JPH043196B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an improved method for producing glutathione using a recombinant plasmid in which a gene for a glutathione biosynthetic enzyme is incorporated into an E. Coli vector. Glutathione is
Tribeptide, which is composed of glutamic acid, cysteine, and glycine, is used as a treatment for a wide range of liver diseases.
It is also an important compound that is frequently used as a reagent.
Conventionally, glutathione has been produced by organic synthesis or by extraction from microorganisms (particularly yeast), but the former has a long and complex reaction process, while the latter requires complicated operations and low intracellular levels. This method cannot necessarily be said to be advantageous due to the content, and there is a desire to develop a more efficient production method.
In view of this current situation, the present inventors combined biochemical methods and genetic recombination technology to transform Escherichia coli to impart glutathione synthesis activity, and cultured this Escherichia coli to achieve high production of glutathione. succeeded in manufacturing the product using sex. Glutathione (hereinafter abbreviated as GSH) is produced by two enzymes, γ-glutamyl-L-cysteine synthase (hereinafter abbreviated as GSH-), which requires adenosine-5'-triphosphate (hereinafter abbreviated as ATP) to react.
It is biosynthesized from glutamic acid, cysteine, and glycine, catalyzed by glutathione synthetase (hereinafter abbreviated as GSH-). already,
The present inventors bred E. coli in which the first enzyme GSH-activity was enhanced through genetic recombination,
It was revealed that this strain was excellent as a GSH-producing strain (Patent Application No. 120546 (1983)).
20188)). As a result of further intensive research into improving this strain, the present inventors succeeded in cloning the gene for GSH-, the second enzyme involved in GSH biosynthesis (hereinafter abbreviated as gsh-), and enhanced GSH-activity. obtained the E. coli strain. The present invention
The present invention relates to a method for enzymatically producing glutathione using a recombinant plasmid with enhanced GSH-activity or a recombinant plasmid with enhanced GSH- and GSH-enzyme activities. Hereinafter, the present invention will be explained in the order of cloning of the GSH gene, cloning of the gene that enhances GSH and both enzyme activities, and a method for producing glutathione using the same. The vector used in the present invention is not particularly limited as long as it is an E.Coli vector plasmid with a relatively high copy number or a phage, but the case where pBR322 and pBR325 plasmids are used will be described. Cloning of GSH-gene In the present invention, the method applied to clone the GSH-gene gsh first involves using E. coli B (E. coli) as a host E. coli.
Agricultural and Biological Chemistry (Agric.Biol.Chem.) strain was used. 45 (g) 2131 (1981) to obtain a revertant strain RC912 of the GSH-deficient strain C912. ), 72 , 619
-629 (1963)] and fragment the chromosomal DNA with an appropriate restriction enzyme, such as Hind. On the other hand, the vector pBR322 was cut with the same restriction enzymes used to fragment the chromosomal DNA and further digested with the same restriction enzymes used to fragment the chromosomal DNA. 196 , 1313−
Treat with alkaline phosphatase according to the method of 1319 (1977). The thus obtained pBR322 treated product is mixed with the previously prepared chromosomal DNA treated product, annealed at 75°C for 5 minutes, and recombinant DNA is prepared using T ₄ DNA ligase. In this process,
There are no restrictions on the type of restriction enzyme that can be used. Furthermore, alkaline phosphatase treatment is not essential. In order to select the target GSH- gene gsh from among the recombinant DNAs prepared in this way, all the recombinant DNA was converted into GSH mutagenized from E.coliB.
- Introduce the activity-defective strain C1001 into concomitant bacterial cells by treatment with calcium ions (Molec. gen. Genet., 124 , 1-10 (1973)). In order to select a strain with the desired gene gsh- from the DNA-introduced strains thus obtained, 80μ of tetramethylthiuram disulfide (hereinafter abbreviated as TMTD) was added.
Minimum agar medium [KH ₂ PO ₄
0.3%, K ₂ HPO ₄ 0.7%, MgSO _{4.7H 2} O 0.01%,
( _NH4 ) _{2SO4 0.1} %, glucose 0.5%, agar 1.5
%] (hereinafter abbreviated as DM medium) and incubated at 37°C.
Incubate for 10 to 40 hours. By selecting large colonies thus generated, the target gene can be isolated.
