JPH0426837B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides adenosine-5'-triphosphate (hereinafter referred to as
It's called ATP. ) as a cofactor for the production of physiologically active substances by enzymatic reactions.

In recent years, due to a review of the current chemical industry, attention has been focused on chemical reactions in living organisms, and many attempts have been made to reproduce chemical reactions in 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. Because 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 the binding reaction, which is a particularly important reaction among biosynthetic reactions, ATP is required as an energy source or a cofactor. At this time, ATP acts as an energy source or a cofactor, and then adenosine-5'-diphosphate (hereinafter referred to as ATP)
It's called ADP. ) or adenosine-5'-monophosphate (hereinafter referred to as AMP), and is consumed. 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, the important technology to overcome this problem is to regenerate and convert ADP and AMP, which are the forms after consumption of ATP, especially AMP that has been consumed at the lowest energy level, into ATP.

However, there are not many known examples of producing substances through such binding reactions while regenerating ATP. For example, JP-A-54-
The only known information is Publication No. 122793. This causes γ to glutamic acid, cysteine, and glycine.
- ATP consumed when glutathione is synthesized by the action of glutamylcysteine synthetase and glutathione synthase is recycled and reused from ADP, which is the form after being consumed by the action of acetate kinase. It does not provide knowledge about regenerating and converting AMP, which has been consumed at the lowest energy level mentioned above, into ATP. On the other hand, there is also the idea of regenerating or replenishing consumed ATP using the glycolytic system of microorganisms, and there are examples of using AMP as a raw material for ATP (for example, Tatsuroku Tochikura et al., Synthetic Organic Chemistry, Volume 39, No. 6, Page 487, 1981). However, this AMP
It is not the figure after ATP is consumed, but it is added separately as an ATP source, and the result of this attempt is
The conversion efficiency of AMP to ATP was extremely poor (ibid.). In other words, even when using the glycolytic system of microorganisms, there was only negative speculation that ATP would be regenerated from AMP, which is the form after consumption.

Conventionally, from this perspective, the concept of a reactor (bioreactor) for synthesizing useful substances by continuously consuming ATP to AMP was considered, and the completion of this system was strongly desired.

In view of these actual circumstances, the present inventors particularly focused on AMP, which is a product decomposed to the lowest energy level.
As a result of intensive research on converting and regenerating ATP into ATP, we found that using a converting enzyme produced by microorganisms whose optimal growth temperature is 50℃ to 85℃, it can be done in a short time.
We discovered that AMP can be converted to ATP with high yield, and that it is possible to regenerate AMP into ATP by controlling the mixing ratio of AMP and ATP under specific conditions, at low cost,
We discovered that it is easy to operate and can convert virtually 100% of AMP into ATP stably over a long period of time.
As a result of further investigation, based on this knowledge, (a) a reaction system that regenerates AMP to ATP, and (b)
By linking ATP to an enzymatic reaction system that synthesizes physiologically active substances, we discovered that physiologically active substances could be synthesized from AMP, which is a product broken down to the lowest energy level, as a raw material, and completed the present invention. .

That is, the present invention provides (a) an enzyme that converts adenosine-5'-monophosphate into adenosine-5'-diphosphate;
adenosine-5'-triphosphate is produced from adenosine-5'-monophosphate in combination with an enzyme that converts it to 5'-triphosphate, and (b) the produced adenosine-
A physiologically active substance is synthesized by an enzymatic reaction using 5'-triphosphate, and then the adenosine-5'-monophosphate produced in the reaction (b) is converted into adenosine-5 by the reaction (a). Production of 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 a physiologically active substance by the enzymatic reaction (b). It is the law.

