JPS59106296A - Preparation of physiologically active substance by composite enzyme process - Google Patents

Abstract

PURPOSE:To prepare a physiologically active substance in high yield, in a short time, by controlling the mixing ratio of adenosine-5'-mono-phosphate (AMP) and adenosine-5'-triphosphate (ATP) to a specific condition. CONSTITUTION:(A) An enzyme (e.g. adenylic acid kinase) converting AMP to adenosine-5'-diphosphate (ADP) is made to react with an enzyme (e.g. acetic acid kinase) converting ADP to ATP to produce ATP from AMP, and (B) a physiologically active substance is synthesized by using the produced ATP. The AMP produced by reaction (B) and the ATP produced by the reaction (A) are mixed at a ratio defined by the formula [ATP and AMP are the concentration (mM); gamma is the ratio of the activity of the immobilized enzyme to convert ADP to ATP to the activity of the immobilized enzyme to convert AMP to ADP, and is >=1] to regenerate AMP to ATP, and the ATP is recycled to the step (B) for the synthesis of the physiologically active substance.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a physiologically active substance by an enzymatic reaction using adenosine-5'-triphosphate (hereinafter referred to as ATP) as a cofactor.

In recent years, due to a review of the current chemical industry, attention has been paid to chemical reactions within living organisms, and many attempts have been made to reproduce chemical reactions within living organisms using reactors in factories. There is. originally.

In order to maintain life in living organisms, many biosynthetic reactions are carried out using enzymes as catalysts.These reactions not only easily produce compounds that are difficult to synthesize using chemical synthesis reactions, but also are energy-saving and non-polluting. It also has elements that respond to society's demands, such as gender.

It is about to become an essential technology in the chemical industry. Among these, some have already reached the level of practical use in technical fields such as hydrolysis reactions and isomerization reactions.

However, in performing binding reactions, which are particularly important reactions among biosynthetic reactions, ATP is required as an energy source or cofactor. At this time, ATP is used as an energy source or a cofactor, and then decomposed into adenosine-5'-diphosphate (hereinafter referred to as ADP) or adenosine-5/-monophosphate (hereinafter referred to as AMP) and consumed. This will result in Therefore, in order to reproduce the binding reaction industrially, it is necessary to supply ATP at low cost, but in reality ATP is an extremely expensive substance.

Therefore, ADP SAM, which is the state after consumption of ATP
P.

Especially when AMP is completely consumed at the lowest energy level.
Regeneration and conversion to e-ATP is an important technology to overcome this problem.

However, the production of substances through such bonding reactions,
There are not many known examples of performing this while regenerating ATP. For example, Japanese Patent Laid-Open No. 54-122798 is known. this is.

ATP consumed when synthesizing glutathione by the action of γ-glutamylcysteine synthetase and glutathione synthetase on glutamic acid, cysteine, and glycine is recycled and reused from ADP, which is the form after being consumed by the action of acetate kinase. However, it remains at that level and does not provide knowledge about regenerating and converting AMP, which has been consumed to the lowest energy level mentioned above, into ATP. - 1,000,000 AT consumed
There is also the idea of regenerating or replenishing P using the glycolytic system of microorganisms, and there is an example of using AMP as a raw material for ATP (for example, Tatsurokube Tochikura et al., Organic Synthetic Chemistry, Vol. 39, No. 6, page 487.

(1981). However, this AMP should be added separately as a %ATP source, not in the form after consumption of ATP.

Moreover, the result of this attempt was that the conversion efficiency of AMP to ATP was extremely poor (ibid.).

In other words, it also uses the glycolytic system of microorganisms.

There were only negative speculations regarding the regeneration of ATP from AMP, which is the form after consumption.

Conventionally, from this point of view, the concept of a reactor (bioreactor) for rapidly consuming ATP to AMP to synthesize useful substances has been considered, and the completion of this system has been strongly desired.

The inventors of the present invention have studied these actual conditions and have conducted intensive research on converting and regenerating AMP, which is a product decomposed to the lowest energy level, into 'ATP'.

