JPS5935591B2 - Manufacturing method of glutathione - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing glutathione, and more specifically,
When producing glutathione by an enzymatic method, adenosine-5'-triphosphate (hereinafter abbreviated as ATP) is present in the reaction system in the form of a soluble polymer carrier, and the conjugate is used in an ATP regeneration system. The present invention relates to a method for producing glutathione by conjugating it with.

Glutathione is a substance useful as a treatment agent for liver diseases, an antidote, and the like.

Conventionally, known methods for producing glutathione include extraction from glutathione-containing bodies, fermentation using microorganisms capable of producing glutathione, organic synthesis, and enzymatic methods using glutathione synthetase.

Among them, methods for producing glutathione by enzymatic methods include a method using dried baker's yeast with excellent membrane permeability (Japanese Patent Publication No. 47-2631), and a method using a kabamilphosphokinase-containing substance, ATP, and cyanate in the reaction system. and a method of conjugating the ATP regeneration system in the presence of phosphoric acid (Japanese Unexamined Patent Publication No. 51-14
No. 4789) are known.

In particular, the latter method is an excellent method in that the amount of ATP added can be saved and glutathione can be produced efficiently because the ATP regeneration system is conjugated. This method involves a problem in that complicated operations are required to separate it from diphosphoric acid (hereinafter abbreviated as ADP) or to recover expensive ATP.

The present inventors have conducted various studies in order to overcome the problems of the conventional method and efficiently produce high-purity glutathione. When producing glutathione, (1) an acetate kinase-containing substance, a polymer-soluble carrier/ATP conjugate, and acetyl phosphate are present in the reaction system, or (2) an acetate kinase-containing substance is used together with the glutathione synthetase-containing substance. Discovered a method in which an inclusion body and a polymer soluble carrier/ATP conjugate are encapsulated in a gel lattice of a polymer, and the inclusion body is made to act on L-glutamic acid, L-cysteine, and glycine in the presence of acetyl phosphoric acid. The present invention has now been completed.

The enzymatic reactions involved in the method of the present invention are broadly classified into the following two reactions.

(1) Glutathione synthetase reaction As shown below, the method of the present invention combines the enzyme reactions of (1) and (2) above, and quantitatively converts ADP from ATP in the glutathione production reaction. ni AT
By regenerating it to P, the reaction proceeds efficiently and easily.
and/or facilitates separation from ADP and facilitates recovery of ATP.

In addition, the part surrounded by dotted lines in the above figure is a schematic representation of an inclusion body in which a glutathione synthetase-containing body, an acetate kinase-containing body, and a polymer soluble carrier/ATP conjugate are encapsulated in a gel lattice of a polymer. This is shown in .

The glutathione synthetase-containing bodies and acetate kinase-containing bodies used in the present invention are microbial cells that produce the corresponding enzymes, cell-free extracts from the cells, enzymes purified from the extracts, and immobilized bacteria. body, enzymes, or processed products thereof.

Glutathione synthetase refers to glutathione synthetase I (γ-L-glutamyl-cysteine synthetase, E, C, 6,3,2,2,) and glutathione synthetase ■ (γ-L-glutamyl-L-cysteine synthetase, Cysteinyl-glycine synthetase, E, C16,3,2
, 3,), and the enzymes include Nizielysia coli, Satucharomyces cerevisiae, Satucharomyces carlspergiensis, Satucharomyces
fragilis, Satucharomyces rouxii, Cansida neetilis, Cansida tropicalis, Schizosatucharomyces bombe, Torulopsis hersatheilis, Torulopsis petrophyllum, and other Pichia spp.
Pletanomyces, Mycotorula, Rhodotorula,
It is included in yeast such as the genus Henzenus and Endomyces.

Also, acetate kinase (E, C, 2, 7, 2, 1,
) are Elysia sp., alkaline earth metals, Serratia sp.
It is included in some bacteria belonging to the genus Shutomonas and Arthrobacterium.

The glutathione synthetase-containing body and the acetate kinase-containing body may be separate bodies having glutathione synthetase activity and acetate kinase activity, or may be a single body having both activities.

For example, 8 N. coli strains (ATCC23226
) has both glutathione synthetase activity and acetate kinase activity, so the cells or cell-free extracts from the cells can be used as glutathione synthetase-containing bodies and acetate kinase-containing bodies.

On the other hand, the polymer soluble carrier/ATP conjugate used in the present invention is preferably one in which the polymer soluble carrier and ATP are covalently bonded via a spacer.

