JPH01132384A - Plasmid and escherichia coli transformed therewith - Google Patents

Abstract

PURPOSE:To efficiently produce thermostable leucine dehydrogenase, by forming a recombinant plasmid obtained by connecting a thermostable leucine dehydrogenase having <=2 megadalton molecular weight to a vector plasmid. CONSTITUTION:Escherichia coli transformed with a recombinant plasmid prepared by connecting a thermostable leucine dehydrogenase having <=2 megadalton molecular weight to a vector plasmid is obtained. This Escherichia coli is Escherichia coli C600-pICR220 strain (FERM P-9327). The Escherichia coli is cultivated in a medium containing a carbon source and a nitrogen source so that the thermostable leucine dehydrogenase can be collected from the culture mixture. In the operation, when the ground solution of the culture mixture is heat-treated, enzyme and protein having no thermostability are thermally denaturated and the thermostable leucine dehydrogenase is selectively obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a novel plasmid containing a heat-stable leucine dehydrogenase gene and a novel N. coli transformed with this plasmid.

(Prior Art) Leucine dehydrogenase is a very important enzyme for clinical test 1 food analysis or as an enzyme for L-leucine synthesis. A microorganism that can produce this leucine dehydrogenase is Bacillus sphaericus, a thermophilic bacterium.
Bacteria of the genus Bacillus, such as Bacterium japonica, are known. However, leucine dehydrogenase obtained from these bacteria typically loses most of its activity within 1 to 3 weeks in an aqueous solution at room temperature, and is said to lack thermostability and long-term stability. It has major drawbacks.

Therefore, in order to maximize the advantages of clinical test analysis methods and L-leucine synthesis methods that use leucine dehydrogenase, it is necessary to use leucine dehydrogenase, which is stable to heat and does not lose its activity for a long time at room temperature. His appearance was eagerly awaited.

Therefore, from this point of view, the applicant has previously conducted intensive research in search of a leucine dehydrogenase that is heat-stable and does not lose its activity for a long period of time. discovered that leucine dehydrogenase with the above properties existed in
9-34884).

However, this thermophilic bacterium belonging to the genus Bacillus has low productivity of heat-resistant leucine dehydrogenase.

The method for obtaining this enzyme efficiently was not fully satisfactory.

On the other hand, bacteria belonging to the genus Escherichia do not originally have any ability to produce leucine dehydrogenase.

Also, plasmids useful for recombinant DNA and microorganisms transformed therewith are well known. for example,
Science (5science) 198.

1056 (1978), plasmid pBR32
It has been described that animal proteins are produced in E. coli into which a plasmid in which a lactose promoter is linked to E. coli.

Furthermore, Japanese Patent Application Laid-open No. 56-5093 discloses that Esch.
Although it has been described that a heat-stable enzyme is prepared using bacteria belonging to the genus Erichia), nothing has been reported regarding the plasmid containing the heat-stable leucine dehydrogenase gene and microorganisms transformed by it. It has not been described, nor has there been any report that its creation was successful.

Furthermore, from this point of view, some of the present inventors have developed a plasmid that can express thermostable leucine dehydrogenase in normal temperature microorganisms and a thermostable leucine dehydrogenase in order to improve the productivity of thermostable leucine dehydrogenase. As a result of intensive research in search of a microorganism that can efficiently produce leucine dehydrogenase and produce scales, the gene for leucine dehydrogenase was isolated from a thermophilic microorganism, and this gene was introduced into known plasmid DNA as a scale. They also discovered that the plasmid obtained by introducing the gene in this manner has the above-mentioned properties, and further discovered that N. coli transformed with this plasmid produces large amounts of heat-stable leucine dehydrogenase. , filed a patent application (JP-A-59-159778, JP-A-59-159775 and JP-A-59-
162882).

(Problems to be Solved by the Invention) The above-mentioned plasmid contains many DNA regions derived from thermophilic microorganisms in addition to the gene for leucine dehydrogenase (
(molecular weight: 3 megadaltons), expression in normal-temperature microorganisms is insufficient. Therefore, the productivity of heat-resistant leucine dehydrogenase in N. coli transformed with this plasmid is still insufficient, and heat-resistant leucine dehydrogenase It was not fully satisfactory for efficiently obtaining leucine dehydrogenase.