Using a converting enzyme produced by microorganisms with an optimal growth temperature of 50°C to 85°C, AMP can be produced in a short time and with high yield.
We discovered that it is possible to convert .

AM by controlling the mixing ratio of AMP and ATP under specific conditions.
We discovered that by regenerating P into ATP, virtually 100% of AMP can be converted into ATP at low cost, with good operability, and stably over a long period of time.As a result of further studies, based on this knowledge ( By connecting a) a reaction system that regenerates AMP to ATP and (b) a reaction system that synthesizes a physiologically active substance using ATP.

Finding 1: Physiologically active substances are synthesized using AMP, which is a product of decomposition into low energy levels.
The invention has been completed.

That is, 1 the present invention provides (a) an enzyme that converts adenosine-57+-phosphate to adenosine-5/-diphosphate, and an enzyme that converts adenosine-5'-diphosphate to adenosine-5'-triphosphate. In combination with an enzyme, adenosine-5'-
Generate adenosine-5'-triphosphate from monophosphate,
(b) Synthesize a physiologically active substance using the produced adenosine-5'-triphosphate; A method for producing a physiologically active substance by a complex enzyme method, characterized in that it is regenerated into 5'-triphosphate and the regenerated adenosine-5'-triphosphate is repeatedly used for the synthesis of the physiologically active substance (b). be.

In the present invention, an enzymatic reaction that uses ATP to synthesize a physiologically active substance (hereinafter referred to as a physiologically active substance reaction system)
The main reaction is the so-called bonding reaction mentioned above, and for example, amino acid to aminoamyl
- Reaction to synthesize peptides and peptide derivatives by RNA synthesis enzymes, reaction to synthesize acetyl-CoA or acyl-CoA from acetic acid or fatty acid and CoA by acetyl-CoA synthetase or acyl-CoA synthetase, pantothenic acid from pantothenic acid and β-alanine Synthetic enzymes 1) Reaction to synthesize L-hanthenic acid, reaction to synthesize saguanylic acid from xanthylic acid and ammonia or glutamine by guanylate synthetase, reaction to synthesize asparagine from aspartic acid and ammonia by asparagine synthetase, carvone Reaction for synthesizing acyl-CoA from acid and CoA by butyryl-CoA synthetase, D
-0-D from alanine and poly(ribitol phosphate) by D-alanyl-poly(ribitol phosphate) synthase
Examples include a reaction to synthesize -alanyl-poly(ribitol phosphate), a reaction to synthesize NAD+ from deamide NAD+ and L-glutamine using NAD'' synthase, and the like.

In the present invention, ATP in the physiologically active substance reaction system is
It is consumed and becomes AMP, but this AMP
is referred to as an ATP regeneration reaction system in which ADP is converted to ATP by combining an enzyme that converts it to ADP and an enzyme that converts to ATP. ). For example, adenylate kinase is used as the enzyme that converts AMP to ADP, and in this case, ATP is used as the phosphate donor to AMP. In addition, many enzymes can be used to convert ADP to ATP, such as acetate kinase, carbamate kinase, creatine kinase, 8-phosphoglycerate kinase, pyruvate kinase, and polyphosphate kinase. Considering the price, conversion activity to ATP, ease of obtaining the enzyme, etc., it is most advantageous to use acetate kinase, and in this case, acetyl phosphate is used as the phosphate donor. In this way, when using adenylate kinase and acetate kinase, ATP and acetyl phosphate are used as phosphate donors for each enzyme, but ATP as a phosphate donor is converted into ATP, which is the final converted product. Since it is possible to recycle and use acetyl phosphoric acid, only acetyl phosphoric acid needs to be supplied as a phosphoric acid donor. It is an advantage of both of these enzymes that such a combination allows for efficient design.

In this way, it is possible to regenerate the produced AMP into ATP by using two types of converting enzymes, and both of these enzymes are produced by microorganisms whose optimal growth temperature is 60°C to 85°C. This is desirable. Such microorganisms include Bacillus stearothermophilus,
Bacillus plebis.

