
of the reaction.
EXAMPLE 10
After the reaction was ini-tiated under the same
condition as in Example ~r, a solution eluted from the
", ~
column after 30 minutes (containing 98% of ATP) was
circulated to feed to the column so that the ratio by
concentration of ATP fed to the column to AMP was 0.043.
As a result, the conversion to ATP was kept in
the range of 98.2 to 98.7% over 10 days after initiation
of the reaction.
EXAMPLE 11
Adenylate kinase and acetate kinase materials
derived from available Bacillus stearothermophilus (sold
by Seikagaku Kogyo Co.) ~ere obtained. Acetyl CoA
synthetase, a material derived from available yeast
(produced by Boehringer Mannheim Co.) was also obtained.
- 27 -
2~i
These three enzymes were immobilized on
Sepharose 4B as follows. Namely, after 5 g of activated
CH-Sepharose 4B (produced by Pharmacia Fine Chemicals)
was washed to swell, 2,000 units of acetate kinase were
added thereto and the reaction was carried out to obtain
1,000 units of irllmobilized acetate kinase. Likewise,
100 units of immobilized adenylase were obtained from
250 units of adenylate kinase, and lO units of immobil-
ized acetyl CoA synthetase were obtained from lOO units
of acetyl CoA synthetase. ~ column for reproduction of
ATP (inside diameter: 1.6 cm, length: lO cm~ was packed
with the immobilized acetate kinase and the immobilized
adenylate kinase. 6 mM of AMP, 0.3 mM of ATP and Z5 mM
of acetyl phosphate which were dissolved in a 25 mM
iMidazole hydrochloride buffer solution containillg 10 mM
magnesium chloride having a pH of 7.5 were fed to the
column at a flow rate of 25 mQ/hour. The reac~ion
temperature in the column was kept at 30C. The result-
ing AlP was sent back to the inlet of the column in an
amount of 5~ (0.3 mM) based on -the concentration of AMP.
Further, another column (inside diameter: l.O
cm, length: 9 cm) was packed with immobilized acetyl CoA
synthetase. As a substrate, 4 mM of potassium acetate,
4 mM of reduction type CoA, lithium salt and 4 mM of
MgCQ2 which were dissolved in a 100 mM imidazole hydro-
- 28 -
:~9'~5
chloride buffer solution having a pH of 7.5 were flown
at a flow rate of 25 mQ/hour, which was mixed ~ith an
ATP solution eluted from the reaction system for produc-
tion of ATP in a ratio of 1:1. The mixture was fed to
a column packed with the immobilized acetyl CoA
synthetase (flow rate: 50 mQ/hour, reaction temperature:
@~ls 37C). Further, AMP was taken out from an ~lutc from
.c h~.;`
the column by means of Dowex l-X8 ~produced by the Dow
Chemical Co.2. After the pH and the concentration were
controlled to desired values, it was fed to the column
for reproduction of ATP by means of a pump. The amount
of the resulting acetyl CoA was measured by a method
which comprises sampling 0.05 mQ of the ~t~ from the
column, adding 3 mQ of 1 m~ 5,5'-dithiobis(2-nitrobenzoic
acid) ~DTNB) ~pH 6.65, phosphoric acid buffer solution),
determining the amount of unreacting SH group by change
in absorbance in 412 nm at room temperature after the
passage of 20 minutes, and calculating therefrom.
As a result, 1.5 mM of acetyl CoA was formed
after 30 minutes from the initiation of the reaction,
and thereafter a stabilized state was maintained over
24 hours.
