JPH01228473A - Production on novel recombinant dna and glutathione - Google Patents

Abstract

PURPOSE:To obtain glutathione in good yield by culturing a microorganism belonging to a specific Escherichia. CONSTITUTION:DNA containing a gene coding gamma-glutamyl-L-cysteine synthetic enzyme derived from Escherichia coli and DNA containing a gene coding glutathione synthetic enzyme are inserted into a bacteriophage vector DNA (e.g., lambdaCl8571121SDNA) and purified to afford a recombinant DNA. Then a microorganism containing the recombinant DNA and belonging to the genus Escherichia having glutathione-producing ability is inoculated into T-Y culture medium and cultured under state where self-renewal of recombinant DNA is not induced and them a bacterial cell is collected from the cultured product and washed with a physiological saline and treated in boiled water for 1-10min to extract the aimed glutathione or a bacterial cell after culture is collected and brought into contact with a reaction liquid containing glutamic acid, cysteine, glycine, adenosine-5'-3-triphosphate and Mg ion at pH6-9 and 20-40 deg.C for several times to provide the aimed glutathione.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel recombinant DNA and a method for producing glutathione.

[Conventional technology]

Traditionally, glutathione is produced by organic synthesis, microorganisms (especially yeast)
Two enzymes involved in glutathione synthesis, γ-glutamyl-cysteine synthase (hereinafter abbreviated as GSH-1), were extracted from bacterial cells and genetically modified.
and glutathione synthase (hereinafter abbreviated as GSH-II) activity is enhanced using Escherichia coli.

Glutathione is a tripeptide consisting of glutamic acid, cysteine, and glycine, and is an important compound frequently used as a therapeutic agent for a wide range of liver diseases and as a biochemical reagent.

[Problems to be Solved by the Invention] However, the former two methods are not necessarily advantageous due to the length and complexity of the reaction process, as well as the complicated operations and the low content in the cells. Furthermore, the latter also has problems such as limited glutathione production due to low GSH-1 activity, and there is a desire to develop a more efficient production method.

[Means for Solving the Problems] Therefore, as a result of various studies to solve the above problems, the present inventors discovered the gene encoding GSH-1 (hereinafter referred to as GSH-1).
SH-1 gene) and G
A recombinant DNA was obtained by inserting a DNA containing a gene encoding SH-II (hereinafter abbreviated as GSH-If gene) into a bacteriophage vector DNA, and this recombinant was transformed into Escherichia (Escherichia).
) A strain having the ability to produce glutathione, which is included in a strain belonging to the genus A. The present invention was completed based on the findings that glutathione can be produced in a higher yield than conventional methods by contacting with adenosine-5'-triphosphate and magnesium ions.

That is, the present invention provides DNA containing a gene encoding T-glutamyl-L-cysteine synthetase and a DNA containing a gene encoding glutathione synthase.
is inserted into bacteriophage vector DNA, and the present invention also relates to DNA containing a gene encoding T-glutamyl-L-cysteine synthase and a gene encoding glutathione synthase. A microorganism belonging to the genus Escherichia containing a recombinant DNA obtained by inserting a DNA containing a gene into a bacteriophage vector DNA and having the ability to produce glutathione is cultured in a medium, and glucthione is collected from the culture, or the microorganism is This is a method for producing glutathione, which is characterized in that glutathione is produced by bringing the microbial cells and/or the microbial cell-treated product into contact with glutamic acid, cysteine, glycine, adenosine-5'-triphosphate, and magnesium ions.

The present invention will be explained in detail below.

First, DNA containing the GSH-n gene, for example, the DNA derived from E. coli, is transformed into bacteriophage vector D.
The preparation of recombinant DNA inserted into NA will be described.

Examples of bacteriophage vector DNA include:
λCl11571121S DNA etc., and the D
NA can be obtained in exactly the same manner as the method described in JP-A-61-152285.

On the other hand, DN containing the GSH-IIn gene derived from E. coli
A is, for example, a recombinant plasmid pBR32 prepared in exactly the same manner as described in JP-A-60-180592.
It can be obtained as 5-gsh II b DNA.

Then plasmid pBR325-gsh II b
For example, restriction enzymes such as ``restriction enzymes'' (manufactured by Boehringer Mannheim Yamanouchi Co., Ltd.) and tlindll (
(manufactured by Herringer Mannheim Yamanouchi) at a temperature of 30°C or higher, preferably at 37°C, and an enzyme concentration of 1 to 1, respectively.
Digestion is performed at 10 units/ral for 20 minutes or more, preferably 30 minutes to 2 hours, to obtain a DNA digest.

This DNA digest is subjected to, for example, molecular cloning, the first
Pages 13-114, Cold Spring Harbor Laboratory
Laboratory) (1982), the digested DNA ends were blunt-ended and mixed with EcoRI linker (Takara Shuzo Co., Ltd.), and this was injected with, for example, T4 DNA ligase (Boehringer Mannheim Co., Ltd.). manufactured by Yamanouchi Co., Ltd.) at a temperature of 4 to 37°C, preferably 4 to 16°C, and an enzyme concentration of 0.1 to 10 units/
ml for 20 minutes or more, preferably 12 to 24 hours, to obtain a recombinant plasmid having EcoRI cleavage sites on both sides of the GSH-11 gene.

Next, the thus obtained recombinant plasmid and bacteriophage vector λclsst1121
Mix S DNA and add restriction enzyme EcoR to this, for example.
I (manufactured by Boehringer Mannheim Yamanouchi Co., Ltd.) at a temperature of 30°C or higher, preferably 37°C, and an enzyme concentration of 1 or more.
10 units/- for 20 minutes or more, preferably 30 minutes to 2
T4 DNA ligase (
(manufactured by Boehringer Mannheim Yamanouchi) at a temperature of 4°
The ligation is carried out at a temperature of 16°C or more, preferably 16°C, and an enzyme concentration of 0.1 to 5 units/m for 1 hour or more, preferably 16 to 24 hours to obtain various recombinant DNAs.

The following method is used to collect the desired recombinant DNA from the mixture of various recombinant DNAs thus obtained.

First, a temperate phage that has the same cohesive ends and immunity as the phage used for the production of recombinant DNA is lysogenized into Escherichia coli in advance. Attachment site to (atta)
After infecting a large amount of temperate phages that do not have a site that recombines with the chromosome of the host bacterium during lysogenization, the recombinant DNA is mixed and kept at a temperature of 20 to 40"C. The recombinant DNA can be incorporated into the host bacterium (helper method).

In addition to the above-mentioned helper method, the calcium chloride method and the like can also be used to similarly incorporate the protein into the host bacteria.

Furthermore, the various recombinant DNAs obtained were subjected to in vitro packaging.
ing) method [Methods in Enzymology (Met)
Hodsin Enzymology) Volume 68, No. 2
pp. 81-298, 1979, Academic Press (
It is also possible to incorporate the recombinant DNA into the host bacterium by preparing bacteriophage particles by wrapping them in a lambda bacteriophage coat protein and infecting the host bacterium.

The host bacteria used here may be any Escherichia coli that is included in the 12 strains of Funatsuri (for example,
AB1157 (E, coli Genetic 5to
ck Center.

Yale University, USA), W3110 (ATCC273
25), W3350 (ATCC27020), 110
Escherichia coli such as 0 (Max-Plank-Inslloo (West Germany)) can be said to be particularly suitable.

By screening microorganisms capable of producing GSH-n from the above-mentioned strains, we determined that Escherichia spp. It is possible to obtain microorganisms belonging to

To obtain purified recombinant DNA from the microorganism thus obtained, for example, the lysogenic bacteria carrying the recombinant DNA are incubated at 40-45°C, preferably at 42-45°C.
After culturing by heat induction at 3°C for 10 minutes or more, preferably 15 to 30 minutes, the host bacteria are cultured by adding, for example, chloroform and shaking at a temperature of 0 to 40°C for 10 minutes to 1 hour. After lysing the body and expelling bacteriophage into the medium, the culture solution was mixed with 7.000-20.00 Or,
The number of bacteria is removed by centrifugation for 10 to 30 minutes at, for example, 20,000 to 40.00 Or, p.