Microorganisms of the genus Bacillus such as Bacillus coagulans, Bacillus thermoproteolyteix, and Bacillus acidocaldarius, microorganisms of Clostridium rot, and microorganisms of the genus Thermoactinomyces.

Microorganisms of the genus Achromobacter, microorganisms of the genus Streptomyces, microorganisms of the genus Micropolyspora, microorganisms of the genus Thermus such as Thermus aquaticus, Thermus thermophilus, Thermus flap, microorganisms of the genus Thermomicrobium, microorganisms of the genus Calburia. microorganisms, etc.

It also includes microorganisms that grow at room temperature into which the genes of these microorganisms have been introduced. Among these microorganisms is adenylate kinase.

Particularly suitable for the production of both enzymes, acetate kinase, is Bacillus stearothermophilus. Both enzymes obtained from this microorganism are easy to purify.

High specific activity. At the beginning of the research, the optimal growth temperature was 6.
It was thought that using converting enzymes produced by microorganisms at temperatures between 0°C and 86°C was not suitable for regenerating ATP at room temperature, but surprisingly it was possible to do so at room temperature in a short period of time, with high yields, and for a long period of time. AMP can be converted to ATP, and these effects far exceed those of enzymes produced by normal-temperature organisms.

In the present invention, to synthesize a physiologically active substance using the above-mentioned physiologically active substance reaction system using AMP as a raw material, first.

What is necessary is to construct a reactor, and as such a reactor, for example, a demolding reactor or a column type reactor can be used. The model reactor can be used particularly effectively when the physiologically active substance is a low molecular weight substance.

At this time, since the enzymes are polymeric substances, each enzyme can be used in the intact state in which it remains in the reactor. Since the generated AMP is a low-molecular substance, it flows out of the reactor, but it can be easily separated from the physiologically active substance by an operation such as ion exchange chromatography, and then returned to the first reactor.

It is possible to regenerate it into ATP. Further, if so-called water-soluble polymerized ATP, which is obtained by introducing a suitable spacer into ATP in advance and bonding it to a water-soluble polymer substance, is used, such separation operation becomes unnecessary. In this case, as a water-soluble polymer substance.

For example, various materials such as polysaccharides such as soluble dextran, polymer derivatives such as polyacrylamide derivatives and polyacrylic acid derivatives, and polyether derivatives such as polyethylene glycol derivatives can be used.

Next, the column reactor can be used with any physiologically active substance. In this case, each enzyme used is carried in a suitable carrier, for example, a polysaccharide derivative such as cellulose, dextran, agarose, etc.

Polystyrene, ethylene-maleic acid copolymer.

Derivatives of vinyl polymers such as cross-linked polyacrylamide, L-alanine and monoglutamic acid copolymers, derivatives of boria-based acids or boria-based acids such as polyaspartic acid, glass, alumina, hydroxyapatite, etc. It may be used by binding, entrapping, or adsorbing it to an inorganic derivative, etc., and filling it in a column as a so-called immobilized enzyme. In this reactor, the produced A
MP flows out of one reactor regardless of whether it is polymerized or non-polymerized, but it can be separated from the physiologically active substance and returned to one reactor in the same manner as described above. In addition, water-soluble polymerized ATP
Since membrane separation is possible, one separation operation can be simplified.

Although the above reactor was explained on the assumption that it would be operated continuously, other reactors could be devised based on this idea, and in some cases, a batch type reactor could be used. It may also be used for batchwise operation.

In the present invention, the above-mentioned reactors are used separately for the physiologically active substance reaction system and the ATP regeneration reaction system.

These can be operated by connecting them, or the physiologically active substance reaction system and the ATP regeneration reaction system can be operated by making them coexist in the same reactor, but it is possible to operate the physiologically active substances efficiently. In order to synthesize both, it is desirable to provide AMP produced in a physiologically active substance reaction system together with ATP to an ATP regeneration reaction system. At this time, it is desirable that the ratio of amounts of AMP to ATP be within the range shown by the linear equation.