You can easily acquire stocks that have gsh. The recombinant DNA of this strain is called pBR322-gsh.
The strain holding this recombinant DNA was transferred to L-medium [yeast extract 0.5%, glucose 0.1%,
After growing to the late log phase in NaCl 0.5%, peptone 1.0% (PH 7.2)], chloramenicol (hereinafter abbreviated as Cm) was added at a concentration of 150 μg/ml for 16 hours. Continue culturing to increase the amount of recombinant DNA within the bacterial cells. After collecting these bacteria, they were collected using the usual method (Nucleic Acid Res., 7, 1513-1517).
(1979)) pBR322−gsh by density gradient centrifugation.
Prepare in large quantities. The molecular weight of the thus obtained recombinant DNA: pBR322-gs is 4.2 megadaltons (Md), and its structure is similar to Himd of pBR322.
A 1.6Md chromosomal DNA fragment derived from the RC912 strain was introduced into this site (Figure 1A). Also Pst
By treating the chromosomal DNA of RC912, a recombinant as shown in Figure 1B can also be obtained. By introducing this recombinant DNA: pBR322-gsh into various mutant strains derived from E. coli, such as RC912 and C600, strains in which GSH-activity is specifically enhanced can be obtained. Cloning of a gene that enhances GSH- and both enzyme activities Next, construction of a strain that simultaneously enhances both GSH- and GSH-activities can be carried out by the following two methods. The first method is to transform pBR322-gsh and pBR322-gsh into mutant strains derived from E. coli.
This is a way to coexist. used here
pBR322-gsh is derived from chromosomal DNA derived from RC912 using restriction enzymes, as described in Japanese Patent Application No. 120546/1983.
Recombinant DNA obtained by treatment with Pst:
pBR322-gsh (having a structure in which an RC912 chromosomal DNA fragment with a molecular weight of 4.7 Md and corresponding to 2.1 Md was inserted into the Pst site of pBR322 (see Figure 2)) is used. The two types of recombinant DNA are introduced into the E. coli-derived mutant strain made compatible using the method described above, but the order of introduction does not matter; in some cases, pBR322-gsh and pBR322-gsh may be mixed and introduced at the same time. Selection of these hybrid plasmid-introduced strains can be carried out using the above-mentioned resistance to TMTD or sensitivity to ambicillin (hereinafter abbreviated as Am) and tetracycline (hereinafter abbreviated as Tc) as indicators. For example, strain C912 mutated from E.coliB
A strain into which pBR322-gsh has been introduced (GSH-activity-deficient strain) can be easily selected as a strain that grows in L-medium containing 20 μm/ml of Tc. Also C912
strain pBR322-gsh (plasmid in Figure 1 A)
The strain into which Am is introduced can be selected as a strain that grows in L-medium containing 20 μg/ml of Am. When using the above-mentioned DM medium instead of L-medium, 10~
It is possible to select bacteria resistant to 20 μg/ml of TMTD. Also, pBR322−gsh, pBR322
The strain introduced with both -gsh and 20 μg/ml of Am
It can be easily selected as a bacterium that grows in L-medium containing 20 μg/ml of Tc and Tc. Thus, two types of recombinant DNA within the same bacterial body:
Combined with pBR322−gsh and pBR322−gsh,
A strain can be obtained in which GSH- and GSH-activity are simultaneously enhanced. A second method for increasing both GSH- and GSH- activities is to link both GSH- and GSH- genes to the same vector and introduce the vector into an E. coli-derived strain. As a method, first
Treat pBR322−gsh with Pst to release it
Isolate the RC912-derived DNA fragment. This fragment can be easily isolated using Methods in Entemology, which can be easily performed by subjecting the Pst-treated product of pBR322-gsh to agarose gel electrophoresis, separating it, and then extracting it from the gel.
Enzymology), 68 , 176-182 (1979). pBR322−, the isolated DNA fragment was treated with Pst.
gsh (plasmid in Figure 1A). This mixture is introduced into a strain derived from E. coli, such as the C912 strain, which has been treated with T ₄ DNA ligase and made compatible by calcium treatment. Target gsh and gsh
recombinant DNA containing the gene (this
Selection of strains harboring pBR322-gsh was performed using strain C912 after transformation scanning.