The bacteriophage was purified by ultracentrifugation for 1 to 3 hours in step 1, and the thus purified bacteriophage was subjected to, for example, Molecule cloning, Cold Spring Harbor Laboratory (Cold)
Spring 1larbor Laboratory
), pp. 76-85 (1982).

In addition, the C to be inserted into the bacteriophage vector DNA
;The number of DNAs containing the 5H-II gene is 1 or more, preferably 1 to 2.

Next, the DNA containing the GSH-1 gene, for example, the DNA derived from E. coli, is transformed into a bacteriophage vector DNA.
The preparation of recombinant DNA inserted into A will be described.

Any bacteriophage vector DNA may be used as long as it has an endonuclease cleavage site only in the genetic information portion of the coat protein, such as bacteriophage EN501S-Tc DNA.

The above bacteriophage EN501S-Tc DNA can be prepared by the following method.
Bacteriophage λCTs, tl121 DNA was obtained in exactly the same manner as in the example described in Publication No. -212781,
In order to facilitate isolation of this bacteriophage DNA, a marker is added to the region upstream or downstream of the late promoter. Any marker may be used, including, for example, a drug resistance marker.

As an example of adding a drug resistance marker, for example, a tetracycline resistance gene (
It is abbreviated as Tc'. ), and this bacteriophage DNA can be easily isolated due to its tetracycline resistance.

Furthermore, tetracycline-resistant Bacterio 77-di DNA, for example, EN1121Tc/xb, can be produced 8 times as described below.

Plasmid pKN206 DN^ [T.
, Ma-suda), etc. Paiol, Kem, (
Agri, Biol.

Chem, ), Vol. 50, pp. 271-278 (198
6) Obtained by the method described. ], for example,
Restriction enzyme PvuII (manufactured by Boehringer Mannheim Yamanouchi Co., Ltd.) at a temperature of 30"C or higher, preferably 37°C
Digestion is performed at an enzyme concentration of 1-10 units/ml for 20 minutes or more, preferably 30 minutes to 2 hours, to obtain a DNA digest.

After mixing EcoRI linker (manufactured by Takara Shuzo Co., Ltd.) with this DNA digest, DNA derived from T4 bacteriophage was added.
A ligase (Boehringer, Mannheim, Yamanouchi, Sha,
(manufactured by) was added at an enzyme concentration of 0.1 to 10 units/-, and the temperature was 4 to 37°C, preferably 4 to 16°C, for 20 minutes or more.
Preferably, the recombinant plasmid p having an EcoRI cleavage site is prepared by ligating for 6 to 24 hours.
KN306 DNA can be prepared. Transformation with recombinant plasmid pKN306 ONA was
Molec.

This can be carried out by the calcium chloride treatment described in J. Gen., Genet, Vol. 124, pp. 1-10 (1973).

In addition, as the host bacteria used here, for example, Escherichia coli 1100 (Max-Plankins institute
(obtained from Heidelberg, West Germany).

From the thus obtained transformed strain, by a conventional method,
For example, plasmid DNA p purified by cesium chloride-ethidium bromide density gradient ultracentrifugation treatment
KN306 can be obtained and this plasmid pKN3
06 ONA, for example EcoRI (Boehringer
(manufactured by Mayheim Yamanouchi Co., Ltd.) at an enzyme concentration of 1 to 10 units I-/d, and the temperature is 4°C or higher, preferably 37°C.
The DNA fragment containing the tetracycline resistance gene is obtained by reacting for 20 minutes or more, preferably 30 minutes to 2 hours, and this DNA fragment is subjected to, for example, Men's in Enzymology.
It can be isolated by agarose electrophoresis treatment as described in Vol. 68, pp. 176-182 (1979).

For example, λclastl121 DNA cut with the restriction enzyme EcoRI and Xba
5′-pAATTGTCT containing a site that identifies I
AGA linker (Beckman PT, UST DN)
(synthesized by synthesizer A), and T4DN
A ligase (Herringer, Mayheim, Yamanouchi, Sha,
bacteriophage λclss containing a tetracycline resistance gene,
1121-Tc/xb can be bred.

Next, this bacteriophage λCl8S? 1121
- DNA is extracted from Tc/xb by a conventional method, for example, by a method such as ToManiatis (Molecular Cloning).
CIo, ning), pp. 76-85 (1982)]
Purify by

Furthermore, we will describe the preparation of bacteriophage DNA that has an endonuclease cleavage site only in the genetic information for biosynthesis of the coat protein of the bacteriophage DNA, and a drug resistance marker is added downstream of the late promoter.

The drug resistance marker may be of any type, but for example, bacteriophage DNA with a Tcr marker attached.
Furthermore, any endonuclease cleavage site may be used to identify only the genetic information portion of the biosynthesis of the capsular protein, and examples thereof include the 171 cleavage site jEJ2ul cleavage site, and the like.

Bacteriophage DNA has an endonuclease cleavage site only in the genetic information for the biosynthesis of this coat protein.
can be obtained in exactly the same manner as in Japanese Patent Publication No. 61-37915. For example, bacteriophage 12-2 DNA obtained in exactly the same manner as the method in item (6) in the examples of JP-A-58-212781, and bacteriophage λgtWES, B DNA (Bethesda Research Laboratory) Re5
1 to 50 units, preferably 5 to 50 units, of a restriction enzyme, such as EcoRl (Boehringer Mannheim Yamanouchi Co., Ltd.)
Temperature 4-37°C for 10 units, preferably 37°C
The digested product is obtained by reacting at C for 20 minutes to 24 hours, preferably 30 minutes to 2 hours, and the digested product is heated using T4 DNA ligase at a temperature of 4°C to 37°C.
A ligation reaction is performed by reacting at 6° C. for 11 to 24 hours, preferably 16 to 24 hours, and this DNA is used to transduce E. coli, eg, 1100. Transduction methods can be used, for example, by D.M. Morrison (D.M.Mo.
rrison) [Men in Enzymology (M
Transduction can be carried out by the calcium chloride treatment method described in Ethos in Enzymology, Vol. 68, pp. 326-331 (1979). With this method,
Bacteriophage 12-2WE that forms plaques on E. coli, for example E. coli 1100 strain, can be obtained. This bacteriophage 12-2WE and bacteriophage φ80 were prepared using Escherichia coli (E.
coli) W3110 (φ80) (ATCC3127
7). 〕 〕Unexamined Japanese Patent Publication No. 58-212
Bacteriophage 12-2-300 DNA, which forms plaques by infecting Escherichia coli, can be obtained in exactly the same manner as described in item 2) in the Examples of Publication No. 781.

Next, we developed a recombinant bacteriophage D that has an endonuclease cleavage site only in the genetic information part of the coat protein.
For example, by combining NA and bacteriophage DNA with a drug resistance marker attached to the downstream region of the late promoter,
hoI (manufactured by Boehringer Mannheim Yamanouchi)
By recombining the bacteriophage DNA using, for example, the genetic information portion of the coat protein, it is possible to breed bacteriophage DNA that has an endonuclease cleavage site only in the genetic information portion of the coat protein and has a drug resistance marker added to the downstream region of the late promoter.

