JPH0249713B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel plasmid containing a gene for thermostable leucine dehydrogenase. Leucine dehydrogenase is a very important enzyme for clinical testing. The microorganism that can produce this leucine dehydrogenase is Bacillus sphaericus, a thermophilic bacterium.
Bacillus bacteria such as B. sphaericus are known. However, the 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 the major drawback of lacking thermostability and long-term stability. It has its drawbacks. Therefore, in order to maximize the advantages of analytical methods for clinical tests using leucine dehydrogenase, it has been eagerly awaited for the emergence of a leucine dehydrogenase that is stable to heat and does not lose its activity for a long period of time at room temperature. Therefore, from this perspective, some of the present inventors conducted intensive research in search of a leucine dehydrogenase that is heat-stable and does not lose its activity over a long period of time. He discovered that leucine dehydrogenase with the above properties exists in bacteria belonging to the genus, applied for a patent, and further developed biochemistry and
Vol. 54, p. 634, (1982). However, this thermophilic bacterium belonging to the genus Bacillus has a low productivity of heat-stable leucine dehydrogenase, and has not been fully satisfactory for efficiently obtaining this enzyme. On the other hand, bacteria belonging to the genus Escherichia originally do not have any ability to produce leucine dehydrogenase. Furthermore, plasmids useful for recombination and DNA genetic engineering are well known. For example, Science Vol. 198, p. 1056 (1978) describes that animal proteins are produced in Escherichia coli into which a plasmid in which a lactose promoter is linked to plasmid pBR322 is introduced. Furthermore, in Japanese Patent Application Laid-Open No. 56-5093, Escherichia genus was transformed by introducing a plasmid (plasmid pBR322 was used as a vector) containing the gene of a bacterium belonging to genus Thermus. Although it has been described that a thermostable enzyme can be prepared using the same bacteria, nothing has been described about a plasmid containing the gene for a thermostable leucine dehydrogenase, and no one has succeeded in creating one. No reports have been made. Therefore, in order to improve the productivity of thermostable leucine dehydrogenase, the present inventors conducted extensive research in search of a plasmid that can express thermostable leucine dehydrogenase in a thermophilic microorganism. isolated the gene for leucine dehydrogenase from , and discovered that this gene can be introduced into known plasmid DNA, and that the plasmid obtained by introducing the gene in this way has the above-mentioned properties,
The invention has been completed. That is, the present invention is a plasmid comprising a vector plasmid and a thermostable leucine dehydrogenase gene derived from the chromosomal DNA of Bacillus stearothermophilus and generated by cleavage with the restriction enzyme SalI. To obtain the plasmid of the present invention, for example, Biochem. Biophys acta 72 , 619-629 (1963)
According to the method described in
It can be prepared by digesting DNA with restriction enzymes and then ligating with ligase. The thermostable leucine dehydrogenase gene used in the present invention is a chromosome of Bacillus stearothermophilus.
It is derived from DNA. Specific examples of stearothermophiles include IFO12550, ATCC7953,
There are 7954, 8005, 10194, 12980, NCA1503, etc. In addition, examples of DNA that functions as a vector include plasmid DNA,
Particularly preferred is plasmid pBR322. Furthermore, examples of restriction enzymes include SalI, and examples of ligases include T4 DNA ligase. Examples of the plasmid of the present invention include, for example, a plasmid in which a leucine dehydrogenase gene derived from the chromosomal DNA of Bacillus stearothermophilus is introduced into plasmid pBR322 by the method described above.
One example is pICR1. 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 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 EocR 2 Hind 1 Pst 2 Sal 4 BamH 2 The name of the restriction enzyme is an abbreviation of the restriction enzyme obtained from the following bacterial species. EcoR ; Escherichia coa Hind ; Haemophilus influenzae Pst ; Providencia styuartii Sal ; Streptomyces alpus 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. The plasmid of the present invention can be introduced, for example, into bacteria that grow at room temperature, and transformed bacteria can be obtained by introducing this plasmid. Examples of bacteria that grow at room temperature include bacteria belonging to the genus Escherichia , with Escherichia coli being particularly preferred. A specific example of Esieritia coli is Esieritia coli.
C600 is mentioned. Furthermore, in order to introduce the plasmid of the present invention into, for example, the above-mentioned bacteria that grow at room temperature, for example, the method described in the Journal of Molecular Biology (J.
