JPS59159778A - Preparation of heat-resistant leucine dehydrogenase - Google Patents

Abstract

PURPOSE:To prepare a heat-resistant leucine dehydrogenase efficiently, by cultivating a mold belonging to the genus Escherichia, transformed with a specific plasmid. CONSTITUTION:A bacterium such as Escherichia coli C600-pICR1 (FERM P 6937) belonging to the genus Escherichia, transformed with a plasmid having a gene of leucine dehydrogenase derived from chromosome DNA of a thermophilic bacterium such as Bacillus stearothermophilus, etc. is cultivated, and a heat-resistant leucine dehydrogenase is collected from the culture.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing thermostable leucine dehydrogenase.

Leucine dehydrogenase is used as an enzyme for clinical testing.

It is a very important enzyme. A microorganism that can produce this leucine dehydrogenase is Bacillus sphaericus, which is a normal-temperature tooth.
) are known to belong to the genus Bacillus. but,
Leucine dehydrogenase obtained from these bacteria usually loses most of its activity within 1 to 3 weeks in an aqueous solution at room temperature, and has a major drawback of lacking thermostability and long-term stability. have shortcomings.

Therefore, in order to maximize the advantages of analytical methods for clinical tests using leucine dehydrogenase, there has been a desire for the emergence of a leucine dehydrogenase that is stable to heat and does not lose its activity for a long time at room temperature.

Therefore, from this perspective, some of the present inventors conducted intensive research in search of a leucine dehydrogenase that is stable to heat and does not lose its activity for a long time, and as a result, a thermophilic Bacillus sp. discovered that a leucine dehydrogenase having the above-mentioned properties existed in a bacterium belonging to the genus, filed a patent application, and published it in Biochemistry, Vol. 1, page 634 (1982).

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 Isscherichia have the ability to produce leucine dehydrogenase (
I don't have it.

In addition, plasmids useful in recombinant DNA genetic engineering and microorganisms transformed thereby are well known.

For example, Science Volume 198,
On page 1056 (1978), plasmid PBR3
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 is introduced.

Furthermore, in Japanese Patent Application Laid-open No. 56-5093, 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.

From this perspective, the present inventors.

As a result of intensive research in search of microorganisms with a high content of heat-resistant leucine dehydrogenase, we discovered that heat-resistant leucine dehydrogenase-producing bacteria belonging to the genus Nizieritia efficiently produce heat-resistant leucine dehydrogenase. Completed the invention.

Specifically, the present invention provides a method for producing heat-stable leucine dehydrogenase, which comprises culturing a heat-stable leucine dehydrogenase-producing bacterium belonging to the genus Nizieritia, and collecting the heat-stable leucine dehydrogenase from the culture. It is.

The bacterium used in the present invention is a bacterium of the genus Nizierichia that can produce heat-stable leucine dehydrogenase, and any such bacterium can be used. Preferred bacteria include, for example, bacteria belonging to the genus Nizierichia that have been transformed with a plasmid containing a gene for heat-stable leucine dehydrogenase. Examples of bacteria belonging to the genus Nigelichia include Nigelichia coli (Es.
cherichia coli). Particularly in the present invention, N. coli C600-plcRl, which is obtained by transforming the known bacterial strain N. coli C600 with plasmid plcR1, is preferred. This strain is
The known N. coli C600 (see nature 2, 1110-1114 (1968)) and
They have the same mycological properties except for the ability to produce heat-resistant leucine dehydrogenase and resistance to ampicillin. This strain is.

Safety is maintained without changing non-transmissible to transmissible or non-pathogenic to pathogenic. especially,
There have been no reports of N. coli having the ability to produce heat-stable leucine dehydrogenase.

From this, Nizierichia coli C600-plcR
Since strain I is considered to be a new bacterial strain, it was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry on February 26, 1981. Its microbial umbrella acceptance number is No. 6937.

To obtain the plasmids used in the present invention, for example, Bioch.
em, Biophysacta) 72,619-6
29 (1963), by digesting the heat-stable leucine dehydrogenase gene and DNA serving as a vector with restriction enzymes, and then ligating them using ligase.