For example, bacteriophage 12-2- obtained above
300 DNA and λcTes71121-Tc/x
b with restriction enzyme XhoI (manufactured by Zenshuzo Co., Ltd.) 1 to 20 units at a temperature of 4 to 37°C, preferably 2 to 37°C.
The reaction is carried out for 0 minutes to 24 hours, preferably 30 minutes to 2 hours, and a digested product is obtained by a conventional method.
using a temperature of 4 to 30°C, preferably 4 to 16°C.
After continuous reaction for 120 minutes to 48 hours, preferably 16 to 24 hours, the above-mentioned D.M. Morrison (
D.

Bacteriophage EN501S-Tc, which forms plaques on this strain, can be obtained by transforming Escherichia coli treated with calcium chloride, for example, Escherichia coli QD5003 strain (obtained from Bonyo University), by the method of I., Morrison).

In this way, it is possible to breed bacteriophage DNA that has an endonuclease cleavage site only in the genetic information portion of the target coat protein and has a drug resistance marker added to the downstream region of the late promoter.

On the other hand, DNA containing the GSH-1 gene derived from E. coli
For example, the recombinant plasmid pHR322 was prepared in exactly the same manner as described in JP-A-59-192088.
-g5hl DNA.

Next, recombinant plasmid pBR322-gsh
For example, add 5 tul of restriction enzyme (manufactured by Boehringer Mannheim Yamanouchi Co., Ltd.) to rDNA at a temperature of 30°C or higher.
Preferably at 37°C1 enzyme concentration 1-10 units/rn
1 for 20 minutes or more, preferably 30 minutes to 2 hours, to obtain a DNA digest. This DNA digest and blood I linker (manufactured by Zenshuzo Co., Ltd.) are mixed, and then, for example, T4 DNA ligase (manufactured by Boehringer Mannheim Yamanouchi Co., Ltd.) is added to the mixture at a temperature of 4 to 37°C, preferably , 4-16°C1 Enzyme concentration 0.1-10 units/ml
The recombinant plasmid pBR322-gshl D
A recombinant plasmid with a ml cleavage site on the NA is obtained.

The recombinant plasmid obtained in this way is processed using a conventional method.
For example, recombinant plasmid DNA purified by a conventional method such as cesium chloride density gradient ultracentrifugation from a transformant obtained by transforming E. coli (E. coli) 100 using a calcium chloride treatment method, etc. get.

This purified recombinant plasmid DNA and plasmid vector, for example, pUc19 (manufactured by Takara Shuzo Co., Ltd.)
For example, the restriction enzyme Pstl (manufactured by Takara Shuzo Co., Ltd.) is added to the mixture at a temperature of 30°C or higher, preferably 37°C.
After digestion at an enzyme concentration of 1 to 10 units/d for 20 minutes or more, preferably 30 minutes to 2 hours, the DNA digest is treated with, for example, T4 DNA ligase (Boehringer Mannheim Yamanouchi Sha Co., Ltd.). ) etc. at a temperature of 4~
37°C1 Preferably 4-16°C1 Enzyme concentration 0.1-
10 units/- for 20 minutes or more, preferably 12-2
The reaction was allowed to proceed for 4 hours to obtain a recombinant plasmid having upper cleavage sites on both sides of the GSH-■ gene.

Next, a recombinant plasmid into which a plurality of GSH-1 genes are integrated is prepared.

DNA containing two GSH-1 genes derived from Escherichia coli can be prepared using a recombinant plasmid pl'lR325-
g5hl-T DNA.

The recombinant plasmid pBR3 thus obtained
25-gshl-I DNA and GSH as described above.
For example, the restriction enzyme lIJ (Takara Shuzo Co., Ltd.
(manufactured by Co., Ltd.) at a temperature of 30°C or higher, preferably 37°C.
Enzyme concentration 1-10 units/m! The DNA is digested for 20 minutes or more, preferably 30 minutes to 2 hours, to obtain a DNA digest.

For example, add T4 DNA ligase (
manufactured by Boehringer Mannheim Yamanouchi Co., Ltd.) at a temperature of 4 to 37°C, preferably 4 to 16°C, and an enzyme concentration of 0.
．． 1-10 units)/m/ for 20 minutes or more, preferably,
By allowing the reaction to occur for 6 to 12 hours, a recombinant plasmid DNA containing a plurality of GSH-1 genes, for example two, and LI cleavage sites on both sides thereof is obtained.

Next, the recombinant plasmid DNA thus obtained and the bacteriophage vector EN described above were used.
501S Tc DNA, for example, restriction enzyme cloth I (Boehringer Mannheim Yamanouchi Co., Ltd.)
(manufactured by Manufacturer) at a temperature of 30°C or higher, preferably 37°C, and an enzyme concentration of 1 to 10 units/ml for 20 minutes or longer, preferably 30°C.
After being digested for 0 minutes to 2 hours, 74 DNA ligase (manufactured by Boehringer Mannheim Yamanouchi Co., Ltd.) is added at a temperature of 4°C or higher, preferably 16°C, and an enzyme concentration of 0.1.
~5 units/- for 1 hour or more, preferably 16-2
After 4 hours of ligation, various recombinant DNAs are obtained.

Hereinafter, a recombinant DNA in which DNA containing the GSH-1 gene was inserted into bacteriophage DNA was prepared in the same manner as the method for preparing the recombinant DNA in which DNA containing the E. coli-derived GSH-n gene was inserted into bacteriophage vector DNA. A recombinant microorganism belonging to the genus Escherichia containing DNA and capable of producing GSH-1 can be obtained, and purified by the method described in, for example, Science, Vol. 202, p. 1279 (1978). body DNA can be obtained.

In addition, G inserted into the bacteriophage vector DNA
The number of DNAs containing the SHI gene is 1 or more, preferably 2 to 8.

DNA containing the GSH-n gene obtained in this way
A recombinant DNA obtained by inserting GSH-n into a bacteriophage vector DNA and a recombinant DNA obtained by inserting a DNA containing the GSH-n gene into a bacteriophage vector DNA are mixed, and a restriction enzyme such as XhoI is added to the recombinant DNA.
(manufactured by Boehringer Mannheim Yamanouchi Co., Ltd.) at a temperature of 30°C or higher, preferably at 37°C, and at an enzyme concentration of 1 to 5.
0 units/mI for 30 minutes or more, preferably 30 minutes to 2
The digested material was treated with T4 DNA ligase (Boehringer Mannheim Yamanouchi Co., Ltd.) at a temperature of 4°C or higher, preferably between 4 and 16°C, and at an enzyme concentration of 0.1.
~10 units/rri for 20 minutes or more, preferably 12
After ligation for ~24 hours, various recombinant DNA
get.

Hereinafter, a microorganism belonging to the genus Escherichia that includes a recombinant DNA obtained by inserting the above C,5H-II gene-containing DNA into a bacteriophage vector DNA and has the ability to produce C,5H-n, and a method for preparing the recombinant DNA will be described. Similarly, it is possible to obtain a microorganism belonging to the genus Escherichia that contains a recombinant DNA in which a DNA containing the GSH-1 gene and a DNA containing the GSH-II gene are inserted into a bacteriophage vector DNA and has the ability to produce glutathione. Also, for example, the above GS
Purified recombinant DNA can be obtained in the same manner as the method for purifying recombinant DNA in which DNA containing the H-1il gene is inserted into bacteriophage vector DNA.

Then, DNA containing the GSH-I gene and GS
A microorganism belonging to the genus Escherichia containing a recombinant DNA obtained by inserting a DNA containing the H-It gene into a bacteriophage vector DNA and having an ability to produce glutathione is subjected to conditions such as temperature that do not induce self-replication of the recombinant DNA. Incubate at 30-35°C for 2-24 hours.

At this time, a bacterial culture medium was used,
For example, TY medium is preferred.

Then, for example, heat induction (temperature 40-4
After inducing self-replication of the recombinant DNA by UV treatment or addition of mitomycin (5°C), the recombinant DNA is further cultured at a temperature at which the host bacteria can proliferate.

