JPS6031480B2 - 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,
Using adenosine-5'-triphosphate (hereinafter referred to as ATP) and a yeast glutathione synthetase-containing substance containing glycolytic enzymes, reactions by glycolytic enzymes and reactions by glutathione synthetase were performed. The present invention relates to a method for efficiently producing glutathione by co-precipitation of glutathione.

Glutathione is a substance useful as a treatment agent for liver diseases, an antidote, and the like. Conventionally, known methods for producing glutathione include an extraction method from a glutathione-containing body, a lotus peel method using microorganisms capable of producing glutathione, an organic synthesis method, and an enzymatic method using glutathione synthetase. Among them, the enzymatic method for producing glutathione is as follows:
Method using dry yeast with good waist permeability (Special Publication No. 47-2
No. 6314), the reaction by glutathione synthase, and the reaction by carbamyl phosphokinase are combined to produce A.
Method for reducing the amount of TP added (Tokukan Sho 51-114478
y), etc.

However, in the former method, [1] Preparation of dry yeast is complicated, [2] Since the produced glutathione remains in the bacterial cells, it is necessary to extract glutathione from the bacterial cells after the reaction, and the rigid ram method is used. There were drawbacks such as the inability to continuously produce glutathione. On the other hand, although the latter method is relatively superior, it requires expensive A in the reaction system.
There was a drawback that it was necessary to add TP. As a result of various studies on enzymatic methods for producing glutathione, the present inventors encapsulated yeast containing ATP and glycolytic enzymes together with a glutathione synthetase-containing substance in a gel lattice of a polymer. When the immobilized product is allowed to act on L-glutamic acid, L-cysteine, and glycine in the presence of glucose or its intermediate metabolites, ATP
They found that glutathione can be produced efficiently without the addition of glutathione, and have completed the present invention.

As is clear from the figure below, the method of the present invention uses yeast containing ATP and glycolytic enzymes and a glutathione synthetase-containing body immobilized in the same gel, and also uses ATP necessary for the glutathione production reaction. ATP* contained in the yeast cell body is substituted for ADP, and ADP, which is converted from ATP as the glutathione production reaction progresses, is converted into ATP by the action of reductase enzymes (glucose assimilation reaction) in the yeast cell body. By regenerating the ATP level,
glutathione production reaction to proceed efficiently.

The area surrounded by dotted lines in the above diagram schematically shows an immobilized product in which yeast containing ATP and various glycolytic enzymes and a glutathione synthetase-containing body are encapsulated in a gel lattice of a polymer. This is what is shown.

Yeast that belongs to the genus Saccharomyces and has the ability to convert glucose used in the present invention (i.e., yeast that belongs to the genus Saccharomyces and contains ATP and glycolytic enzymes), that is, the ability to convert glucose into CO2 and ethanol. Any yeast can be used as long as it has the following properties; for example, Saccharomyces cerevisiae IF02044 is preferred.

In addition, glycolytic enzymes include glucokinase, glucose phosphate isomerase, fructose phosphate kinase,
These include fructose diphosphate aldolase, glycerate aldehyde-3-phosphate dehydrogenase, glycerate phosphate kinase, glycerate phosphate mutase, pyruvate phosphate hydratase, and pyruvate kinase. enzyme, pyruvate kinase, etc. are involved in the reaction that converts ADP to ATP. On the other hand, examples of the glutathione synthetase-containing body used in the present invention include microorganisms that produce the enzyme, cell-free extracts from the microorganism, enzymes purified from the extract, and especially those containing the enzyme. yeast (e.g., Saccharomyces cerepigee '02044) and Escherichia coli (e.g., Escherichia coli IJATCC2322).
6) etc. Here, glutathione synthetase refers to glutathione synthetase 1 (y-L-glutamyl-L-cysteine synthetase, E.C.6.3).
・2.2) and glutathione synthase U (y-L-glutamyl-L-cysteinyl-glycine synthetase, E
・C・6・3・2・3)

Further, when the yeast containing the ATP and the various enzymes of the rice cracking system contains the glutathione synthetase, the yeast can also be used as a glutathione synthetase-containing product. An immobilized product in which yeast containing ATP and shark sugar-based enzymes and a glutathione synthetase-containing body are encapsulated in a gel lattice of a polymer is a polymer that is used for the preparation of immobilized microorganisms and immobilized enzymes. It can be easily prepared by acrylamide gel entrapment method, carrageenan gel entrapment method, agar gel entrapment method, polyvinyl alcohol gel entrapment method, etc.

For example, when employing the polyacrylamide gel entrapment method, the above-mentioned two-component water suspension includes an acrylamide monomer, a crosslinking agent (e.g., N·N'-methylenebisacrylamide), and a polymerization accelerator (e.g., 6- (dimethylamino)
The desired immobilized product can be prepared by adding a polymerization initiator (e.g., potassium persulfate) and gelation. In addition, when adopting the carrageenan gel entrapment method, the above two components are suspended in an aqueous carrageenan solution, and this suspension is brought into contact with a gelling agent (for example, potassium chloride) to gel, thereby obtaining the desired immobilized product. It can be prepared. The immobilized product prepared as described above was mixed with glucose, glucose-6-phosphate,
fructose-6-phosphate or fructose-1.6
- Glutathione can be produced by acting on L-glutamic acid, L-cystine and glycine in the presence of diphosphoric acid. The above reaction is carried out in an appropriate buffer (for example, a phosphate buffer of about 0.02 to 0.0M).
Magnesium ions from 1 to 50 mM, especially from 5 to 20
PH5-8, especially 6-7, temperature 2.
Preferably, the temperature is 0 to 4000C, particularly 30 to 360C.

