JPH074239B2 - Method for producing thermostable leucine dehydrogenase - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing thermostable leucine dehydrogenase.

(Prior Art) Leucine dehydrogenase is a very important enzyme as a clinical laboratory enzyme. As a microorganism capable of producing this leucine dehydrogenase, a bacterium belonging to the genus Bacillus such as Bacillus sphaericus, which is a normal-temperature bacterium, is known. However, 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 lacks thermostability and long-term stability. It has a major drawback.

Therefore, in order to maximize the advantages of the analysis method of the clinical test using leucine dehydrogenase, the appearance of leucine dehydrogenase, which is stable to heat and does not lose its activity at room temperature for a long time, has been earnestly desired.

Therefore, as a result of some previous studies conducted by some of the present inventors for a leucine dehydrogenase having a property of being stable to heat and not losing activity for a long time, as a result, a thermophilic Bacillus genus was obtained. It was discovered that leucine dehydrogenase having the above-mentioned properties is present in a bacterium belonging to the category I and applied for a patent (JP-A-59-348841).

However, this thermophilic bacterium belonging to the genus Bacillus has a low productivity of thermostable leucine dehydrogenase, and is not sufficiently satisfactory to efficiently obtain this enzyme.

On the other hand, bacteria belonging to the genus Escherichia have no ability to produce leucine dehydrogenase at all.

In addition, plasmids useful for tissue DNA genetic engineering and microorganisms transformed with them are well known. For example, Science 198, 1056 (1978)
Describes that animal proteins are produced in Escherichia coli in which a plasmid in which a lactose promoter is ligated is introduced into plasmid pBR322.

Further, in JP-A-56-5093, a genus Escherichia transformed by introducing a plasmid having a gene of a bacterium belonging to the genus Thermus (the plasmid pBR322 is used as a vector) is introduced. It has been described that a thermostable enzyme is prepared using a bacterium belonging to it, but nothing is described about a plasmid having a gene for thermostable leucine dehydrogenase and a microorganism transformed by the plasmid. Also, there is no report that the creation was successful.

Further, as a result of earnest research for a microorganism having a high thermostable leucine dehydrogenase content from such a viewpoint, some of the present inventors found that a thermostable leucine dehydrogenase-producing bacterium belonging to the genus Escherichia was found. It was found that the thermostable leucine dehydrogenase was efficiently produced, and a patent application was filed (Japanese Patent Laid-Open No. 59-159778).

(Problems to be Solved by the Invention) With the thermostable leucine dehydrogenase-producing bacterium belonging to the genus Escherichia, the productivity of the thermostable leucine dehydrogenase is not yet sufficient. It was not enough to obtain it efficiently.

(Means for Solving Problems) Therefore, as a result of earnest research for a microorganism having a high thermostable leucine dehydrogenase content, the present inventors belong to the anaerobic and thermophilic Clostridium genus. It was found that a bacterium belonging to the genus Escherichia transformed with a recombinant plasmid in which a leucine dehydrogenase gene derived from a bacterium is ligated to a vector plasmid efficiently produces thermostable leucine dehydrogenase. completed.

That is, the present invention provides an Escherichia coli transformed with a recombinant plasmid in which a leucine dehydrogenase gene derived from an anaerobic and thermophilic bacterium belonging to the genus Clostridium is ligated to a vector plasmid.
The gist is a method for producing a thermostable leucine dehydrogenase, which comprises culturing a bacterium belonging to the genus hia) and collecting the thermostable leucine dehydrogenase from the culture.

The bacterium used in the present invention belongs to the genus Escherichia transformed with a recombinant plasmid in which a leucine dehydrogenase gene derived from an anaerobic and thermophilic bacterium belonging to the genus Clostridium is ligated to a vector plasmid. It is a bacterium, and any such bacterium can be used. Examples of preferred bacteria include, for example, Escherichia coli C600 known from recombinant plasmid pICD242 in which an anaerobic and thermophilic bacterium belonging to the genus Clostridium and a leucine dehydrogenase gene are ligated to a vector plasmid.
Escherichia coli C600-pICD242 transformed with E. coli. This strain is known as Escherichia coli C600.
[See Nature 217 , 1110 to 1114 (1968)] and has the same mycological properties except that it has a thermostable leucine dehydrogenase-producing ability and ampicillin resistance. This strain does not change non-transmissibility to transmissibility,
In addition, safety is maintained without changing non-pathogenicity to pathogenicity. In particular, there has been no report of Escherichia coli having a thermostable leucine dehydrogenase-producing ability that incorporates a leucine dehydrogenase gene derived from a bacterium belonging to the genus Clostridium, which is anaerobic and thermophilic. From this, the Escherichia coli C600-pICD242 strain is considered to be a new strain and was deposited on February 21, 1987 at the Institute for Microbial Technology, Ministry of International Trade and Industry, Institute of Industrial Science and Technology. The microorganism accession number is 9213.