The thermostable leucine dehydrogenase gene preferably used in the present invention includes a leucine dehydrogenase gene derived from the chromosomal DNA of a thermophilic microorganism. This thermophilic microorganism is Thermus.
Examples include bacteria belonging to the genus Bacillus and thermophilic bacteria belonging to the genus Bacillus, and bacteria belonging to the thermophilic genus Bacillus are particularly preferred. Among them, Bacillus stearothermophilus has high leucine dehydrogenase activity.
ste, 1rother-mophilus) is preferred. A specific example of stearothermophilus is
IFO12250, ATCC7953, 7954°8
005, 10194.12980. Examples include NCAl3O3.

In addition, 9 DNAs have a role as a vector.
For example, plasmid DNA1y<, and plasmid pBR322 is particularly preferred. In addition, as a restriction enzyme, 1
Examples include Sal I, and examples of ligases include T4ONA ligase.

Using this method, the gene for leucine dehydrogenase derived from the chromosomal DNA of Bacillus stearothermophilus was introduced into plasmid pB11322.
l is obtained. This plasmid pIcR1 was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry on February 23, 1981, but this plasmid was not deposited.

Next, the physicochemical properties of this plasmid pIc121 will be shown.

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

(2) Has 5th-order cleavage sensitivity to the following restriction enzymes.

Restriction enzyme Number of cleavage sites EcoRI 2 Hind I [I Pst I 2 Sal 1 4 Bamll I 2 The name of the restriction enzyme is an abbreviation of the restriction enzyme obtained from the secondary bacterial species.

EcoRI; N. coli II s n d III; Haemophilus influenzae Pst I; Providencia stuartii Sal I; Streptomyces alpus Bam1
ll; The number of cleavage sites with the Bacillus amyloliquefaciens restriction enzyme is 1, determined from the number of fragments that can be separated by digesting plasmid plcR1 in the presence of an excess of restriction enzyme and subjecting the digested product to agarose gel electrophoresis. Ru.

(3) Molecular weight is approximately 6 megadaltons.

As a carbon source for the nutrient medium used for culturing the bacteria in the present invention, for example, glycose.

Sucrose, fructose, starch hydrolyzate.

Organic acids such as molasses, sulfite pulp waste liquid, acetic acid, lactic acid, alcohols, fatty acids, glycerin, etc. that can be assimilated by the bacteria used can be used, and as a nitrogen source, for example, ammonium sulfate.

Inorganic or organic substances such as ammonium chloride, ammonium phosphate, amino acids, peptone, meat extract, yeast extract, etc. can be used. Additionally, inorganic salts such as potassium, sodium, phosphoric acid, and zinc.

Iron, magnesium, manganese, copper, calcium.

Various salts such as cobalt, trace metal salts as necessary.

Corn staple liquor, vitamins, nucleic acids, etc. may be used, and common nutrient media for bacteria can be used.

Using these media, the bacteria in the present invention were grown at 20°C
45°C9 preferably 35°C to 40°C, optimally 37°C
for about 10 to 20 hours to adjust the pH to 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 a purification method in this case, 9 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 may be 2, 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.

According to the present invention, heat-stable leucine dehydrogenase can be obtained easily in large quantities.

Very useful.

The thermostable leucine dehydrogenase obtained by the present invention is described in Biochemistry, Vol. 54. Page 634 (1982)
It has the same physicochemical properties as the thermostable leucine dehydrogenase described in .

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

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

Amino acids, nucleic acids (Amino Ac1d and Nu
cleic 27.84-88 (1973)), namely, 100 μmole of glycine-KCI of pnil, 3.
- + 1.25 μmole NAD in KOII buffer
A mixture containing 10 μmole of leucine was prepared, and an appropriate amount of crude enzyme extract was added to the mixture to make the final volume 0.8+nl. Increase per 340 nm
This was done by measuring the increase in absorbance of .

Moreover, % in Examples and Reference Examples indicates volume %.

Reference example 1 (Chromosome DN of al. Bacillus stearothermophilus
Separation of A.

From Bacillus s1 allothermophilus F012250 strain, Biochemistry etho Biophysica acta (
Biochem Biophys acta) 72.
Chromosomal DNAΔ was isolated according to the method described in 619-629 (1963).

First, Bacillus stearothermophilus IFO122
50 strains were placed in glycerol medium (polypeptone 10 g/j
2. Yeast extract 2.5 g/j! , meat extract 2g/
Il.

Glycerol 2 g/j2, sodium chloride 5 g
/ ' + Lin fl! , 2 potassium 2 g/it,
Magnesium sulfate 0.1 g/β, biotin 4 μs
/ It + and adjusted to pH 7,21) at 24, 55
After shaking culture for 12 hours at °C.

