JPH0514555B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to restriction endonuclease MroI and a method for producing the same.

[Prior art] Type 1 restriction enzymes are deoxyribonucleic acid (DNA)
It is a highly specific restriction endonuclease that recognizes and cleaves a specific base sequence in a chain.

Such type-1 restriction enzymes have attracted attention for their excellent specificity and are widely used in the field of genetic engineering. That is, in genetic manipulation and genetic structure analysis research, etc., in which DNA is cut at a specific position to extract a desired gene and then incorporated into microorganisms such as Escherichia coli, Bacillus subtilis, or yeast, to produce a desired product in large quantities. The first restriction enzyme plays an extremely important role.

On the other hand, since there are various types of DNA that serve as substrates for the above restriction enzymes due to diversity in their base sequences, etc., various types of restriction enzymes with different specificities are required, and a variety of high-quality restriction enzymes are required. It is desired that enzymes be supplied at low cost. To date, various types of restriction enzymes have been isolated from protists such as bacteria, and about 100 types are already known, and their enzymatic chemical properties have been clarified.

[Problems to be Solved by the Invention] An object of the present invention is to provide a novel restriction endonuclease with extremely high specificity that recognizes and cleaves a specific base sequence in a DNA strand, and a method for producing the same. It's about doing.

[Means for Solving the Problems] The first restriction enzyme of the present invention is a restriction endonuclease having the following physical and chemical properties.

(a) Action and substrate specificity Base sequence in double-stranded deoxyribonucleic acid: 5'-T↓ CCGGA-3'3'-AGGCC↑T-5' (where A is adenosine, G is guanosine, and T is thymidine) , C represent cytidine) and cut at the position of the above arrow.

(b) Optimum PH 6.5-8.5 (c) Stable PH 6.0-9.0 (d) Optimum temperature 25-35°C In addition, the present invention cultivates restriction endonuclease MroI-producing bacteria belonging to the genus Micrococcus in a nutrient medium; This is a method for producing a restriction endonuclease, the gist of which is to collect restriction endonuclease MroI having the following physical and chemical properties from a substance.

(a) Action and substrate specificity Base sequence in double-stranded deoxyribonucleic acid: 5'-T↓ CCGGA-3'3'-AGGCC↑T-5' (where A is adelisine, G is guanosine, and T is thymidine) , C represent cytidine) and cut at the position of the above arrow.

(b) Optimum PH 6.5 to 8.5 (c) Stable PH 6.0 to 9.0 (d) Optimal temperature 25 to 35°C [Function] The present inventors used Micrococcus
While searching for restriction enzymes (restriction endonucleases) as part of biochemical research on bacteria of the genus Micrococcus roseus S.
(Micrococcus roseus S) was found to produce a novel restriction endonuclease MroI.
The present invention has now been completed. The above-mentioned bacterial strain has been deposited with the Institute of Microbiology, Agency of Industrial Science and Technology (accession number: Microbiological Research Institute No. 8996).

It has been well known that two or more types of restriction endocleases with different specificities may be produced from the same microorganism. It is also well known that the above-mentioned situation is a major hindrance to industrialization because it is difficult to separate two or more types of enzymes from each other. Therefore, in the industrial production of restriction endonuclease-producing microorganisms, the current situation is that it is desired to develop microorganisms that are highly productive and produce a single enzyme.

In contrast, the restriction endonuclease of the present invention
Since MroI (hereinafter sometimes referred to as the present enzyme) is produced singly by the above-mentioned bacteria, the strain producing the present enzyme can be said to be the optimal strain for industrial production. In addition, this enzyme has novel specificities as described below, so it is extremely effective as a biochemical reagent for studying the structure and function of genetic DNA, as well as for analysis of genetic immunity and genetic recombination research. It provides important guidelines.

As the microorganism (the enzyme-producing bacteria) used in the present invention, any enzyme-producing bacteria belonging to the genus Micrococcus can be used, but the aforementioned Micrococcus roseus S is most suitable.

