JPH0198487A - Novel plasmid, novel bacterium transformed with said plasmid and enzymatic production of s-lactoylglutathione using said bacterium - Google Patents

Abstract

PURPOSE:To produce S-lactoylglutathione, by bringing Escherichia coli transduced with a structural gene of glyoxalase I derived from Pseudomonas putida into contact with a substrate containing methylglyoxal and glutathione. CONSTITUTION:Chromosome DNA of Pseudomonas putida IFO3738 is digested with restriction enzyme Sau3AI and connected to pBR322 digested with BamHI by T4 ligase to give pGI318. The plasmid is further treated with HindIII to give pGI423. Escherichia coli C600 strain is transduced with these plasmids to give C600 (pGI318) (FERM P-3638) and C600 (PGI423) (FERM P-3639). These strains are cultivated in a medium, the prepared cell or a treated material of the cell is brought into contact with a solution of a substrate containing methylglyoxal and glutathione to give S-lactoylglutathione.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Application] The present invention relates to a novel plasmid, a novel microorganism transformed with the same, and a method for producing S-lactoylglutathione using the same.

[Prior Art] Glyoxalase I (
glroxalasa +) is classified as enzyme number E1.4.4.1.5 by the Enzyme Committee of the International Union of Biochemistry, and its systematic name is lactoylglutathione lyase (Lactoylglutathione lyase).
It is an enzyme called ylglutations 12age).

This enzyme catalyzes the conversion reaction of methylglyoxal and glutathione to S-lactoylglutathione as shown in the reaction formula below.

Methylglyoxal + glutathione - hemimercaptal → S-lactoylglutathione This enzyme has been reported to exist in various organs of humans and rats, as well as in bacteria such as yeast, mold, actinomycetes, pseudomonas, and Escherichia coli. [Proteins, nucleic acids, enzymes; 31.10
10. (1988)],]@On the other hand, S-lactoylglutathione, which is biosynthesized by the action of glyoxalase, has an excellent anti-inflammatory effect that is equal to or better than that of glutathione as a physiological action, and is accepted as an anti-inflammatory agent. It is a useful substance (Japanese Patent Application No. 82-1010114).

As a method for producing S-lactoylglutathione,
The simplest method is considered to be biosynthesis from glutathione and methylglyoxal using the catalyst of glyoxalase I produced by microorganisms.

[Problems to be solved by the invention] However, glyoxalase produced by microorganisms! Due to its low activity, it is not possible to directly use the hedron or the bacterial cell-treated product for the reaction, and in order to efficiently produce S-lactoylglutathione, the report of Lacquer [J, BIol, C
he*,, Idandan4. As seen in [Reference], it was first necessary to purify glyoxase from bacterial cell extracts and increase its activity.

In view of this situation, the present inventors developed glyoxalase!
As a result of various studies aimed at finding microorganisms with excellent productivity, we isolated a DNA fragment carrying the genetic information for glyoxalase I from the chromosomal DNA of Pseudomonas putida, which has relatively high glyoxalase I activity. Ia succeeded in inserting a DNA fragment carrying the genetic information of glyoxalase I into a vector and introducing this recombinant DNA into Escherichia coli, and the microorganism thus obtained was an excellent glyoxalase! It was discovered that the present invention has production capability, and the present invention was completed.

[Means for Solving the Problems] That is, the present invention provides (1) a plasmid containing a DNA fragment carrying the genetic information of glyoxalase I obtained from Pseudomonas putida, and (2) a plasmid containing the 8A plasmid in Escherichia and coli. and (3) a method for producing S-lactoylglutathione by culturing the microorganism and contacting the resulting two or two treated products with a substrate solution containing methylglyoxal and glutathione.

Hereinafter, the present invention will be explained in detail.