EXAMPLE 12
A column (inside diameter: 1.8 cm, length: 12
cm) was packed with 2,000 units of immobilized acetate
kinase, 200 units of immobilized adenylate kinase and
- 29 -
:~ 94~S
lO units of immobilized acetyl CoA synthetase which wereobtained by the same methods as in Example 11, and 4 mI~
of A~IP, l.0 mM of ATP (25% based on the concentration of
AMP)s 25 mM of acetyl phosphate, 2.5 mM of potassium
acetate and 2.5 mM of reduction type CoA lithium salt
which were dissolved in a 100 m?I imidazole hydrochloride
buffer solution containing lO mM magnesium chloride were
fed thereto at a flow rate of 50 mQ/hour. The reaction
temperature in the column was kept at 37C. A~IP was
separated from the solution eluted from the column by
the same method as in Example X and it was sent back to
. ,.!r
the inlet for substrates. After 40 minutes from the
initiation of the reaction, the formed acetyl CoA became
1.6 mM, and thereafter a stabilized state was kept over
15 hours.
E~AMPLE 13
To the same column for reproduction of ATP as
in Example 11, 6 mM of A~IP, 0.05 mM of ATP and 25 m~I of
acetyl phosphate which were dissolved in a 25 m~
imidazole hydrochloride buffer solution con*aining lO ~M
of magnesium chloride having a pH of 7.5 were fed at a
flow rate of ZS mQ/hour. A part of the solution eluted
from the column was sent back to the inlet of the column
for reproduction of ATP by the same method as in Example
11 in an amount of 0.05 mM as a concentration of ATP.
- 30 -
Then, synthesis of ace~yl CoA was carried Ollt by the
same column packed with immobilized acetyl CoA synthetase
as in Example 11.
As a result, l.0 mM o-f acetyl CoA was formed
after 30 minutes from the initiation of the reaction,
~nd thereafter a stabilized state was main~ainea over
Z4 hours.
EXA~PLE 14
Asparagine synthetase was produced from Lacto-
bacillus arabinosus ATCC ~014 by carrying out ammoniumsulfate fractionation, calcium phosphate gel. treatment
and gel filtration.
Using the same reaction system for reproduction
of ATP as in Example 1, 4 mM of AMP, 0.3 mM of ATP (7.5
based OII the CGIlCetltration of A~IP) and 16 mM of acetyl
phosphate which were dissolved in a 25 mM imidazole
hydrochloride buffer solution containing lO mM manganese
chloride having a pH of 7.5 were fed, and ATP was
reproduced.
Further, lO0 units of asparagine synthetase
were dissolved in a lO0 mM tris-hydrochloride buffer
solution containing 4 mM of manganese chloride, and the
resulting solution was enclosed in a membrane type
reactor having an inside volume of 50 mQ using an ultra-
filtration membrane having a molecular weight of 30,000.
- 31 -
1 A 100 mM tris-hydrochloride buffer solution
containing 4 mM man~anese chloride in which 4 mM of
ammonium chloride and 4 mM of L-aspartic acid were
dissolved was mixed with an ATP solution obtained from
the reaction system for reproduction of ATP in a ratio of
1:1 (by volume), and the resulting mixture was fed to
the above-described reactor at a flow rate of 25 mQ/hour.
The reaction temperature in this case was kept at 37 C.
AMP was separated from the eluate by the same method as
in E~ample 11, which was sent back to-the reaction system
~or reprod~ction of ATP. The amount of the formed L-
asparagine was determined by applying the eluate to a
high performance liquid chromatographic apparatus.
As conditions for the chromatographic apparatus
in this case, Shimadzu Zorbax ODS was used as the column
and a mixture of 0.01M sodium acetate (pH 4.5~/acetonitrile
(55/45 by volume~ was used as an eluate at a flow rate of
1 mQ/min, and detection was carried out by measuring an
absorbance at 210 nm.
As a result, 1.5 mM of L-asparagine was formed
after 30 minutes from the initiation of the reaction,
and thereafter a stabilized state was maintained over
15 hours.
~'
1 EXAMPI,~
Tyrosyl t-RNA synthetase was produced from
saclllus stearothermophilus: Deposition No. 5141 in
Fermentation Research Institute by purifying through
chromatography of DEAE-CELI,ULOSE* (produced by Whatman
Ltd.), hydroxyapatite (.sold by Seikaga]cu Kogyo Co.) and
DEAE-SEPHADEX* (.produced by Pharmacia Fine Chemicals),
ammonium sulfate (fractionation and chromatography of
hydroxyapatite, DEAE-SEPHADEX* and SEPH~DEX G-150*
(produced by Pharmacia Fine Chemicals).