On the other hand, as a method for culturing the above-mentioned producing bacteria, the most common method currently in use may be adopted. For example, in a medium optionally containing polypeptone, meat extract, amino acids, casein decomposition products, yeast extract, malt extract, etc. as a nitrogen source; glucose, glycerol, etc. as a carbon source; phosphate, sulfate, magnesium salt, etc. as an inorganic salt, A method of inoculating the above-mentioned production bacteria and culturing with aeration at a temperature of about 25 to 35° C. using a jar fermenta or the like can be mentioned. Note that the pH may drop during culturing, but in this case, it is of course possible to culture while neutralizing with alkali.

Then, bacteria are collected from the logarithmic phase to the early stationary phase,
After washing the bacterial cells with an appropriate buffer, a cell-free extract is obtained by sonicating the cells and centrifuging them.

The obtained cell-free extract was subjected to streptomycin sulfate treatment, polyethyleneimine treatment, or ammonium fractionation, and after dialysis, ion exchange chromatography using phosphocellulose or DEAE cellulose, or affinity chromatography using heparin-cepharose was performed. This enzyme can be purified by E or Cephacryl S-200 gel filtration method.

The enzyme thus obtained has the following physicochemical properties.

(1) Action and substrate specificity The action and substrate specificity of this enzyme were investigated according to the following procedure.

First, plasmids are used as substrates for enzymatic reactions.
pBR322, adenovirus-2DNA and λ-
An enzymatic reaction was carried out using DNA, and the generated site after the enzymatic reaction was subjected to agarose gel electrophoresis.

The results are shown in FIG. 1, and an agarose gel electropherogram of DNA fragmented by the action of this enzyme was obtained. As a result, this enzyme has λ-
24 DNA locations, 8 adenovirus-2 DNA locations
It was found that pBR322DNA was cut at one location.

Next, we clarified the base sequence recognized by this enzyme and the cleavage mode.

First, 50 μg of plasmid pBR322 was cut with this enzyme, and the cut DNA fragment was treated with alkaline phosphatase to remove the phosphoric acid at the end of the DNA fragment. Then (γ− ³² P)−
ATP was treated with polynucleotide kinase and the 5' end was labeled with radioactive phosphate. Thereafter, using a DNA fragment labeled with radioactive phosphoric acid, the base sequence near the site cleaved by this enzyme was determined by the Nuxam-Gilbert method.

This result is schematically shown in Figure 2. As is clear from the results in Figure 2, this enzyme is 5'-
Recognize the nucleotide sequence of TCCGGA-3' and mark the arrow
It was found that it was necessary to cut at the Y position (see Figure 2).

Next, the cutting situation shown in FIG. 2 was confirmed according to the following procedure.

First, λ-DNA was completely digested with this enzyme. After treating this with alkaline phosphatase to remove the terminal phosphate of the DNA fragment, polynucleotide kinase and γ-
The ends of the DNA fragments were labeled with radioactive phosphate using ³² P-ATP. After that, it is digested to mononucleotides with P1 nuclease, and PEI-
Analysis was performed using a cellulose thin layer chromatography plate. As a result, the labeled 5'-mononucleotide was found to be cytosine, indicating that it was cleaved at the positions indicated by arrows X and Y in FIG.

Although the present invention has the above-mentioned action and substrate specificity, it was recognized that it also has the following physical and chemical properties.

(2) Optimal PH PH6.5-8.5 (3) Stable PH PH6.0-9.0 (4) Optimal temperature 25-35℃ (activity decreases at 40℃) (5) Salt concentration NaCl, KCl concentration The activity of this enzyme is maintained up to 300mM, but is inhibited above that level.

(6) The metal salt MgCl is essential, and a concentration of 10mM is optimal, but it is inhibited at concentrations above 20mM. or
It is also activated by MnCl ₂ , but to a lesser extent than Mg ²⁺ .

