JPH0525479B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for converting AMP to ATP using adenylate kinase and acetate kinase.

In living organisms, many biosynthetic reactions are carried out using enzymes as catalysts to maintain life.
ATP is required as an energy source or a cofactor for particularly important binding reactions. At this time, ATP acts as an energy source or a cofactor and is then broken down into ADP or AMP and consumed. On the other hand, recently, attempts have been made to industrially reproduce in-vivo reactions in reactors in factories. This is a result of a review of the modern chemical industry, which has brought attention to the energy-saving and non-polluting properties of biological reactions, and it is becoming an essential technology, especially in the field of fine chemicals. It has already been successfully put into practical use in technical fields such as isomerization reactions. However, as mentioned above, the binding reaction consumes an expensive substance called ATP as an energy source or a cofactor, so it is at least economically unviable, which hinders its practical application. That is, ADP, which is the product from which ATP is consumed,
The key technology to overcome this problem is to regenerate and convert AMP, especially AMP that has been consumed at the lowest energy level, into ATP.

From this point of view, various studies have been conducted on the regenerative conversion of ATP. For example, ATP is produced in living organisms through reactions such as glycolysis, and attempts to utilize this production are known. That is, consumed using microbial cells.
There are things that regenerate and replenish ATP.
However, this method has not yet reached a practical level due to side reactions and poor conversion efficiency. on the other hand,
Attempts have also been made to utilize ATP converting enzymes, although they are not enzymes produced by microorganisms with an optimal growth temperature of 50°C to 85°C as in the present invention. For example, Whitesides et al. obtained enzymatic conversion of adenosine.
AMP is converted to ATP by conventional adenylate and acetate kinases (Journal of the American Chemical Society, vol. 100,
No. 1, p. 304, 1978). However, although these examples seem to be similar to the present invention, they require a long time for reaction, the conversion efficiency is not very high, and they cannot be used for long-term operation at the chemical industrial level. It is something.

The present inventors have conducted intensive research on converting and regenerating AMP, which is a product decomposed to the lowest energy level, into ATP.
Using the converting enzyme produced by stearothermophilus, the conversion enzyme can be used in a short period of time, with high yield, and stably for a long period of time.
They discovered that AMP can be converted to ATP and completed the present invention. The present invention will be explained in more detail below.

The present invention consists of converting AMP into ADP and converting the ADP generated here into ATP. Adenylate kinase is used as the enzyme that converts AMP to ADP, and ATP is used as the phosphate donor to AMP. Then ADP
Acetate kinase is used as the enzyme to convert ATP to ATP, and acetyl phosphate is used as the phosphate donor in this case. In this way, when using adenylate kinase and acetate kinase, ATP and acetyl phosphate are used as phosphate donors for each enzyme, but
Since ATP, which is the final converted product, can be recycled, only acetyl phosphate needs to be supplied as a phosphate donor. Another advantage of both enzymes is that such systemization allows for efficient design.

As described above, it is possible to convert AMP to ATP by using two types of converting enzymes, but this alone is not practical. That is, these enzymes need to be produced by Bacillus stearothermophilus. It also includes microorganisms that grow at room temperature into which the genes of these microorganisms have been introduced. Both enzymes obtained from this microorganism are easy to purify and have high specific activities.

In this way, the present invention was first completed by using converting enzymes produced by Bacillus stearothermophilus, but when using these enzymes, membrane reactors, column reactors, etc. are used. be able to. In the case of a membrane reactor
Since ATP is a low-molecular substance and the enzyme is a high-molecular substance, the enzyme of the present invention can be used as is. At this time, the produced ATP can flow out of the reactor due to the action of the membrane, and the enzyme can remain in the reactor due to the action of the membrane, so the conversion of AMP to ATP is continuous. It can be done. In the case of a column reactor, the enzyme used is carried in a suitable carrier, e.g. derivatives of polysaccharides such as cellulose, dextran, agarose, etc., derivatives of vinyl polymers such as polystyrene, ethylene-maleic acid copolymers, cross-linked polyacrylamide, etc. , L-alanine-L-glutamic acid copolymer, polyamino acid or polyamide derivatives such as polyaspartic acid, derivatives of inorganic substances such as glass, alumina, hydroxyapatite, etc., It can be used by filling a column as a so-called immobilized enzyme. In this case, AMP can be continuously converted to ATP by flowing the reaction solution through the column.