To obtain the plasmid used in the present invention, for example, Biochimica et Biophysica Actor (Biochimica
et Biophysica Acta) 72 , pp. 619-629 (1963), the thermostable leucine dehydrogenase gene and DNA having a role as a vector are digested with a restriction enzyme, and then ligase is used. It can be prepared by coupling.

The thermostable leucine dehydrogenase gene used in the present invention includes anaerobic and thermophilic Clostridium (Cl
It is necessary that the gene is a leucine dehydrogenase gene derived from a bacterium belonging to the genus ostridium. Among them, Clostridium thermoaceticum, which has a high activity of leucine dehydrogenase, is preferable, and specifically, AN28-4 strain (on February 21, 1987, Ministry of International Trade and Industry Institute of Industrial Technology, Institute of Microbial Technology) Deposited. The microorganism accession number is 9214).

In addition, examples of DNA having a role as a vector include plasmid DNA, and particularly plasmid pBR232
Is preferred. Moreover, as a restriction enzyme, for example, Hind III
Examples of the ligase include T4 DNA ligase.

By this method, a plasmid pICD242 is obtained in which the gene for leucine dehydrogenase derived from the chromosomal DNA of Clostridium thermoaseticum AN28-4 is introduced into the plasmid pBR322. This plasmid pICD242 was deposited on February 21, 1987, at the Institute for Microbial Technology, Ministry of International Trade and Industry, and the plasmid was not accepted.

Next, the physicochemical properties of this plasmid pICD242 are shown.

(1) A thermostable leucine dehydrogenase can be expressed in a room temperature microorganism.

(2) As shown in FIG. 1, it has the following cleavage sensitivities to the following restriction enzymes.

Restriction enzyme number of cleavage sites EcoR I 3 Hind III 2 Pst I 3 Sal I 5 BamH I 1 The name of the restriction enzyme is an abbreviation of the restriction enzyme obtained from the following bacterial species. EcoR I; Escherichia coli Hind III; Haemophilus influenzae Pst I; Providencia futuarti Sal I; Streptomyces albus BamH I; Bacillus amyloliquefaciens The number of restriction enzyme cleavage sites is excessive. The plasmid pICD242 is digested below and the digest is subjected to agarose gel electrophoresis and determined from the number of separable fragments.

(3) The molecular weight is about 5.8 megadalton.

As the carbon source of the nutrient medium used for culturing the bacterium in the present invention, for example, glucose, sucrose, fructose, starch hydrolyzate, molasses, sugars of sulfite waste liquor, organic acids such as acetic acid and lactic acid, and the like are used. Alcohols, fatty acids and glycerin that can be assimilated by bacteria can be used, and as the nitrogen source, for example, inorganic or organic substances such as ammonium sulfate, ammonium chloride, ammonium phosphate, amino acids, peptone, meat extract and yeast extract can be used. Further, as inorganic salts, for example, potassium, sodium, phosphoric acid, zinc, iron, magnesium, manganese, copper, calcium, cobalt, and other salts, if necessary, trace metal salts, corn steep liquor, vitamins, nucleic acids Etc. may be used, and a general nutrient medium for bacteria can be used.

Using these media, the bacteria of the present invention can be treated at 20 ° C to 45 ° C.
C., preferably 35.degree. C. to 40.degree. C., optimally 37.degree. C. for about 10 to 20 hours, pH 7.0 to 7.4, optimally 7.2, and aerobically cultivated.

Next, the thermostable leucine dehydrogenase of the present invention is collected from the obtained culture, but at any stage such as culture, isolated viable cells, processed product of isolated cells, crude enzyme extract, purified enzyme, etc. Can be collected at. As a purification method in that case, a usual enzyme purification method can be used. In particular, in the present invention, if the disrupted solution is heat-treated prior to collecting the thermostable leucine dehydrogenase, the thermostable enzyme or protein is heat-denatured to selectively heat-resistant leucine dehydrogenase. Advantageously, the enzyme is obtained. The conditions for this heat treatment may be, for example, a temperature of 50 to 80 ° C. for 5 to 30 minutes. After the treatment in this way, the thermostable leucine dehydrogenase may be obtained by separation and purification, but 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 and JP-A-59-159778.

