JPH05153993A - Production of diadenosine polyphosphate - Google Patents

Abstract

PURPOSE:To extremely raise enzyme action and to obtain diadenosine polyphosphate industrially and advantageously in producing diadenosine polyphosphate by using a specific enzyme by carrying out the reaction in the presence of an organic solvent. CONSTITUTION:An amino acid, ATP, ADP, ppppA, etc., are subjected to enzyme reaction in the presence of maximum 100mM (preferably 10nM-1mM) of RNA synthase of aminoacyl (preferably isoleucyl tRNA synthase) in an organic solvent (methanol or ethanol having about 50% concentration range) in a tris buffer solution to give objective diadenosine polyphosphate.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing diadenosine polyphosphate using an aminoacyl-tRNA synthetase, and more particularly to a method for industrially advantageous production of diadenosine polyphosphate.

[0002]

2. Description of the Related Art Diadenosine polyphosphate (hereinafter referred to as Apn
It is abbreviated as A. ), As described by J. Luthje in the Klinische Wochenschrift, 67, 17-327 (1989) magazine, the effect on DNA synthesis, the effect on blood coagulation,
It is a substance that has various physiological actions such as action on blood vessels and is expected to be used as a drug and a drug raw material.

As a method for obtaining this ApnA, adenosine triphosphate (hereinafter abbreviated as ATP) and amino acid, and optionally adenosine diphosphate (hereinafter abbreviated as ADP) and adenosine are catalyzed by an aminoacyl-tRNA synthetase. A method of reacting with tetraphosphoric acid (hereinafter abbreviated as ppppA) or the like to synthesize ApnA is known. An example of this is the European Journal of Biochemistry [Eur.J.Biochem., 12
6,135-142 (1982)] in Ottmer Goerlich (Ottm
ar GOERLICH) et al. describe a method for synthesizing Ap3A and Ap4A using an aminoacyl-tRNA synthetase. In addition, biochemistry [Biochemistry, 21, 5273-5279
(1982)] Pierre Plateau in the magazine
, ATP ₅ mM, MgCl ₂ 10 mM, isoleucine 2.5 mM, ZnCl ₂ 0.1 mM, Tris-
To an aqueous solution containing 20 mM HCl (pH 7.8), 10-
10,000 nM (1.2u / ml-1,200u / m
Aminoacyl-tRNA synthetase is added so that it becomes 1), and the mixture is reacted at 37 ° C. for 10 hours.
A method of obtaining A has been reported. All of these conventional methods carry out the synthesis reaction of ApnA in an aqueous solution containing a buffer such as Tris buffer.

[0004]

In the conventional method as described above, the catalytic activity of aminoacyl-tRNA synthetase is low, and as a result, the yield of ApnA per unit enzyme is low, which is an obstacle to implementation on an industrial scale. It was. An object of the present invention is to provide a method capable of enhancing the catalytic properties of aminoacyl-tRNA synthetase and efficiently producing ApnA by devising a reaction system.

[0005]

Means for Solving the Problems As a result of intensive studies to solve such problems, the present inventors have found that an organic solvent can be used and reached the present invention.

That is, the gist of the present invention is a method for producing ApnA, which is characterized in that an organic solvent is allowed to coexist when producing ApnA using an aminoacyl-tRNA synthetase.

The present invention will be described in detail below. In the production method of the present invention, the conventional method for synthesizing ApnA described above can be used except that an organic solvent is allowed to coexist.
That is, aminoacyl-tRNA synthetase and ATP, AD
P, ppppA, amino acids, and magnesium, manganese whose main purpose is to facilitate the reaction and suppress the inactivation of the enzyme,
It may be carried out by mixing a divalent cation such as zinc and the like, and further coexisting with an organic solvent.

The aminoacyl-tRNA synthetase used in the present invention may be any aminoacyl-tRNA synthetase as long as it can synthesize ApnA, and it can be applied regardless of the specificity or origin of the enzyme. Among them, isoleucyl tR
It is particularly preferable to use NA synthase, leucyl tRNA synthetase, lysyl tRNA synthetase, phenyl tRNA synthetase, phenylalanyl tRNA synthetase, ceryl tRNA synthetase or histidyl tRNA synthetase because of high enzymatic activity.