The conjugate is prepared by introducing a spacer into ATP to make an ATP analogue, and covalently bonding this to a polymer-soluble carrier.

Examples of spacers include alkylene diamines such as ethylene diamine, tetramethylene diamine, hexamethylene diamine, octamethylene diamine, decamethylene diamine, glycine, β-alanine, 5-aminopentanoic acid,
Preferred examples include aminocarboxylic acids such as 7-aminehebutanoic acid, 9-aminononanoic acid, and 11-aminoundecanoic acid.

As the polymer-soluble carrier, those which are water-soluble and have a molecular weight of about 10' or more are preferably used, such as polysaccharides such as dextran and starch,
Examples include water-soluble polymers such as polyethyleneimine, polyvinyl alcohol, and polyamino acids.

As a method for introducing a spacer into ATP, for example, A
TP is reacted with iodoacetic acid to form N6-carboxymethyl-ATP, which is then reacted with a spacer such as an alkylene diamine or an aminocarboxylic acid (Eur.
, J.Biochem,,53゜481-48
6 (1975)], or reacting ATP with bromine to form C8-bromo-ATP, which is then reacted with a spacer such as an alkylene diamine, an aminocarboxylic acid (Eur, J. Biochemo, J., 265-274). (1977')].

Thus, for example, N6-((w-aminoalkyl)carbamoylmethyl)-ATP, N'-((w-carboxyalkyl)carbamoylmethyl)-ATP, C8-
(w-aminoalkylamino)-ATP, CB
ATP analogs such as (w-carboxyalkylamine)-ATP can be prepared.

If desired, these ATP analogs can further have a spacer introduced therein to lengthen the side chain (arm).

In order to covalently bond the ATP analog obtained above to a polymer-soluble carrier, depending on the type of ATP analog and carrier, the following methods can be used: the bromcyan method, epichlorohydrin method, diepoxide method, carbodiimide method, etc., which are used to prepare the immobilized enzyme. This can be done by law etc.

For example, when using N6-[(w-aminoalkyl)carbamoylmethyl)-ATP or C8-(w-aminoalkylamino)-ATP and using a polysaccharide such as dextran as a carrier, the bromcyan method, epichlorohydrin method, diepoxide method, etc. law etc. can be adopted.

Also, as ATP analogs, N'-(: (w-carboxyalkyl)carbamoylmethyl]-ATP and C8-
When using (w-carboxyalkylamine)-ATP and polyethyleneimine as a phase, a carbodiimide method can be employed.

In addition, when using polyethyleneimine as a carrier, AT
Preferably, the conjugate is further formylated after conjugation with the P analog to increase the water solubility of the conjugate.

Furthermore, a glutathione synthetase-containing body, an acetate kinase-containing body, and the polymer soluble carrier obtained above, A
Methods for preparing inclusion bodies in which a TP conjugate is encapsulated within a gel lattice of a polymer include the polyacrylamide gel entrapment method, which is used for the preparation of immobilized enzymes or microorganisms;
Methods such as an agar gel entrapment method and a carrageenan gel entrapment method can be employed.

For example, when using the polyacrylamide gel method, acrylamide and N-N'-
This can be carried out by copolymerizing methylene-bis-acrylamide.

Next, the method for producing glutathione according to the present invention will be specifically explained.

The first aspect is to add L-glutamic acid, L-cysteine and glycine to L-glutamic acid, L-cysteine and glycine in the presence of an acetate kinase-containing body, a polymer soluble carrier, an ATP conjugate and acetyl phosphate;
This is a method for producing glutathione by causing a substance containing glutathione synthetase to act.

The above reaction is preferably carried out in a suitable buffer at pH 5.0 to 8.0, preferably pH 6.0 to 7.5, and at a temperature of 25 to 50°C, preferably 30 to 40°C.

In addition, 5.0 to 50% of magnesium ions were added to this reaction solution.
It is preferable to add about mM.

The amount of the polymer soluble carrier/ATP conjugate (as ATP) to be present in the reaction solution is preferably about twice the concentration of the three types of amino acids.

Further, the amount of acetyl phosphoric acid is preferably about 1.5 times the amount of the above-mentioned conjugate (as ATP).

In addition, if the enzyme source glutathione synthetase-containing body and/or acetate kinase-containing body contains acetyl phosphate in a sufficient amount to express acetate kinase activity, it is not necessary to add acetyl phosphate separately. There isn't.