(a) Plasmid and its preparation The novel plasmid of the present invention takes a form that can proliferate in cultured cells, such as the colicin E1 factor, which is known as an extrachromosomal gene (plasmid) of a microorganism belonging to the genus Escherichia. Glyoxalase in plasmids (extrachromosomal DNA or vector DNA)! This is a plasmid that incorporates a DNA fragment involved in the production of glyoxalase I prepared from the chromosomal DNA of a microorganism that produces the vector. Some DNA parts other than the part may be missing.1For example, Co1E+ strain, 9M89 strain, PBR322.
strains, pscIol strains, R8 strains, lambda phage strains, and the like.

Glyoxalase! DNA fragments involved in the production of can be prepared from the aforementioned yeasts, molds, actinomycetes, large intestine 1, Pseudomonas bacteria, etc., but chromosomal DNA fragments prepared from Pseudomonas bacteria are particularly advantageously used.
For example, a DNA fragment prepared from the chromosomal DNA of Pseudomonas putida IFO3738 can be used.

In addition, any known method can be used to incorporate the chromosomal DNA fragment into the vector DNA. For example, the chromosomal DNA is cut at a specific site by treatment with an appropriate restriction enzyme (Endonualea-ia), and then the same method is used. A method is used in which the vector DNA is mixed with the previously treated vector DNA and religated using ligase. As vector DNA,
pBR322 plasmid was used, and chromosome D prepared from the Pseudomonas putida IFO3738 was added to it.
By incorporating the NA fragment, a new plasmid par
318 is obtained.

The restriction enzyme cleavage map of the pGl 318 plasmid is shown in Figure 1.As is clear from Figure 1, this plasmid is located at the restriction enzyme site B of the pBR322 plasmid DNA.
The Pseudomonas putida rFO37 on a Peng losite
38 glyoxalases! It is a circular molecule with an 8 kb base pair in which a DNA fragment carrying the genetic information involved in the production of pGl is incorporated (see Figure 2).
318 plasmid DNA with restriction enzyme) tin dI
5, which was processed with HindlI and sandwiched between HindlI+ sites.
3k b replication and glyoxalase! NA fragments unnecessary for gene expression were deleted to create pGl 423, a reduced plasmid of PCR 318 (see Figure 1).

(b) Preparation of microorganisms The thus obtained conjugate of the chromosomal DNA fragment and vector DNA can be transformed using known transformation methods.

, for example, shot gun method (5hot gun method)
tbc+d) into a recipient microbial cell, a transformed strain having both the desired genetic traits and the traits of the vector DNA can be obtained.

Recipient bacteria include Escherichia coli aeoo and Escherichia coli HB.
IO+, same OP, 5upF, same xl? 78, same L
E392 etc. [Molecular Cloning Laboratory Manual
ing A LaboratortXanual) p
Microorganisms commonly used in this type of technical field are advantageously used. A typical example of this is the Escherichia coli ceoo strain (Molecular Cloning Laboratory). No1ecular C1o++-ing
＾Laboratory Manual) p5G
See 4 (+1182); genetic trait F-, tbi-1, t
hr-1, IeuB6, 1acYI, tanA21゜m
upE44.1-L The above plasmid pc! is added to this Escherichia coli ceoo strain. The microorganisms obtained by the transformation method by introducing 318 or pc+423 are new microorganisms, and are respectively Escherichia coli 0800 (po!318).
[! 5chsrichia coli 0800 (p
Gl 318)], Z Scherich 7 ◆ko
Li C[lQO(pGl 42G )[E
nch*ric h Ia c.

0600 (pGI 423) 1 was called 1982.
It was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on October 7Ei. !RM P-9639), the Escherichia coli 08 thus obtained
00 (PCI 318) and Escherichia coli C
Comparing the mycological properties of 800 (P(il 423)) with those of the Escherichia coli ceoo strain, which is a direct recipient of DNA, it was found that the former two strains had high glyoxalase I production ability and ampicillin resistance; They are identical except that the latter does not have these properties.

(c) Production step of S-lactoylglutathione The transformed strain obtained in Cb) has strong glyoxalase! Since it has activity, S-lactoylglutathione can be produced by using bacterial cells or a processed product of the bacterial cells as an enzyme source and bringing the enzyme source into contact with a substrate solution containing methylglyoxal and glutathione to cause an enzymatic reaction. can be manufactured.