All of these methods are made possible or facilitated by using the enzyme produced by Bacillus stearothermophilus of the present invention. In other words, in the case of conventional enzymes in membrane-type reactors, the enzyme activity lasts only for a very short time in an unimmobilized state, making it unsustainable for continuous use, while in the case of column-type reactors, Although the duration of enzyme activity is increased by immobilization, the level of activity is extremely low compared to that of the immobilized enzyme of the present invention, and the immobilization operation is troublesome because it is easily deactivated during immobilization. There were also some difficulties. These problems could be solved all at once by using the enzyme of the present invention, and this is a noteworthy advantage achieved by the present invention.

The enzyme used in the present invention is an enzyme produced by Bacillus stearothermophilus. Therefore, common sense would have predicted that enzyme activity would not appear near room temperature, which would be extremely disadvantageous in use.
Surprisingly, it was found that AMP can be efficiently converted to ATP in an extremely short time even at room temperature.
Even more surprisingly, near the optimal growth temperature of the microorganisms that produce these enzymes, AMP is instead converted into ATP.
It has been found that it is difficult to efficiently convert the growth to 5°C, and that the optimum growth temperature is 5°C or lower. That is, in carrying out the present invention, AMP is replaced by ATP.
It is preferable to set the temperature for conversion to around room temperature to 5° C. or lower, which is the optimal growth temperature for enzyme-producing microorganisms.

In carrying out the present invention, the pH is near neutral,
That is, a range of 6.5 to 11, preferably 6.5 to 9.0, more preferably 7 to 8 is used. As the buffer solution, common ones suitable for these pHs can be used. For example, around 7, phosphate,
Examples include imidazole salts.

By using the present invention, it has become possible to efficiently and stably convert AMP to ATP for a long period of time using a reactor as described above. Therefore, it becomes possible to operate a so-called bioreactor in which the binding reaction that is carried out in the body as mentioned above is carried out as a chemical industrial reaction outside the body. For example, the most important reactions in living organisms, such as protein synthesis reactions and peptide synthesis reactions by amino acid activating enzymes, require ATP as an energy source, and the expensive ATP used in these cases is
Since it is consumed and degraded into AMP, it is practically essential to convert and regenerate AMP into ATP when performing this reaction in vitro. According to the present invention, it becomes possible to put such a reaction into practical use, and its value is immeasurable in industry.

In addition, as above, the desired synthesis reaction and ATP
When combining this with a regeneration reaction, a system is also required to separate the produced AMP from the target product, the reaction product of the phosphate donor, etc. This will be made possible through technologies such as chromatography, and will be completed as a system that combines the desired reaction system, ATP regeneration system, and AMP separation system. In addition, at this time, if ATP is made into a water-soluble polymer, AMP (in this case, water-soluble polymerized AMP)
It is also possible to omit or simplify the separation system.

Although the present invention can be applied extremely advantageously to the recycling of ATP in this way, from a different perspective,
It can also be seen as a method for producing ATP using AMP as a raw material. In other words, ATP is an important substance as a medicine and is produced at an industrial level. However, the current fermentation method has problems such as easy formation of by-products and poor productivity, and ATP
The price also had to rise. However, according to the present invention, it is possible to eliminate such problems. The present invention also includes such objects.

Although described in detail above, the present invention will now be described in more detail with reference to Examples.