[Example] Using Micrococcus roseus S as the enzyme-producing bacterium, polypeptone (manufactured by Daigo Nutritional Chemical Co., Ltd.) 1
%, meat extract (manufactured by Kyokuto Co., Ltd.) 1%, NaCl 0.5%, and enzyme extract (manufactured by Daigo Nutrient Chemical Co., Ltd.) 0.3%. Erlenmeyer flask), bacterial weight (wet bacterial cells)
7.23g of this enzyme-producing bacteria was obtained. The obtained bacterial cells were suspended in 40 ml of phosphate buffer (PH7.0), and 200μ
g/ml lysozyme - 1mM _NaN3 (25μg/ml
me) Add to RMSF, react at 0℃ for 30 minutes, then add salt to make 0.4M NaCl, and perform ultrasonication (200W, 20 minutes).
A supernatant was obtained by ultracentrifugation at 27,000 rpm for 60 minutes.
Add 10mM phosphate buffer to the obtained supernatant and make approximately
It was diluted to 0.2M NaCl. The diluted enzyme solution was adsorbed onto a phosphocellulose (Watmann P11) column (1.5 x 18 cm), washed with 7 volumes of buffer, and then eluted with a 0.2 to 1.0 M NaCl gradient.

The results are shown in FIG. 3, and as is clear from FIG. 3, enzyme activity was detected in the elution fraction at a NaCl concentration of around 0.34 to 0.44M.

Next, the eluted enzyme solution was placed in a dialysis tube and concentrated against polyethylene glycol 6000 powder, and further dialyzed against 10mM phosphate buffer (containing 50% glycerose) to obtain a 0.6me enzyme solution. When the enzyme activity of the obtained enzyme solution was measured, it was found that approximately
It was 3000 units. The enzyme activity was measured according to the method described below. That is, 10mM Tris-HCl buffer (PH7.5), 10mM _MgCl2 , 100mM NaCl, 7
In a reaction solution containing mM mercaptoethanol and 0.01% BSA, 1.0 μg of λ-DNA was reacted at 35° C. for 60 minutes, and the amount of enzyme that completely degraded DNA was defined as 1 enzyme unit.

Approximately 3,000 units of enzyme solution was obtained from 7.23 g of bacterial cells in this manner, and the obtained enzyme solution was completely free of any non-specific DNA degrading enzymes such as phosphatase.

[Effects of the Invention] As described above, according to the present invention, a novel restriction endonuclease with extremely high specificity that recognizes and cleaves a specific base sequence in a DNA strand can be obtained, and The production method also has high industrial value.

[Brief explanation of the drawing]

Figure 1 is an agarose gel electrophoresis diagram when this enzyme is applied to various substrates, Figure 2 is a schematic diagram showing the base sequence before and after cleavage when this enzyme is applied to plasmid pBR322, and Figure 3 The figure is a graph showing the results when this enzyme solution was eluted by phosphocellulose column chromatography.

Claims (1)

[Scope of Claims] Restriction endonuclease MroI characterized by having the following physical and chemical properties. (a) Action and substrate specificity Base sequence in double-stranded deoxyribonucleic acid: 5'-T↓ CCGGA-3'3'-AGGCC↑T-5' (where A is adenosine, G is guanosine, and T is thymidine) , C represent cytidine) and cut at the position of the above arrow. (b) Optimum PH 6.5-8.5 (c) Stable PH 6.0-9.0 (d) Optimum temperature 25-35℃ 2. Cultivate restriction endonuclease MroI-producing bacteria belonging to the genus Micrococcus in a nutrient medium, and extract the following from the culture. A method for producing restriction endonuclease MroI, which comprises collecting restriction endonuclease MroI having physicochemical properties. (a) Action and substrate specificity Base sequence in double-stranded deoxyribonucleic acid: 5'-T↓ CCGGA-3'3'-AGGCC↑T-5' (where A is adelisine, G is guanosine, and T is thymidine) , C represent cytidine) and cut at the position of the above arrow. (b) Optimum PH 6.5-8.5 (c) Stable PH 6.0-9.0 (d) Optimum temperature 25-35°C 3. Claim No. 1 in which the restriction endonuclease MroI-producing bacterium belonging to the genus Micrococcus is Micrococcus roseus S. A method for producing the restriction endonuclease MroI according to item 2.