In the present invention, the enzymatic reaction (hereinafter referred to as a physiologically active substance reaction system) that uses ATP to synthesize a physiologically active substance is mainly the binding reaction described above. Reaction to synthesize peptides and peptide derivatives by t-RNA synthetase, acetyl-CoA from acetic acid or fatty acid and CoA
Reaction to synthesize acetyl-CoA or acyl-CoA using synthetic enzyme or acyl-CoA synthetase, reaction to synthesize L-pantothenic acid from pantoic acid and β-alanine using pantothenic acid synthase, guanylate synthase from xanthylic acid and ammonia or glutamine reaction to synthesize guanylic acid from aspartic acid and ammonia, reaction to synthesize asparagine using asparagine synthetase from aspartic acid and ammonia, reaction to synthesize acyl-CoA from carboxylic acid and CoA using butyryl-CoA synthetase, and reaction to synthesize acyl-CoA from D-alanine and poly(ribitol phosphate). By D-alanyl-poly(ribitol phosphate) synthase,
Examples include a reaction to synthesize O-D-alanyl-poly(ribitol phosphate) and a reaction to synthesize NAD ⁺ from deamide NAD ⁺ and L-glutamine using NAD ⁺ synthetase.

In the present invention, in the physiologically active substance reaction system,
ATP is consumed and becomes AMP, but
This AMP is converted to ATP by combining an enzyme that converts to ADP and an enzyme that converts to ATP (hereinafter referred to as the ATP regeneration reaction system). This AMP
For example, adenylate kinase is used as the enzyme that converts into ADP, and in this case, ATP is used as the phosphate donor to AMP. Also,
Many enzymes can be used to convert ADP to ATP, such as acetate kinase, carbamate kinase, creatine kinase, 3-phosphoglycerate kinase, pyruvate kinase, and polyphosphate kinase, but the price of the phosphate donor Considering the conversion activity to ATP, the 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 acertyphosphate are used as phosphate donors for each enzyme.
Since ATP, which is the final converted product, can be recycled, only acetyl phosphate needs to be supplied as a phosphate 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 generated AMP into ATP by using two types of converting enzymes, but both of these enzymes are produced by microorganisms whose optimal growth temperature is 50°C to 85°C. This is desirable. Such microorganisms include microorganisms of the genus Bacillus such as Bacillus stearothermophilus, Bacillus brevis, Bacillus coagulans, Bacillus thermoproteoliticus, Bacillus acidocaldarius, microorganisms of the genus Clostridium, and Thermoactinomyces. microorganisms of the genus Achromobacter, microorganisms of the genus Streptomyces, microorganisms of the genus Micropolispor, microorganisms of the genus Thermus such as Thermus aquatix, Thermus thermophilus, Thermus flavus, microorganisms of the genus Thermomicrobium; Examples include microorganisms and microorganisms of the genus Calderia. It also includes microorganisms that grow at room temperature into which the genes of these microorganisms have been introduced. Among these microorganisms, Bacillus stearothermophilus is particularly suitable for producing both adenylate kinase and acetate kinase enzymes. Both enzymes obtained from this microorganism are easy to purify and have high specific activities. At the beginning of the research, it was thought that using converting enzymes produced by microorganisms whose optimal growth temperature is 50℃ to 85℃ would not be suitable for ATP regeneration at room temperature, but surprisingly, it was possible to reproduce ATP at room temperature. Stable for a long time with high yield
It can convert AMP to ATP, and these effects far exceed those of enzymes produced by normal-blooded organisms.

In order to synthesize a physiologically active substance using the above-mentioned physiologically active substance reaction system using AMP as a raw material in the present invention, it is first necessary to construct a reactor. Examples of such a reactor include, for example, a membrane reactor, Column type reactors can be used. A membrane reactor can be used particularly effectively when the physiologically active substance is a low molecular weight substance. At this time, since enzymes are polymeric substances, each enzyme can be used while remaining in the reactor. The generated AMP is a low-molecular substance, so it flows out of the reactor, but it can be easily separated from physiologically active substances using ion exchange chromatography and other procedures, and then returned to the reactor to be regenerated into ATP. is possible. Furthermore, if a so-called water-soluble polymerized ATP, in which a suitable spacer is introduced into ATP in advance and bound to a water-soluble polymer substance, is used, such a separation operation becomes unnecessary. In this case, water-soluble polymer substances include, for example, polysaccharides such as soluble dextran, vinyl polymer derivatives such as polyacrylamide derivatives and polyacrylic acid derivatives,
Various types of 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 may be supported in a suitable carrier, for example, a derivative of a polysaccharide such as cellulose, dextran, agarose, etc., a vinyl polymer such as polystyrene, ethylene-maleic acid copolymer, cross-linked polyacrylamide, etc. derivatives, derivatives of polyamino acids or polyamides such as L-alanine L-glutamic acid copolymers, polyaspartic acid, etc., glass,
It may be used by binding, entrapping or adsorbing it to inorganic derivatives such as alumina, hydroxyapatite, etc. and filling it in a column as a so-called immobilized enzyme. In this reactor, the produced
AMP, whether polymerized or non-polymerized, flows out of the reactor, but it can be separated from the physiologically active substance and returned to the reactor in the same manner as described above. Also,
Since water-soluble polymerized ATP can be separated through a membrane, the separation operation can be simplified.