The apparatus for carrying out the above ATP regeneration reaction system is a patented apparatus consisting of a substrate supply section, a reaction tank containing the above two types of converting enzymes, an automatic sampling section 1 analysis device, and a control section. is desirable. The substrate supply section is composed of, for example, an AMP tank, an ATP tank, and an acetyl phosphate tank, and each is supplied to the enzyme reaction tank at a constant flow rate and concentration by the operation of a variable amount liquid feeding device such as a metering pump and an automatic control valve. The substrate can be delivered. The automatic sampling section can automatically sample a certain amount of the reaction solution from the enzyme reaction tank at preset time intervals and send it to one analysis device. Analyzers are AMP and ADP.

The control unit calculates each concentration of ATP, and based on this value, controls the operation of the variable amount liquid delivery device and automatic control valve to control the amount of ATP and acetyl phosphate to be supplied to the physiologically active substance reaction system. Can be adjusted.

Next, in order to connect and operate the physiologically active substance reaction system and the ATP regeneration reaction system using separate reactors, the following method can be used, for example. first,
For the reactor of ATP regeneration reaction system.

AMP from 0.1 μM to 4M, preferably from 1 μM to 2M
, more preferably 10 μM to 500 mM, acetyl phosphate 0.1 μM to 500 mM, preferably 1 μM to 4
00mM, more preferably 10μM to 800mM,
And as shown in equation (a), ATP is compared to AMP,
Although it varies depending on the quantitative ratio of the ATP regeneration reaction system, preferably 4% or more is supplied together with AMP from one end of the reactor, so that the conversion from AMP to ADP-ATP can be carried out in one reactor.

The reaction solution eluted from the reactor can be analyzed with a suitable analysis system to determine the concentrations of AMP, ADP, and ATP and the conversion rate to ATP. At this time, the flow rate varies depending on the size of one reactor, but for example, 0
．． A suitable flow rate between 8 μe/hour and 1×tO'g/hour can be selected. A portion of this reaction solution (ATP concentration is AM
It is desirable that the P concentration be 4% or more. ) is circulated back to the reactor inlet, and the initially supplied ATP liquid is stopped. If most of the remaining reaction solution is a substance other than ATP in one reaction solution, such as acetic acid, which does not inhibit the physiologically active substance reaction system, the concentration is adjusted and the physiologically active substance is directly transferred to the reactor for the physiologically active substance reaction system. Supply it by mixing it with the substrate of the reaction system,
In addition, in the case of inhibition, after removing the inhibitor using an appropriate separation means such as an ion exchange resin, a solution containing the substrate for the physiologically active substance reaction system is added to one end of the reactor for the physiologically active substance reaction system. It can be supplied by mixing with At this time, the flow rate varies depending on the size of the reactor.

For example, from 08μe/hour to lXl0J? / time may be selected. This substrate concentration varies depending on the physiologically active substance, but if the solubility of the substrate is lower than the ATP concentration supplied from the ATP regeneration reaction system, the solubility at that substrate concentration will be the upper limit, and the solubility of the substrate will be lower than the ATP concentration supplied from the ATP regeneration reaction system. If the concentration is higher than the ATP concentration, the concentration equivalent to the ATP concentration is the highest concentration of the substrate. In the latter case, if the supplied ATP is concentrated, the synthesis reaction can be carried out at a higher concentration; however, in this case, a concentration operation is involved, resulting in a discontinuous method. The reaction product C (physiologically active substance) and AMP are separated from the eluate in the reactor using a suitable separation means, such as an ion exchange resin.

AMP is returned to one end of the reactor of the ATP regeneration reaction system.

All you have to do is regenerate it to ATP again.