(Means for Solving the Problems) Therefore, in order to improve the productivity of heat-stable leucine dehydrogenase, the present inventors developed a plasmid that can be efficiently expressed in normal-temperature microorganisms, and a heat-stable leucine dehydrogenase. As a result of intensive research in search of a microorganism with a high content, we found that a recombinant plasmid in which a leucine dehydrogenase gene derived from a thermophilic microorganism with a specific molecular weight and a specific promoter linked upstream thereof to a vector plasmid has the above-mentioned properties. and Escherichia coli transformed with this plasmid.
The present invention was completed based on the discovery that a thermostable leucine dehydrogenase (Namdan) can be efficiently produced.

Specifically, 1 the present invention provides a recombinant plasmid in which a heat-stable leucine dehydrogenase gene with a molecular weight of 2 megadaltons or less is linked to a vector plasmid, and a recombinant plasmid in which a heat-stable leucine dehydrogenase gene with a molecular weight of 2 megadaltons or less is linked to a vector plasmid. Escherichia coli (Escherichia coli) transformed with the recombinant plasmid
The main point is the juxtaposition).

To obtain the plasmid of the present invention, 1.
Biochim.

Biophys, Acta) 72,619-629
(1963), a heat-stable leucine dehydrogenase gene with a molecular weight of 2 megadaltons or less, and a DNA that serves as a promoter and vector.
and the Journal in Molecular Biology (
J.

Mo1. Biol, )96171-184 (197
It can be prepared by digesting with restriction enzymes and then ligating with ligase according to the method described in 4).

The above-mentioned DNA that functions as a vector is 1
For example, D such as pBR322 derived from N. coli
Examples of promoters include tryptophan promoter, tack promoter, and lactose promoter, with tryptophan promoter being particularly preferred. Examples of restriction enzymes include Sal I and HindII, and examples of ligases include T4 DNA ligase.

The heat-stable leucine dehydrogenase gene used in the present invention must have a molecular weight of 2 megadaltons or less, and to obtain this gene, for example, a method such as the following can be adopted. . That is, first of all, Bacillus stearothermophilus, which is a thermophilic bacterium, is particularly preferable, and IFO12 is a specific example.
550゜ATCC7593,7594,8005,10
149, 12980), chromosomal DNA containing the leucine dehydrogenase gene of Clostridium, etc.
A is prepared and introduced into vector plasmid pBR322 to create a plasmid containing the leucine dehydrogenase gene. Next, extract the foreign gene region from this plasmid, and transfer this extracted foreign gene region to M
13 into a phage vector and determine the DNA base sequence according to the method described in "Proteins, Nucleic Acids, and Enzymes J29294-306 (1986). Based on this DNA base sequence, the leucine dehydrogenase gene is encoded. 2
Identify the DNA region of megadalton or less and remove unnecessary regions by restriction enzyme treatment and agarose gel electrophoresis.
DNA regions smaller than megadaltons can be collected.

Using this method, for example, plasmid pTcR2, in which the leucine dehydrogenase gene and tryptophan promoter derived from the chromosomal DNA of Bacillus stearothermophilus IFO12550 strain were introduced into plasmid pBR322.
20 is obtained. This plasmid-7 = pIcR220 was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry on April 13, 1986.

This plasmid was not deposited.

Next, the physicochemical properties of this plasmid pIcR220 will be shown.

(1) Heat-stable leucine dehydrogenase can be expressed in normal-temperature microorganisms.

(2) As shown in Figure 1, it has first-order cleavage sensitivity to the following restriction enzymes.

Restriction enzyme Number of cleavage sites EcoRI 3 HindI[I Pst I 5al I 2 The name of the restriction enzyme is an abbreviation of the restriction enzyme obtained from the 9th bacterial species.

E CORI; Nizierichia coli Hind I Plasmid pIcR220 was digested under the following conditions and the digest was subjected to agarose gel electrophoresis.

Determined from the number of separable fragments.

(3) Molecular weight is approximately 4 megadaltons.