Although the above-mentioned reactor has been explained on the assumption that it will be operated continuously, other reactors can be devised based on this idea, and in some cases, batch-type reactions may also be possible. It may also be operated in batches using a container.

In the present invention, the physiologically active substance reaction system and ATP
It is also possible to use the regeneration reaction system and the above-mentioned reactor separately, and operate these by connecting them,
It is also possible to operate the physiologically active substance reaction system and the ATP regeneration reaction system by making them coexist in the same reactor as described above, but in order to efficiently synthesize physiologically active substances, it is necessary to It is desirable to provide the AMP produced in the ATP regeneration reaction system together with ATP. At this time, it is desirable that the ratio of amounts of AMP to ATP be within the range shown by the following formula.

0.15×5×γ ² /γ ² ×AMP≧ATP≧0.04 ×5＋γ ² /γ ² ×AMP (a) However, AMP is the concentration of AMP (mM), and ATP is
It represents the concentration of ATP (mM), and γ is the ratio of the immobilized enzyme activity of the enzyme that converts ADP to ATP and the immobilized enzyme activity of the enzyme that converts AMP to ADP, and represents a positive number of 1 or more.

The apparatus for carrying out the above ATP regeneration reaction system is one that is equipped with a substrate supply section, a reaction tank containing the above two types of converting enzymes, an automatic sampling section, an analyzer, 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 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 the analyzer. The analyzer is AMP,
The control unit calculates each concentration of ADP and ATP, and based on this value, controls the operation of the variable amount liquid feeding device and automatic control valve, and supplies AMP and ATP to the ATP regeneration reaction system.
and the amount of acetyl phosphate 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, in the reactor of the ATP regeneration reaction system,
AMP 0.1μM to 4M, preferably 1μM to 2M, more preferably 10μM to 500mM, acetyl phosphate 0.1μM to 500mM, preferably 1μM to 400mM,
More preferably, 10 μM to 300 mM, and as shown in equation (a), ATP to AMP is supplied together with AMP from one end of the reactor, depending on the amount ratio of the ATP regeneration reaction system, but preferably 4% or more. However, the conversion of AMP→ADP→ATP can be carried out in the reactor. The reaction solution eluted from the reactor is analyzed using an appropriate analysis system, and AMP, ADP,
Each concentration of ATP and the conversion rate to ATP can be determined. At this time, the flow rate varies depending on the size of the reactor, but an appropriate flow rate can be selected, for example, from 0.3 μ/hour to 1×10 ⁶ /hour.
A portion of this reaction solution (ATP concentration is 4% of AMP concentration)
The above is desirable. ) is circulated back to the reactor inlet,
Stop the supply of ATP liquid that was initially supplied.
If most of the remaining reaction solution does not inhibit the physiologically active substance reaction system, such as acetic acid, other than ATP in the reaction liquid, adjust the concentration and directly transfer the physiologically active substance to the reactor for the physiologically active substance reaction system. When supplying or inhibiting the reaction system by mixing it with the substrate,
After removing inhibitors using an appropriate separation means such as an ion exchange resin, the inhibitor may be mixed with a solution containing a substrate for the physiologically active substance reaction system and supplied to one end of the reactor for the physiologically active substance reaction system. At this time, the flow rate varies depending on the size of the reactor, but for example,
An appropriate flow rate between 0.3 μ/hour and 1×10 ⁶ /hour may be selected. This substrate concentration differs depending on the physiologically active substance, but the solubility of the substrate is
If the ATP concentration supplied from the regeneration reaction system is lower than the solubility of the substrate, the upper limit is the solubility of the substrate, and if the solubility of the substrate is higher than the ATP concentration supplied, the solubility of the substrate is the same as the ATP concentration. The highest concentration of In the latter case, if the supplied ATP is concentrated, the synthesis reaction can be carried out at a higher concentration, but in this case, a concentration operation is involved, resulting in a discontinuous method. The reaction product (physiologically active substance) and AMP are separated from the eluate in the reactor using an appropriate separation means, such as an ion exchange resin, and the AMP is returned to one end of the reactor of the ATP regeneration reaction system and converted to ATP again. Just play it.