1 as a medium used for culturing uninvented signs
Either a synthetic medium or a natural medium containing a carbon source, nitrogen source, and inorganic substances, which are normally used as a growth medium for Escherichia coli, can be used. Various carbohydrates such as glucose, sucrose, fructose, starch, starch hydrolysates, and molasses can be used as carbonaceous sources;
The amount used is 0.5 to 5. In addition, nitrogen sources include various inorganic and organic ammoniums such as ammonium sulfate, phosphorous ammonium, charcoal ammonium, vinegar/fed ammonium, or peptone, yeast extract, cornstap liquor, and casein. Nitrogen organic substances such as hydrolysates can be used, and the amount used is 0.5 to 2.0! Further, as the inorganic substance, for example, phosphorus-poor potassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, manganese sulfate, etc. can be used, and the amount used is preferably about 0.005 to 1.0z. The method can use conditions such as pH, temperature, and oxygen supply that are usually suitable for the growth of Escherichia microorganisms, but when the microorganisms grow and reach the maximum number of microorganisms, that is, logarithmic growth. It is preferable to grow until the vk stage, and the culture temperature is usually 30 to 37 degrees Celsius.
, the pH condition is P) in the range of 15 to 8, especially around neutrality.

Examples of the processed bacterial cells used in the above enzymatic reaction are 1. For example, processed bacterial cells obtained by drying or treating viable bacterial cells with an organic solvent such as toluene, and cell extracts obtained by ultrasonicating viable bacterial cells. In addition to enzymes obtained by purifying liquids and extracts, immobilized bacterial cells or enzymes obtained by known immobilization methods such as polyacrylamide gel entrapment method and carrageenan gel entrapment method. can also be used.

As a reaction solution for producing S-lactoylglutathione, methylglyoxal is added to 5 to 10111, and 5 to 10
Glutathione at 0 m, 20-100 mN buffer (P
Add the treated bacterial cells to H6-9) as an enzyme source. A temperature range of 25 to 40°C and a reaction time of 30 to 120 minutes are sufficient.

After stopping the reaction by adding 0.5 to 1.0% trichloroformic acid to the reaction solution, S-lactoylglutathione in the supernatant increases the absorbance to 240 nn or uses glyoxalase ■. It can be measured by enzymatic method.

In this way, S-lactoylglutathione is generated and accumulated in the reaction-completed solution, but the generated S-lactoylglutathione can be isolated by a known method such as ion-exchange resin treatment. resin(
For example, Rohm and Haas Amberlight fR
120B, conducts to I (type). S-lactoylglutathione can be obtained by eluting the adsorbed S-lactoylglutathione with 0.1N sodium hydroxide, adding 50% ethanol to the eluate, and isolating the precipitated crystals.

Hereinafter, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to this in any way.

Example 1 (1) Preparation of chromosomal DNA having genetic information for glyoxalase I-producing ability.

N. putida IFO3738, which has the ability to produce glyoxalase I, was grown in LB medium [(g/l):
Peptone 10.0, yeast extract 5.0. Mail
5.0゜glucose 1.0 adjusted to pH 7.0] After culturing with shaking in 200ml at 30℃ for 5 hours and collecting the bacterial cells in the late logarithmic growth stage, D by the phenol method.
Chromosomal DNA was extracted and purified by the NA extraction method to obtain 900 grams of chromosomal DNA.

(2) Preparation of vector DNA DNA of pBR322 plasmid having ampicillin resistance and tetracycline resistance used as a vector was prepared as follows.

Escherichia carrying pBR,322 as a plasmid
+Add 50 eg/intestine I of chloramphenicol-
Ta. The supernatant was treated with 2-butanol to concentrate the DNA, then treated with ribonucleolytic enzyme at 37°C for 3 hours, and then subjected to cesium chloride-ethidium bromide equilibrium density gradient centrifugation to extract pBR322 plasmid DNA.
450 rugs were obtained.