(4) A heat-stable leucine dehydrogenase gene having a molecular weight of 2 megadaltons or less, preferably about 1 megadalton or less, and more preferably 0.77 megadaltons is inserted downstream of the promoter in the same direction.

The N. coli of the present invention has the above-mentioned heat-resistant leucine dehydrogenase gene with a molecular weight of 2 megadaltons or less,
N. coli was transformed with a recombinant plasmid in which a promoter was linked to the vector plasmid upstream of the vector plasmid. Biology (J, Mo
1゜Biol, ) 53.159-162' (1970
), the above plasmid pIcR220 treated with calcila chloride is brought into contact with N. coli C600 at a temperature around 0°C.

An example of N. coli transformed as described above is N. coli C600-pIcR220 strain into which plasmid pIcR220 has been introduced.

This strain is known as N. coli C600 (Nature 217, 1) 10-1) 14
(1968)] and the extra-punctate, which has heat-stable leucine dehydrogenase-producing ability and ampicillin resistance, has the same mycological properties. This strain maintains safety without changing from non-transmissible to transmissible or from non-pathogenic to pathogenic. especially.

There have been no reports of N. coli having the ability to produce a heat-stable leucine dehydrogenase, in which a heat-stable leucine dehydrogenase gene with a molecular weight of 2 megadaltons or less is inserted downstream of the N. coli promoter. From this, it is thought that N. coli C600-pIcR220 strain is a new strain.

It was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry on April 13, 1986. The microorganism accession number is 9327.
This is the number.

For example, as a carbon source for a nutrient medium used for culturing N. coli of the present invention.

Glucose, sucrose, fructose, starch hydrolyzate, molasses, sugars from sulfite pulp waste liquor.

Organic acids such as acetic acid and lactic acid, as well as alcohols, fatty acids, and glycerin that can be assimilated by the bacteria used can be used, and nitrogen sources such as ammonium sulfate, ammonium chloride, ammonium phosphate, amino acids, peptone, meat extract, and yeast can be used. Inorganic or organic substances such as extracts can be used. Furthermore, there are 2 inorganic salts such as potassium and sodium.

Phosphate, zinc, iron, magnesium, manganese, copper.

Salts such as calcium and cobalt, trace metal salts, corn staple liquor, vitamins, nucleic acids, etc. may be used as necessary, and general nutrient media for bacteria may be used.

Using these media, the N. coli of the present invention is cultured at 20°C to 45°C, preferably 35°C to 40°C, optimally at 37°C, for about 10 to 20 hours, and at a pH of 7.0 to 7.4.
．． Optimally, it may be cultured aerobically at 7.2.

Next, the thermostable leucine dehydrogenase of the present invention is collected from the obtained culture.

Isolated live bacterial cells 1 Processed product of isolated bacterial cells, crude enzyme extract.

It can be collected at any stage, including purified enzymes. As the purification method at that time, two common enzyme purification methods can be used. In particular, in the present invention, if the crushed solution is heat-treated before collecting heat-stable leucine dehydrogenase, enzymes and proteins that are not heat-stable are thermally denatured, thereby selectively producing heat-stable leucine dehydrogenase. This is advantageous because enzymes are obtained.

The conditions for this heat treatment are 9, for example, at a temperature of 50 to 80°C for 5 to 30 minutes. After being treated in this way, the enzyme may be separated and purified to obtain a heat-stable leucine dehydrogenase, or it can be used as it is as an enzyme solution.

The thermostable leucine dehydrogenase obtained by the present invention has the same physicochemical properties as the thermostable leucine dehydrogenase described in JP-A-59-34884.

In other words, it exhibits first-order physical and chemical properties.

(a) Catalyze the following reaction.

L-leucine + NAD” (:: α-ketoisocaproic acid + NADH + NH, “+H” L-leucine (100%), L-valine (98%) as substrate for oxidative deamination reaction
%), L-isoleucine (73%), and α-ketoisocaproic acid (100%) and α-ketoisovaleric acid (167%) as substrates for reductive amination reactions.

These include α-ketovaleric acid (86%) and α-ketobutyric acid (47%).