Furthermore, when operating a physiologically active substance reaction system and an ATP regeneration reaction system in coexistence in one reactor, this can be applied only when the substrates of both enzyme systems do not mutually inhibit the enzyme reaction system. The substrate concentration for both enzyme reaction systems is that generated in the ATP regeneration reaction system.
It is desirable to select it based on the relationship between ATP concentration and the solubility of the substrate in the physiologically active substance reaction system. For example,
When the solubility of the substrate in the physiologically active substance reaction system is lower than the ATP concentration, it is generated in the ATP regeneration reaction system.
AMP, so as to match the ATP concentration to the substrate concentration.
The concentration of ATP and acetyl phosphate can 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. The amount of ATP supplied together with AMP to the ATP regeneration reaction system is twice as much as when using separate reactors as described above and when these are connected.
It is more preferable to add 4 times, and still more preferably 5 times or more. The flow rate varies depending on the size of the reactor, as in the case of separate reactors, but for example, from 0.3 μ/hour to 1×10 ⁶
/ time can be selected. Also,
As mentioned above, the reaction solution flowing out of the reactor is separated into AMP and physiologically active substances using an appropriate separation means such as an ion exchange resin, and the AMP is returned to the inlet of the reactor as a substrate and reused. can do.

In carrying out the present invention, the pH used varies depending on the enzyme used, but is generally in the vicinity of neutrality, that is, in the range of 6 to 11, preferably 6.5 to 9.0. 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 can be selected from room temperature to 50°C. If an enzyme produced by a microorganism whose optimal growth temperature is between 50°C and 85°C is used in the ATP regeneration reaction system, the ATP regeneration reaction system can be run at a higher temperature, but the temperature must be below the optimal growth temperature of 5°C. is desired.

According to the present invention, using a reactor as described above,
It has become possible to carry out enzymatic reactions using ATP as a cofactor continuously, extremely efficiently and economically. Therefore, it has become possible to operate a so-called bioreactor, which performs the binding reaction that takes place in the body as a chemical industrial reaction outside the body, as described above. 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 For adenylate kinase and acetate kinase, commercially available preparations derived from Bacillus stearothermophilus (Seikagaku Corporation) were used, and for acetyl-CoA synthase, a commercially available preparation derived from yeast (manufactured by Boehringer Mannheim) was used. It was used.

These three types of enzymes are made into cepharose as follows.
It was fixed on 4B. That is, after washing and swelling 5 g of activated CH-Sepharose 4B (manufactured by Pharmacia),
2000 units of acetate kinase were added and reacted to obtain 1000 units of immobilized acetate kinase. Similarly, 100 units of immobilized adenylate kinase were obtained from 250 units of adenylate kinase, and 10 units of immobilized acetyl-CoA synthase were obtained from 100 units of acetyl-CoA synthase. Immobilized acetate kinase and immobilized adenylate kinase were packed into an ATP regeneration column (inner diameter 1.6 cm, length 10 cm), and 6mMAMP dissolved in 25mA imidazole hydrochloride buffer PH7.5 containing 10mM magnesium chloride;
0.3mM ATP, 25mM acetyl phosphate was fed to the column at a flow rate of 25ml/hr. The reaction temperature inside the column was maintained at 30°C. The ATP generated here is AMP
Only 5% (0.3mM) of the concentration was returned to the regeneration column inlet.