Example 1, Comparative Example 1 0.5mM ATP, 0.5mM AMP, 1.2mM acetyl phosphate, 0.04% sodium azide and 10mM
Bacillus stearothermophilus strain UK788 (Feikoken Bacterial Serial No. 5141,
1000 units and 200 units of acetate kinase and adenylate kinase, respectively, obtained from the optimal growth temperature (60°C) were dissolved and placed in a 200 ml reaction glass vessel. While stirring, this solution was pumped using a peristaltic pump into a hollow fiber eyelid with a molecular weight cutoff of 10,000 (H1P10 type manufactured by Amicon), and the solution that passed through the hollow fiber eyer was returned to the reaction glass container. The partially filtered solution is received in another container, and 0.5mM ATP, 0.5mM AMP,
50mM imidazole-hydrochloric acid buffer containing 1.2mM acetyl phosphate, 0.04% sodium azide and 10mM magnesium chloride, PH7.5 (substrate solution) was replenished into the reaction glass container (Amicon hollow fiber system DC2 type or CH4 ). In this example, the flow rate of the reaction solution to the hollow fiber eyebar was 2 liters per hour, and the overrate and substrate solution replenishment rates were 200 ml per hour.
The operation temperature was room temperature for 7 days. From AMP
The conversion rate to ATP was quantitatively analyzed by high performance liquid chromatography using a PNH ₂ -10 packed column. (Example 1) Separately, Escherichia coli (Escherichia coli,
Similar experiments were conducted using acetate kinase (manufactured by Boehringer Mannheim) with an optimal growth temperature of 37°C and adenylate kinase from pig muscle of non-microbial origin (manufactured by Boehringer Mannheim). (Comparative Example 1) As a result, in Example 1, after only 10 minutes
ATP conversion rate reaches 98% and after 7 days
The ATP conversion rate was also maintained at 98%. On the other hand, in Comparative Example 1, the ATP conversion rate reached 72% after 10 minutes, but the conversion rate gradually decreased, and after 5 days it almost reached 72%.
ATP conversion rate has become 0%.

Example 2 Activated CH-Sepharose 4B (manufactured by Pharmacia) 5g was added to 1mM hydrochloric acid 1000ml on a glass filter.
After repeating washing and swelling three times with 50mM boric acid buffer, 100ml of 50mM borate buffer, PH8.3, Bacillus.
Add 1 ml of the above buffer solution and 5000 units of acetate kinase obtained from Stearothermophilus strain NCA1503 (Feikoken Bacteria No. 4778, optimum growth temperature 60°C), and gently shake at 40°C. Reacted for 30 minutes. Pass the reaction mixture through a glass filter,
Then, the obtained immobilized acetate kinase complex was
50mM Tris-HCl buffer containing 0.5MKCl, PH
8.0, 3 times with 200 ml each, and further washed 3 times with 200 ml each of 25 mM imidazole-hydrochloric acid buffer, pH 7.5. The units of immobilized acetate kinase were 2500 units. When the above procedure was repeated using 500 units of adenylate kinase instead of acetate kinase, a complex with 200 units of adenylate kinase immobilized was obtained. Both these complexes were packed into a glass column with an inner diameter of 2 cm and a length of 10 cm.
Contains 0.5mM ATP, 0.5mM AMP, 1.2mM Acetyl Phosphate and 10mM Magnesium Chloride
50mM imidazole-hydrochloric acid buffer, PH7.5, 200
The liquid was passed from the top of the column at a flow rate of ml/hour. The temperature of the column and the feed solution to the column are both 37
It was kept at ℃. When the ATP concentration in the reaction solution eluted from the bottom was quantified by high-performance liquid chromatography, AMP was no longer detected 15 minutes after the solution was passed through the column, and the concentration was 98.2% ATP and 1.8%.
of ADP was detected.

Claims (1)

[Scope of Claims] 1. A method for converting adenosine monophosphate to adenosine triphosphate, characterized by carrying out the following steps simultaneously and in succession. (a) A step of introducing a reaction solution containing adenosine monophosphate and a phosphate donor into a reactor in which Bacillus stearothermophilus-derived acetate kinase and Bacillus stearothermophilus-derived adenylate kinase are present ( b) converting adenosine monophosphate to adenosine triphosphate by the action of the acetate kinase and the adenylate kinase; and (c) draining the reaction solution containing adenosine triphosphate from the reactor. 2 The step (c) is a step in which the reaction solution containing adenosine triphosphate is filtered through an ultrafiltration membrane, the solution that does not pass through the filtration membrane is returned to the reactor, and only the solution that passes through the filtration membrane is discharged. A conversion method according to claim 1. 3 The reactor in step (a) is a column filled with Bacillus stearothermophilus-derived acetate kinase immobilized on a carrier and Bacillus stearothermophilus-derived adenylate kinase immobilized on a carrier. A conversion method according to claim 1.