The temperature at this time does not necessarily need to be maintained at a temperature that induces self-replication of the recombinant DNA, and can be appropriately selected and employed within a range where the host bacteria can proliferate.

After inducing self-replication of the new recombinant DNA in this manner, it is cultured for, for example, 2 to 6 hours to obtain a culture.

Next, the production of glutathione will be described.

In this way, the bacterial cells were collected from the culture, and the -0.85 degree
After washing with 1% physiological saline, this was made into an aqueous suspension at 1%
Glutathione in the E. coli cells can be extracted by treatment in boiling water at 00°C for 1 to 10 minutes, preferably 1 minute. In addition, after collecting the bacterial cells after the above culture, the bacterial cells are treated as described below, and then treated with glutamic acid, cysteine, glycine, magnesium ions, ATP, and preferably in the presence of an appropriate ATP regeneration system. Glutathione can be produced by the reaction. Examples of such bacterial cell treatments include organic solvent-treated bacterial cells, surfactant-treated bacterial cells, sonicated bacterial cells, cell-free extracts obtained by centrifugation after sonication, or cell-free extracts obtained by centrifugation after sonication, or cell-free extracts obtained by centrifugation after sonication. Examples include bacterial cells and enzymes.

In this case, usable organic solvents include acetone, toluene, and ether, and surfactants include Triton xioo, dodecyl sulfate, and cetyltrimethylammonium bromide.

In addition, for immobilization of bacterial cells or enzymes, polyacrylamide gel, alginate gel, photocurable resin, and other DEA
E-cellulose, DEAE-cefazo-x, etc. can also be used as carriers. Further, as the ATP regeneration system used in such a reaction, the acetate kinase reaction and carbamate kinase reaction of Escherichia coli, or the glycolytic system of microorganisms are suitable. For the glutathione production reaction, the enzyme-containing substance is mixed with 5-40mM noglutamic acid (preferably 20mM
), 5-40mM nocysteine (preferably 20mM)
, 5-50mM glycine (preferably 20mM) 1
~30mM magnesium ion (preferably 101011
I, 20°C to 40″C (preferably 37°C) in a reaction solution containing 1 to 20mM ATP (preferably 10mM)
The reaction can be carried out at a pH of 6 to 9 (preferably 7.5) by contacting for several hours. In this reaction system, when acetate kinase reaction is used as an ATP regeneration system, acetyl phosphate is 5 to 40mM (preferably 20d
) may be added. Since E. coli has strong acetate kinase activity, there is no need to add an acetate kinase source.

Gluction produced in the extraction or reaction solution as described above can be easily isolated using a conventional ion exchange resin column. First, adjust the pH of the extract or reaction solution to 3 with sulfuric acid.
．． After adjusting to 0, this is passed through a cation exchange resin such as Diaion PK-22811'' and eluted with 0.5M ammonium hydroxide.The pH of the eluate is adjusted to 4 with sulfuric acid.
．． 5, conduction is conducted to an anion exchange resin, such as Deolite A2 (CII, COO-type). After eluting the adsorbed glutathione with 0.5M sulfuric acid, crystalline glutathione can be isolated and obtained by adding ethanol to 50%.

〔Effect of the invention〕

As described above, according to the present invention, a recombinant DNA is obtained by inserting a DNA containing the GSH-1 gene and a DNA containing the GSH-II gene into batatteriophage vector DNA, and this recombinant DNA is
Either a strain belonging to the genus Cherichia that has the ability to produce glutathione is cultured in a medium, and glutathione is collected from the culture; The present invention is extremely useful because glutathione can be produced in a higher yield than conventional methods by contacting with glycine, adenosine-5-3 phosphate, and magnesium ions.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 [1] Recombinant plasmid pBR322gsh I [
e DNA 8-cycle preparation (1) Escherichia coli (E, coli) 1100 (pBR32
Preparation of recombinant plasmid pBR325-gshU b
Escherichia coli that retains DNA C6C600
Recombinant plasmid pBR325 from r-(E, coliC600r-m-) (Feikoken Bacterial Serial No. 7471)
-gsh II b DNA (Unexamined Japanese Patent Publication No. 18059/1983)
No. 2 Publication R), T. Maniatis (T, Man
i-atis) and other methods [Molecular Cloning, Cold Spring Harbor Laboratory (Cold Spr.
ing 1 labor laboratory)
, pp. 86-96 (1982)] and purified and isolated the recombinant plasmid pBR325-gsh.
1 mg of I[b DNA was obtained.

This recombinant plasmid pBR325-gsh n
b 2 μg of DNA, 1 unit of old Bam (manufactured by Boehringer Mannheim Yamanouchi Co., Ltd.) and tlindllll
(Boehringer Mannheim Yamanouchi Co., Ltd.) l unit, 50mM) Lis-〇CI (p'H7,
5) / 20μ of solution containing 10mM MgClz/1m dithiothreitol/100mM NaCl
! After reacting for 1 hour at a temperature of 37°C, DNA fragments were separated by agarose gel electrophoresis using a conventional method, and the gel containing the DNA fragments was cut out and packed into a dialysis tube. Electrophoresis is performed again to remove the DNA fragments present in the gel, and GSH
-DNA fragment containing the It gene gsh U bh
6 μg of O was obtained.

Then, plasmid pRR322 (Bethesda Research Laboratories)
h Laboratories, Inc.) 0.5μ
g, Bam old 1 unit and Hindn [1 unit, 50mM) Lis-ICI (pH 7,5)/
10mM MgC1z/1mM dithiothreitol/
20μ of a solution containing 100mM NaCl! Temperature 3 inside
After reacting at 7°C for 1 hour, the DNA fragments obtained as described above were mixed with gsh II bh O,5II g and T4 DNA ligase (Boehringer Mannheim).
Yamanouchi Co., Ltd.) l units were added respectively, and the temperature was 16°.
Recombinant plasmid pBR32 was incubated with C for 12 hours.
0.8 μg of various recombinant plasmid DNA containing 2-gsh U bh DNA was obtained.

Next, using the thus obtained recombinant plasmid pBR322-gsh It bh DNA, Escherichia coli (E. coli) 1100 cells were treated with calcium chloride.
(obtained from Max-Plank-Institut West Germany, Heidelberg), by Jay Bakchiliol, (
J, Bacteriol, ), Volume 119, No. 107
2-1074 (1974), grown on 'ry medium containing 20 μg/ml ampicillin (hereinafter abbreviated as Ap), and tetracycline (hereinafter abbreviated as Ap). It is abbreviated as Tc.)20
E. coli (E, colt) 1100 (pBR322), an ungrown transformed strain on TY medium containing μg/-
-gshUbh) was obtained.

(2) Recombinant plasmid pBR322-gsh I
Preparation of I e DN^ Then, E. coli 110 l 100 (p
BR322-II bh) to recombinant plasmid pBR322-g by the method described in item [1] (1).
Purify and isolate sh II bh DNA and prepare 1 mg of DNA.
I got it.

This pBR322-gsh II bh DNA 5
ug was treated with 5 units of BsmHI as described above, followed by phenol treatment and ethanol precipitation in the usual manner. The resulting precipitate was dissolved in 20 μl of water and subjected to molecular cloning.
Cloning), pp. 113-114, Cold Spring Harbor Laboratory (Cold S
pring Harbor Laboratory)
(1982), E. coli DNA
Klenou fragment of polymerase I
ow Fragment of E, coli DNA
Polymerasel) (manufactured by Zenshuzo Co., Ltd.), further added 10 ug of "Original Linker", and treated with 1 unit of T4 DNA ligase as described above,
A recombinant plasmid pBR322-gsh ne DN^4ug having EcoRI cleavage sites on both sides of the GSH-■ gene was obtained on plasmid pBR322.

