JPH0514554B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] This invention relates to type restriction enzymes and methods for producing the same, and particularly to double-stranded deoxyribonucleic acid (double-stranded DNA).
base sequence within the molecule. 5'...G-↓G-A-T-C-C...3'3'...C-C-T-A-G-↑G...5' (However, in the formula, A represents adenosine, This invention relates to Gcein, a type of restriction enzyme that recognizes Gcein (G represents guanosine, T represents thymidine, and C represents cytidine (the same applies hereinafter)) and cleaves it at the position indicated by the arrow, and a method for producing the same. [Prior art] Restriction enzymes are strain-specific endonucleases that cut and eliminate foreign DNA that invades cells as viral DNA or naked DNA. I recognize it, but
Types that cut DNA nonspecifically, types that recognize a specific base sequence of double-stranded DNA and cut it at that specific position, and types that cut DNA at a specific position that is different from the recognized base sequence. A type that exhibits characteristics intermediate between the above-mentioned types and the other types is known. Among these, type restriction enzymes can be used in combination with several restriction enzymes with different specificities to systematically cut long DNA molecules and obtain uniform DNA fragments containing the target gene. . For this reason, these types of restriction enzymes are useful for recombinant
An enzyme used in DNA technology to extract DNA fragments that contain the necessary genetic information and to introduce the extracted DNA fragments into the site where the genetic information is expressed in an active form, as well as for gene analysis. It is an extremely important enzyme in genetic engineering-related technology. By the way, in the field of research on type restriction enzymes, many enzymes with specificity in recognition base sequences and cleavage positions have been isolated to date, and their oxygen chemical properties have also been clarified.
Recognizes the base sequence 5'...G-↓G-A-T-C-C...3'3'...C-C-T-A-G-↑G...5' in the DNA molecule. Regarding the type of restriction enzyme that cuts this at the position of the arrow, for example, Bacillus amyloliquefaciens H (Bacillus amyloliquefaciens H)
BamH obtained from amyloliquefaciensH)
{Roberts, RJ, Wilson, GA and Young, F.
E., Nature, 256 , 82-84 (1977), and
Geoge, J. and Cnirikjian, J.G., Proc. Natl.
AcadSci.USA, 79, 2432-2436 (1982)} and this isoschizomer of BamH.
Ali obtained from Acetobacter liquefaciens IAM 1834 (Japanese Unexamined Patent Publication No. 175881/1981) has been reported. However, the DNA that is the subject of research in recombinant DNA technology and the base sequences of that DNA are extremely diverse, which requires the development and production of restriction enzyme types with various specificities, as well as the need for dilution. It has excellent enzymatic chemical properties such as high stability against salt concentration, wide stability range against salt concentration, and high thermal stability, and is cheaper and easier to use in terms of ease of production. There is a need for the development of type restriction enzymes. [Problems to be solved by the invention] Therefore, as part of physiological and chemical research on acetic acid bacteria, the present inventors focused on the fact that certain types of acetic acid bacteria produce restriction enzymes, and investigated the enzyme chemical properties and structure. As a result of extensive research into type restriction enzymes that have excellent properties such as ease of use, we have discovered that certain bacteria belonging to the genus Gluconobacter have been found to have a nucleotide sequence of 5'...G- in the molecule of double-stranded DNA. ↓G-A-T-C-C...3'3'...C-C-T-A-G-↑G...5' The present invention was achieved by discovering that it was possible to produce a compound (named .) and establishing a method for its purification. Therefore, an object of the present invention is to provide a Gcein-type restriction enzyme that has excellent properties in terms of stability against dilution, stability range against salt concentration, oxygen chemical properties such as thermal stability, and ease of production. Another object of the present invention is to provide a method for producing this type of restriction enzyme Gcein. [Means for Solving the Problems] That is, the present invention is directed to culturing a restriction enzyme Gcein-producing bacterium belonging to the genus Gluconobacter, and a restriction enzyme collected from the resulting culture, which is a double-stranded deoxyribonin. Recognizes the base sequence 5'...G-G-↓G-T-C-C...3'3'...C-C-T-A-G-↑G...5' in the nucleic acid molecule. Gcein is a type of restriction enzyme that cleaves this at the position indicated by the arrow, and is a type of restriction enzyme that is used to collect the above-mentioned restriction enzyme Gcein from the obtained bacterial cells by culturing Gcein-producing bacteria belonging to the genus Gluconobacter. This is the manufacturing method. The microorganisms used in the present invention include restriction enzymes belonging to the genus Gluconobacter, which is an acetic acid bacterium.
All Gcein-producing bacteria can be used, but for example, Gluconobacter cerinus, which is preserved by the Fermentation Research Institute and can be freely distributed to third parties.