A second aspect of the present invention is to add a glutathione synthetase-containing substance, an acetate kinase-containing substance, and a polymer-soluble carrier/ATP conjugate to L-glutamic acid, L-cysteine, and glycine in the presence of acetyl phosphate. This is a method for producing glutathione by causing inclusion bodies encapsulated within a combined gel lattice to act.

The above reaction is preferably carried out in a suitable buffer at pH 4.5 to 8.5, preferably pH 6.0 to 7.5, and at a temperature of 20 to 60°C, preferably 40 to 50°C.

In addition, 5.0 to 1100 magnesium ions were added to the reaction solution.
It is preferable to add about rrL.

The amount of acetyl phosphate present in the reaction solution is about the same molar amount as the polymer soluble carrier/ATP conjugate (as ATP) in the inclusion body, or about 2 times the concentration of the three amino acids that are the substrates.
About double that is preferable.

In addition, the glutathione synthetase-containing body in the inclusion body and (
or) If the acetate kinase-containing body contains a sufficient amount of acetyl phosphate to exhibit acetate kinase activity, it is not necessarily necessary to separately add acetyl phosphate.

In addition to the batch method, the method using the above-mentioned inclusion bodies can also be carried out by a column method.

That is, glutathione can be continuously produced by filling a column with inclusion bodies and passing a substrate solution through the column.

According to the method of the present invention described above, the following advantages can be obtained.

(1) The polymer soluble carrier/ADP conjugate converted from the polymer soluble carrier/ATP conjugate by the expression of glutathione synthetase activity is regenerated into the former conjugate by the expression of acetokinase activity, resulting in glutathione production. The reaction can proceed efficiently.

(2) Because ATP is bound to a polymer-soluble carrier,
After the reaction is completed, glutathione and the conjugate can be easily separated.

In particular, when using an inclusion body containing an enzyme-containing body and a polymer-soluble carrier/ATP conjugate, the enzyme, ATP, etc. will not be mixed into the reaction solution containing glutathione, making it easy to separate and purify glutathione. It's extremely easy.

(3) The polymer-soluble carrier/ATP conjugate or the inclusion body in which the conjugate is encapsulated can be easily recovered from the glutathione-containing solution and can be reused.

Particularly when inclusion bodies are used, continuous production of glutathione is possible by employing a column method.

The present invention will be explained below with reference to Examples.

Example 1 (1) Preparation of polymer soluble carrier/ATP conjugate Method of Mosbach et al.
53, 481-486 (1975))
(6-aminehexyl)carbamoylmethyl nee ATP
(referred to as ATP analog).

On the other hand, dextran (molecular weight 4
X10') 0.5f? Dissolve in 2.0 rul of water, 10
Adjust the pH to 11 with IJ (normal hydroxide).

To this is added 0.3 mmol of bromo cyanide and the pH i is maintained with lO normal sodium hydroxide.

When no decrease in pH is observed, 0.3 mmol of the above ATP analog is added, and the mixture is allowed to react at room temperature with stirring for about 16 hours.

After the reaction is completed, the reaction solution is thoroughly dialyzed against water or chromatography is performed using a column packed with Sephadex G-10 to obtain the dextran/ATP analog conjugate 0.45? get.

The ATP analog binding amount of this sample is 140 per gram of the sample.
, 1 μmol.

In addition, by setting the amount of bromo cyanide used in the above reaction to 0.1 mmol or 0.2 mmol, the amount of ATP analog bound is 46.4 μmol/? or 58.2 μmol/
Obtain a standard specimen.

(2) Preparation of enzyme-containing body Glucose 1.0%, dibasic potassium phosphate 0.5%, yeast extract 1.0%, peptone 1.0%, inner extract 0.5%
% and magnesium sulfate 0.1% (pH 7
, 0) and 8 N. coli strains (ATCC23226
) and cultured at 30°C for 16 hours with shaking.

After culturing, cool and centrifuge to collect bacterial cells.

After washing this once with physiological saline, 5rrLM Tris.
Suspend in hydrochloric acid buffer and sonicate.

Centrifugation is performed at 25,000XP for 30 minutes, and the resulting supernatant is used as a source of glutathione synthetase and acetate kinase in the following reaction.

(3) Glutathione production reaction Glycine 5771M - L-Glutamic acid 5mM.

L-cysteine 5mM 1 Magnesium chloride 10mM,)
Dextran A prepared in (1) above was added to a leather solution consisting of 100 ml of lithium-hydrochloric acid buffer (pH 7.0).
10 mM of TP analog conjugate and the enzyme solution prepared in (2) above (1.5-2.0 my/rul as protein amount)
) and react at 37°C for 3 hours.