This can be easily carried out using bacterial cells that can grow on a DM-agar medium containing 10 μg/ml of TMTD (see Figure 3).
Thus, on vector pBR322, GSH-,
A recombinant DNA containing both GSH- genes, gsh and gsh, was obtained: pBR322-gsh. By introducing this recombinant DNA into an E. coli-derived strain, GSH- and GSH- activities were simultaneously enhanced. You can obtain bacterial strains. GSH− and GSH
- Another way to simultaneously enhance the activities is to use pBR325 instead of vector pBR322. GSH−, GSH− on pBR325
The method of incorporating the two genes gsh and gsh is as follows.
The basic value is the same as in the case of pBR322 described above. The procedure is as shown in Figure 4, pBR322-
DNA fragments containing gsh are extracted from gsh by Pst treatment. Similarly, Hind pBR322−gsh
Process to extract DNA fragments containing gsh. These two types of DNA fragments are inserted into the Pst site and Himd site of pBR325 to prepare a recombinant DNA having gsh on pBR325: pBR325-gsh. In this case, since pBR325 has a Cm resistance gene, selection of transformed strains becomes extremely easy. By introducing pBR325-gsh into various strains derived from E. coli, GSH- and GSH-
It is possible to obtain microbial cells in which the activities of the following are simultaneously enhanced. Method for producing glutathione GSH−, GSH− and
Using E. coli with enhanced GSH− activity
Production of GSH can be carried out as follows. Bacterial strains with enhanced GSH- or both GSH- and GSH- activities are grown in nutrient media containing carbon sources, nitrogen sources, inorganic salts, etc., or minimal media (DM).
culture medium, etc.) and culture by shaking. Oxygen sources include crucose, fructose, glycerol,
In addition to inorganic nitrogen such as ammonium chloride, ammonium nitrate, ammonium sulfate, and (urea), organic nitrogen such as yeast extract, peptone, and meat extract can be used as the nitrogen source. It is also effective to add magnesium chloride, manganese chloride, etc. as trace metal elements.
The concentration of the carbon source used is 0.1-5%, preferably 0.5
%, the concentration of the nitrogen source is 0.05-5%, preferably 0.5%,
Further, it is preferable to add trace metal elements in an amount of about 0.005%. Culture temperature is 20-40℃, preferably 37℃,
Further, the culture pH is preferably 6 to 8, preferably 7. In this way, the bacterial cells were collected from the cultured solution, and once 0.85%
The GSH in the E. coli cells can be extracted by using physiological saline as a washing solution or water suspension and treating this in boiling water at 100° 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 treated with glutamic acid, cysteine, glycine, magnesium ions, ATP, and preferably appropriate ATP.
By reacting in the presence of a regeneration system, GSH
can be manufactured. 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. Examples include bacterial cells and enzymes. In this case, usable organic solvents include acetone, toluene, and ether, and surfactants include Triton x 100, dodecyl sulfate, and cetinitrimethylammonium bromide. Also,
For immobilization of bacterial cells or enzymes, other DEAEs such as polyacrylamide gel, carrageenan gel, alginate gel, photocurable resin, cellulose, DEAE etc.
- Cephadex and the like can also be used as carriers. 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. The GSH production reaction is performed by converting the enzyme-containing material into 10-100mM curtamic acid (preferably
80mM), 5-40mM cysteine (preferably
20mM), 5-50mM glycine (preferably
20m), 1-30mM magnesium ions (preferably 10mM), 1-20mM ATP (preferably
10mM) at 20°C to 40°C (preferably
(37°C) The reaction pH is 6 to 9 (preferably 7.5) and the reaction can be carried out by contacting for several hours. In this reaction system, when using acetate kinase reaction as an ATP regeneration system, 5-40mM of acetyl phosphate
(preferably 20mM) may be added. Since E. coli has strong acetate kinase activity,
There is no need to add an acetate kinase source. GSH produced in the extraction or reaction solution as described above can be easily isolated using an ordinary ion exchange resin column. First, extract or reaction solution
After adjusting the pH to 3.0 with sulfuric acid, it is passed through a cation exchange resin, such as Diaion PK-228H ⁺ , and eluted with 0.5 M ammonium hydroxide. After adjusting the pH of the eluate to 4.5 with sulfuric acid, conductive to an anion exchange resin, such as Deolite A2 (CH ₃ COO ^- type). After eluting the adsorbed GSH with 0.5M sulfuric acid,
Crystalline GSH can be isolated and obtained by adding ethanol to 50%. The present invention will be specifically explained below with reference to Examples. In addition, pBR322-gsh, both pBR322-gsh and pBR322-gsh, pBR322-gsh,
and pBR325-gsh, respectively, were transferred into RC912, and the strains were each transferred to the National Institute of Microbiology, Agency of Industrial Science and Technology, with accession number No. 336 (FERM).