(Examples) Next, the present invention will be specifically described with reference to Examples.

Incidentally, the activity of the heat resistance of the leucine dehydrogenase, amino acids, nucleic acids [Amino Acid and Nucleic Acid. 27, 84~88 (1
973)] and the measuring method of leucine dehydrogenase activity. That is, in 120 μmole of glycine-KCl-KOH buffer at pH 10.5, 2.5 μmole of NAD and 20 μmole of L were added.
-Prepare a mixture containing leucine, add an appropriate amount of crude enzyme extract to the mixture to make the final volume 0.8 ml, and increase the reduced NAD at 25 ℃ or 55 ℃ per unit time by 340 nm. Was measured as an increase in the absorbance.

In addition,% in the examples and reference examples indicates% by volume.

Reference Example 1 (a) Chromosome of Clostridium thermoaseticum
DNA isolation.

From Clostridium thermoaseticum AN28-4 strain (Microtechnology Research Institute, No. 9124), Biochi-mica et Biophysica Acta
Chromosomal DNA was isolated according to the method described in Volume 72 , pages 619 to 629 (1963).

First, Clostridium thermoasetycum AN28-4
Strains trypticose 17g / l, phyton 3g / l, glucose 6
g / l, sodium chloride 2.5g / l, sodium thioglycolate 0.5g / l, L-cystine 0.25g / l, sodium sulfite 0.1g
After statically culturing at 60 ° C. for 24 hours in Medium 2 adjusted to pH 1 / l and pH 7.2, the cells were collected by centrifugation.

Next, 12 mg of lysozyme was added to 6 ml of Saline-EDTA.
The solution (0.15M Nacl and 0.1M EDTA) was included and adjusted to pH 8.0. ), And the collected strain was added to this solution and stirred well. This was heated at 37 ° C for about 10 minutes, and when the cells started to lyse, they were immediately frozen.

50 ml of Tris-SDS buffer (10 mg / ml
0.1M Tris buffer adjusted to pH 9.0 containing SDS and 0.1M Nacl. ) Was added and stirred, and the mixture was heated to 60 ° C. and completely lysed.

To this lysate, 56 ml of 80% phenol was added and shaken for about 20 minutes to perform phenol extraction to remove contaminating proteins. 2 volumes of cold ethanol was added to the extracted crude DNA solution and the fibrous precipitate was wound up with a glass rod.
After dipping in 10 ml each of 80 and 90% ethanol for several minutes, 20 ml of dilute saline-citrat (saline-citrat)
e) Dissolve in a solution (prepared with 0.015M NaCl, 0.0015M Na ₃ -citric acid), and add concentrated saline-citrate solution (1.5M
Prepared in NaCl, 0.15M Na ₃ -citric acid. ) Is added to the crude D
NA solution was prepared.

Diluted the crude DNA solution in 500 [mu] g / ml level, ribonuclease A [R NaseA (Sigma)] was 50 [mu] g / ml, ribonuclease T ₁ [R NaSEt ₁ (Sigma)] was to be 30 [mu] g / ml The mixture was heated at 37 ° C for 30 minutes. 80% of the same amount after cooling
Phenol was added, phenol extraction was performed, and the extracted DNA was recovered by ethanol precipitation.
Dissolve it in 20 ml of the rate solution, and then add the concentrated saline-citr
A chromosomal DNA extract was prepared by adding 2 ml of the ate solution.

(B) Preparation of vector-plasmid pBR322.

The Escherichia coli C600 strain into which the plasmid pBR322 (manufactured by Bethesda. Research Laboratories) was introduced was transformed into 2 L-
Medium (polypeptone 10g / l, yeast extract 5g / l, glucose 1
Adjusted to pH 7.2 with g / l and sodium chloride 5g / l. ), Aerobically cultivated at 37 ° C until the logarithmic growth phase, then 10 ml of chloramphenicol solution (prepared with ethanol to give 3.6 mg / ml) was added, and further aerobically incubated at 37 ° C for 15 minutes. The plasmid pBR322 was propagated.