(b) An oligomeric enzyme with a molecular weight of approximately 300,000 and consisting of 6 identical subunits of approximately 49,000.

(C) Optimum pH: 10 to 1) for deamination reaction, and 8 to 9 for amination reaction.

(d) Heat resistance It is not inactivated even after heat treatment at 70°C for 30 minutes.

(Example) Next, the present invention will be specifically explained using examples.

Furthermore, the activity of heat-stable leucine dehydrogenase is as follows.

Amino acid and nuclear acid (Am
ino Ac1d and Nucleic
Ac1d) 2 7. 8 4-88 (1
The method for measuring leucine dehydrogenase activity described in 973) was followed. That is, 100 μmo at pH 10.5
le glycine-MCI-KOH buffer at 1°25
A mixture containing p mole of NAD and 10 u mole of L-leucine was prepared, and an appropriate amount of crude enzyme extract was added to the mixture to make a final volume of 0.8 ml. This was done by measuring the increase in absorbance at 340 nm per unit time.

Further, % in Examples and Reference side indicates volume %.

Example 1 (a) Chromosomal DN of Bacillus stearothermophilus
Separation of A.

From Bacillus stearothermophilus IF012550 strain, Biochimica et Biophysica acta (
Biochim, Biophys.

Chromosomal DNA was isolated according to the method described in Acta, ) 72, 619-629 (1963).

Mas, Bacillus stearothermophilus IF0125
50 plants were treated with polypeptone 10g/4. Yeast extract 2.5g
/β8 meat extract 2g/j! , Glycerol 2g/L Sodium chloride 5g/L Monopotassium phosphate 2g/L
Dipotassium phosphate 2g/j2° Magnesium sulfate 0.1
g/E, biotin 4 μg/It and pi 7.2
After culturing with shaking at 155° C. for 12 hours in medium 21 prepared in 2008, the bacteria were collected by centrifugation.

Next, add 12 mg of Norizozyme to 6 ml of sali.
ne)-EDTA solution (0,15M NaCl and 0.I
Contains MEDTA.

Adjusted to pl+ 8.0. ), and the collected bacterial strain was added to this solution and stirred well. This was heated at 37°C for about 10 minutes, and when the bacterial cells began to lyse, it was immediately frozen.

Add 50 ml of Tris-5O5 buffer (
Long/m1sDs and 0. 0.1M) Lys buffer adjusted to pH 9.0 containing IM NaCl. ) and stir.

The mixture was further heated to 60°C to completely lyse the bacteria.

Add 56ml of 80% phenol to this lysate.

The mixture was shaken for about 20 minutes and subjected to phenol extraction to remove contaminant proteins. Add twice the volume of cold ethanol to this extracted crude DNA solution and roll up the fibrous precipitate with a glass rod. After several minutes of immersion in 10 ml each of 0.80.90% ethanol, 20 ml of dilute saline-citrate solution (0.015 M NaCl, 0.0015 M Na1) was added.
- Prepared in citric acid. ) and further concentrate 5aline.
-citrate solution (1,5M NaCl1,0,15
Add 2 ml of M Na3-citrate) to the crude DN.
Solution A was prepared.

Dilute this crude DNA solution to about 500 μg/ml.

Ribonuclease A (RNaseA (manufactured by Sigma)
] was added at a concentration of 50 μg/ml, and the mixture was heated at 37° C. for 30 minutes. After cooling, add 5 equivalents of 80% phenol,
Phenol extraction was performed, the extracted DNA was recovered by ethanol precipitation, and the above diluted 5aline-citrat
Dissolve in 20ml of e solution, and add the above concentrated 5al
A chromosomal DNA extract was prepared by adding 2 ml of ine-citrate solution.

(b) Preparation of vector plasmid pBR322.

Plasmid pBR322C Bethesda Research Laboratories
oratories)] was introduced.
coli C600 strain was grown in 21 L-medium (polypeptone 10
g/jl! , yeast extract 5g/I! , glucose 1 g
/ta, add 5g/# of sodium chloride, pH 7
, prepared in 2. ) was cultured aerobically at 37°C until early logarithmic growth phase, and then 10 ml of chloramphenicol solution (3
, prepared with ethanol to a concentration of 6 mg/ml. ) was added thereto, and further aerobically cultured at 37°C for 15 minutes to propagate plasmid pBR322.