Furthermore, when a physiologically active substance reaction system and an ATP regeneration reaction system are operated together in one reactor, this can be applied only when the substrates of both enzyme systems do not inhibit each other's enzyme reaction systems. The substrate concentration for both enzyme reaction systems is ATP
It is desirable to select it based on the relationship between the ATP concentration produced in the regeneration reaction system and the solubility of the substrate in the physiologically active in situ reaction system. For example, when the solubility of the substrate in the physiologically active substance reaction system is lower than ATP 8 degrees, the ATP produced in the ATP regeneration reaction system is
AMP, so as to match the TP 9 degrees to the substrate concentration.
The ATP and acetyl phosphate concentrations may be selected. Conversely, if the solubility of the substrate in the physiologically active substance reaction system is higher than the ATP concentration, it is desirable to adjust the substrate concentration to the ATP concentration generated in the ATP regeneration reaction system. A
The amount of ATP supplied together with AMP to the TP regeneration reaction system is twice, more preferably four times, even more preferably five times or more, than when using the separate reactors and connecting them. It is preferable to add. As for the flow velocity,
As with the case of separate reactors, it varies depending on the size of one reactor, but for example, from 0.8 μe/hour to 1×10
An appropriate flow rate between 'e/hour can be selected. Further, the reaction solution flowing out from one reactor is separated into AMP and physiologically active substances using an appropriate separation means, such as an ion exchange resin, as described above.

AMP can be recycled by returning it to the inlet of the reactor as a substrate.

In carrying out the present invention, the pH will vary depending on the enzyme used, but will generally be around neutral, i.e. 6-11.
．． Preferably a range of 6.5 to 9.0 is used. As the buffer solution, common ones suitable for these pHs can be used. For example, around 7, phosphates, imidazole salts, triethanolamine salts, etc. can be cited.

The temperature for carrying out the present invention is room temperature to 50°C.
You can choose from a range of When using an enzyme produced by a microorganism whose optimal growth humidity is 50°C to 85°C in the ATP regeneration reaction system, the ATP regeneration reaction system can be carried out at a higher temperature, but the temperature must be below the optimal growth temperature of 5°C. is desired.

According to the present invention, it has become possible to carry out an enzyme reaction using ATP as a cofactor continuously, extremely efficiently and economically using the above-mentioned reactor.

Therefore, it is possible to perform the binding reaction that takes place in the body as described above as a chemical industrial reaction outside the body.
This makes it possible to operate a so-called bioreactor. In particular, the fact that it has become possible to put into practical use the most important reactions in living organisms, such as protein synthesis reactions and peptide synthesis reactions using amino acid activating enzymes, is of immeasurable industrial value.

Next, the present invention will be specifically explained using examples.

Example 1 Adenylate kinase and acetate kinase were obtained from commercially available Bacillus
Specimen derived from Stearothermophilus (Seikagaku Kogyo Sales)
was used, and a commercially available yeast-derived preparation (manufactured by Boehringer Mannheim) was used as the acetyl-CoA synthase.

These eight enzymes were immobilized on Sepharose 4B as follows. That is, activated CH-Sepharose 4B (
After washing and swelling 5f (manufactured by Pharmacia).

2000 units of acetate kinase were added and reacted to obtain 1000 units of immobilized acetate kinase. Similarly, 2
From 50 units of adenylate kinase to 100 units of immobilized adenylate kinase, 100 units/)
(7) 10 units of immobilized acetyl-CoA synthetase were obtained from 7 cetyl-CoA synthase. Immobilized acetate kinase and immobilized adenylate kinase were added to an ATP regeneration column (
Fill the tube with an inner diameter of 1.6 cm and a length of 10 cm.

6mMAMP dissolved in 25mM imidazole, hydrochloric acid buffer pH 7.5 containing 10mM magnesium chloride,
08mM ATP, 25mM acetyl phosphate
The column was fed at a flow rate of e/h. The reaction temperature inside the column was maintained at 80°C. The ATP produced here was increased to 5% (0.8 mM) relative to the AMP concentration and returned to the inlet of the regeneration column.

Further, immobilized acetyl-CoA synthetase was packed into another column (inner diameter 1.0 cm + length 9 cm). As a substrate,
4mM potassium acetate + 4mM reduced COA dissolved in 100mM imidazole hydrochloride buffer, pH 7.5,
The lithium salt, 4 mM MgC112, was flowed at a flow rate of 25 me/hr.