After the reaction, the reaction is stopped by keeping it at 100° C. for 1 minute, and glutathione in the supernatant is measured using glutathione reductase.

The results are shown in Table 1 below.

Example 2 In (3) of Example 1, a predetermined amount of acetyl phosphoric acid is added to the reaction solution, and the reaction is carried out in the same manner as in (3) of Example I.

The amount of glutathione produced in the reaction solution is shown in Table 2 below.

Example 3 (1) Preparation of polymer soluble carrier/ATP conjugate N
Synthesize 6-carboxymethyl-ATP.

On the other hand, aminocarboxylic acid H2N-(CH2)n-C00
1 mmol of H (where n=4.6.10) is suspended in a mixture of 20 rul of dioxane and 1 ml of concentrated sulfuric acid, and the mixture is reacted overnight under saturation with isobutylene.

After the reaction, add powdered bicarbonate of soda to adjust the pH of the reaction solution to 8.0.
and ethyl ether/acetic acid ethyl ester (1:IV
/V) Extract 3 times with 50 rul.

The extracts are combined and concentrated under reduced pressure to obtain an aminocarboxylic acid with a protected carboxyl group as a sleeve (
yield 10-20%).

0.1 mm IJ mole of this sample is added to 2 m of tetrahydrofuran.
1 ml of a 0.2M concentration N6-carboxymethyl-ATP aqueous solution and 0.3 ml of carbodiimide.
Add mmol and hold at room temperature for 16 hours.

Then, it was concentrated to dryness under reduced pressure to give anisole of about 0.2 mm! J
mol and 5 m of trifluoroacetic acid are added and reacted at room temperature for 20 minutes to remove the protecting group of the carboxyl group.

After concentrating under reduced pressure, 100 rrLl of ethyl ether/ethyl acetate (1: IV/V) is added to dissolve the precipitate in water.

Next, chromatography was performed using a DOWEX 1×2 (chlor type) column to obtain N6-C(w-carboxyalkyl)carbamoylmethyl)-ATP (hereinafter referred to as AT
(referred to as P analog) is 0.07 mi each! About J mol is obtained.

Using 0.22 mmol of polyethyleneimine as a polymer-soluble carrier, this was reacted with 0.07 mmol of the ATP analog obtained above in the presence of 0.2 mmol of carbodiimide (volume 2+7 LA') for 16 hours at room temperature with stirring. let

After the reaction, polyethyleneimine/ATP is obtained by performing dialysis or chromatography in the same manner as in Example 1 (1).
Analog conjugates are obtained on the order of 0.165' each.

The unreacted imino group of this conjugate was removed using the method of Rykes et al. (B
iochem, B 1ophys, Acta, 2
86.260-268 (1972)).

Thus, a formylated polyethyleneimine/ATP analog conjugate is obtained.

(2) Glutathione production reaction In (3) of Example 1, the formylated polyethyleneimine/ATP analog conjugate obtained in (1) above was used instead of the dextran/ATP analog conjugate, and ).

The amount of glutathione produced in the reaction solution is shown in Table 3 below.

Example 4 (1) Preparation of polymer soluble carrier/ATP conjugate Lowe's method (Eur, J. Biochem, 73
, 256-274 (1977)) synthesize C3-(6-aminehexylamino)-ATP (referred to as ATP analog).

On the other hand, dextran (molecular weight 4
A dextran/ATP analog conjugate is prepared in the same manner as in Example 1 (1) using X10').

(2) In (3) of glutathione production reaction Example 1, using the dextran/ATP analog conjugate obtained in (1) above as the conjugate,
The reaction is carried out in the same manner as in Example 1 (3).

The amount of glutathione produced in the reaction solution is shown in Table 4 below.

Example 5 Dextran prepared in the same manner as in Example 1 (1)
ATP analog conjugate (ATP analog binding amount: 46.
6 μmole/g conjugate preparation) of Example 1 (
10 mM of acetyl phosphate is added to the substrate solution of 3), and the reaction is carried out in the same manner as in Example 1 (3).

After the reaction, the mixture was kept at 100°C for 1 hour, and then the precipitate was removed by centrifugation.

The supernatant is ultrafiltered using Amicon UM-10 Membrane to recover the dextran/ATP analog conjugate.

The recovered conjugate preparation is used again in the glutathione production reaction.