Next, the bacteria collected by centrifugation were added to 80ml of TE-sucrose buffer (200μ/ml sucrose, 20mM
0.05 containing EDTA and adjusted to pH 8.0
M) Lys buffer. ), further add 8 ml of Norizozyme solution (prepared to 5 mg/ml with the above TE-sucrose buffer), and further add 28 ml
1 of 5M NaCl solution and 4 ml of 40 mg/ml sD
s solution was added.

After reacting this mixture at 37° C. for 2 hours, crude plasmid DNA was separated by centrifugation.

Next, phenol treatment was performed by adding +'A volume 1 of 80% phenol to remove contaminant proteins. The extracted crude plasmid was precipitated and collected using cold isopropanol, and further added to TE buffer (20mM adjusted to pH 7.5, containing 0.14M NaCl, 1mM EDTA) Lis buffer. ) dissolved in Add 2 mg of RNase A to this mixture.
was added and reacted for 2 hours at 37°C, and contaminant RNA was removed by phenol treatment in the same manner as above. This extracted crude plasmid was recovered by precipitation with 2 times the volume of ethanol. This was further dissolved in 10 ml of the above TE buffer, contaminant RNA was further removed by agarose gel filtration, and the resulting crude DNA was recovered again by ethanol precipitation.

This precipitate was dissolved in 23.1 ml of 0.02M) Lys buffer (p
Adjusted to H8.0. ) and further dissolve 23.7 g of cesium chloride and 0.6 ml of ethidium bromide solution (1
Adjusted to 0 mg/ml. ) and ultracentrifuged for about 40 hours to separate plasmid DNA, and then ethidium bromide was removed with n-butanol.

This isolated plasmid was added to 0.01M TE buffer (0.0
0, OIM) Lys buffer adjusted to pH 7,5 containing 1 mM EDTA. ) by dialysis.

A purified plasmid pBR322 was obtained.

(C) Creation of plasmid pICR2.

0 μg of Bacillus stearothermophilus chromosomal DNA obtained by method (a) and restriction enzyme 5alI (manufactured by Takara Shuzo Co., Ltd.) 30-Lnit + 7mM (7) MgC
12゜150mM NaCl, 0.2mM EDTA,
7mM 2-methylcaptoethanol, 0.01%
pH 7.5 containing BSA (bovine serum albumin)
into 100 μl of 10 mM Tris buffer prepared in
After digesting the DNA by reacting at 37°C for 30 minutes,
The substrate was heated at 65° C. for 5 minutes to inactivate it, and the digested DNA fragments were precipitated and recovered with cold ethanol.

Next, plasmid pBR322 obtained by method (b)
5al13 units of restriction enzyme were added to 3 μg of the mixture and reacted in the same buffer as above at 37°C for 10 hours, and the digested plasmid DNA was recovered in the same manner as above. Mixing the thus obtained digested chromosome and plasmid DNA,
6.6 m using T4 DNA ligase (manufactured by Takara Shuzo Co., Ltd.)
MgC1z, 10mM DTT, 6EIM AT
Plasmid pICR2 containing the leucine dehydrogenase gene was obtained by reacting at 13° C. for 16 hours in a 66 mM Tris buffer containing P and adjusted to pH 7.6 and recombining the digested DNA.

(d) Identification of a thermostable leucine dehydrogenase gene.

25 μg of plasmid pICR obtained by method (e) was treated with 7 mM Mg using 20 units of restriction enzyme 5alI.
Cl□.

150mM NaCl, 0.2mM EDTA, 7m
M 2-methylcaptoethanol, 0.1 +ng/
mj! Plasmid pICR2 treated with 5alI was obtained by reacting with 10mM Tris buffer adjusted to pH 7.5 containing BSA at 37°C for 15 hours, and this plasmid pICR2 was treated with 5alI.
ICR2 and lambda phage DN as a molecular weight marker
1 of A (indnI digested fragment) and 1 μg of each at the same time.
The electrophoresis was performed at a constant voltage of 7 V per cm for 3 to 4 hours. Next, irradiate it with an ultraviolet lamp, determine the band of the target fragment, cut out the gel of that part, and heat it for 50 minutes at 65℃.
Heat treatment was performed for 1 minute to dissolve the agarose, and the chromosomal DNA of Bacillus stearothermophilus containing the leucine dehydrogenase gene was treated with phenol and ethanol.
Separated areas.