BP-336, FERM P-
6731)), Microtechnical Research Institute No. 6732 (FERM
P-6732) FERM P-6733 (FERM P-6733) and FERM BP-337 (transferred from FERM BP-337, FERM P-6734)
Deposited as. Example 1 (Preparation of pBR322-gsh and strain retaining it) E.coli B
(ATCC23226)) Mutant strain induced by mutation
After growing RC912 in L-medium to the logarithmic growth phase, the cells are collected and washed once with physiological saline (0.85%). From 1 g (wet weight) of the bacterial cells thus obtained, the phenol method [Biochm, Biophys, Acta, 72 ,
619-629 (1963)] to extract chromosomal DNA, approximately 1.6
mg of DNA was obtained. Hind 1 μg of this chromosomal DNA
Fragment by processing at 37°C for 30 min and separate Hind.
After treatment for 2 hours, the method of Ullrich et al. (Science
1 μg of vector plasmid pBR322 treated with alkaline phosphatase in 196, 1313-1319 (1977).
and treated with _T4 DNA ligase at 10°C for 16 hours to prepare recombinant DNA. To select the desired recombinant DNA: pBR322: gsh, all the obtained recombinants were introduced into the compatible cells of the GSH-deficient strain C1001, which was mutated from E. coli B, and a strain carrying pBR322-gsh was obtained. For selection, spread on DM medium containing 80 μg/ml of TMTD. After culturing at 37°C for 40 hours, the GSH- gene gsh was isolated by harvesting large colonies.
A strain containing a recombinant DNA: pBR322-Gsh was obtained. A large amount of pBR322-gsh was prepared from the bacterial cells by density gradient centrifugation. The restriction structure of the obtained recombinant DNA was as shown in Figure 1A. The molecular weight is 4.2Md, and a 1.6Md RC912-derived DNA fragment is inserted into the Hind site of vector pBR322. In this example, Pst is used instead of Hind.
similar recombinant DNA by using
"Figure 1 B" that can be obtained. In this case, the molecular weight of the recombinant DNA is 8.0Md and the Pst of pBR322
A DNA fragment corresponding to 5.4Md derived from RC912 is incorporated at this site. thus obtained
pBR322-gsh (Fig. 1A)) was introduced into various mutant strains derived from E. coli B. For introduction into mutant strains, the advanced calcium treatment method [Molec.gen.Genet., 124 ,
1-10 (1973)], and the transformed strains were selected using L-medium containing 20 μg/ml ambicillin or 10 μg/ml to 80 μg/ml.
This can be done as a resistant strain for each drug using DM medium containing g/ml of TMTD. Table 1 shows the GSH- and GSH-activities of the thus obtained transformed strain. In addition, each enzyme activity was carried out using a bacterial cell extract prepared from bacterial cells in logarithmic growth phase in DM medium. Enzyme activity was also determined by the Journal of General Microbiology (J.Gen.
Microbiol) 128 . 1047-1052 (1982). [Table] Example 2 The strains listed in Table 1 of Example 1 were inoculated into 500 ml of DM medium, and cultured with shaking at 37°C for 3 hours. The thus obtained bacterial cells in the logarithmic growth phase are collected and washed once with 0.85% physiological saline. This bacterial cell is resuspended in water to make a 50 ml/ml solution. 0.5ml of this suspension
Heat at 100°C for 1 minute to extract glutathione from the bacterial cells. The amount of glutathione thus obtained was as shown in Table 2. [Table] Assay method: Analytical Biochemistry (Anal.Biochem.) 27 , 502-522 (1969) Example 3 (Bacterial strain having both pBR322-gsh and pBR322-gsh) Bacterial strain C912/ described in Table 1 of Example 1 pBR322−
gsh. C1001/pBR322-gsh and RC912/
After each pBR322-gsh was made compatible by calcium ion treatment, the recombinant DNA:
pBR322-gsh was introduced into the same strain.