(3) Vector insertion of chromosomal DNA fragment 1.5p of the chromosomal DNA obtained in (1) was taken and digested by adding restriction enzyme Sau 3^■ and reacting at 37°C for 1.5 hours. on the other hand,(
1.0 μg of the vector DNA obtained in 2) was completely digested with the restriction enzyme Bag Hl, and each of the six reaction mixtures was incubated at 85°C.
, After heat treatment for 10 minutes, both reaction solutions were mixed and T4
DNA strands were ligated using phage-derived DNA ligase at 10°C for 16 hours. After heat treatment at 65°C for 5 minutes, twice the volume of ethanol was added to the reaction solution and the mixture was heated for 120 minutes.
℃ for 1 hour [centrifuged at 135,000 x g to collect the precipitate, which was diluted to 5.0 μg.

Dissolve in 0.15 ml of Tris-HCl buffer (pH 17.5).

This was used as a DNA solution.

(4) Transformation with a plasmid carrying a gene involved in the production of glyoxalase I After growing Escherichia coli C800 in 50 liters of LBL soil until the mid-log phase (00, . - 〇, 3), Tris buffer (50 m
ll, pH 7,0) and then the same buffer 1.51
resuspended in. Add 1 ml of the DNA solution Q obtained in (3) to 0.2 ml of this suspension, hold at 0°C for 30 minutes, and immediately apply a heat pulse at 42°C for 2 minutes to transfer the plasmid DNA to cells. The 0 that was taken into the
This cell suspension was separately inoculated into the LB medium, and incubated at 37°C.
After shaking culture for 3 hours to perform a transformation reaction, a strain with 7-ampicillin resistance and high glyoxalase activity was isolated and transformed into Escherichia Fist: I IJ 0800 (p.
GI 318) (Feikoken Bibori No. 91138) was obtained.

Example 2 (1) Preparation of pGI 318 Transformed strain Escherichia coli ce described in Example 1
pGI 318 plasmid DNA was prepared from oo (par 31B) by the method described in Example 1 (2).

(2) Construction of pGI 423 and transformation with the plasmid The pat 318 plasmid DNA obtained in (1) was completely cleaved with the restriction enzyme H+aal[, and this solution was subjected to 7 galose gel electrophoresis (agarose 0.0.90 V). and an 8.5 kb D containing the glyoxalase gene part.
The NA band was cut out under ultraviolet irradiation, the gel was placed in a dialysis tube, and electrophoresis was performed again.
A was extracted. The extract was treated with 2-butanol to obtain DN.
After concentrating A and removing ethidium bromide, T
4 phage-derived DNA ligase at 10°C for 16
A time DNAfi ligation reaction was carried out to construct the pGI 423 plasmid. Next, Example! Escherichia coli ceoo was made to have DNA uptake ability in the same manner as in (4), and then the above pGI 423 was taken up. The thus obtained strain was cultured in LB medium containing 50 pg/ml of 7-ampicillin, and the resulting colonies were isolated to obtain the transformed strain Escherichia coli 0800 (pGl
423) (Feikoken ΔKyo No. 9639).

Example 3 Transformed strain Escherichia coli C8 described in Example 2
00 (POT 423) (, Microtechnical Research Institute No. 1111
No. 39).

Escherichia coli CIIGO (pBR322) and Pseudomonas putida IFO3738 were grown in Davis-Mingioli's minimal medium C (g/I) on a 5. H, PO43,0, K2 1 (PO47,0, (111
1,)2Lso4 +, 0, Mg5O+ ・7Hλ
00.1] Shaking culture was performed at 30° C. for 18 hours in a 500 ml flask containing 100 intestines. This was collected by centrifugation, washed, and then adjusted to pH 7.0 with lO
- Suspended in Asun Ugly Buffer 1 (1++1) and disrupted by ultrasonication at 0°C for 5 minutes. Centrifuged at 25,0 OOX g for 30 minutes to collect the supernatant, and its glyoxalase! activity was measured. The results are shown in Table 1.

Table 1 Glyoxalase in Table 1! Activity is enteral o1e/intestinal in
/mg protsin is shown.