[2 Breeding of bacteriophage λcTas71121s (]) Breeding of bacteriophage λCll5?1121 Bacteriophage λclas
1121 was bred.

In addition, the λCIas71121 bacteriophage was obtained by incubating the phage with Escherichia coli (E. coli) 1100 cells using a conventional method.
(obtained from MaxPIank4nstftut Heidelberg, West Germany, the same strain is used hereafter), namely E, colt 1100 (λCl8
5?1121) (FERM
BP-133)] has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.

(2) Breeding item of bacteriophage λcI 5syl121s [2] Bacteriophage λ obtained in Q)
DNA was extracted from clsstl121 by a conventional method, and 2.1 μg of this DNA was cut with 10 units of "2R1" (manufactured by Boehringer Mannheim Yamanouchi Co., Ltd.).
To the NA fragment was added 0.4 μg of a DNA fragment obtained by previously cutting λcIss7 Sam7 DNA (obtained from Washington, Inc.) with 1 unit of the above EcoRI, then 1 unit of T4 DNA ligase was added, and the mixture was incubated at a temperature of 7°C. C for 48 hours to obtain a mixture of recombinant DNA, which was further subjected to in vitro packaging method [Methods in Enzymology].
Enzy-Hology) Volume 68, Nos. 281-29
8 pages, 1979, Academic Press (Academ)
ic Press] to obtain bacteriophage particles wrapped in bacteriophage coat protein.

Next, the bacteriophage particles thus obtained were spread on a tryptone agar medium using Escherichia coli QD5003 (obtained from Bonyo University, hereinafter the same strain is used) as an indicator bacteria, and after culturing at a temperature of 37°C for 16 hours, the resulting Among the plaques obtained, when E. coli 1100 described in item [2] (1) is used as an indicator bacteria, it does not form plaques, and when E. coli D5003 is used as an indicator bacteria, it has the property of forming plaques. , and only the DNA portion downstream of the late promoter P'r site and reaching its cohesive end is
There is one RI cleavage site, and the S gene involved in bacteriolysis is a mutated DNA fragment (λ
cI□7Sam? We isolated and obtained bacteriophage λCIasyl121S, which has the ability to lyse host bacteria due to its ability to lyse host bacteria.

[3] Recombinant bacteriophage λEN1121S-, in which a GSH-II gene fragment is inserted into the endonuclease cleavage site present in the DNA region downstream of the late promoter of λcIasyl121s and ending with its cohesive end under the control of the NA sequence necessary for translation. G II preparation item [2] Bacteriophage λc obtained in (2)
lsstl121s DNA 10 II g and
Item [1] Recombinant plasmid p obtained in (2)
3 μg of BR322-gsh If eDNA was mixed, and this was mixed with 50 yeast Tris-0Ct (pH 7,4)/10
50μ of a solution with a composition of 0mM NaCl/10mM Mg5O! After adding 50 units of EcoRI and allowing it to react at a temperature of 37"C for 2 hours, phenol extraction and ethanol precipitation were carried out in a conventional manner to obtain a precipitate.
H7,4)/10mM dithiothreitol/10mM
It was added to 8 μl of a composition solution of MgC1z10.1mM ATP and allowed to react at a temperature of 4°C for 18 hours to perform a ligation reaction.

0 μg of the obtained DNA was subjected to in vitro packaging.
Prepare bacteriophage particles wrapped in λ bacteriophage coat protein, and add E. coli 1100 (109 cells/rnl, 0.5 u) to 50 μl of this phage particle solution.
42) was added and incubated at a temperature of 30"C for 30 minutes, and then bacteriophage λcb2 (1010
cells/m, 0.1 rnl) and slowly heated at 30°C for 30 minutes (this operation kills non-lysogenic bacteria), plated on a T-Y agar medium, and heated at 32°C for 16 Cultured for hours.

In addition, bacteriophage λcb2 has a -12
(λ) (ATCC12435), A, D, Kaiser, Pyrology (Vi
vol. 3, p. 24 (1957) and Gee Kellenberger (G.

Kellenberger et al., J. Mol. Paiol (J.

Mo1. Biol, ), Volume 3, pp. 399-408 (
It was prepared by the method described in (1961).

Among the strains cultivated and grown as described above, λc
A lysogenic bacterium using recombinant bacteriophage DNA carrying the GSH-II gene on the restl121s DNA was searched for using the following method, and several strains of lysogenic bacteria carrying the GSH-II gene on the bacteriophage DNA were obtained.

The lysogenic bacteria obtained above, that is, λcb2-resistant and temperature-sensitive bacteria, were cultured at a temperature of 32°C using T-Y medium.
0.5 ml of the culture solution obtained by shaking culture for 16 hours was
Klett units were inoculated into 10-TY medium in a 0 ml Erlenmeyer flask.
When the temperature reached about 100, the temperature was raised to 43°C and shaken for 25 minutes, and then the temperature was lowered again to 32°C and shaking was continued for about 3 hours.

1 ml of the culture fluid thus obtained was subjected to ultrasonic disruption treatment and used as a cell extract, published in the Journal of General Microbiology (J.

Gen, Microbiol, ), Volume 128, No. 1
GS according to the method described on pages 047-1052 (1982).
The enzymatic activity of H-II was measured, and strains with high activity were selected. This highly active lysogen was cultured using Klett Units (Klett) at 32°C using 10mZ TY medium.
After culturing with shaking until the number of units (units) reached approximately 100, it was heated at a temperature of 43°C for 20 minutes, and further incubated with shaking at a temperature of 37°C for 3 hours to obtain a culture solution, from which an equal volume of phenol was added. /chloroform mixed solvent
Extract A, precipitate the resulting DNA with ethanol, and extract D.
Got NA.

The DNA thus obtained was mixed with Tris-HCl (pH
7,5) / 1mM EDTA composition dissolved in 1+nl of solution, 4uf of JI solution, 10mM) Lis-ncI
(pH7,4)/100mM NaC+/10mM
In addition, 50 units (7) were added to 30 μl of a solution with the composition of Mg5On/1mM dithiothreitol.
EcoRT and 20 μg RNase A (Sigma
The DNA fragments were digested at 37°C for 1 hour, subjected to agarose gel electrophoresis, and the size of the DNA fragments was analyzed.

As a result, all lysogenic bacteria with high GSH-n activity had approximately 1.
6 Kbp recombinant plasmid pBR322-gs
A DNA fragment derived from h n eDNA was detected.

As described above, Escherichia coli (E coli) 1100 (E
N1121S-G■) was isolated and the desired recombinant bacteriophage λEN1121S-G II DN was prepared.

[41 Recombinant plasmid pUc19-gsh U
Isolation of kk DNA (1) Escherichia coli (Lcoli) 1100 (plloo (
Preparation of pBR322- Recombinant plasmid pBR32
2-Colon carrying gsh I DNA'CIfRC9
12 (E, coli RC912)
Recombinant plasmid pBR322-g from No. 731)
shI DNA (described in JP-A-59-192088) was purified and isolated by the method described in item [11(1)].
NAImg was obtained.

This recombinant plasmid pBR322-gsh I
Mix 5 μg of DNA with 5 units of Stu I (manufactured by Herringer Mannheim Yamanouchi Co., Ltd.), digest at a temperature of 37"C for 1 hour, and then perform phenol treatment and ethanol precipitation in a conventional manner to obtain a precipitate. The substance was dissolved in 20μ! of water and this was dissolved in 20μ! of water, which was published in Molecular Cloning, pp. 113-114, Cold Spring Harbor.
Laboratory (Cold Spring Harbor)
The Klenow Fragment of E. coli DNA polymerase I (1982)
E.coli DNA polymerase T) was added, and 10 μg of blood linker-IO was added.
Recombinant DNA ApBR322-gsh was prepared by treating one unit of DNA ligase at a temperature of 16°C for 16 hours.
Recombinant plasmid pB with blood I cleavage site on I
R322-gsh I k DNA was obtained.