A transformed strain carrying both gsh and pBR322-gsh was constructed. GSH− of these transformed strains,
GSH-activity and glutathione content within the bacterial cells were as shown in Table 3. [Table] Example 4 (Preparation of pBR322-gsh and strain retaining it) After treating 50 μg of pBR322-gsh with Pst,
Separate the generated DNA fragments by agarose gel electrophoresis. The gel containing the small fragments was cut out, placed in a dialysis tube, and subjected to electrophoresis again.
DNA fragments present in the gel are brought out of the gel. In this way, a DNA fragment containing about 5 μg of gsh was obtained. Next, 1 μg of pBR322-gsh is treated with Pst, 1 μg of the gsh-containing DNA fragment prepared above is added, and the mixture is treated with T ₄ DNA ligase. Thus
A recombinant DNA containing both gsh and gsh genes is obtained on pBR322: pBR322-gsh. This pBR322-gsh• is introduced into an E. coli-derived strain by calcium treatment. The transformed strain is
It can be easily selected by picking large colonies growing on DM medium containing 20 μg/ml of TMTD. The GSH of the thus obtained reversible strain
-, GSH- activity and GSH content are as shown in the table below. [Table] Example 5 (Preparation of pBR325-gsh and the strain harboring it) 50 μg of pBR322-gsh was treated with Pst to obtain 4 μg of a DNA fragment containing gsh in the same manner as in Example 4. On the other hand, vector pBR325 was treated with Pst,
Add 1μg of DNA fragment containing gsh to this 1μg.
Treat with _T4 DNA ligase. To obtain the desired recombinant DNA: pBR325-gsh, transform C912 strain with the ligase-treated product, and transform pBR325-gsh.
strain containing 20μg/ml TMTD and 5μ
Colonies grown on DM medium containing g/ml tetracycline were obtained. Next, a large amount of pBR325-gsh is prepared from this strain by density gradient centrifugation. This recombinant DNA: pBR325-gsh
In order to incorporate it into gsh, first pBR322−gsh
(50 μg) was treated with Hindd, and as in Example 4,
The fragments are separated by electrophoresis and contain gsh
Approximately 7 μg of DNA fragments were obtained. 1μg of this DNA fragment
Mix with 1 μg of pBR325-gsh treated with Hind and treat with _T4 DNA ligase. Thus,
Recombinant DNA containing both gsh and gsh genes on pBR325: pBR325−gsh・in
Can be synthesized in vitro. This is introduced into various strains derived from E. coli B by a calcium treatment method. Selection of transformed strains can be performed by picking large colonies in a DM medium containing 80 μg/ml TMTD and 2 μg/ml chloranephenicol. The GSH-, GSH-activity and GSH content of the thus constructed strain containing pBR325-gsh were as shown in the table below. [Table] Example 6 Example 2 Strain RC912/pBR322-gsh listed in Table 2
, strain RC912/pBR322− as described in Example 3 Table 3
gsh, -gsh, strain RC912/ as described in Example 4 Table 4
pBR322-gsh. and the strain RC912/pBR325-gsh. listed in Table 5 of Example 5 are cultured in DM medium in the same manner as in Example 2. After collecting the cells in the logarithmic growth phase, they were washed once with 0.85% physiological saline, and then suspended in 5mM Tris-HCl buffer (PH7.5).
Crush for 5 minutes at 90KHz, then crush at 15.000 rpm, 30
Centrifuge for minutes. The bacterial cell extract obtained in this way is
20mML-glutamic acid, 20mML-cysteine,
20mM glycine, 10mM magnesium chloride,
37 in a reaction solution containing 10mM ATP, 10mM acetyl phosphate, 50mM Tris-HCl buffer (PH7.0).