Next, 80 ml of the bacteria collected by centrifugation was added to TE-sucrose buffer (200 mg / ml sucrose, containing 20 mM EDTA, pH 8.0.
0.05M Tris buffer prepared in. ), And then 8
Add 1 ml of lysozyme solution (prepared with the above TE-sucrose buffer so that it will be 5 mg / ml), and add 28 ml of 5M.
NaCl solution and 4 ml of 40 mg / ml SDS solution were added.

After allowing this mixed solution to react at 37 ° C. for 2 hours, the crude plasmid DNA was separated by centrifugation.

Next, 1/2 volume of 80% phenol was added and phenol treatment was performed to remove contaminating proteins. The extracted crude plasmid was recovered by precipitation with cold isopropanol, and the TE buffer (0.14M NaCl, containing 1 mM EDTA, pH 7.5) was added.
0 mM Tris buffer. ) Dissolved in. 2 mg R in this mixture
NaseA was added and reacted at 37 ° C. for 2 hours, and contaminating RNA was removed by phenol treatment in the same manner as above. The extracted crude plasmid was recovered by precipitation with 2 volumes of ethanol. This is further dissolved in 10 ml of the above TE buffer, and contaminated RNA is further removed by agarose gel filtration.
The obtained crude DNA was recovered again by ethanol precipitation.

This precipitate was dissolved in 23.1 ml of 0.02 M Tris buffer (adjusted to pH 8.0), 23.7 g of cesium chloride and 0.6 ml of ethidium bromide solution (prepared to 10 mg / ml) were added, and the time was about 40 hours. The plasmid DNA was separated by ultracentrifugation, and then ethidium bromide was removed with normal butanol. 0.01M of this isolated plasmid
TE buffer (0.01M adjusted to pH 7.5 with 0.1mM EDTA)
Tris buffer. ), The purified plasmid pBR322 was obtained.

(C) Creation of plasmid pICD242.

10 μg of chromosomal DNA of Clostridium thermoaseticum obtained by the method of (a) and 30 units of restriction enzyme Hind III (Takara Shuzo) were added to 7 mM MgCl ₂ , 150 mM NaCl, 0.2 mM ED
TA, 7 mM 2-mercaptoethanol, 0.01% BSA in pH 7.
Add to 100 μl of 10 mM Tris buffer prepared in 5 and mix at 37 ℃.
After incubating for 5 minutes to digest the DNA, heat at 65 ° C for 5 minutes to inactivate Hind III, and digest with cold ethanol.
The fragment was recovered by precipitation.

Next, the plasmid pBR3223 μg obtained by the method of (b)
The restriction enzyme Hind III 3 units was added to the reaction mixture and the mixture was reacted in the same buffer as above at 37 ° C. for 10 hours, and the digested plasmid DNA was recovered by the same method as above. The digested chromosomes thus obtained and the plasmid and DNA were mixed, and using T4 DNA ligase (Takara Shuzo Co., Ltd.), the pH was adjusted to 7.6 containing 6.6 mM MgCl ₂ , 10 mM DTT, 66 μM ATP, and the pH was adjusted to 7.6. The plasmid pICD242 was obtained by allowing the reaction to proceed for 16 hours and re-ligating the digested DNA.

(D) Transformation.

First, the host strain Escherichia coli C600r ^- m ^- strain is cultured in 50 ml of the above L-medium, collected by centrifugation, and then 50 ml.
Was suspended in 0.1 M MgCl ₂ solution, and further centrifuged to finally suspend it in 2.5 ml of 0.1 M MgCl ₂ solution.

In 0.2 ml of the suspension of the Escherichia coli C600r ^- m ^- strain thus obtained, the plasmid pIC obtained by the method of (c)
0.1 ml of a mixture containing D242 was added and treated at 0 ° C. for 30 minutes and then at 42 ° C. for 2 minutes.

Next, 3 ml of the above-mentioned L-medium was added thereto, and the mixture was incubated at 37 ° C for 1 hour, and further, L-agar medium containing ampicillin (prepared to 15 µg / ml) (15 g of agar per L-medium was added). After culturing the resulting colonies at 37 ° C. in L-agar medium containing tetracycline (prepared to 25 μg / ml), the colonies that did not grow were found to find the plasmid pICD242. Introduced Escherichia
E. coli C600-pICD242 was obtained.