Bacteria were collected by centrifugation.

Next, 12mg of lysozyme was added to 6ml of saline (sali).
ne)-EDTA solution (0,15M NaCl and 0.
1 M [contains EDTA.

Adjust to pH 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-5OS buffer (
1%SOS and 0. pl+9.0 containing I M NaCl
0.1M 1-Lys buffer. ) was added, stirred, and 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 phenol extraction was performed to remove contaminant proteins. Add twice the volume of cold ethanol to this extracted crude DNA solution, roll up the fibrous precipitate with a glass rod, and add 10 ml each of 70%, 80%, and 90% ethanol.
After soaking for several minutes in sequence, add 20 ml of diluted lint-citrate (5aline-citrate).
) solution (0,015M NaCl, 0,0015M
Prepared as Na5-citric acid. ) and further thicken 5a
line-citrate solution (1,5M NaCl
,0.15M Naa-citric acid. ) 2ml
In addition, a crude DNA solution was prepared.

This crude DNA solution was diluted to 500 μg/ml, and ribonuclease [RNa5e (manufactured by Sigma)] was added at 50 μg/ml.
μg/ml+ RNA5eT+ (manufactured by Sigma) at 3
It was added at a concentration of 0 μg/ml and heated at 37° C. for 30 minutes. After cooling, 1 equivalent of 80% phenol was added to perform phenol extraction, and the extracted DNA was recovered by ethanol precipitation.

Furthermore, it was dissolved in 20 ml of the above dilute 1aline-citrate solution, and then the above concentrated 5aline-citrate solution was dissolved.
A chromosomal DNA extract was prepared by adding 2 ml of citrate solution.

Preparation of fbl vector plasmid audience R322.

Plasmid pBR322 (Betheada Re5
Nizierichia coli strain C600, which had been introduced with ``arch Laboratories'', was grown in 2z Noshiichi medium (polypeptone 10 g/It, yeast extract 5 g/J2.
Adjust to pl+7.2 with glycose Ig/j2° sodium chloride 5 g/β. ) until early logarithmic growth phase.
After aerated culture at 37°C, 10 ml of chloramphecol solution (prepared with ethanol to give 3.6 mg/m+) was added, and further aerated culture was performed at 37°C for 15 minutes to propagate plasmid pBR322.

Next, the bacteria collected by centrifugation were added to 80ml of TE-sucrose buffer (20% sucrose, 20mM ED).
0.05M containing TA and prepared at p118.0)
Squirrel buffer. ), add 8 ml of Norizozyme solution (prepared to 5 mg/ml with the above TE-sucrose buffer), and add 28 ml of 5M N
ACl solution and 4 ml of 4% SOS solution were 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 2 volumes of 80% phenol to remove contaminant proteins. The extracted crude plasmid was precipitated and recovered using cold isopropanol, and then T
E buffer (0,14M MaCL 1 mM EDTA
20mM) Lys buffer prepared at pl+7.5 containing: ) was dissolved. Add 2mg of RNA5 to this mixture.
e was added, the mixture was reacted at 37°C for 2 hours, 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. Add this to another 10m1 of the above T
Contaminated RN was dissolved in E buffer and filtered through agarose gel.
A was further removed, and the resulting crude DNA was recovered again by ethanol precipitation.

23.1 m1 (7) of cono precipitation 0.02 M h!
Dissolve in J.S. laxative solution (manufactured by 11, pH 8.0), add 23.7 g of cesium chloride and 0.6 ml of ethidium bromide solution (adjusted to 10 mg/ml), and ultracentrifuge for about 40 hours. Plasmid DNA was separated by this, and ethidium bromide was first removed with n-butanol. This isolated plasmid was added to 0. '0
1 M TE buffer (containing 0.1 mM EDTA pH
7.5 ml of 0.01M Tris buffer. ), purified plasmid pBR322 was obtained.

(Creation of Cl plasmid plcR1.

5 μg of the chromosomal DNA of Bacillus stearomorphus obtained by the method in (a) and 20 units of restriction enzyme Sal I (manufactured by Takara Shuzo Co., Ltd.) were mixed with 7 mM MgCh,
150mM NaCl, 0,2mM EDTA, 7m
M 2-methylcabutoethyl, 0.01%BS
p117.5b containing A, [Pour it into 100 μl of the prepared 1°1 Tris buffer, react at 37°C for 2.5 hours to digest the DNA, then heat at 65°C for 5 minutes,
was inactivated and digested with cold ethanol to precipitate and collect the digested DNA fragments.