Then, ATP (purity: 98%) was reproduced from
AMP, a catalytic amount ~5% based on the concentration
of AMPl of ATP and acetyl phosphate by t,he same method
as in Example 1, and it was used for the following re-
action.
150 mg of the above-described purified tyrosyl
t-RNA synthetase, 200 mg of magnesium chloride, 51 mg of
ATP (purity: ~8~1, 0.5 mg of L-tyrosine, lOO.units of
pyrophosphatase (.produced by Boehringer Mannheim) and
0~005 mg of dithiothreitol were dissolved in 70 mQ of a
20.mM HEPES buffer solution ~pH: 8.0), and they were
allowed to react at 4C for 15 minutes to obtain a re-
action mixture. To the resulting reaction mixture 2 g of.
L-phenylalanine methyl ester was added and well blended.
The mixture was allowed to stand for 1 day with maintain-
*Trade Mark
- 33 -
... ~'~, , '
. - , - . .
ing the reaction temperature at 30C to carry out the
reaction.
To the resulting reacting solution, 100 m~ of
acetone was added. After precipitates were removed by
centrifugal separation, the supernatant was concentrated
to about 10 mQ by an evaporator and processed by a high
per-formance liquid chromatographic apparatus to separate
a reaction product. As conditions for the chromatograph-
ic apparatus in -this case, ~ Bondapak C18 ~produced by
~aters Associates) was used as the column and development
was carried out using a solvent mixture of 50 m~l
phosphoric acid buffer solution ~p~I 7.0)/acetonitrile
(~85/15) and detection was carried out by measuring an
, absorbance ~ 210 nm.
As a result, 0.22 mg of L-tyrosyl-L-phenyl-
alanine methyl ester was obtained. Further~ AMP eluted
in the void section was separated at the same time by
the same method as in Example 11 and sent back to the
reaction system for reproduction of ATP to reproduce ATP.
EXAMPLE 16
I~Iethionyl t-RNA synthetase was obtained as a
crude enzyme solution ~purity: 10%) from available
baker's yeast (produced by Oriental Yeast Co.~ by operat-
ing with cellulose phosphate column chromatbgraphy.
- 34 -
Then, ATP (purity: 98%) was reproduced from
AMP, a catalytic amount (5% based on the concentration
of AMP) of ATP and acetyl phosphate by the same method
as in Example 1, and it was used for the following reac-
tion.
1 g of the above-described crude methionyl-t-
RNA syn-thetase, 10 mg of magnesium chloride, 21 mg of
ATP (purity: 98%), 0.5 mg of L-methionine, 5 units of
pyrophosphatase (produced by Boehringer ~;lannheim) and
lG 20 mQ of mercaptoethanol were added to 15 mQ of a 50 m~l
2~5-dimethylimidazole bu~fer solution having a pH o-f 9Ø
After being allowed to react by the same mehtod as in
Example 15~ the reaction mixture was treated with
Sephadex G-75 and elution was carried out with a HEPES
buffer solution (pH: 8.0). 30 mQ of a fraction in the
void section was collected and the reaction mixture was
isolated. To the isolated mixture, 0.5 g of L-leucine
ethyl ester was added in a solid state and the reaction
was carried out at 25C for 4 hours. To the resulting
reaction product, 30 mQ of acetone was added. After the
formed precipitates were removed by centrifugal separa-
tion, the supernatant was concentrated to about 10 mQ by
an evaporator and separation was carried out- by the same
method as in Example 15 to obtain 0.92 mg of L-methionyl-
L.-leucine ethyl ester.
- 35 -
Further, ATP was reproduced from A?IP by the
same method as in Example 15.
While the invention has been described in
detail and with reference to specific embodiments
thereof, it will be apparent to one skilled in the art
that various changes and modifications can be made
therein without departing from the spirit and scope
thereof.
- 36 -
