JPH0357746B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel Escherichia coli having the ability to produce heat-stable leucine dehydrogenase. Leucine dehydrogenase is a very important enzyme for clinical testing. Microorganisms that can produce this leucine dehydrogenase include Bacillus and Bacillus sphaericus, which are thermophilic bacteria.
Bacillus bacteria such as Bacillus sphaericus 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 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 leucine dehydrogenase with the above properties existed in bacteria belonging to the
Published in Vol. 54 , p. 634 (1982). However, this thermophilic bacterium belonging to the genus Bacillus has a low productivity of heat-stable leucine dehydrogenase, and it has not been sufficient to efficiently obtain this enzyme. On the other hand, bacteria belonging to the genus Escherichia originally do not have any ability to produce leucine dehydrogenase. Plasmids useful in recombinant DNA genetic engineering and microorganisms transformed with them are also well known. For example, Science 198
Volume, p. 1056 (1978), plasmid PBR322
It has been described that animal proteins are produced in E. coli into which a plasmid linked to a lactose promoter has been introduced. Furthermore, in Japanese Patent Application Laid-Open No. 56-5093, a plasmid of the genus Escherichia was transformed by introducing a plasmid containing a gene of a bacterium belonging to the genus Thermus (plasmid PBR322 was used as a vector). Although it has been described that a thermostable enzyme is prepared using the same bacteria, nothing has been written about a plasmid containing the gene for heat-stable leucine dehydrogenase or a microorganism transformed by it. No,
Nor has there been any report that its creation has been successful. Therefore, the present inventors conducted extensive research in search of a microorganism that can efficiently produce heat-stable leucine dehydrogenase, and as a result, they transformed it with a plasmid containing the gene for heat-stable leucine dehydrogenase. The present invention was completed based on the discovery that Escherichia coli produces large amounts of heat-stable leucine dehydrogenase. That is, the present invention provides a strain of Escherichia leucine dehydrogenase having the ability to produce a heat-stable leucine dehydrogenase that has been transformed with a plasmid containing a gene for a heat-stable leucine dehydrogenase derived from the chromosomal DNA of Bacillus stearothermophilus. The main topic is Escherichia coli. To obtain the plasmid used in the present invention, for example, Biochem. Biophysacta 72 , 619-629
(1963), by digesting the heat-stable leucine dehydrogenase gene and DNA serving as a vector with restriction enzymes, and then ligating them using ligase. Examples of the thermostable leucine dehydrogenase gene used in the present invention include the leucine dehydrogenase gene derived from the chromosomal DNA of Bacillus stearothermophilus. Specific examples of Bacillus stearothermophilus include IFO12550, ATCC7953, 7954,
There are 8005, 10194, 12980, NCA1503, etc. Furthermore, examples of DNA that functions as a vector include plasmid DNA, and plasmid pBR322 is particularly preferred. Examples of restriction enzymes include Sal, and examples of ligases include T4 DNA ligase. As a pramid preferably used in the present invention, there is a plasmid pICR1 in which a gene for leucine dehydrogenase derived from the chromosomal DNA of Bacillus stearothermophilus is introduced into pBR322 by the method described above. This plasmid
On February 23, 1981, pICR1 was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, but this plasmid was not deposited. Next, the physicochemical properties of this plasmid pICR1 will be shown. (1) Heat-stable leucine dehydrogenase can be expressed in room-temperature microorganisms. (2) Has the following cleavage sensitivity to the following restriction enzymes. Restriction enzyme Number of cleavage sites EcoR 2 Hind 1 Pst 2 Sal 4 BamH 2 The names of restriction enzymes are abbreviations of restriction enzymes obtained from the following bacterial species. EcoR; Escherichia coli Hind; Haemophilus influenzae Pst; Providencia styuartii Sal; Streptomyces albus BamH; Bacillus amyloliquefaciens , the digest is subjected to agarose gel electrophoresis and determined from the number of separable fragments. (3) The molecular weight is approximately 6 megadaltons. Using this pramid pICR1,
To transform E. coli, e.g.