One can mention FO 3260. This strain was developed by the present inventors as Gluconobacter industrius FO.
The bacterium was deposited as 3260 at the Fermentation Research Institute, but later it was classified as Gluconobacter cerinus along with other strains of Gluconobacter bacteria. {Y.Yamada ana M.Akita, J.Gen.Appl.
Microbiol., 30 , 115-126 (1984)}. In the present invention, the above-mentioned restriction enzyme Gcein-producing bacteria can be cultured by a known method known as a culture method for acetic acid bacteria. In other words, carbon sources such as glucose, glycerol, and ethanol, nitrogen sources such as ammonium nitrate and ammonium chloride, various phosphates, inorganic salts such as magnesium sulfate and calcium chloride, and natural organic nutrients such as yeast extract, malt extract, and peptone. Usually, under aerobic conditions such as shaking culture, the pH of the medium is 5 to 7, the culture temperature is 25 to 30°C, and the culture time is 24 to 48 hours, so that tooth growth reaches the early stationary phase. Do this until you reach . In addition, as a method for separating and purifying the target restriction enzyme Gcein from the culture obtained after culturing,
A conventionally known method can be employed, and for example, the following method can be used. First, the bacterial cells collected by centrifuging the culture solution are washed with an appropriate buffer such as tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), and then sonicated to disrupt the cells. Centrifuge to remove the precipitate and obtain a cell-free extract. Next, this cell-free extract was treated with sloreptomycin sulfate and further fractionated using ammonium sulfate, followed by heparin cepharose CL-6B, diethylaminoethyl (DEAE) cepharose CL-6B,
The type restriction enzyme Gceil of the present invention can be obtained by combining methods such as affinity chromatography such as Cephadex G-200, ion exchange chromatography, and gel filtration.
Purification is carried out. The type restriction enzyme Gceil of the present invention produced in this way has the base sequence 5'...G-↓G-A-T-C-C...3'3'... in the molecule of double-stranded DNA. It is a type of restriction enzyme that recognizes C-C-T-A-G-↑G...5' and cleaves it at the position indicated by the arrow. Acetobacter liquefaciens AM
It is an isoschizomer of Ali obtained from 1834, and its stability against dilution,
The above-mentioned BamH and
Demonstrates better characteristics than Ali. [Examples] Hereinafter, the present invention will be explained in detail based on Examples and Test Examples. (1) Example (Production of Gcein-type restriction enzyme) Gluconobacter selinus
cerinus) FO 3260 with potato extract 20% by weight,
Yeast extract 1.55% by weight, glycerol 1.5% by weight,
Glucose 0.5% by weight, peptone 1.5% by weight, agar
5 ml of a slant medium containing 2.0% by weight and 0.7% by weight of calcium carbonate was inoculated and cultured at 30°C for 48 hours.
Next, the obtained culture was mixed with 1.5% by weight of glycerol.
It was added to a nutrient medium 10 with a pH of 6.8 consisting of 0.5% by weight of glucose, 0.5% by weight of peptone, 0.5% by weight of yeast extract, and 0.1% by weight of malt extract, and cultured for an additional 48 hours at 30°C while shaking on a rotary shaker. Bacterial cells in the early stationary phase were collected using a centrifuge. 32g of the obtained bacterial cells were mixed with 10mM Tris-HCl (PH
7.5) and in the presence of 10mM 2-mercaptoethanol, the bacterial cells were disrupted by sonication at 180W for 6 minutes, and after centrifugation, NaCl was added to the supernatant solution to make it 100mM, and finally to 1% by weight. Streptomycin was added to remove nucleic acids, and the mixture was allowed to stand for 30 minutes, followed by centrifugation to remove the precipitate and obtain a supernatant (purification step 1). Ammonium sulfate was added to the supernatant thus obtained while stirring until it became a 35% solution by weight, and the precipitate was removed by centrifugation.
Ammonium sulfate was added to the supernatant to make a 60% solution by weight, and the mixture was allowed to stand overnight. After standing overnight, add buffer A (50mM) to the centrifuged precipitate.
Tris-HCl (PH7.5), 7mM magnesium chloride, and 7mM 2-mercaptoethanol were added to dissolve and dialyzed overnight (purification step 2). At night, 40 ml of the obtained dialysate was equilibrated with buffer A containing 100 mM NaCl, and then heparin-sepharose was added.
Chromatography column packed with CL-6B (diameter 1.3
cm, height 8 cm) and pre-adsorbed with 100mM NaCl.