Next, plasmid pBR322 obtained by method (b)
113 units of restriction enzyme was added to 1 μg, and the mixture was 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. The digested chromosome and plasmid DNA thus obtained were mixed, and T
4ONA ligase (Takara Shuzo Co., Ltd.) was used.

6.6mM MgCh, 10mM DTT, 66
Digestion D
Plasmid plcR1 was obtained by recombining NA.

(d) Transformation.

First, the host bacteria N. coli CC600r strain was
Culture in 0 ml of the above Shiichi medium, collect the bacteria by centrifugation, and then add 50 ml of 0. It was suspended in IM MgCh solution and further centrifuged to give a final solution of 2.5 ml.
1 MMgCl, suspended in solution.

Nizierichia coli CC60 obtained in this way
Add 0.1 ml of the mixture containing the plasmid pIcR1 (obtained by method C1) to 0.2 ml of the suspension of the 0r strain,
After processing at ℃ for 30 minutes, it was processed at 42℃ for 2 minutes.

Next, 3 ml of the above-mentioned Shishitsu medium was added to this, and cultured at 37°C for 1 hour.
Added 5g of agar. ) After culturing at 37°C, the resulting 20 knees were further treated with tetracycline (25 μg/
Prepare to ml. ) By finding colomies that did not grow after culturing on an agar medium containing plasmid pl.
N. coli C600-pIc introduced with c1
Rl was obtained.

Next, Nizierichia coli C600-p obtained in this way
Ampicillin (15 Mg/m
Prepared to l. ) above glycerol medium 100 containing
ml was cultured with shaking at 37°C for 16 hours. This was collected by centrifugation, washed, and then mixed with 5 ml of 0.01% 2-mercaptoethanol and adjusted to pH 7.4.
The cells were suspended in 01M phosphate buffer, disrupted by sonication at 0°C for 5 minutes, and centrifuged to obtain a crude enzyme extract.

The heat-stable leucine dehydrogenase activity of the crude enzyme extract thus obtained was measured and found to be 0.201 units/mg protein. This is [lNA donor Bacillus stearothermophilus IP0122]
The activity was 10 times higher than that of the 50 strain leucine dehydrogenase (0,010 units/mg protein).

In addition, the crude enzyme extract containing this leucine dehydrogenase is
2 to p117 containing 0.01% mercaptoethanol.
When heat-treated at 70° C. for 20 minutes in 10 mM phosphate buffer of No. 2, it had residual activity of 80% or more.

Example 1 Nizierichia coli C600-plcR obtained in Reference Example 1
The L strain was cultured with shaking at 37° C. for 15 hours in 100 ml of the above glycerol medium containing ampicillin (adjusted to 15 μg/ml). After culturing, collect bacteria by centrifugation,
．． pH 7.4 with 0.1% 2-mercaptoethanol
Suspend in 5 ml of 0.01 M phosphate buffer prepared in
The bacterial cells were disrupted by ultrasonication for about 5 minutes.

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 0.800 units/mg of protein, and by heat-treating the crude enzyme extract at 70°C for 30 minutes, the specific activity was increased about 4 times compared to before heat treatment. . This is 1 original Bacillus stearothermophilus IFO12
Compared to the specific activity of leucine dehydrogenase of 250 strains, approximately 4
It was equivalent to 0 times.

Patent applicant: Unitika Co., Ltd.

Claims (5)

[Claims] (1) A heat-resistant leucine dehydrogenase-producing bacterium belonging to the genus Hscherichia was cultured. A method for producing heat-stable leucine dehydrogenase, which comprises collecting heat-stable leucine dehydrogenase from a culture. (2) Claim 1, wherein the heat-resistant leucine dehydrogenase-producing bacterium belonging to the genus Nidierythia is a bacterium belonging to the genus Nidierythia transformed with a plasmid having a gene for heat-stable leucine dehydrogenase. manufacturing method. (3) The production method according to claim 2, wherein the thermostable leucine dehydrogenase gene is a leucine dehydrogenase gene derived from the chromosomal DNA of a thermophilic microorganism. (4) The production method according to claim 3, wherein the thermophilic microorganism is a thermophilic bacterium belonging to the genus Bacillus. (5) Bacteria belonging to the thermophilic genus Bacillus are
Bacillus tea
ro thermoph i 1us).