Incubate at ℃ for 2 hours. The amount of glutathione thus produced in the reaction solution was as shown in the table below. [Table] Example 7 Strain RC912/pBR325- listed in Table 5 of Example 5
gsh・peptone 1.0%, yeast extract 1.0%,
Meat extract 0.5%, glycose 1.0%, magnesium sulfate, heptahydrate 0.01%, monopotassium phosphate 0.5%,
Medium containing chloramphenicol 20 μg/ml (PH
7.0) Inoculate 1000ml and culture with aeration and shaking at 30℃ for 20 hours. After collecting the bacteria and washing them once with physiological saline, 28 g (wet weight) of viable bacteria were suspended in 15 ml of 30% potassium chloride solution, and 13 ml of 33.5% acrylamide monomer and 20% N,N'- Add 2 ml of methylenebisacrylamide, 5 ml of 5.0% β-dimethylaminopropionitrile and 6 ml of 6.5% potassium persulfate, and leave at 20°C until a gel is formed. Next, this immobilized bacteria was molded into a cube with sides of 2 mm and washed with physiological saline to form the immobilized E. coli RC912/pBR325−.
Obtain gsh・80g. Contains no plasmids
Immobilized bacterial cells were prepared for the RC912 strain in the same manner. 2.5g of the prepared fixed bacterial cells were mixed with 80mML-glutamic acid, 20mML-cysteine, 20mM glycine,
25mM Magnesium Chloride, 20mM ATP, 20mM
A shaking reaction was carried out at 37°C in 5 ml of a reaction solution containing acetyl phosphate and 25 mM potassium phosphate buffer (PH7.0), and the amount of glutathione produced over time was determined as shown in the table below (conversion The percentage indicates the conversion rate from L-cysteine). [Table] Example 8 Strain RC912/pBR325- listed in Table 5 of Example 5
gsh. and RC912 strains are cultured in the same medium as in Example 7. After collecting the bacterial cells (wet weight: 10 g), wash once with physiological saline, suspend in 10 ml of physiological saline, and warm to 37°C. Add and mix 20 ml of a 3.1% carrageenan aqueous solution (37°C), and drop this mixture into a 2% potassium chloride aqueous solution from a nozzle to prepare a spherical gel with a diameter of about 3 mm. 2.5 g of this immobilized bacterial body was added to 37 ml of the same reaction solution as in Example 7.
The shaking reaction was carried out at .degree. C., and the amount of glutathione produced over time was quantified, as shown in Table 8 (the conversion rate indicates the conversion rate from L-cysteine). Furthermore, after 4 hours of reaction, the immobilized bacterial cells were filtered off, and the same reaction solution was used repeatedly to quantify the amount of glutathione produced after 4 hours of reaction. The results are shown in Table 9. Almost the same results were obtained even when the concentrations of ATP and acetyl phosphate in the reaction solution were varied. [Table] [Table] [Brief explanation of drawings] [Table] [Table]

Claims (1)

[Claims] 1. Has a restriction enzyme map shown in (i) below obtained by treating E. coli chromosomal DNA with Hind.
Glutathione synthase gene (gsh) contained in the 1.6Md gene fragment and has the restriction enzyme map shown in (ii) below obtained by treating E. coli chromosomal DNA with Pst.
γ-glutamyl contained in the 2.1Md gene fragment
L-cysteine synthase gene (gsh) E. coli carrying a recombinant plasmid incorporating both genes or only the gsh gene is cultured, and glutathione is collected from the culture, or the treated E. coli cells are treated with glutamic acid, cysteine, glycine, adenosine-5'- A method for producing glutathione, which comprises producing glutathione by contacting it with triphosphate and magnesium ions. 2. The production method according to claim 1, characterized in that an adenosine-5'-triphosphate regeneration system is conjugated to the reaction system. 3. The production method according to claim 1 or 2, wherein the adenosine-5'-triphosphate regeneration system is an acetate kinase reaction, a carbamate kinase reaction, or a bacterial or yeast glycolytic reaction system. . 4. The production method according to claim 1, wherein the bacterial cell-treated product is a cell-free extract, a cell suspension, or an immobilized bacterial cell or enzyme of Escherichia coli. 5. The manufacturing method according to claim 4, which comprises immobilizing in a gel-like body. 6. The manufacturing method according to claim 5, wherein the gel-like carrier is a polyacrylamide gel.