Mol. Biol.) 53 , 159-162 (1970), the plasmid of the present invention may be brought into contact with the above-mentioned bacterium treated with calcium chloride at a temperature around °C. Examples of bacteria transformed as described above include
An example is Escherichia coli C600-pICR1 strain (Feikoken Bacterial Serial No. 6937) into which plasmid pICR1 has been introduced. This strain is known as Escherichia coli C600 [see Nature 217 , 1110-1114 (1968). ]
It has the same mycological properties except that it has the ability to produce heat-resistant leucine dehydrogenase and is resistant to ampicillin. This strain maintains safety without changing from non-transmissible to transmissible or from non-pathogenic to pathogenic. As described above, the plasmid of the present invention can be used to transform useful microorganisms, such as bacteria that grow at room temperature, to impart heat-resistant leucine dehydrogenase production ability, and the transformed bacteria can be endowed with heat-resistant leucine dehydrogenase production ability. Leucine dehydrogenase can be easily obtained in large quantities, making it very useful. The thermostable leucine dehydrogenase obtained by the present invention is described in Biochemistry, Vol. 54 , p. 634, 4 (1982).
It has the same physical and chemical properties as the heat-stable leucine dehydrogenase described in 2010). Next, the present invention will be specifically explained using examples. The activity of heat-stable leucine dehydrogenase is based on amino acids, nucleic acids [Amino Acid and Nucleic Acid].
27, 84-88 (1973)], that is, the method for measuring leucine dehydrogenase activity described in
In 100 μmole of glycine-KCl-KOH buffer,
Prepare a mixture containing 1.25 μmole of NAD and 10 μmole of L-leucine, add an appropriate amount of crude enzyme extract to the mixture to make a final volume of 0.8 ml, and prepare reduced NAD at 25°C or 55°C. This was done by measuring the increase per unit time as the increase in absorbance at 340 nm. Moreover, % in Examples and Reference Examples indicates volume %. Example 1 (i) Chromosome of Bacillus stearothermophilus
DNA isolation. bacillus stearothermophilus
From the IFO12250 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/, potassium phosphate 2g/, dipotassium phosphate 2g/, magnesium sulfate 0.1
After culturing with shaking at 55°C for 12 hours,
Bacteria were collected by centrifugation. Next, 12 mg of lysozyme was added to 6 ml of saline-EDTA solution (0.15 M NaCl,
Contains 0.1 MEDTA and adjusted to pH 8.0. ),
The collected bacterial cells were 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 then 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 several minutes of immersion in 10 ml of 90% ethanol each, 20 ml of dilute saline-citrate solution (0.015M NaCl,
Prepared as 0.0015M Na ₃ - phosphoric acid. ) and then add concentrated Saline-citrate solution (1.5M NaCl,
Prepared as 0.15M Na ₃ -phosphoric acid. ) and add 2 ml of
A crude DNA solution was prepared. This crude DNA solution was diluted to about 500 μg/ml and treated with liponuclease [R Nase (manufactured by Sigma)].
50μg/ml, 30μ of R NaseA (Shimada)
g/ml and heated at 37°C for 30 minutes. After cooling, add an equal amount of 80% ethanol, perform phenol extraction, collect the extracted DNA by ethanol precipitation, and add the diluted Saline-
Dissolve in 20ml of citrate solution and add the above concentration.
By adding 2 ml of Saline-citrate solution,
A chromosomal DNA extract was prepared. (ii) Preparation of vector plasmid pBR322. Plasmid pBR322 (Betheada Research
Laboratories)
coli C600 strain in 2 liters of L-medium (polypeptone 10
g/, yeast extract 5g/, glycose 1
Adjusted to pH 7.2 with 5 g/g/ of sodium chloride. ) and aerated at 37°C until logarithmic growth phase, then 10ml of chloramphenicol solution (prepared with ethanol to give 3.6mg/ml).
was added and cultured with aeration at 37°C for 15 hours to propagate plasmid pBR322. 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 37°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.