The amount of aminoacyl-tRNA synthetase used varies depending on the time required to complete the synthesis. The concentration at the time of use is not particularly limited, and the maximum is 100 mM (1.2 x 10
^It can be used in the range of up to ⁶ u / ml). Usually 1n
M-2 mM (1.2 u / ml-2.4 × 10 ³ u / m
l), and more practically, 10 nM to 1 mM (12 u /
It is preferable to use in the range of (ml to 1.2 × 10 ³ u / ml).

These aminoacyl-tRNA synthetases are mainly obtained by extraction and purification from E. coli, Bacillus stearothermophillus, yeast and the like. , ApnA
The origin and type of the aminoacyl-tRNA synthetase are not particularly limited as long as they are capable of synthesizing. Practically, it is preferable to use those derived from Escherichia coli or Bacillus stearothermophilus from which an enzyme is easily obtained.

The manufacturing method of the present invention requires addition of ATP as a raw material as another component. The concentration of ATP used is 1 μM or more, and practically 1 mM or more. Furthermore, ADP, ppppA, etc. may be added, if necessary. These concentrations are 1 μM or more, and practically 1 mM or more.

Further, in order to smoothly proceed the reaction,
Pyrophosphatase can be added, and metal ions such as magnesium ion, manganese ion, calcium ion, cobalt ion, and cadmium ion, and amino acid can be added.

The buffer solution is one that dissolves amino acids, aminoacyl-tRNA synthetase, ATP, ADP, ppppA, etc., maintains the enzymatic activity, is mixed with an organic solvent, and has a predetermined pH. Any material may be used as long as it is available. As such a concrete example,
Examples thereof include Good's buffer such as Tris buffer and Hepes buffer, imidazole buffer, triethanolamine buffer, acetate buffer, phosphate buffer and the like. pH
Is pH 3 to pH 10, preferably pH 4 to p
H9, optimally pH 5 to pH 8.

As the organic solvent used in the present invention, any organic solvent may be used, and among them, alcohols or polyhydric alcohols are particularly desirable. Furthermore, methanol, ethanol, propanol, ethylene glycol, polyethylene glycol, glycerol and the like are particularly preferable.

The concentration range of the organic solvent used in the reaction solution is preferably 2% to 90%, more preferably 20% to 60%, most preferably about 50%.

The production method of the present invention is carried out by allowing the above-mentioned components to coexist in the same reactor, for example. The reactor used at this time may be any one as long as it can smoothly proceed the reaction, and the size and shape of the reactor are selected depending on the amount of enzyme, the concentration of the substrate solution, the pH, the reaction temperature and the like. do it. As the shape of such a reactor, for example, a batch reactor, a membrane reactor, a column reactor or the like can be used.

The enzyme to be used may be a suitable carrier, for example, a derivative of polysaccharide such as cellulose, dextran, agarose, etc., a derivative of vinyl polymer such as polystyrene, ethylene-maleic acid copolymer, cross-linked polyacrylamide, etc. L-alanine, L-glutamic acid copolymer, polyamino acid or amide derivative such as polyaspartic acid, inorganic material derivative such as glass, alumina, hydroxyapatite, etc. are bound, entrapped or adsorbed, so-called immobilization It can also be used as an enzyme.

Next, the reaction method will be described, taking the case of producing ApnA in a batch type reactor as an example. ATP, ADP, ppppA, amino acid, metal ion, buffer solution and the like are added to the reaction tank in the above concentration range, and further aminoacyl-tRNA synthetase is added in the above concentration range. Stirring may be performed to smoothly carry out the reaction. From the viewpoint of maintaining enzyme activity, the temperature during synthesis is generally preferably 0 ° C to 70 ° C, and most preferably 30 ° C to 50 ° C.

The thus-synthesized ApnA is purified by a widely used method such as ion exchange chromatography to isolate Ap4A.

[0020]

EXAMPLES The present invention will now be described in detail with reference to typical examples, but the present invention is not limited thereto. The aminoacyl-tRNA synthetases used in the following examples were obtained by self-purification.

Isoleucyl tRNA synthetase is
E. coli JE5506 self-cloned as described by Makoto Kawakami et al. In Letters [FEBS letters, 185, 162-164 (1985)].
-From pkD15 (Microtechnology Research Institute No. 11950), after pretreatment such as removal of nucleic acid, purified by DEAE-Sepharose and hydroxyapatite column chromatography, and the enzyme which became electrophoretically almost single was obtained. Using.