Repeat this operation three times.

The amount of glutathione produced in the reaction solution is shown in Table 5 below.

Example 6 (1) Preparation of inclusion bodies Dextran prepared in the same manner as in Example 1 (1)
ATP analog conjugate (ATP analog binding amount: 14
3 μmole/f? Conjugate preparation) 0.42S' and bacterial cells obtained in the same manner as in (2) of Example 1 (wet bacterial cells)
Add 0.7 P to a 30% aqueous potassium chloride solution (2TI'Ll) and homogeneously suspend the solution.

Acrylamide 1 in this suspension? and N-N'-methylene-bis-acrylamide 0.05y dissolved in 2M water, 5.0% β-(dimethylamino)propioni) IJ1 0.5 rIL1 and 51.0% potassium persulfate 0 Add .5 ml and allow to gel.

This gel was suspended in physiological saline, a surfactant cation S was added to give a concentration of 0.05%, and the gel was pressed for 60 minutes.
．． Transferred into 1M acetate buffer (pH 6,0) and incubated at 4°C for 20
Leave it for a while.

The gel thus obtained is subjected to the following reaction.

(2) Glutathione production reaction Glycine 5rrLM1L-Glutamic acid 5rrLM.

L-cysteine 5rILM, magnesium chloride 25rr
2rnl of substrate solution consisting of LM, acetyl phosphate 6771M and Tris-HCl buffer (pH 7,0) 100mM
1 g of the gel prepared in (1) above was added to the solution, and the mixture was shaken and reacted at 37°C for 3 hours.

Incidentally, the reaction was carried out in the same manner in the case where acetyl phosphoric acid was not added.

The amount of glutathione produced in the reaction solution is shown in Table 6 below.

Example 7 A glutathione production reaction is carried out in the same manner as in Example 6 (2), and after 3 hours of reaction, the gel is collected by filtration.

The glutathione production reaction is performed again using the recovered gel.

Repeat the mastication and reaction operations 4 times. The amount of glutathione produced in the reaction solution is shown in Table 7 below.

Example 8 Gel 5.02 prepared in the same manner as in Example 6 (1) was packed into a column (1c1rLxlQcm), and 5mM of glycine and 5mM of L-glutamic acid were added from the bottom of the column.
, L-cysteine 5mM, magnesium chloride 10mM,
Acetyl phosphate 6mM and Tris-HCl buffer (pH 7)
, 0) A substrate solution consisting of 100 mM is flowed at various flow rates.

The amount of glutathione produced in the effluent is shown in Table 8 below.

Example 9 Gel 5 prepared in the same manner as in Example 6 (1). Of?
was packed into a column (1mXI Om), and the same substrate solution as in Example 8 was added from the bottom of the column at S, V, = 0.1hr.
-1 to run continuously for a long period of time.

The amount of glutathione produced in the effluent is shown in Table 9 below.

Claims (1)

[Claims] 1. When producing glutathione by reacting L-glutamic acid, L-cysteine, and glycine with a glutathione synthetase-containing substance, an acetate kinase-containing substance, a molecule-soluble carrier, and adenosine-5' are added to the reaction system. - A method for producing glutathione, comprising the presence of a triphosphate conjugate and acetyl phosphate. 2. Claim 1, wherein the polymer soluble carrier/adenosine-5'-triphosphate conjugate is a polymer soluble carrier and adenosine-5'-triphosphate bonded via a spacer through a covalent bond. Manufacturing method described. 3. The method according to claim 1, wherein the acetyl phosphate is acetyl phosphate contained in the glutathione synthetase-containing body and/or the acetate kinase-containing body. 4 When producing glutathione by allowing a glutathione synthetase-containing body to act on L-glutamic acid, L-cysteine, and glycine, an acetate kinase-containing body and a polymer soluble carrier/adenosine-5'-triphosphate are added together with the glutathione synthetase-containing body. A method for producing glutathione, which comprises allowing an inclusion body in which an acid conjugate is encapsulated in a gel lattice of a polymer to act on L-glutamic acid, L-cysteine, and glycine in the presence of acetyl phosphoric acid. 5. Claim 4, wherein the polymer soluble carrier/adenosine-5-trisic acid conjugate is a polymer soluble carrier and adenosine-5'-triphosphate bound by a covalent bond via a spacer. Manufacturing method described. 6. The method according to claim 4, wherein the acetyl phosphate is acetyl phosphate contained in the glutathione synthetase-containing body and/or the acetate kinase-containing body.