This recombinant plasmid pBR322-gsh I
E. coli 1 treated with calcium chloride using k DNA
100 is transformed by the method described in item [1] (1) and grows on TY medium containing 20 M/d of Ap, but cannot grow on TY medium containing 20 ug/rd of Tc. convertible strain, i.e. Escherichia coli (E.coli)
110l100 (pBR322-Ik) was obtained.

(2) E. coli JMlol (pUc1
Preparation of Escherichia coli (E. coli) 1100 (pBR3
22-gsh 1 k) to recombinant plasmid pB
R322-gsh r k DNA as item [1] (1
The DNA was purified and isolated by the method described in ) to obtain 1 mg of DNA.

The recombinant plasmid pBR322 thus obtained
-gsh Ik DNA 5 ug is used (
5 units (manufactured by Herringer Mannheim Yamanouchi Co., Ltd.) and digested at 37°C for 1 hour, the mixture was subjected to agarose gel electrophoresis using a conventional method, and the DNA
Got a piece.

The gel containing this DNA fragment was cut out, packed into a dialysis tube, and subjected to electrophoresis again to remove the DNA present in the gel.
The NA fragment was taken out of the gel to obtain a DNA fragment gsh I kp containing 2 μg of the GSH-I gene.

Next, plasmid pHc19 (manufactured by Zenshuzo Co., Ltd.) lμ
The DNA fragment gsh obtained as described above was mixed with 5 units of Pstl (manufactured by Boehringer Mannheim Yamanouchi Co., Ltd.) and digested at a temperature of 37°C for 1 hour.
1 μg of T4 DNA ligase (manufactured by Boehringer Mannheim Yamanouchi Co., Ltd.) was added and ligated by reacting at a temperature of 16°C for 12 minutes. A recombinant plasmid I) UCl3-gsh I kpDNA containing Ml cleavage sites on both sides of the gene was obtained.

Next, using the thus obtained recombinant plasmid pHc19-gsh I kp DNA, calcium chloride-treated Escherichia coli (E. coli) JMIOI
(purchased from Zenshuzo ■) by the method described in item [1] (1) above, and transformed T containing 20 ug/- of Ap.
-Y grown on medium, 5-bromo-4-chloro-3
- A transformed strain that forms white colonies on TY medium containing indolyl-β-D-galactopyranoside and isopropyl-β-D-thiogalactopyranoside,
Escherichia coli (E, coli) JMIOI (pUc19-
gsh T kp) was obtained.

(3) Recombinant plasmid pUc19-gsh U
Preparation of kk DNA Escherichia coli (E. coli) JMIOI (pUc19-g
recombinant DNApUc19-gsh I kp) by the method described in item [1] (1)
The DNA was purified and isolated to obtain 1 mg of DNA.

In addition, recombinant plasmid pBR322-gsh I
A recombinant plasmid pBR325-gsh1.in which two GSH-1 genes were integrated was prepared from DNA according to the method described in the Examples of JP-A-60-180592. IDN
A was prepared and the above Escherichia coli (E. coli) was treated with calcium chloride using this recombinant plasmid DNA.
1100 was transformed by the method described in item [1] (1), but it could not grow on TY medium containing 20 ug/ml of Ap, and 20 ug/ml of chloramphenicol! T containing
- A transformed strain, Escherichia coli (E, col) that grows on Y medium.
i) 1100l100 (pBR325-, I) was obtained.

Escherichia coli (E coli) 1100 thus obtained
(pBR325-gshl I) to recombinant plasmid pBR325-gsh1. I DNA, item [1
] (1) Purification and isolation was performed by the method described in (1) to obtain DNA 1■.

Then, recombinant DNA ApBR325-gsh
A mixture of 5 μg of DNA and 5 units of Shi II (Boehringer Mannheim Yamanouchi Co., Ltd.) was digested at 37°C for 1 hour, and the DNA fragments were separated by agarose gel electrophoresis using a conventional method. , the DN
The gel containing the A fragment was cut out, packed into a dialysis tube, and electrophoresed again to remove the DNA fragments present in the gel from the gel, resulting in a DNA fragment gsh1. 2 μg of Ibg was obtained.

Recombinant plasmid pUc1 purified as described above
9-gsh I kp lug and III (
5 units (manufactured by Boehringer Mannheim Yamanouchi Co., Ltd.) were mixed and digested at a temperature of 37°C for 1 hour, and the above DNA fragment gsh1. I bg 1 tlg was added, and a unit of T4 DNA ligase (manufactured by Boehringer Mannheim Yamanouchi Co., Ltd.) was added and ligated by reaction at a temperature of 16°C for 12 hours, and the ligated DNA was treated with calcium chloride. Said Escherichia coli (E, coli) 1
100 by the method described in item [1] (1) to obtain a transformed strain, Escherichia coli (E, coli) 1100 (pUc
19-gshl, 1kk) was isolated.

The recombinant plasmid DNA from the transformant strain thus obtained was transferred to NewClick Acids Research (N
ucleic Ac1ds Research), No. 7
A recombinant plasmid in which two GSH-■ genes have been integrated among the X and recombinant plasmid DNAs detected by isolation by the alkaline extraction method described in Vol. Ha, G
It is detected as recombinant plasmid DNA larger than recombinant plasmid DNA in which one S H-1 gene has been integrated. That is, it is detected as recombinant plasmid DNA with short electrophoretic isolation in agarose electrophoresis. In this way, a recombinant plasmid pUc19-gsh1 containing two GSH-fiff genes. I k
k DNA was obtained.

(4) Recombinant plasmid pUc19-gsh1. I
kk I] Isolation of NA Escherichia coli (E. coli) 1100 (pUc19-gsh
Recombinant plasmid pUC19-gsh1. I k
1 mg of k DNA was obtained.

[51 Breeding of bacteriophage EN501S-Tc (
1) Breeding of bacteriophage λCl8S71121 Breeding of bacteriophage λClB571121 [
This phage was isolated from Escherichia coli (E.
, coli) 1100 (Max-Plank-Ins
(obtained from Heidelberg, West Germany), lysogenic bacteria obtained by lysogenization, that is, E. coli 110
0(λClll5?1121)
No. (FERM BP-133)) has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology. ] was prepared from this bacteriophage λCIas, 1121 by the method of T. Maniatis et al.
bacteriophage λCl1lS? 112
1 DNA was obtained.

(2) Bacteriophage λcIestl121-Tc
Next, bacteriophage λcI obtained in this way
1 μg of sstl121 DNA and 10 units of Ec
oRI (manufactured by Takara Shuzo Co., Ltd.) in 50mM To'J-HCl buffer? &(100mM NaCl and 10mM Mg5
Bacteriophage λclas, 1121 was reacted for 1 hour at 37°C in O <containing pH 7,4) and then subjected to phenol extraction and ethanol precipitation using a conventional method.
0.8 μg of EcoRI digested DNA was obtained.

On the other hand, plasmid pKN206 DNA (ToMasuda et al., Agri Paiol Chem, (Agri.

Biol, Chem,), Volume 50, Nos. 271-27
It was obtained by the method described on page 8 (1986). ]
10 units of 118g PvuII (manufactured by Takara Shuzo Co., Ltd.) were added to 10mM Tris-HCl buffer a, (50mM
After reacting for 2 hours at 37°C in NaCl, 10mM MgSO4, and 1mM dithiothreitol (pH 7.4), phenol extraction and ethanol precipitation were performed using a conventional method to transfer blood to plasmid pKN206 ON. .8 μg was obtained.