The electrophoresis conditions at this time were 2.51 E as a buffer solution.
An 89mM Tris buffer containing DTA and 89mM boric acid and adjusted to pH 8.3 was used, and 8μ/ml LMP agarose [Besseda] was added to this Tris buffer as a gel.
Research Laboratories (Bethesda Re5
[manufactured by ach Laboratories] and
Ethidium bromide adjusted to 0.5 mg/l was added.

Next, the separated DNA was transferred to M13 phage vector using T4.
The DNA was ligated using DNA ligase, and the base sequence of the DNA was determined by the dideoxy method according to the method described in Proteins, Nucleic Acids, and Enzymes, Takeshi 294-306 (1984). That is, D corresponding to the N-terminal 10 amino acid sequence determined by Edman degradation of purified leucine dehydrogenase.
The NA base sequence and the amino acid corresponding to each codon were translated, and the corresponding start codon (A T G) and stop codon (T G A) were determined.

The results are shown in FIG.

From this, the leucine dehydrogenase gene of the above plasmid plcR2 has the restriction enzyme XhoI as shown in FIG.
(Xanthomonas forsicola)
As holcicola)] was found to be located in the region downstream from the cleavage site (X in Figure 3).

(e) Creation of plasmid plcR220.

As mentioned above, since we were able to identify the leucine dehydrogenase gene of plasmid pICR2, we first removed the unnecessary region of the gene [the unnecessary region is shown in pT
As indicated by CR2, the cleavage site of H (Hind ■),
region sandwiched between the cleavage site of X (XhoI)].

That is, the plasmid plcI'125Mg obtained above
20 each of the restriction enzymes HindllI and XhoI
The unit was incubated at 37°C in 10mM Tris buffer adjusted to pH 7.5 containing 7mM MgCIz, 150mM NaCl, 0.2mM EDTA, 7mM 2-methylcaptoethanol, 00lq/mj2 BSA.
The reaction was then carried out for 15 hours.

After precipitating and collecting the DNA with cold ethanol.

Agarose gel electrophoresis was performed. This electrophoresis is
After reacting for 3 to 4 hours at a constant voltage of 7 cm per cm,
The target flagmate was determined by irradiating it with an ultraviolet lamp. Next, cut out that part of the gel and incubate it at 65°C for 5 minutes.
Agarose was dissolved by heat treatment for 1 minute, and unnecessary regions of the leucine dehydrogenase gene were removed by phenol treatment and ethanol treatment.

As a result, the molecular weight of the leucine dehydrogenase gene is 0
．． It is 77 megadaltons, and the number of bases is 1290bp.
Met.

Next, in order to insert the tryptophan promoter derived from N. coli, I μg of a 9 obp base (manufactured by Pharmacia) encoding the tryptophan promoter.
The tryptophan promoter sandwiched between the cleavage sites of HindI [(H in Figure 3) and 5alI (S in Figure 3) was incubated with the above Tris buffer at 37°C for 15 minutes using restriction enzyme 5al12 units. (portion H3 indicated by the vertical line in FIG. 3) was produced. Next, this promoter and the plasmid plcR2 obtained above were mixed, and T4DN
Using A ligase (manufactured by Takara Shuzo Co., Ltd.), 6.6mM
A plasmid pIcR220 containing a leucine dehydrogenase gene with a molecular weight of 0.77 megadaltons was obtained by reacting with a 66mM lith buffer prepared in pu'7.6 containing 10mM DTT and 66μM ATP.

Example 2 Plasmid pIcR220 obtained in Example 1 was used to transform N. coli.

First, a host strain, N. coli C600r-m-, was cultured in 50 ml of the above Shiichi medium, collected by centrifugation, and then suspended in 50 ml of 0.1 M MgCIz solution.