Furthermore, when the immobilized product is used repeatedly or continuously for a long period of time, it is preferable to add nicotinamide adenine dinucleotide to the reaction system. Glucose, glucose-6-phosphate, fructose-6-phosphate or fructose-1 present in this reaction system
・The concentration of 6-diphosphoric acid is 0.1 to IM, especially 0.3
~0.8M is preferable.

Furthermore, the higher the concentration of the three types of amino acids that are substrates, the greater the amount of glutathione produced, but generally it is preferably about 5 to 50 mM, particularly about 20 to 30 mM. The above reaction can be carried out not only by a batch method but also by a column method. Especially when using a column method, glutathione can be produced continuously. The present invention will be explained below with reference to Examples.

Example 1 {1} Preparation of immobilized product Glucose 0.5%, yeast extract 1%, beptone 1%,
Medium consisting of 0.5% meat extract and 0.5% salt (pH 7)
．． Inoculate Saccharomyces cerevisiae 02044 onto 0) and culture with shaking at 30°C for 1 hour.

After culturing, collect the bacteria and wash once with physiological saline. Twenty grams of the wet bacteria thus obtained were suspended in a 30% aqueous potassium chloride solution. Acrylamide monomer 5 is added to this suspension.
and N・N′-methylene-bis-acrylamide 0.
Add a solution of 25 minutes dissolved in 15 hours of water. 5% to this
Add G-(dimethylamine)propionitrile 6,
Furthermore, a solution of 0.4 ml of potassium persulfate dissolved in 6 ml of water is added to form a gel. Thus gel 50 yen (wet weight)
get. '2' To the gel (wet weight) obtained in [1] above, L-glutamic acid, L-cysteine and glycine each low M, magnesium chloride 10mM, phosphate buffer (pH 7.5) 10mM, and the following Add a substrate solution consisting of glucose at the concentration shown in the table to make the total volume 20 or more.

This was reacted at 30q0 for 5 hours with shaking. The amount of glutathione thus produced is shown in Table 1 below.
Table 1 Example 2 Gel 5 (wet weight) prepared in the same manner as [1] of Example 1 was added with three types of amino acids (L-glutamic acid, L-cysteine and glycine) at the concentrations shown in Table 2 below, Magnesium chloride 10mM, phosphate buffer (pH 7.5) 100
M, and a substrate solution consisting of 0.8 M glucose to make a total amount of 20 skins.

This was reacted at 30° C. for 5 hours with shaking. The amount of glutathione thus produced is shown in Table 2 below. Table 2 Example 3 To a gel (wet weight) prepared in the same manner as in {1) of Example 1, 10 mM each of L-glutamic acid, L-cysteine and glycine, 10 M of magnesium chloride, and phosphate buffer were added. (pH 7.5) 100mM, glucose 0.9M, and nicotinamide adenine dinucleotide 5x10
-Add a substrate solution consisting of 6M to make a total volume of 20ml.

This was reacted for 5 hours under shaking at 30 oo. After the reaction, the gel is collected, washed, and reacted again in the same manner as above. The amount of glutathione produced when this operation was repeated is shown in Table 3 below. Table 3 Example 4 Gel 7 (wet weight) prepared in the same manner as in Example 1 (1) was packed into a column (1.2 cm x 14 arcs).

To this column, 20mM each of L-glutamic acid, L-cysteine, and glycine, 10mM magnesium chloride, 0.1M phosphate buffer (pH 7.1), and 0.9M glucose were added.
, and nicotinamide adenine dinucleotide 10
-6M substrate solution at a space velocity (S.V.) of about 0.
25, and continuously reacted at 30q C. The amount of glutathione in the effluent is shown in Table 4 below. Table 4 Example 5 Gel 52 (wet weight) prepared in the same manner as in {1} of Example 1 was added with low M each of L-glutamic acid, L-cysteine and glycine, 10 M of magnesium chloride, and a phosphate buffer (pH 7). .5) Add a substrate solution consisting of fructose-1,6-diphosphate of 100 M and the concentration shown in Table 5 below to bring the total volume to over 20.

This was reacted at 30 qo with shaking for 5 hours. The amount of glutathione thus produced is shown in Table 5 below.
Table 5 Example 6 {1} Twenty days of wet bacteria of Saccharomyces cervizier IF02044 obtained in the same manner as in Example 1 [1] were air-dried at 30° C. for 2 hours.

In this way, about 5 to 6 days of air-dried bacterial cells are obtained. Suspend 2 parts of the air-dried bacterial cells in 10 parts of water. A solution of 1 part carrageenan dissolved in 40 parts water is added to this and mixed. This mixed solution was dropped into a 1% potassium chloride aqueous solution to obtain a spherical gel with a diameter of approximately 50 mm (wet weight). ■ Add 10 mM of the gel obtained above (green weight) to 10 mM each of L-glutamic acid, L-cystic acid, and glycine, 10 mM magnesium chloride, 100 mM phosphate buffer (pH 7.5), 0.8 M glucose, and A substrate solution consisting of 1% potassium chloride is added to bring the total volume to over 20.

Claims (1)

[Scope of Claims] 1. When producing glutathione enzymatically by reacting L-glutamic acid, L-cysteine and glycine with a glutathione synthetase-containing substance, the above-mentioned yeast belonging to the genus Satucharomyces and having the ability to assimilate glucose is used. An immobilized product encapsulated in a gel lattice of a high molecular weight polymer together with a glutathione synthase-containing substance is
- in the presence of phosphoric acid, fructose-6-phosphate or fructose-1,6-diphosphate, L-glutamic acid, L
- A method for producing glutathione, characterized in that it is made to act on cysteine and glycine. 2. The production method according to claim 1, wherein the glutathione production reaction is carried out by a column method. 3. The manufacturing method according to claim 1 or 2, wherein the high molecular weight polymer is polyacrylamide gel or carrageenan gel.