The results for Escherichia coli 0800 (pGI318) (Feikoken ΔFl No. 94138) were almost the same as those for Escherichia coli CθGo (pGl 423) (1,703 μzen01/sin/ag protein
) was obtained.

Example 4 The cell-free extract prepared in Example 2 was diluted with 7.2, /
I-Methylglyoxal, 30.7g/I-Glutathione, 50 plates were incubated with phosphorous H1s buffer (pH 7,0) to perform the biosynthesis of S-lactoylglutathione.The protein concentration in the reaction solution was O,lsg.
/-1, reaction time BO minutes 1, reaction temperature was 37°C.

The results are shown in Table 2.

Table 2 In Table 2, the conversion rate of S-lactoylglutathione to glutathione is
38 and Escherichia coli C800 (pBR322)
were low at 7.8 and 1.3, respectively, while Escherichia coli 0800 (pG[423) had a score of 82.3, which was improved to 1.3.

In Escherichia coli C800 (pGI 318), Escherichia Φ coli 01!00 (pGI 423
), S-lactoylglutathione could be produced at an exchange rate (90.0 ml).

[Brief explanation of the drawing]

Figure 1 shows pGI 318 plasmid and pGI 42 plasmid.
Figure 2 is a diagram showing the preparation process of 3 plasmids;
Restriction enzyme breakpoint maps of the I 318 plasmid and pGI 423 plasmid are shown. (For convenience, it is shown as a straight line.)

Claims (11)

[Claims] (1) DNA obtained by treating the chromosomal DNA of a microorganism that has the ability to produce glyoxalase I with restriction enzymes
fragment, a natural DNA fragment related to said DNA fragment by nucleotide substitutions, nucleotide deletions, nucleotide insertions and nucleotide sequence inversions, and other mutations, or encoding said glyoxalase I; A plasmid containing a DNA fragment obtained by semi-synthesis, which gives a host the ability to produce glyoxalase I. (2) The plasmid according to claim 1, wherein the host is a microorganism belonging to the genus Escherichia. (3) Microorganisms belonging to the genus Escherichia are
The plasmid according to claim 2, which is coli C800. (4) The plasmid according to claim 1, wherein the DNA fragment is a DNA fragment prepared from the chromosomal DNA of a microorganism belonging to the genus Pseudomonas. (5) The plasmid according to claim 4, wherein the microorganism belonging to the genus Pseudomonas is Pseudomonas butida IFO3728. (6) The plasmid according to claim 1, wherein the plasmid is a pGI318 plasmid. (7) The plasmid according to claim 1, wherein the plasmid is a pGI423 plasmid. (8) DNA obtained by treating chromosomal DNA of a microorganism capable of producing glyoxalase I with restriction enzymes
fragment or related to said DNA fragment by nucleotide substitutions, nucleotide deletions, nucleotide insertions, nucleotide sequence inversions and other mutations, and which encodes said glyoxalase I.
A microorganism transformed with a plasmid which is a plasmid containing a natural or semi-synthetically obtained DNA fragment which is an NA fragment and which gives the host the ability to produce glyoxalase I. (9) The transformed microorganism is Escherichia coli C6
00 (pGI318). (10) The transformed microorganism is Escherichia coli C.
600 (pGI423). (11) DNA obtained by treating the chromosomal DNA of a microorganism capable of producing glyoxalase I with restriction enzymes
A fragment, or a DNA that is related to the DNA by nucleotide substitutions, nucleotide deletions, nucleotide insertions, nucleotide sequence inversions, and other mutations and encodes the glyoxalase I.
DNA fragments obtained naturally or semi-synthetically
A microorganism transformed with a plasmid containing the A fragment that gives the host the ability to produce glyoxalase I is cultured, and the resulting bacterial cells or the processed product of the bacterial cells are treated with methylglyoxal and glutathione. S-lactoylglutathione produced by contacting with a substrate solution containing S-lactoylglutathione is collected.
Method for producing lactoyl glutathione.