After washing with 10 columns of buffer A containing 100
The type restriction enzyme Gceinl was eluted with a linear gradient in buffer A containing ~600 mM NaCl. The target type restriction enzyme Gcein is approximately 250-350mM NaCl
(purification step 3). Furthermore, the obtained solution containing the restriction enzyme Gcein was dialyzed overnight with buffer A, and 30.5 ml of the obtained dialysate was transferred to a chromatography column packed with EAE Sepharose CL-6B equilibrated with buffer A in advance. (diameter 1.3 cm, height 8 cm), buffer solution A1
After washing for 0 column minutes, the restriction enzyme Gcein was eluted with a linear gradient of buffer A containing 0 to 500 mM NaCl.
It was eluted into a solution of NaCl concentration (purification step 4). Next, the resulting solution of the restriction enzyme Gcein-containing fraction was placed in a dialysis tube, soaked in polyethylene glycol 6000, and concentrated. Next, 100m in advance
The enzyme solution was applied to a gel filtration column (diameter 1.3 cm, height 25 cm) packed with Sephadex G-200 equilibrated with buffer A containing 100 mM NaCl.
Type restriction enzyme in buffer A1 column containing NaCl
The Gcein-containing fraction was eluted. The solution of the fraction containing this type of restriction enzyme Gcein was placed in a dialysis tube again, soaked in polyethylene glycol 6000, concentrated, and diluted with buffer B {10mM Tris-HCl (PH7.5), 7mM
It was purified by overnight dialysis against M magnesium chloride, 7 mM 2-mercaptoethanol, and 50% V/V glycerol (purification step 5). In addition, each operation of all the above purification steps 1-5 was performed at the temperature of 4 degreeC or less. Type restriction enzyme Gcein thus obtained
Use 5-10μ of 10mM Tris-HCl (PH
7.5), 7mM magnesium chloride, 7mM 2-
A total of 50μ of a mixture consisting of mercaptoethanol and 1μg of λDNA was prepared and reacted at 37°C for 1 hour. To stop the reaction, 5μ of a solution of 1% by weight sodium dotecyl sulfate (SDS) and 10mM ethylenediaminetetraacetic acid (EDTA) was added. Added. The resulting reaction mixture was subjected to agarose gel electrophoresis.
Enzyme activity was examined based on the presence or absence of λDNA cleavage.
Regarding the enzyme activity of this type of restriction enzyme Gcein,
The amount of enzyme that completely cleaves 1 μg of λDNA in 1 hour was defined as 1 unit. For agarose gel electrophoresis, 1% by weight agarose, 89mM Tris-boric acid (PH8.3), 2.5m
Using a plate gel consisting of M EDTA and 500 μg/ethidium bromide, electrophoresis buffer {89 mM Tris-boric acid (PH8.3), 2.5 mM EDTA
and 500 μg/ethidium bromide} at a constant voltage of 5.5 V per cm for about 4 hours, and then the cleavage of the λDNA was confirmed under a 302 nm UV lamp. Type restriction enzyme Gcein obtained as above
Other cleavage patterns did not change even after 24 hours of reaction, and other activities such as non-specific DNase were not included. The degree of purification and total activity in the above purification steps 1 to 5 were as shown in Table 1. (2) Test example (confirmation of enzymatic chemical properties of type restriction enzyme Gcein) Double digestion
Test with known type restriction enzyme BamH
pBR322DNA, 1μg of E. coli plasmid pBR322DNA, 10mM Tris
- mixture of HCl (PH7.5), 7mM 2-mercaptoethanol and 7mM magnesium chloride
No oxygen added in 30μ medium at 37℃ for 2 hours.
The reaction was carried out with the addition of only the type restriction enzyme BamH, only the purified type restriction enzyme Gcein, and the addition of a mixture of type restriction enzymes BamH and Gcein, and the type restriction enzyme Gcein was recognized by the same agarose gel electrophoresis method as above. The base sequence and cleavage position were investigated. The results showed that both enzymes recognized and cut pBR322DNA at exactly the same position.
From this, the type restriction enzyme Gcein has a base sequence within the molecule of double-stranded deoxyribonucleic acid. It recognizes and cleaves -G-G...5', and was thought to have an isocismal relationship with BamH. Determination of recognition cleavage site pBR322DNA was cut with the type restriction enzyme Gcein, treated with phoshatase to remove the phosphate at the cut end, and the cut end was labeled with ^32P using [γ- ³² P]ATP. This was treated with the type restriction enzyme Hind, and the resulting fragment (approximately 346 base pairs) was sequenced using the Maxam-Gilbert method. As a result, as shown in Figure 1,
The base sequence from that cut was found to be 5'-G-AT-C-C-A-C-A-G... Furthermore, as shown in FIG. 2, the terminal base was found to be G. From the above results, the type restriction enzyme Gcein is
Base sequence in the molecule of double-stranded deoxyribonucleic acid 5'...G-↓G-A-T-C-C...3'3'...C-C-T-A-G-↑G...5 ′ and cut it at the position of the arrow. Number of cleavage sites for various substrate DNAs E. coli phage λDNA, E.