The extracted crude plasmid was collected by precipitation with cold isopropanol, and further TE buffer (0.14M
20mM Tris buffer prepared to PH7.5 containing NaCl, 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 ethanol precipitation by adding twice 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 with ethanol. This precipitate was dissolved in 23.1 ml of 0.02M 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 (containing 0.1mM EDTA.PH
Purified plasmid pBR322 was obtained by dialysis with 0.01M Tris buffer prepared in 7.5. (iii) Creation of plasmid pICR1. 5μg of Bacillus stearothermophilus chromosomal DNA obtained by method (i) and restriction enzyme Sal
(manufactured by Takara Shuzo Co., Ltd.) 20 units, 7mM Mg
_Cl2 , 150mM NaCl 0.2mM EDTA 7mM2−
Mercaptoethanol, PH with 0.01% BSA
Pour into 100μ of 10mA Tris buffer prepared in step 7.5, react at 37℃ for 2.5 hours to digest DNA, heat at 65℃ for 5 minutes to inactivate Sal , digest with cold ethanol, and extract DNA fragments. was collected as a precipitate. Next, the plasmid obtained by method (ii)
Add 3 units of restriction enzyme Sal 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.), 6.6mA
PH containing _MgCl2 , 10mM DTT, 66μM ATP
7.6 in 66mM Tris buffer at 13°C.
Plasmid pICR1 was obtained by reacting for 19 hours and recombining with the digested DNA. Reference Example 1 In order to examine the expressivity of plasmid pICR1 obtained in Example 1, the following transformation treatment was performed. (iv) Transformation First, host bacteria Escherichia coli C600r−
The m-strain was cultured in 50 ml of the above L-medium, collected by centrifugation, suspended in 50 ml of 0.1M MgCl ₂ solution, and further centrifuged to reach a final concentration of 2.5
ml of 0.1M _MgCl2 solution. Esieritia coli obtained in this way
Add 0.1 ml of the mixture containing plasmid pICR1 obtained by method (iii) to 0.2 ml of suspension of C600r-m- strain, and
After processing for 30 minutes at 42°C for 2 minutes.
Next, 3 ml of the above L-medium was added, cultured at 37°C for 1 hour, and ampicillin (15 μg/ml) was added.
Prepared to. ) containing L-agar medium (L-medium 1
Added 15g of agar per serving. ), the resulting colonies were further cultured on L-agar medium containing tetracycline (adjusted to 25 μg/ml), and colonies that did not grow were found. - E. coli C600-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 had more activity than culture. Additionally, 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%. Next, plasmid pICR1 was isolated from this strain in the same manner as in (iii) above, and the properties of plasmid pICR1 were examined by horizontal agarose gel electrophoresis. 2.5mM as a buffer using electrophoresis
Using 89mM Tris buffer containing EDTA-Na and 89mM boric acid and adjusted to pH 8.3, the gel was prepared by adding 0.7% agarose and ethidium bromide adjusted to 0.5mg/ml to this buffer.
In addition, plasmid pICR1 and plasmid pBR322,
Furthermore, lambda phage as a molecular weight marker
DNA Hind (manufactured by Takara Shuzo Co., Ltd.) digested fragments of approximately 1μ each
gDNA was 1 cm wide on the same agarose gel at the same time.
Each gel was run for 3 to 4 hours at a constant applied voltage of 7V. Next, the band was determined using an ultraviolet lamp and the size of plasmid pICR1 was determined, and the molecular weight was approximately 6.
It was Mega Dalton. Also, plasmid
The molecular weight of pBR322 was 2.8 megadaltons. We also used this plasmid pICR1 with the restriction enzyme EcoR
, Hind , BamH , Pst , and Sal under appropriate conditions for each restriction enzyme, and plasmid
pICR1 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 a result, plasmid pICR1 contained the following cleavage-sensitive sites. It was found that it has. Restriction enzyme Number of cleavage sites EcoR 2 Hind 1 Pst 2 Sal 4 BamH 2 In addition, plasmid pICR1 has a Bacillus stearothermophilus at the Sal cleavage site.
It is clear that the chromosomal DNA fragment containing the leucine dehydrogenase gene of strain IFO12250 and plasmid pBR322DNA are bound together.

Claims (1)

[Claims] 1 Chromosomal DNA of Bacillus stearothermophilus
A plasmid consisting of a vector plasmid and a heat-stable leucine dehydrogenase gene that can be produced by cutting with the restriction enzyme SalI.