Then, Pvu I of plasmid pKN206 DNA
0.8 μg of I digest, 5' end phosphorylated EcoR
I Linker - [New England Biolapse (N
ewEngland Biolabs) ・Manufactured by) 1
8 g and 1 unit of T4 DNA ligase (manufactured by Boehringer Mannheim Yamanouchi) at 66 mM l-
Lys-HCl buffer (6,6mM MgC1z, 10mM
Contains dithiothreitol and 10mM ATP, pH 7,
5), the reaction was carried out at a temperature of 4'C for 200 hours to obtain 8 μg of plasmid pKN306 ON8.

The thus obtained plasmid pNK306 DNA was used by DoM Morrison (DoM).
) Method [Men's Iso Enzymology (Meth
ods in Enzymology), Volume 68, No. 3
26-331 (1979)], Escherichia coli (E. coli) DHl (ATCC3
3849) strain and transformed E. coli DI+ 1 (pK
N306) was obtained.

In exactly the same manner as in item [1] (1) above, plasmid p
1200 μg of KN306 DNA was obtained, and 3 g of this DNA fragment and 50 units of EcoRI (manufactured by Zenshuzo Co., Ltd.) were added to 50 mM 1-Lis-HCl buffer (100 mM N
aC1 and 10mM Mg5O.

After reacting at a temperature of 37°C for 2 hours in a pH 7.4 solution, RoC, A, Y
ang) etc. [Methods in Enzymology (
Methods in Enzymology, Vol. 68, pp. 176-182 (1979)],
A few Kb of D containing the tetracycline resistance gene
3.5 μg of NA fragment was collected.

1 μg of the above 2.3 Kb DNA fragment, synthesized 5'-p
AATTGTCTAG linker (synthesized using a PLUSI DNA synthesizer manufactured by Beckman).

) 1 μg of bacteriophage λ cut with EcoRI
cls5tl121 DNA 1 tl g and T4
DNA ligase (Heringer, Mannheim, Yamanouchi,
12 units (manufactured by Co., Ltd.) were incubated at 4°C in 66mM Tris-HCl buffer (containing 6.6mM MgSO4, 10mM dithiothreitol and 10mM ATP, pH 7.5).
E. coli (E. coli) 1100 treated with calcium chloride was treated with the DNA obtained by reacting with C for 200 hours at the D.M.
) to transform Escherichia coli (E. coli).
1100 (λcles, 1121-Tc/xb) was obtained.

(3) Breeding of bacteriophage EN501S-Tc Escherichia coli (E. coli) 1100 (λcles7112
1-Tc/xb) to Tea Maniais (T, M
[Molecular Cloning, No. 76-
85 (1982)] bacteriophage λCIas71121-Tc/xb DNA 2
μg and Nishi 1 (Zen Shuzo Co., Ltd.) 10 units for 50
mixed in mM Tris-HCl buffer (containing 100mM NaC+ and 10mM MgO, pH 7.4);
After reacting at a temperature of 37°C for 1 hour, phenol extraction and ethanol precipitation were performed using a conventional method to extract the bacteriophage λCl11S71121-Tc/xb DNA
1.6 μg of ho I digest was obtained.

On the other hand, 1 μg of bacteriophage 12-2 DNA and 1 μg of bacteriophage λgt WES/λB DNA obtained in exactly the same manner as in item (6) in the examples described in JP-A-58-212781 [Bethesda Research Laboratory] -(Bethesda Re5earc
h Laboratory) 0 company/manufactured] 1 μg and EcoRI (Zen Shuzo Co., Ltd./manufactured) 10 units in a 50 m
M) in Lis-HCl buffer (containing 100mM NaCl and 10mM MgSO4, pH 7.4) at temperature 3.
After reacting at 7°C for 1 hour, phenol extraction and ethanol precipitation were performed using a conventional method to obtain 166 μg of the digested product.

1.6 μg of the thus obtained digest and T4 DNA
Ligase 1 unit was added to 66 mM Tris-HCl buffer (6
, 6mM MgClz, 10mM dithiothreitol and 10mM ATP, pH 7.5) at a temperature of 4°C for 16 hours, and the resulting DNA was used to incubate E. coli 1100 treated with calcium chloride.・M Morrison (D, M, Morrison)
Escherichia coli (E. coli) 11 was transformed by the method of
Bacteriophage 12- forming plaques on 00
Got 2WE.

Bacteriophage 12-2W obtained as described above
E and bacteriophage φ80 [JP-A-58-212
Escherichia coli (E, coli) W3110 (φ80) (ATC
C31277)] was published in JP-A-58-212781.
Escherichia coli 11 was prepared by the method described in item (2) in the examples of the publication.
00 to obtain a lysate, and this lysate was used to infect Escherichia coli (E, coli) W3110 (φ80), which is a lysogenic bacterium.
ATCC 31277) to obtain bacteriophage 12-2-300 that forms plaques.

Then, from bacteriophage 12-2-300, the aforementioned T. maniatis (T. Mon1atis)
Bacteriophage 12-2- prepared by the method of
2 μg of 300 DNA and 20 units of -Xho I were added to 50 mM) Lys-HCl buffer (100 mM Na
After reacting for 1 hour at 37°C in C1 and 10mM Mg5Oa containing (pH 7.4), phenol extraction and ethanol precipitation were performed using a conventional method to obtain a digested product of 1.6μ
I got g.

Bacteriophage 12-2-300 DNA described above
1 μg of Xho I digest of bacteriophage λclss described above, Xho of 1121-Tc/xb DNA
I digest lag and 1 unit of T4 DNA ligase in 66mM) Lis-HCl buffer (6.6mM MgCl
After reacting at a temperature of 16°C for 16 hours in a solution containing 10mM dithiothreitol and 10mM ATP, pH 7.5, the resulting DNA was used to incubate the D.M. Escherichia coli (E. coli) QD5003 treated with calcium chloride according to the method
(obtained from Bonyo University) was transformed into Escherichia coli (E, c
oli) Bacteriophage EN501S-Tc forming plaques on QD5003 strain was obtained.

[6] Recombinant bacteriophage λEN501S in which a GSH-1 gene pre-fragment is inserted into the endonuclease cleavage site present in the gene information part for producing the coat protein of batatteriophage FN501S-Tc under the control of the NA sequence necessary for translation. -Production items of GI2 [5] (3)
Bacteriophage EN501STc DN obtained in
A 108g and item [4] Recombinant plasmid obtained in (4) I) LiCl2-gshl, Ikk
Mix 3ug of DNA and add this to 1QmM Tris-
11cI (pH 7,5)/10mM MgCl□
/1 fermented dithiothreitol was added to 50 µp of a solution of the composition, and further 50 units of "I" (manufactured by Boehringer Mannheim Yamanouchi Co., Ltd.) were added, and the mixture was incubated at a temperature of 37°C for 2 hours.
After being allowed to react for a period of time, a precipitate was obtained by phenol extraction and ethanol precipitation using a conventional method.
z/] OmM dithiothreitol 10, 1mM ATP
A solution with the composition of
DNA ligase (Boehringer, Mannheim, Yamanouchi,
Co., Ltd.) was added thereto and allowed to react at a temperature of 4''C for 18 hours to carry out a ligation reaction.

0 μg of the obtained DNA was wrapped in λ bacteriophage coat protein by an in vitro packaging method to prepare bacteriophage particles, and 50 μg of this phage particle solution was added.
1 of the E. coli 1lOOclO9/rnl, 0.5
μA) was added and incubated at 30°C for 3 hours, and then treated with bacteriophage λcb2 in the same manner as described in item [3]. I got bacteria.

These lysogens were each incubated at 32°C using T-Y medium.
After culturing with shaking for 6 hours, 0.5 m of the culture solution was inoculated into 10-TY medium in a 150 m Erlenmeyer flask, and when the number of talent units reached approximately 100, the temperature was lowered to 43°. After raising the temperature to 32°C and shaking for 25 minutes, the temperature was lowered again to 32°C and shaking was continued for about 3 hours.