Next obtained Escherichia coli C600-pICD24
Ampicillin (prepared to 15 μg / ml) from 2 colonies
Glycerol medium from above (polypeptone 10 g / l,
Yeast extract 2.5g / l, meat extract 2g / l, glycerol 2g / l, sodium chloride 5g / l, 1 potassium phosphate 2g / l, 2 potassium phosphate 2g / l, magnesium sulfate 0.1g / l, biotin 4μg / l The culture was performed with shaking in 100 ml at 37 ° C. for 16 hours. After collecting the cells by centrifugation and washing, 0.01% of 5 ml
0.01 containing 2-mercaptoethanol to pH 7.4
The cells were suspended in M phosphate buffer, sonicated at 0 ° C. for 5 minutes to crush the cells, and centrifuged to obtain a crude enzyme extract.

The activity of the thermostable leucine dehydrogenase of the crude enzyme extract thus obtained was measured and found to be 8.9 units / mg protein. This is 8 more than the activity of leucine dehydrogenase (0.010 unit / mg. Protein) of Clostridium thermoaseticum AN28-4 strain which is a DNA donor.
There was more than 00 times the activity.

In addition, the crude enzyme extract containing this leucine dehydrogenase is 2
-When it was heat-treated at 70 ℃ for 20 minutes in 10 mM phosphate buffer of pH 7.2 containing 0.01% of mercaptoethanol, it had a residual activity of more than 80%.

Example 1 and Comparative Example 1 Escherichia coli C600-pICD242 strain (Microtechnology Research Institute No. 9213) obtained in Reference Example 1 was treated with 100 ml of the glycerol medium containing ampicillin (prepared to 15 μg / ml) at 37 ° C. At 16
The culture was shaken for an hour. After culturing, collect the cells by centrifugation to 0.0
Adjusted to pH 7.4 containing 1% 2-mercaptoethanol
The cells were suspended in 5 ml of 0.01 M phosphate buffer and sonicated for about 5 minutes to disrupt the cells. When the enzyme activity of the disrupted cell suspension was measured, the leucine dehydrogenase activity per medium 1 was
It was found to be 9500 units, which is shown in Comparative Example 1 below.
It is active for about 10 times more than that of, and it is clear that productivity is significantly improved.

After that, a crude enzyme extract was obtained by centrifugation, the crude enzyme extract was heat-treated at 70 ° C for 30 minutes, then centrifuged, and the leucine dehydrogenase activity of the supernatant was measured. It has the activity of mg / protein and contains 70% of crude enzyme extract.
Heat treatment at 30 ° C for 30 minutes improved the specific activity about 5 times compared to before heat treatment.

Next, it was purified by preparative electrophoresis using 7.5% acrylamide and high-performance liquid chromatography using a Super Rose 12 gel chromatographic column (Farmasia, two columns connected in series) to obtain a specific activity of 116 units / mg / protein. The thermostable leucine dehydrogenase of was obtained.

This enzyme gave a single band by 7.5% acrylamide gel electrophoresis at pH 9.4, which was similar to the properties of conventional leucine dehydrogenase derived from Bacillus stearothermophilus.

For comparison, Escherichia coli C600-pICR1 (Microtechnological Research Institute No. 6937) in which a gene derived from Bacillus stearothermophilus was integrated was used in accordance with a known document (Japanese Patent Laid-Open No. 59-159778) to prepare the glycerol medium. 16 at 37 ℃ in 100 ml
After shaking culture for a period of time and collecting cells by centrifugation, 0.01% of 2
-Suspended in 5 ml of 0.01 M phosphate buffer solution containing mercaptoethanol and adjusted to pH 7.4, and crushed the cells by ultrasonic treatment for about 5 minutes to obtain a crushed solution. When the activity of leucine dehydrogenase was measured for this disrupted solution, it was 10 per medium.
It was 00 units (Comparative Example 1).

(Advantages of the Invention) According to the present invention, a large amount of thermostable leucine dehydrogenase can be obtained, and the leucine dehydrogenase can be obtained in a large amount, which is extremely useful in the field of clinical test reagents and the like.

[Brief description of drawings]

FIG. 1 is a restriction enzyme map of the plasmid pICD242 used in the present invention. S: Sal I, E: EcoR I, P: Pst I, B: BamH I, H: Hind III

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12R 1: 145)

Claims (1)

[Claims] 1. An anaerobic and thermophilic Clostridium (Cl
Escherichia transformed with a recombinant plasmid in which a leucine dehydrogenase gene derived from a bacterium belonging to the genus ostridium is ligated to a vector plasmid.
A method for producing a thermostable leucine dehydrogenase, which comprises culturing a bacterium belonging to the genus and collecting the thermostable leucine dehydrogenase from the culture.