Further, immobilized acetyl-CoA synthetase was packed into another column (inner diameter 1.0 cm, length 9 cm). As substrates, 100mM imidazole hydrochloride buffer, 4mM potassium acetate dissolved in PH7.5, 4mM reduced CoA,
The lithium salt, 4mM _MgCl2, was flowed at a flow rate of 25ml/hr and eluted from the ATP regeneration system.
Mix 1:1 with ATP solution and immobilize acetyl
It was supplied to a CoA synthetase column (flow rate 50 ml/hour, reaction temperature 37°C). Furthermore, the anion exchange resin Dowex 1-X8 (the
(manufactured by Dow Chemical Company) to separate AMP,
After adjusting the pH and concentration to predetermined values, it was supplied to the ATP regeneration column using a pump. Acetyl CoA produced
Collect 0.05ml of the eluate from the column,
Add it to 3 ml of 1mM 5,5'-dithiobis-(2-nitrobenzoylic acid) (DTNB) (PH6.65, phosphate buffer) and quantify the unreacted SH group by the change in absorbance at 412 mm after 20 minutes at room temperature. Calculated.

As a result, 1.5mM acetyl-CoA was produced 30 minutes after the start of the reaction, and remained stable for the next 24 hours.

Example 2 Immobilized acetate kinase obtained in the same manner as Example 1
2000 units, immobilized adenylate kinase 200 units, and immobilized acetyl-CoA synthetase 10 units in one column (inner diameter 1.8 cm, length 12 cm)
Contains 10mM Magnesium Chloride
Dissolved in 100mM imidazole hydrochloride buffer PH7.5
4mM AMP, 1.0mM ATP (25% relative to AMP concentration)
%), 25mM acetyl phosphate, 2.5mM potassium acetate, and 2.5mM reduced CoA lithium salt were supplied at a flow rate of 50ml/hour. The reaction temperature in this column is 37
It was kept at ℃. AMP was separated from the eluate from the column in the same manner as in Example 1 and returned to the original substrate supply section.

40 minutes after the start of the reaction, the acetyl CoA produced
The concentration reached 1.6mM and remained stable over the next 15 hours.

Example 3 Acetate kinase derived from commercially available Escherichia coli (manufactured by Boehringer Mannheim) and adenylate kinase derived from commercially available yeast (manufactured by Oriental Yeast)
An experiment was conducted under the same conditions as in Example 1.

As a result, the acetyl-CoA produced 40 minutes after the start of the reaction reached 1.1mM, but after that it gradually decreased to 15mM.
It tended to decrease by half after hours.

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

Using the same ATP reproduction system as in Example 1,
4mMAMP dissolved in 25mM imidazole hydrochloride buffer, PH7.5 containing 10mM manganese chloride;
0.3mMATP (7.5% relative to AMP concentration) and 16mM acetyl phosphate were supplied to regenerate ATP.

Furthermore, 100 units of asparagine synthase was dissolved in 100 mM Tris-HCl buffer containing 4 mM manganese chloride, and the solution was sealed in a membrane reactor with a capacity of 50 ml using an ultrafiltration membrane with a molecular weight of 30,000.

This was combined with 4mM ammonium chloride dissolved in 100mM Tris-HCl buffer containing 4mM manganese chloride;
Mix 4mML-aspartic acid and ATP solution from the ATP regeneration system at a ratio of 1:1, and adjust the flow rate to 25ml/1:1.
Supplied on time. The reaction temperature at this time was maintained at 37°C. AMP was separated from the eluate in the same manner as in Example 1 and returned to the ATP regeneration 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 used were a Shimadzu Zorbax ODS column, an eluent of 0.01 M sodium acetate (PH4.5)/acetonitrile (55/45), a flow rate of 1 ml/min, and detection at 210 nm. This was done using absorbance.

As a result, 1.5mM of L-asparagine was produced 30 minutes after the start of the reaction, and remained stable for the next 15 hours.