Of Molecule Biology (J.Mol.Biol)
53, 159-162 (1970), by contacting the above Escherichia coli C600 treated with calcium chloride with plasmid pICR1 at a temperature around 0°C. An example of E. coli transformed as described above is E. coli C600-pICR1 strain into which plasmid pICR1 has been introduced. This strain is a known strain of Escherichia coli.
C600 [see Nature 217 , 1110-1114 (1968)]
It has the same mycological properties except that it has a heat-resistant leucine dehydrogenase production ability and ampicillin resistance. This strain remains safe without changing from non-transmissible to transmissible or from non-pathogenic to pathogenic. In particular, there have been no reports of Escherichia coli having the ability to produce heat-stable leucine dehydrogenase. From this, the Escherichia coli C600-pICR1 strain is considered to be a new strain, so 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 accession number is No. 6937. Carbon sources for the nutrient medium used for culturing E. coli of the present invention include, for example, glucose, sucrose, fructose, starch hydrolyzate, molasses, sugars from sulfite pulp waste liquid, organic acids such as acetic acid and lactic acid, and even organic acids such as acetic acid and lactic acid. Alcohols, fatty acids, glycerin, etc. that can be assimilated by the bacteria used can be used, and as nitrogen sources, inorganic or organic substances such as ammonium sulfate, ammonium chloride, ammonium phosphate, amino acids, peptone, meat extract, yeast extract, etc. can be used. moreover,
Examples of inorganic salts include potassium, sodium,
phosphoric acid, zinc, iron, magnesium, manganese,
Salts such as copper, calcium, covarna, etc., trace metal salts, corn staple liquor, vitamins, nucleic acids, etc. may be used as necessary, and general nutrient media for bacteria can be used. Using these media, Escherichia coli of the present invention is grown at 20°C to 45°C, preferably 35°C to 40°C,
Optimally, it may be cultured aerobically at 37°C for about 10 to 20 hours at a pH of 7.0 to 7.4, optimally 7.2. The culture thus obtained can be used as it is as an enzyme source for heat-stable leucine dehydrogenase, but crude enzyme extracts, purified enzymes, isolated live bacterial cells, and processed isolates can also be used as enzyme sources. It can be used as Next, the thermostable leucine dehydrogenase of the present invention is collected from the obtained culture,
It can be collected at any stage, including culture, isolated live bacterial cells, processed isolated bacterial cells, crude enzyme extracts, and purified enzymes. As a purification method at that time, a usual enzyme purification method 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. Advantageously, enzymes are obtained.
The conditions for this heat treatment may be, for example, a treatment 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 Escherichia coli of the present invention is very useful because it can easily produce large amounts of heat-stable leucine dehydrogenase. The thermostable leucine dehydrogenase obtained by the present invention is described in Biochemistry, Vol. 54 , p. 634 (1982).
has the same physicochemical properties as the described thermostable leucine dehydrogenase. Next, the present invention will be specifically explained using examples. The activity of heat-stable leucine dehydrogenase is
Amino Acid and Nucleic Acid
The method for measuring leucine dehydrogenase activity described in Nucleic Acid 27, 84-88 (1973), that is, PH
Prepare a mixture containing 1.25 μmole of NAD and 10 μmole of L-leucine in 100 μmole of glycine-KC1-KOH buffer from 11.3, add an appropriate amount of crude enzyme extract to the mixture, and make up the final volume. is 0.8ml,
The increase in reduced NAD per unit time at 25°C or 55°C was measured as the increase in absorbance at 340 nm. Moreover, % in Examples and Reference Examples indicates volume %. Reference example 1 (a) Chromosome of Bacillus stearothermophilus
DNA isolation. bacillus stearothermophilus
From the IFO12550 strain, Biochem Biophys
Chromosomal DNA was isolated according to the method described in Acta) 72 , 619-629 (1963). First, Bacillus stearothermophilus
IFO12250 strain was grown in glycerol medium (polypeptone 10g/, yeast extract 2.5g/, meat extract 2g/, glycerol 2g/, sodium chloride 5g/, dipotassium phosphate 2g/, magnesium sulfate 0.1g/, biotin 4μg/,
After culturing with shaking at 55°C for 12 hours (adjusted to pH 7.21), the bacteria were collected by centrifugation. Next, 12 mg of lysozyme was added to 6 ml of saline-EDTA solution (0.15 M NaCl and 0.1 M NaCl).