Leucyl tRNA synthetase was pretreated from Bacillus stearothermophilus NCA-1503 strain, purified by DEAE-sepharose, phosphocellulose, and phenylcellulofine column chromatography, and electrophoresed. The enzyme which became almost single was used.

Lysyl tRNA synthetase was pretreated with baker's yeast purchased from Oriental Yeast Co.
An enzyme which was purified by AE-sepharose, phenylcellulofine, red A-sepharose, and blue A-sepharose column chromatography and became electrophoretically almost single was used.

Phenyl-tRNA synthetase is Escherichia coli K-12.
An enzyme which was purified from the strain by column chromatography of DEAE-Sepharose, phenylcellulofine, phosphocellulose, red A-Sepharose, and blue A-Sepharose and became electrophoretically almost single was used.

The ceryl tRNA synthetase was pretreated from Bacillus stearothermophilus NCA-1503 strain, and then DEAE-sepharose, phosphocellulose,
An enzyme which was purified by column chromatography of phenylcellulofine, red A-sepharose, and phosphocellulose and became electrophoretically almost single was used.

Histidyl tRNA synthetase was purified from Bacillus stearothermophilus NCA-1503 strain by pretreatment and then purified by column chromatography of DEAE-sepharose, phosphocellulose, red A-sepharose and phosphocellulose. , An enzyme that became electrophoretically almost single was used.

The unit (u) in aminoacyl-tRNA synthetase means the amount of enzyme which produces 1 × 10 ⁻¹¹ mol of amino acid hydroxamate per minute in the commonly used method for measuring aminoacyl-tRNA synthetase activity by hydroxamate. 1u. The principle of this activity measuring method is as follows.

[0028]

Example 1 Examples 1 to 6 showed examples in which the Ap4A synthesis rate was increased by adding various organic solvents to the reaction system. ATP initial concentration 15 mM, magnesium chloride 30 mM, imidazole 100 mM (pH 7.5), zinc chloride 0.5 mM, various organic solvents of methanol, ethanol, isopropanol, ethylene glycol, polyethylene glycol and glycerol or water 10% as a control and isoleucyl tRNA. The synthase 2,920 u / ml and isoleucine 5 mM were mixed on a 100 ml scale, and the Ap4A synthesis reaction was performed in batch at 40 ° C. The amount of Ap4A synthesized in the reaction was measured to determine the amount of Ap4A synthesized per unit time. The results are shown in Table 1.

Examples 2 to 6 The isoleucyl-tRNA synthetase and isoleucine obtained in Example 1 were replaced with leucyl-tRNA synthetase 36,600 u / ml.
And leucine 5 mM (Example 2), lysyl tRNA synthetase 200 u / ml and lysine 5 mM (Example 3), phenylalanyl tRNA synthetase 1,100 u / ml and phenylalanine 5 mM (Example 4), seryl tRNA synthetase 1 , 510 u / ml and serine 5 mM (Example 5), histidyl tRNA synthetase 860 u / ml and histidine 5
The reaction was performed in the same manner as in Example 1 except that mM (Example 6) was used to determine the initial rate of Ap4A. The results are shown in Table 1.

[0031]

[Table 1]

From the results of Examples 1 to 6, it was shown that the catalytic action of aminoacyl-tRNA synthetase was improved by adding an organic solvent.

Example 7 In Examples 7 to 9, an example was shown in which the addition rate of isoleucyl-tRNA synthetase Ap3A, Ap4A and Ap5A was particularly increased by the addition of ethylene glycol. The operation method is the same as in the first embodiment described above. ATP initial concentration 4 mM, A
DP initial concentration 16 mM, magnesium chloride 30 mM, imidazole 100 mM (pH 7.5), isoleucine 5 m
M, zinc chloride 0.5 mM, ethylene glycol each concentration from 0 to 90%, isoleucyl tRNA synthetase 1, 2
50u / ml, 100ml scale, 40 ° C, Ap3A
The initial rate of the synthetic reaction was determined.