Example 5 Tyrosyl-tRNA synthetase was obtained from Bacillus stearothermophilus microtechnological research No. 5141.
DEAE-Cellulose (manufactured by Watmann), Hydroxyapatatite (Seikagaku Corporation), DEAE-
It was purified into a single product through chromatography using Cephadex (manufactured by Pharmacia), ammonium sulfate fractionation, hydroxyapatite, DEAE-Sephadex and Cephadex G-150 (manufactured by Pharmacia).

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

150 mg of the above purified tyrosyl-tRNA synthetase, 200 mg of magnesium chloride, 51 mg of ATP (98% purity),
0.5 mg of L-tyrosine, 100 units of pyrophosphatase (manufactured by Boehringer Mannheim), and 0.005 mg of dithiothreitol in 20 mM PES buffer.
The mixture was dissolved in 70 ml (PH8.0) and reacted at 4°C for 15 minutes to obtain a reaction mixture. The resulting reaction mixture was
2 g of phenylalanine methyl ester was added, mixed well, and the reaction temperature was maintained at 30° C. and allowed to react for one day.

Next, 100ml of acetone was added to the resulting reaction solution,
After removing the precipitate by centrifugation, the supernatant was concentrated to about 10 ml using an evaporator and applied to a high performance liquid chromatography device to separate the product. The conditions for the chromatography apparatus at this time were to use Microbonta Pack C ₁₈ (manufactured by Waters) as the column and 50mM phosphate buffer (PH7.0)/acetone trile (85/15) as the developing solvent. Detection was done by absorbance at 210 nm.

As a result, 0.22 mg of L-tyrosyl-L-phenylalanine methyl ester was obtained. At the same time, AMP eluted into the void fraction was further separated in the same manner as in Example 1 and returned to the ATP regeneration system.
Reproduced ATP.

Example 6 Methionyl-tRNA synthetase was obtained as a crude enzyme solution (purity 10%) from commercially available baker's yeast (manufactured by Oriental Yeast Co., Ltd.) by cellulose phosphate (manufactured by Watmann) column chromatography.

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

1 g of the above crude methionyl-tRNA synthetase, 10 mg of magnesium chloride, 21 mg of ATP (98% purity), 0.5 mg of L-methionine, 5 units of pyrophosphatase (manufactured by Boehringer Mannheim) and 20 ml of mercaptoethanol were added to 50 mM 2,5-dimethylimidazole. In addition to 15 ml of buffer pH 9.0, Example 5
After reacting in the same manner as above, the reaction mixture was subjected to Sephadex G-75, eluted with Hepes buffer (PH8.0), and 30 ml of void volume fractions were collected to isolate the reaction mixture. 0.5 g of L-leucine ethyl ester was added as a solid to the isolated mixture, and the mixture was reacted at 25° C. for 4 hours. After adding 30 ml of acetone to the obtained reaction product and removing the resulting precipitate by centrifugation, the supernatant was concentrated to about 10 ml using an evaporator and separated in the same manner as in Example 5 to obtain L-methionyl-L-leucine ethyl. 0.92 mg of ester was obtained.

In addition, it was regenerated into AMP and ATP in the same manner as in Example 5.

Claims (1)

[Scope of Claims] 1 (a) Adenosine-5'-monophosphate
An enzyme that converts 5'-diphosphate and adenosine-
producing adenosine-5'-triphosphate from adenosine-5'-monophosphate in combination with an enzyme that converts 5'-diphosphate to adenosine-5'-triphosphate;
(b) Synthesize a physiologically active substance by enzymatic reaction using the generated adenosine-5′-triphosphate, and then (b)
The adenosine-5'-monophosphate produced by the reaction (a) is regenerated into adenosine-5'-triphosphate, and the regenerated adenosine-5'-triphosphate is converted into (b).
1. A method for producing a physiologically active substance by a complex enzyme method, characterized in that it is repeatedly used for the synthesis of a physiologically active substance by an enzymatic reaction. 2. A patent claim in which adenosine-5'-monophosphate produced by the reaction in (b) is subjected to the reaction in (a) together with adenosine-5'-triphosphate to regenerate it into adenosine-5'-triphosphate. The manufacturing method according to item 1.