This was mixed 1:1 with the ATP solution eluted from the ATP regeneration system and supplied to the immobilized acetyl-CoA synthase column (flow rate 50 me/hour 1 reaction temperature 37°C).
. Furthermore, AMP was fractionated from the eluate from the column using an anion exchange resin DOWEX 1-X8 (manufactured by The Dow Chemical Company), and after adjusting the pH and concentration to predetermined values,
A pump fed the ATP regeneration column. To determine the amount of acetyl-CoA produced, collect 0.05 mg of the eluate from the column and add 8 mg of 1mM 5 * 5'-dithiobis-(2-nitropenzoylic acid) (DT) (pH 6.65, phosphate buffer g). 41 after addition and 20 minutes at room temperature.
Calculation was made by quantifying unreacted SH groups based on a change in absorbance over 2 mm.

As a result, 1.5 mM acetyl-CoA was produced 80 minutes after the start of the reaction, and remained stable for the next 24 hours.

Example 2 Immobilized acetate kinase 2000 obtained in the same manner as Example 1
unit, immobilized adenylate kinase 200 units,
and 10 units of immobilized acetyl-CoA synthetase were packed into one column (inner diameter 1.8<em>% and length 12 cm).
4mMAMP dissolved in 100mM imidazole, hydrochloric acid buffer pH 7.5 containing 10mM magnesium chloride
, 25% for 1.0mM ATP CAMP concentration
), 25mM acetyl phosphate, 2.5mM potassium acetate, 2.5mM reduced CoA lithium salt at 50me
/hour flow rate. The reaction temperature in this column was set to 8
It was kept at 7°C. As in Example 1, AMP was separated from the eluate from the column and returned to the original substrate supply unit.

40 minutes after the start of the reaction, the acetyl CoA produced was 1.
The concentration reached 6mM and remained stable for the next 15 hours.

Example 8 An experiment was conducted under the same conditions as in Example 1 using a commercially available acetate kinase derived from Elysia coli (manufactured by Boehringer Mannheim) and a commercially available yeast-derived adenylate kinase (manufactured by Oriental Yeast).

As a result, acetyl Co produced 40 minutes after the start of 1 reaction
A reached +1-1 mM, but after that it gradually decreased and tended to decrease by half after 15 hours.

Example 4 Asparagine synthase was purified from Lactobacillus arabinosus ATCC8014 through ammonium sulfate fractionation, calcium phosphate gel, and gel filtration.

Using the same ATP regeneration system as in Example 1, 25 mM imidazole hydrochloric acid buffer containing 10 ml of manganese chloride, p
4mMAMP dissolved in H7,5, 0.8mM A
TP (7.5% for AMP 4 degrees) and 16mM acetyl phosphate were supplied to regenerate ATP.

In addition, 100 units of asparagine synthase was dissolved in 100mM Lis-HCl buffer containing 4mM manganese chloride and sealed in a model reactor with an internal capacity of 50me using an ultrafiltration membrane of 1 molecular weight go, oo.

This + 4mM ammonium chloride dissolved in 100mM) Lis-HCl buffer containing 4mM manganese chloride, 4mM
A from L-aspartic acid and ATP regeneration system
The TP liquid was mixed at a ratio of 1:1 and supplied at a flow rate of 25 me/hour. The reaction temperature at this time was maintained at 87°C.

AMP was separated from the eluate in the same manner as in Example 1.

Returned to ATP reproduction system. Further, the produced L-asparagine was quantified by directly applying the eluate to a high performance liquid chromatography device.

The conditions for the chromatography equipment at this time were to use a Shimadzu Solpax ODS column, and to use a 0.01M eluate.
Sodium acetate (pH 4,5)/acetonitrile (55
/45), the flow rate was 1 me/min, and the detection was 21
Absorbance was measured at 0 nm.

As a result, 80 minutes after the start of 1 reaction, 1.
The concentration was 5mM and remained stable for the next 15 hours.

Example 5 Tyrosyl-tRNA synthetase was obtained from Bacillus stearothermophilus Kouken Bacteria No. 5141.

DEAE-cellulose (manufactured by Whatman), hydroxyapatite (Seikagaku Corporation), DEAE-Sephadex (manufactured by Pharmacia) chromatography, ammonium sulfate fractionation, hydroxyapatite,
DEAE-Sephadex and Sephadex G-15
0 (manufactured by Pharmacia) and purified into a single product.