coli plasmid pBR322DNA, E.Coli phage φ×174RF DNA and animal virus SV 40
Using DNA, the type restriction enzyme Gcein of the present invention is allowed to act on these substrate DNAs, and after the enzyme reaction is completed, the product is treated with agarose gel electrophoresis to determine the type restriction enzyme Gcein of the present invention for each of the above substrate DNAs. The effect and substrate specificity of the compound were investigated. The results were as shown in Table 2. Optimal PH and PH stability range 1μg of λDNA, 7mM magnesium chloride and 7mM 2-mercaptoethanol for 10m
Each reaction solution was prepared by adding M sodium acetate (PH4), piperazine hydrochloride (PH5), histidine hydrochloride (PH6), imidazole hydrochloride (PH7), Tris-HCl (PH8) or ethanolamine (PH9) Using 50μ, the optimum PH and PH stability range of the type restriction enzyme Gcein were investigated by a conventional method. The result is that the optimal PH is 7.5 to 9.5, and the PH
It was found that the stability range was 5.5 to 10, which was extremely wide. Optimal temperature and temperature stability 1μg of λDNA, 10mM Tris-HCl (PH7.5),
The optimal temperature and temperature stability of the restriction enzyme Gcein were investigated by a conventional method using 50 µ of a standard reaction solution consisting of 7 mM magnesium chloride and 7 mM 2-mercaptoethanol. The results showed that the optimal temperature was 35 to 40°C, and the temperature stability was up to 50°C when kept warm for 5 minutes, indicating that it was extremely stable. Salt requirement and optimal salt concentration By changing the concentration of the reaction solution of the above standard reaction system,
Add NaCl and type restriction enzyme using the usual method.
The salt requirement and optimal salt concentration of Gcein were investigated.
The results showed that the optimal salt concentration was 0 to 150mM NaCl, and since the type restriction enzyme Gcein reacted even at a salt concentration of 0, no particular salt requirement was observed, and the enzyme was found to be easy to use. Stability against dilution The amount of enzyme added to the above standard reaction system (the amount of enzyme that completely cleaves 1 μg of λ DNA in a 10 minute reaction at 37°C) was reduced to 1/5, and the stability was investigated by reacting for 60 to 120 minutes. . The results showed that λDNA was completely cleaved in all reaction systems, and it was found to be extremely stable against dilution. Requirement for divalent metal salt The metal salt magnesium chloride in the standard reaction system was reacted in a reaction system in which 1mM EDTA was added instead of calcium chloride, manganese chloride, cupric chloride, and cobalt chloride. The requirements for valent metal salts were investigated. The results showed some reaction only in the presence of manganese chloride. Molecular weight Molecular weight was measured by gel filtration method. Result is,
It was 25000±5000. Comparison with known restriction enzymes Al and BamH
A comparison with Ali and BamH is as shown in Table 3.

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[Table] [Effects of the Invention] According to the present invention, enzyme chemical properties such as stability against dilution, optimal salt concentration range, and thermal stability are excellent, production is relatively simple, and double-stranded Recognizes the base sequence 5'...G-G-↓G-T-C-C...3'3'...C-C-T-A-G-↑G...5' in the molecule of deoxyribonucleic acid It is possible to provide a restriction enzyme, Gcein, which cleaves Gcein at the position indicated by the arrow, and a method for producing the same.

[Brief explanation of the drawing]

Figure 1 is a photograph showing polyacrylamide gel electrophoresis of pBR322 DNA treated with the type restriction enzyme Gcein of the present invention, and Figure 2 is a PEI-cellulose thin layer chromatography of pBR322 DNA treated with the type restriction enzyme Gcein of the present invention. This is a photo showing E.

Claims (1)

[Claims] 1. Restriction enzyme belonging to the genus Gluconobacter
It is a restriction enzyme collected from the culture obtained by culturing GceinI-producing bacteria, and has the base sequence in the molecule of double-stranded deoxyribonucleic acid 5'...G-↓G-A-T-C-C... 3'3'...C-C-T-A-G-↑G...5' (However, in the formula, A represents adenosine, G represents guanosine, T represents thymidine, and C represents cytidine.) Restriction enzyme GceinI recognizes and cuts it at the position indicated by the arrow. 2 Type restriction enzyme belonging to the genus Gluconobacter
1. A method for producing a restriction enzyme, which comprises culturing a GceinI-producing bacterium and collecting the above-mentioned restriction enzyme Gcein from the resulting culture.