After further centrifugation, 2.5 ml of 0.1
M was suspended in MgCl2 solution.

Nizierichia coli C600r obtained in this way
0.1 ml of the mixture containing plasmid pIcR220 obtained by method (e) was added to 0.2 ml of the suspension of -m- strain.

After processing at 0°C for 30 minutes, it was processed at 42°C for 2 minutes.

Next, 3 ml of the above-mentioned Shishiichi medium was added to this, and cultured at 37°C for 1 hour.
Added 15g of agar. ) at 37°C, and by finding the resulting colonies, the plasmid pIcR2
20 introduced N. elizia co IJ C600-p
IcR220 was obtained.

Next, Nizierichia coli C600-p obtained in this way
Ampicillin (15g) was obtained from the lc+2220 colony.
Adjusted to g/ml. ) and the above glycerol medium (polypeptone Log/N, yeast extract 2.5 g/β, meat extract 2 g/j2. 2g/l, sodium chloride 5g/It, monopotassium phosphate 2g/C dipotassium phosphate 2g/l
.

Magnesium sulfate 0.1 g/β, biotin 4 μg/l
(adjusted to pH 7.2) with 100 ml at 37°C.
Shaking culture was performed for 6 hours. This is centrifuged to collect bacteria.
After washing, 0,0 containing 5 ml of 0.1 mir/m 7!2-mercaptoethanol and adjusted to pH 7,4.
The cells were suspended in 1 M phosphate buffer, disrupted by sonication at 0°C for 5 minutes, and centrifuged to obtain a crude enzyme extract.

When the activity of heat-stable leucine dehydrogenase in the crude enzyme extract thus obtained was measured, it was found to be 450 units)/g.
・It was a wet bacterial cell. This is approximately 90 times more active than the leucine dehydrogenase activity of the DNA donor Bacillus stearothermophilus strain F012550 (5.0 units/g of wet bacterial cells), and it is also -Leucine dehydrogenase activity of pICR1 strain (
The activity was about 9 times higher than that of 50 units/g (wet bacterial cells).

In addition, the crude enzyme extract containing this leucine dehydrogenase is
pH containing 0.1■/ml 1% of 2-mercaptoethanol
When it was heat-treated at 70°C for 20 minutes in 10mM phosphate buffer of No. 7,2, it had a residual activity of 90% or more.

Next, plasmid pIcR220 was isolated from this strain in the same manner as in Example 1 (b), and the properties of plasmid plcR220 were examined by horizontal agarose gel electrophoresis.

As a buffer solution used for electrophoresis, 2.5mM EDT
A-Na, 89mM Tris buffer containing 89mM boric acid prepared in pus, a was used as a gel, and this buffer was supplemented with 7μ/ml of agarose and 0.5mg/I.
To this, plasmid plcR220 and plasmid pB were added.
R322 and 1 μg each of the base ndlIr (manufactured by Takara Shuzo Co., Ltd.) digested fragment of lambda phage DNA as a molecular weight marker were simultaneously grown on the same agarose gel.
per cm.

Migration was carried out for 3-4 hours with a foot applied voltage of 7v. Next, the band was determined using an ultraviolet lamp, and the plasmid plcR220
As a result of examining the size, the molecular weight was approximately 4 megadaltons.

In addition, this plasmid pIcR220 was reacted under suitable conditions for each restriction enzyme Ec, and plasmid pIcR220 was reacted with the restriction enzyme Ec under appropriate conditions.
R220 was digested.

Digested samples obtained with each restriction enzyme were subjected to agarose gel electrophoresis in the same manner as above, and the number of cleavage sites determined by the number of cleavage sites by each restriction enzyme was determined.
As shown in FIG. 1, lcR220 has the following cleavage sensitivity.

Restriction enzyme Number of cleavage sites EcoRI 3 HindI [[I Pst I l 5al I 2 Furthermore, plasmid pIcR220 is
.

At the 3alI cutting site, a chromosomal DNA fragment containing the leucine dehydrogenase gene of Bacillus stearothermophilus strain IF012550, a tryptophan promoter derived from N. coli and plasmid pKK322
It is clear that it is bound to DNA.