Next, in the same manner as in Example 1, AMP and catalyst amount CAMP
ATP (purity 98%) was regenerated from ATP (5% for 111 degrees) and acetyl phosphoric acid, and this was used in the following reaction.

150mf of the above purified tyrosyl-tRNA synthetase, 200mf of magnesium chloride, 51mg of ATP (
0.5 ml of L-tyrosine (purity 98%), 100 units of pyrophosphatase (manufactured by Boehringer Mannheim), 0.005 mg of dithiothreitol at 20 mM
Dissolve in Hepes buffer Tome (pH 8,0) and 4°C.
The mixture was reacted for 15 minutes to obtain a reaction mixture. L-phenylalanine methyl ester 2f was added to the obtained reaction mixture, mixed well, and kept at a reaction temperature of 80° C. for one day to react.

Next, 100 mg of acetone was added to the obtained reaction solution.

After removing the precipitate by centrifugation for 1 iJft, the supernatant was concentrated to about 10 me using an evaporator and subjected to a high performance liquid chromatography device to separate the product. The conditions for the chromatography equipment at this time are that the column is Microbontapack C.
18 (manufactured by Waters) was used.

The developing solvent was 50 mM phosphate buffer (pH 7,0)/acetone trile (85/15), and detection was performed at 210 n
The absorbance was measured at m.

As a result, 0.22 ml of L-tyrosyl-phenylalanine methyl ester was obtained. Furthermore, AMP eluted in the void fraction at the same time was further separated in the same manner as in Example 1,
ATP was regenerated by returning it to the ATP regeneration system.

Thereafter, the temperature remained stable for 24 hours.

Example 6 A crude enzyme solution (purity 1
0%).

Next, in the same manner as in Example 1, AMP and catalyst ffi(A
ATP (purity 98%) was regenerated from ATP (5% relative to MP concentration) and acetyl phosphoric acid, and this was used in the following reaction.

The above crude methionyl-tRNA synthetase If, magnesium chloride 10mf, ATP 21mg (purity 98%)
)% L-methionine, 5 units of pyrophosphatase (manufactured by Peringer Mannheim), and 20 mg of mercaptoethanol were added to 15 mg of 50 mM 2.5-dimethylimidazole buffer, pH 9.0, and reacted in the same manner as in Example 5. After that, one reaction mixture was subjected to Sephadex G-75, eluted with Hepes buffer # (pH 8,0), 80 mg of both void volumes were collected, and the reaction mixture was isolated. 0.51 L-leucine ethyl ester was added as a solid to the isolated mixture, and the mixture was reacted at 25°C for 4 hours. Acetone 80me was added to the obtained reaction product, and the resulting precipitate was removed by centrifugation.

The supernatant was concentrated to about 10 mg using an evaporator and separated in the same manner as in Example 5 to obtain 0.92 mg of L-methionyl-L-leucine ethyl ester.

Further, AMP was regenerated into ATP in the same manner as in Example 5. Thereafter, the temperature remained stable for 24 hours.

Agent Iizo Kodama

Claims (2)

[Claims] (1) (a) An enzyme that converts adenosine-5'-monophosphate to adenosine-5'-diphosphate and adenosine-5'-diphosphate;
adenosine-5'-triphosphate is produced from adenosine-5'-monophosphate in combination with an enzyme that converts diphosphate to adenosine-5'-triphosphate, and (b) the produced adenosine-5 A physiologically active substance is synthesized using '-triphosphoric acid, and then the adenosine produced by the reaction (b) is
5'-monophosphate is converted to 7denosine-5 by the reaction (a).
1. A method for producing a physiologically active substance by a complex enzyme method, characterized in that it is regenerated into '-triphosphate and the regenerated adenosine-5'-triphosphate is repeatedly used for the synthesis of the physiologically active substance (b). (2) Adenosine-5'- produced by the reaction of (b)
Monophosphate with adenosine-5'-triphosphate (a)
2. The production method according to claim 1, wherein adenosine-5'-triphosphate is regenerated by subjecting it to the reaction.