Examples 8 and 9 The initial rate of the Ap4A synthesis reaction was determined in the same manner as in Example 7 except that the ATP initial concentration of 4 mM in Example 7 was changed to 10 mM and ADP was not added (Example 8). ).
Ap was carried out in the same manner as in Example 7 except that the ADP initial concentration of 16 mM in Example 7 was changed to ppppA initial concentration of 16 mM.
The initial rate of the 5A synthesis reaction was determined (Example 9). Example 7
The results of ~ 9 are summarized in Table 2.

[0035]

[Table 2]

From these results, it was found that the synthesis of ApnA was promoted by adding an organic solvent. Further, it has been found that it is most efficient to use an organic solvent having a concentration of about 50%.

Example 10 ATP initial concentrations of 0.1 mM, 0.2 mM and 0.5 m
The reaction was performed using M, 1.0 mM, and 2.0 mM. MgCl ₂ was used in a molar concentration twice the initial ATP concentration. Ethylene glycol concentration 0%, 10%, 20%, 30%, 40% for each ATP initial concentration
Imidazole 100 mM (pH 7.5),
100m of isoleucine 5mM, zinc chloride 0.5mM, isoleucyl tRNA synthetase 120u / ml
The mixture was mixed at 1 scale and reacted at 40 ° C., and the amount of Ap4A synthesis in each case was measured to determine the initial rate. From this, the rate parameter of the Ap4A synthesis reaction was determined. The results are shown in Table 3.

[0038]

[Table 3]

From these results, it was found that the Michaelis constant Km of the reaction parameters was decreased and the maximum velocity Vmax was increased by adding the organic solvent. Thus, it was shown that the catalytic reaction itself of the enzyme was increased by adding the organic solvent.

Example 11 Examples 11 to 13 below show actual examples in which Ap4A production was carried out by adding an organic solvent and using a small amount of enzyme.
ATP initial concentration 3 mM, ADP initial concentration 12 mM, magnesium chloride 30 mM, imidazole 100 mM (pH 7.
5), isoleucine 5 mM, zinc chloride 0.5 mM, ethylene glycol concentration 50%, isoleucyl tRNA synthetase 1,650 u / ml, 100 ml scale, A at 40 ° C.
Production of p3A was performed.

Example 12 ATP initial concentration 15 mM, magnesium chloride 30 mM, imidazole 100 mM (pH 7.5), isoleucine 2.5 mM, zinc chloride 0.5 mM, ethylene glycol 50%, isoleucyl tRNA synthetase 270 u / ml, 1
Production of Ap4A was carried out on a 00 ml scale at 40 ° C.

Example 13 ATP initial concentration 3 mM, pppA initial concentration 12 mM, magnesium chloride 30 mM, imidazole 100 mM (pH
7.5), isoleucine 5 mM, zinc chloride 0.5 mM,
Ethylene glycol concentration 50%, isoleucyl tRNA synthetase 1,650u / ml, 100ml scale, 40 ° C
Was used to produce Ap5A. The results of Examples 11 to 13 above are summarized in Table 4.

[0043]

[Table 4]

From these results, it was found that by adding an organic solvent, the production of ApnA can be carried out without any trouble.

Example 14 An example in which Ap4A was produced on an industrial scale based on the above examples was compared with the case where ethylene glycol was added and the case where ethylene glycol was not added. ATP initial concentration 15 mM, magnesium chloride 30 mM, imidazole 100 mM (pH 7.
5), isoleucine 0.1 mM, zinc chloride 0.5 mM,
Ethylene glycol 50%, isoleucyl tRNA synthetase 270u / ml, 400l scale, Ap4A at 40 ° C
Was carried out. The results are shown in Table 5. Also,
Table 5 also shows the results of the conventional reaction not containing ethylene glycol.

[0046]

[Table 5]

From these results, it was found that ApnA can be produced on an industrial scale with remarkable advantages in terms of the amount of enzyme used, the conversion rate, and the yield.

[0048]

EFFECTS OF THE INVENTION According to the production method of the present invention, the catalytic action of aminoacyl-tRNA synthetase is dramatically increased, and Ap
Since the yield of nA is also improved, Apn can be used on an industrial scale.
A can be produced.

Claims (1)

[Claims] 1. A method for producing diadenosine polyphosphate, which comprises coexisting an organic solvent when producing diadenosine polyphosphate using aminoacyl-tRNA synthetase.