Contains EDTA and adjusted 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-SDS buffer to the frozen cells, and add 0.1 M Tris buffer containing 10 mg/ml SDS and 0.1 M NaCl to a pH of 9.0. ) was added, stirred, and further heated to 60°C to completely lyse the bacteria. 56 ml of 80% phenol was added to this lysate and shaken for about 20 minutes to perform phenol extraction and remove contaminant proteins. This extracted crude
Add twice the volume of cold ethanol to the DNA solution, roll up the fibrous precipitate with a glass rod,
After sequential immersion in 10 ml of 90% ethanol for several minutes each, 20 ml of dilute saline-citrate solution (0.015 M NaCl, 0,
Prepared as 0015M Na ₃ -citric acid. ) and then add concentrated saline-citrate solution (1.5M NaCl,
Prepared to 0.15M Na ₃ -citric acid. ) was added to prepare a crude DNA solution. Dilute this crude DNA solution to about 500 μg/ml, add liponuclease [R NaseA (manufactured by Sigma)] to 50 μg/ml, and R NaseT ₁ (manufactured by Sigma).
was added at a concentration of 30 μg/ml and heated at 37° C. for 30 minutes. After cooling, add an equal amount of 80% phenol to perform phenol extraction, collect the extracted DNA by ethanol precipitation, and add the above diluted saline.
A chromosomal DNA extract was prepared by dissolving the DNA in 20 ml of -citrate solution and adding 2 ml of the above concentrated saline-citrate solution. (b) Preparation of vector plasmid pBR322. Plasmid pBR322 (Batheada Research
Laboratories)
E. coli C600 strain was aerated and cultured at 37°C in L-medium 2 (adjusted to pH 7.2 with 10 g of polypeptone, 5 g of yeast extract, 1 g of glycose, and 5 g of sodium chloride) until early logarithmic growth. , 10ml of chloramphenicol solution (adjusted with ethanol to give a concentration of 3.6mg/ml)
was added, and the plasmid pBR322 was further propagated by aerated culture at 37°C for 15 minutes. Next, collect the bacteria by centrifugation into 80ml of TE-
Sucrose buffer 200mg/ml Sucrose,
Contains 20mM EDTA and adjusted to PH8.0
0.05M Tris buffer. ) and add 8 ml
lysozyme solution (add the above to 5 mg/ml)
Prepared with TE-sucrose buffer. ), then add 28 ml of 5M NaCl solution and 4 ml of 40
mg/ml SDS solution was added. After reacting this mixture at 27°C for 2 hours, crude plasmid DNA was separated by centrifugation. Next, phenol treatment was performed by adding 1/2 volume of 80% phenol to remove contaminant proteins.
This extracted crude plasmid was precipitated and recovered with cold isopropanol, and further TE buffer (0.14M
20mM Tris buffer adjusted to PH7.5 containing MaCl, 1mM EDTA. ) dissolved in 2 mg of RNase was added to this mixture, and the mixture was allowed to react 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. This was further dissolved in 10 ml of the above TE buffer and filtered through agarose gel to remove contaminants.
RNA was further removed, and the resulting crude DNA was recovered again in ethanol precipitation. This precipitate was dissolved in 23.1 ml of 0.02 M Tris buffer (PH
Adjusted to 8.0. ), add 23.7 g of cesium chloride and 0.6 ml of ethidium bromide solution (adjusted to 10 mg/ml), and isolate the plasmid DNA by ultracentrifuging for about 40 hours. Ethidium bromide was removed. This isolated plasmid
0.01M TE buffer (PH containing 0.1mM EDTA)
0.01M Tris buffer prepared in 7.5. ), purified plasmid pBR322 was obtained. (c) Creation of plasmid pICR1. 5μg of Bacillus stearothermophilus chromosomal DNA and restriction enzyme sal obtained by method (a)
(manufactured by Takara Shuzo Co., Ltd.) 20 units, 7mM MgCl ₂ ,
PH7.5 containing 150mM NaCl, 0.2mM EDTA, 7mM 2-methylcaptoethanol, 0.01% BSA
into 100μ of 10mM Tris buffer prepared in
After reacting at 37°C for 2.5 hours to digest the DNA, heating at 65°C for 5 minutes to inactivate Sal.