Reference example 1. Comparative Example 1 Nizierichia coli C600-pICR obtained in 28-Example 2
220 strain was cultured with shaking at 37° C. for 16 hours in 100 ml of the above glycerol medium containing ampicillin (adjusted to 15 μg/ml) and 3-IAA (adjusted to 15 μg/ml).

After culturing, bacteria were collected by centrifugation and adjusted to pH 7.4 containing 0.1 μ/mβ of 2-mercaptoethanol.
The cells were suspended in 5 ml of 1 M phosphate buffer and disrupted by ultrasonication for about 5 minutes. When the enzyme activity of the cell suspension was measured, the specific activity of leucine dehydrogenase was 4.5.
It was found that the protein was approximately 10 times more active than Comparative Example 1 below, and it is clear that the productivity was significantly improved.

Thereafter, a crude enzyme extract was obtained by centrifugation, and the crude enzyme extract was heat-treated at 70°C for 30 minutes, centrifuged, and the leucine dehydrogenase activity of the supernatant was measured.
It had an activity of 36 units/mg protein, and by heat-treating the crude enzyme extract at 70°C for 30 minutes, the specific activity was increased about 8 times compared to before heat treatment.

Next, it was purified by preparative electrophoresis using 7.5% acrylamide and high performance liquid chromatography using a Superlow 12 gel chromatography column (manufactured by Pharmacia, two columns connected in series) to a specific activity of 55 units/■.・Protein thermostable leucine dehydrogenase was obtained.

This enzyme gave a single band in 7.5% acrylamide gel electrophoresis at pH 9.4, and compared to conventional Bacillus
The properties were the same as those of leucine dehydrogenase derived from Stearothermophilus.

For comparison, N. coli C600-pI incorporating a gene derived from Bacillus stearothermophilus
cRl (Feikoken Bibori No. 6937) was cultured with shaking in 100 ml of the glycerol medium at 37°C for 16 hours according to a known document (Japanese Patent Laid-Open No. 59-159778), and the bacteria were collected by centrifugation. 0,0 prepared to pH 7,4 containing 0.1 ■/m It of 2-mercaptoethanol.
The cells were suspended in 5 ml of 1M phosphate buffer, and the cells were disrupted by ultrasonication for about 5 minutes to obtain a disrupted solution. When the leucine dehydrogenase activity of this crushed solution was measured, it was found to be 0.5 units/■ protein (Comparative Example 1).

(Effects of the Invention) The plasmid of the present invention has the leucine dehydrogenase gene itself (with a molecular weight of 2 megadaltons or less) from which the DNA region derived from a thermophilic microorganism has been removed, so it can be used as a useful microorganism as described above.

Bacteria that grow at room temperature can be transformed to be endowed with the ability to produce large amounts of heat-resistant leucine dehydrogenase. Also,
The N. coli of the present invention transformed with this plasmid produces a large amount of heat-stable leucine dehydrogenase, and is therefore extremely useful in fields such as clinical test reagents.

[Brief explanation of the drawing]

Figure 1 is a restriction enzyme map of the plasmid plcR220 of the present invention, Figure 2 is a diagram showing the base sequence of the leucine dehydrogenase gene used in the present invention, and Figure 3 is a diagram showing the nucleotide sequence of the leucine dehydrogenase gene used in the present invention. FIG. 3 shows a method for creating pIcR220. S: 5all, E: EcoRI, P: PstI. H: HindIII, X: Xhol A pr: ampicillin resistance, Ptrp: )
Libutophane promoter, LeuDH: leucine dehydrogenase A: adenine, T: thymine, C: cytosine, G
Nigeanin patent applicant Yu-Tei Riki Co., Ltd.

Claims (2)

[Claims] (1) A recombinant plasmid in which a heat-stable leucine dehydrogenase gene with a molecular weight of 2 megadaltons or less is linked to a vector plasmid. (2) Escherichia coli (\Escherichiacoli\) transformed with a recombinant plasmid in which a heat-stable leucine dehydrogenase gene with a molecular weight of 2 megadaltons or less is linked to a vector plasmid.