Among the culture fluids obtained in this manner, 1- was subjected to ultrasonic disruption treatment and used as a cell extract.

Gen, Microbiol, ), Volume 128, No. 1
According to the method described on pages 047-1052 (1982),
The enzymatic activities of cs and tl-r were measured, and strains with high activity were selected. DNA was extracted from this highly active lysogenic bacterium in the same manner as described in item [31] and incubated at 37°C for 2 hours with 10 units of "Skin" (manufactured by Herringer Mannheim Yamanouchi Co., Ltd.). The DNA fragments were digested and subjected to agarose electrophoresis to size the DNA fragments.

As a result, the lysogen with high GSH-I activity retained a DNA fragment of approximately 4 bp derived from pUc19-gshl and Ikk.

As described above, Escherichia coli (B, coli) 1100 (EN
501SG I2) was isolated, and the desired recombinant bacteriophage λEN501S-GI2 was constructed.

[71 Same - G S H on Hatateriophage DNA
Production of bacteriophage 501Gr2 x G11l carrying GSH-II a gene and GSH-II a gene (1)
E. coli 1100 (501G 12 x GIII)
Bacteriophage ENI obtained in Preparation Item 3
I21SfdlDNA log and bacteriophage EN501SG 12 D obtained in item [61
Mix 10μg of NA and add 50mM)
HCI (pH 7,5) /10mM MgC1z/1mM
A solution with a composition of dithiothreitol/100mM NaCl was added to 50 μC, and 50 units of Xhol (manufactured by Boehringer Mannheim Yamanouchi Co., Ltd.) was added.
After reacting at a temperature of 37°C for 2 hours, phenol extraction and ethanol precipitation were performed using a conventional method to obtain a precipitate, which was mixed with 50 mM Tris-11cI (pH 7,4)/1
0mM MgC1z/10mM dithiothreitol 10
, 1mM ATP, and 2 units of T4 DNA ligase (Boehringer Mannheim Yamanouchi Co., Ltd.) were added at a temperature of 4°C.
The ligation reaction was carried out by allowing the mixture to react for 18 hours.

0 μg of the obtained DNA was wrapped in λ bacteriophage coat protein using an in vitro packaging method to prepare bacteriophage particles, and 50 μl of this phage particle solution was added.
E. coli 1100 (Max-Plank4nstit)
ute West Germany, manned from Heidelberg) (10'/m
/, 0.5 µm was added, and the mixture was incubated at a temperature of 30°C for 2 hours.
After treatment with 2, lysogenic bacteria were obtained.

Lysogen containing recombinant DNA is 1.5 μg/m
/ is selected as a strain that does not grow in a medium containing Tc and lacks the ability to form plaques. A method for detecting recombinant DNA from the selected bacterial strain containing recombinant DNA is shown below.

Lysogenic bacteria containing the selected recombinant DNA -
Inoculated into Y medium rat, about 1 Klett unit
When it reached 00, the temperature was raised to 43°C and 25
After shaking for a minute, the temperature was lowered to 32°C again and shaking was continued for about 3 hours.

Add 50 μl of chloroborum to the culture solution thus obtained.
was added, and shaking was continued for 20 minutes at a temperature of 32°C (this operation causes dissolution), followed by phenol extraction and ethanol precipitation in a conventional manner to extract DNA.

The DNA thus obtained was transformed into Ec as described above.
As a result of treatment with oRI and hr and analysis of the size of DNA fragments by agarose electrophoresis, it was found that the recombinant DNA held by the selected lysogen was pUc19-gshl, which contains two GSH-1 genes, and I kk and gs
It was found that a DNA fragment derived from pBR322-gsh II e containing the HU gene was retained.

As described above, Escherichia coli (E, coli) 1100 (5
01GI 2xGI11) was isolated.

In addition, Escherichia coli (E, coli) 1100 (501GI
2
It has been deposited as 3).

(2) Recombinant bacteriophage 501GI2XG
Isolation of nIDNA The lysogen obtained above, namely Escherichia coli (E, coli) H
oo (501c12 5 containing TY medium 30mZ
0 - volume Erlenmeyer flask, cultured with shaking at a temperature of 32°C until the number of Klett units reached approximately 100, heat induced at a temperature of 42°C for 15 minutes, and then incubated at a temperature of 37°C. A culture solution was obtained by performing shaking culture for 2 hours.

Add 500 μl of chloroform to this culture solution and lyse the bacteria by this operation. ), a lysate was obtained, and Science (Sc
(1978), Volume 202, Page 1279 (1978
) to this lysate 2I11/ in the same manner as described above.
0.25 MEDTA/ 0.5 M l-lith) I
Add 400μ℃ of a solution containing C1 (pH 9,0) / 2.5% sodium dodecyl sulfate, heat at 70℃ for 30 minutes, add 500uffi of 8M potassium acetate solution, and heat on ice for 15 minutes. After leaving it alone,
Perform centrifugation at 12,000g for 20 minutes, collect the supernatant, add twice the amount of ethanol to the supernatant, and
Recombinant bacteriophage 501G I2
500 μg of NA was obtained.

(3) Recombinant bacteriophage EN1121SG
nDNA, bacteriophage EN501SG I
2 DNA and bacteriophage 501GI2 x
GSH-I and GSH-11 activities of the bacterial cells obtained by culturing the lysogenic bacteria of GIII DN Hachiyo.
(EN1121SGII), Escherichia coli 1100 (EN5
01SG I 2) and E. coli 1100 (501G
I2 x G11l) c7) possessing aSHi and G
SH-II activity is shown in Table 1.

In addition, each enzyme activity was carried out using a bacterial cell extract prepared from bacterial cells cultured in TY medium according to the method described in item [6] above.

Enzyme activity was also reported in the Journal of General Microbiology.
ralMicrobiology), Volume 128, 1
The method described on pages 047-1052 (1982) was used.

Table 1 Example 2 Glutathione-producing active lysogen, Escherichia coli 1100 (solGI2x GIII
) was cultured using TY medium according to the method described in item [61], and E. coli 1100 and E. coli 1100/
pBR322-gshLI-II b was cultured in TY medium at 37°C with shaking for 20 hours, and the thus obtained bacterial cells were harvested, and 0.85% (W/V) physiological 1 g of bacterial cells obtained by washing once with saline was added to 2 ml of 0.0.
5mM Tris-11c with 5mM cysteine
The suspension was suspended in I (pH 7,0) buffer and further subjected to ultrasonication treatment in a conventional manner to obtain 80mM L-glutamic acid, 20mM L-cysteine, 20mN glycine, 25mM magnesium chloride, 20mM ATP, 20mM acetyl phosphate and 25+11M. Potassium phosphate (pH 7.2
) Add reaction solution 79- containing buffer solution and incubate at 37°C for 2 hours.
The glutathione production activity calculated from the amount of glutathione thus produced in the reaction solution was
Shown in the table.

(Margins below this page)

Claims (2)

[Claims] (1) A novel recombinant DNA characterized in that DNA containing a gene encoding γ-glutamyl-L-cysteine synthase and DNA containing a gene encoding glutathione synthetase are inserted into bacteriophage vector DNA. . (2) Contains recombinant DNA in which DNA containing a gene encoding γ-glutamyl-L-cysteine synthase and DNA containing a gene encoding glutathione synthetase is inserted into bacteriophage vector DNA, and has glutathione production ability. A microorganism belonging to the genus Escherichia is cultured in a medium, and glutathione is collected from the culture, or the microorganism cells and/or the microorganism cell-treated product are cultured with glutamic acid, cysteine, glycine,
1. A method for producing glutathione, which comprises producing glutathione through contact with adenosine-5'-3 phosphate and magnesium ions.