Digested DNA fragments were precipitated and collected by quenching with cold ethanol. Next, the plasmid obtained by method (b)
Add restriction enzyme Sal3 units to pBR3221μg, react in the same buffer as above at 37℃ for 10 hours, and digest the plasmid in the same manner as above.
DNA was recovered. The thus obtained digested chromosome and plasmid DNA were mixed and T4DNA
Using ligase (manufactured by Takara Shuzo Co., Ltd.), 6.6mM
PH containing MgCl ₂ , 10mM DTT, 66μM ATP
7.6 in 66mM Tris buffer at 13°C.
Plasmid pICR1 was obtained by reacting for 19 hours and recombining the digested DNA. Example 1 Escherichia coli was transformed using the plasmid pICR1 obtained in Reference Example 1. First, the host bacterium Escherichia coli C600r ^- m ^-
The strain was cultured in 50 ml of the above L-medium, collected by centrifugation, suspended in 50 ml of 0.1M MgCl ₂ solution, further centrifuged, and finally 2.5 ml of 0.1M
Suspended in _MgCl2 solution. Esieritia coli obtained in this way
Add 0.1 ml of the mixture containing plasmid pICR1 obtained by method (c) to 0.2 ml of suspension of C600rm strain, and bring to 0°C.
After processing for 30 minutes at 42°C for 2 minutes. Next, add 3 ml of the above L-medium to this, and
Incubate at ℃ for 1 hour, and add ampicillin (15μ
Adjusted to g/ml. ) containing L-agar medium (15 g of agar added per L-medium) at 37°C.
After culturing the resulting colonies on an L-agar medium containing tetracycline (prepared at 25 μg/ml), colonies that did not grow were found to be E. coli C600- into which plasmid pIC1 had been introduced. pICR1 was obtained. Next, Esieritia coli obtained in this way
A colony of C600-pICR1 was cultured with shaking in 100 ml of the above glycerol medium containing ampicillin (adjusted to 15 μg/ml) at 37° C. for 16 hours. This was centrifuged to collect bacteria, and after washing, 5 ml of
Contains 0.01% 2-mercaptoethanol, PH7.4
Suspend in 0.01M phosphate buffer prepared at 0°C.
The bacterial cells were disrupted by ultrasonication for 5 minutes, and a crude enzyme extract was obtained by centrifugation. When the activity of heat-stable leucine dehydrogenase in the crude enzyme extract thus obtained was measured,
It was 0.201 units/mg protein. This is 10 times higher than the leucine dehydrogenase activity (0.010 units/mg protein) of the DNA donor Bacillus stearothermophilus IFO12250 strain.
It was more than twice as active. In addition, this crude enzyme extract containing leucine dehydrogenase contains 0.01% 2-mercaptoethanol.
When heat treated at 70°C for 20 minutes in 10mM phosphate buffer with pH 7.2, it had residual activity of over 80%. Reference example 2 Esieritia coli CC600− obtained in Example 1
The pICR1 strain was cultured with shaking at 37° C. for 15 hours in 100 ml of the aforementioned glycerol medium containing ampicillin (adjusted to 15 μg/ml). After culturing, collect the bacteria by centrifugation and add PH containing 0.01% 2-mercaptoethanol.
Suspend in 5 ml of 0.01M phosphate buffer prepared in 7.4,
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, and it was found to be 0.800 units/ mg of protein, and by heat-treating the crude enzyme extract at 70°C for 30 minutes, the specific activity increased approximately 4 times compared to before heat treatment. This was equivalent to about 40 times the specific activity of leucine dehydrogenase of the original Bacillus stearothermophilus IFO12550.

Claims (1)

[Claims] 1 Chromosomal DNA of Bacillus stearothermophilus
Escherichia coli, which has the ability to produce heat-stable leucine dehydrogenase, was transformed with a plasmid containing the heat-stable leucine dehydrogenase gene derived from Escherichia coli.