JPH03216188A - New restriction enzyme and production thereof - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a novel restriction enzyme useful for genetic manipulation, etc., and a method for producing the same. [Prior Art] Restriction enzymes are endonucleases that recognize a specific base sequence on deoxyliponucleic acid ([lNA) and cleave the double strands within or near this sequence by 9J. This enzyme has high substrate specificity and reproducibility in its enzymatic activity, and is useful for genetic manipulation such as single gene extraction, base sequence analysis, protein structure analysis, and mass production of genetic materials. It is essential. It also has important industrial significance for future elucidation and treatment of genetic diseases, artificial gene conversion, etc.

Restriction enzymes have been isolated from various microorganisms, and to date, approximately 100 types are known, depending on the base sequence they recognize and the cleavage mode, but these restriction enzymes account for only half of the theoretically possible types. Only a few have been discovered.

E. Purpose of the Invention The present inventors have conducted research on a large number of restriction enzyme-producing bacteria with the aim of developing effective restriction enzymes. As a result, a bacterium belonging to the genus Bacteroides was found to be able to absorb the existing algae-derived restriction enzyme Spll (Public Notice 63-qa:・16
It was discovered that the company was producing Einsimmer (No. 1).

We discovered that this bacterium can be cultured for a shorter time than algae and has a higher productivity of restriction enzymes, and that the restriction enzyme BvuI produced by this bacterium has a wider stability than the restriction enzyme SplI. The present invention has been completed. That is, the object of the present invention is to provide a restriction enzyme B▼ul that has a wider range of salt stability than the conventionally used restriction enzyme SPII, and a highly productive and simple industrial production method. [A Thousand Steps to Solve the Problems] The first invention of the present invention relates to a novel restriction enzyme BvuI having the following enzymatic chemical properties. (a) Action and Substrate Specificity Restriction enzyme BvuI is an enzyme that recognizes the T 3 base sequence in two-button deoxyliponucleic acid and cleaves it in the direction of the arrow.

To determine the recognition site of the restriction enzyme BvuI, Escherichia coli phage DNA, animal virus Ad2 DNA. E. coli phage φX174 RF DNA. E. coli phage M13 mpl8 RF DNA. Animal 4 Rus SV40 DNA, Escherichia coli plasmid'pBR322
The number of cleavages of each I]NA in DNA was investigated. As a result, λ DNA was cleaved at 1 site, Ad2 0NA at 4 sites, and φXl74 RF DNA at 2 sites, while other DNAs were not cleaved.

This is 7 7-/cus data (Gene, Vol.1
0, P-371.1980), it was shown that the wood enzyme recognizes and cleaves 5-CGTACG-3'. This is a restriction enzyme SplI (Kawamu
ra, M. ．． etal. , Nucleic Acic
js Research, Vol1. +4, P-188
5, 1986). Therefore, when we tried double titration of restriction enzyme B▼uI and restriction enzyme Spl+ using E. coli Farsi DNA, there was no change in the cleavage fragment, confirming that restriction enzyme 13vu1 was an isoschizomer of restriction enzyme SplI. It was done. The cutting point was determined using the following method. First, the Escherichia coli phage DNA was cut with the restriction enzyme BvuI, and the phosphoric acid at the cut end was removed with alkaline phosphatase.

Next, radioactive phosphate was added to the 5° end of the DNA fragment using polynucleotide forceinase and [γ-32P]adenosine triphosphate.

This radioactive phosphate-added DNA fragment is digested with restriction enzyme Ac.
Among the newly generated fragments, two fragments of 150 and 489 bp were separated and fractionated by polyacrylamide gel electrophoresis. When we examined the base sequence from the 5'' end of these two fragments using the Maxam-Gill method, the restriction enzyme BvuI obtained from these experiments recognized the base sequence T and cut it at the position indicated by the arrow. (b) Optimal conditions for enzyme reaction and optimal pH for enzyme stability:
The optimal p++ of the restriction enzyme Bvu I was 8.0. Stable pH: The restriction enzyme BvuI maintained an activity of 10 oz between pH 7.0 and 9.5 when treated at 4°C for 24 hours. Optimal temperature: The optimal temperature for the restriction fermentation test BvuI was 37°C.

Stable temperature: 100$ of activity was maintained even after heating at 50°C for 5 minutes.

Stable salting degree: 100% activity was shown at sodium chloride concentrations of 50 to loO+*M. (c) Molecular weight: TSKgel G3000SW G
It was 47,000 by gel filtration using lass (manufactured by Tosoh Corporation). The second invention of the present invention is characterized in that a restriction enzyme BvuI-producing bacterium belonging to the genus Bacteroides is cultured in a nutrient medium, and restriction enzyme 13vuI is collected from the culture. The microorganism used in the present invention belongs to the genus Bacteroidetes.
Any vuI-producing bacterium may be used, but for example, Bacteroides Purgatus S-1 isolated from human feces.
5 (Bacteroides vulgatus S-
+5 Microtechnical Research Institute No. 11125] is suitable. The wood fungus had the following physiological properties.

l obligate anaerobes 2 spore-forming -, Gram-negative bacilli (0.3-0.5 x 2.5 - 5. OwIl) 3
Growth promotion by bile Generation of hydrogen sulfide gas Motility Pseudo-acid reduction Indole generation Zelatin liquefaction + + 4 Succinic acid/acetic acid fermentation Beptone, yeast extract, 5 carbon sources with Fildes liquid glucose medium Assimilable arabinose +, xylose lipose -, glucose mannose + fructose galactose + sucrose maltose + cellobiose lactose + trehalose melibiose +, raffinose meletitose - sorbitol mannitol - inositol chrycerol Due to the above physiological properties, wood fungi are known as ``the world of intestinal bacteria - anaerobic''. "Isolation and Identification of Bacteria" (written by Tomozoku Mitsuoka), 7 Hekuteke Ides Purgatus (Bacteraides vulgaris)
gatus). There are no restrictions on the culture method, and any culture method that can be grown using the commonly used culture method for bacteria of the genus Pacteroides may be used. For example, sugars such as glucose and sucrose are used as carbon sources, peptone, amino acids, yeast extract, etc. are used as nitrogen sources, and sodium chloride, magnesium chloride, potassium phosphate, etc. are used as other inorganic salts.

Growth is also improved by adding a few percent of blood components. Extraction and purification of this enzyme can be performed by a method according to a general restriction enzyme purification method.

That is, the cultured bacterial cells are collected according to a conventional method, and the bacterial cells are disrupted by a method such as ultrasonication. After disruption, obtain a cell-free extract using methods such as centrifugation. This extract is purified by a combination of chromatography methods such as ion exchange chromatography, gel filtration, and affinity automatography to obtain restriction enzyme 13vuI.

The activity of BvuI was measured as follows.

IOIIM Tris-HCl, pH 7.5. 7 layers M2
- To a reaction system consisting of DNA containing mercabutoethanol, 7+++M magnesium chloride, 50mM sodium kg chloride, enzyme was added to bring the total volume to 50 μl, and 3
The reaction was carried out at 7°C for 1 hour. Add 1$ SOS (sodium dodecyl sulfate) to the reaction solution.
The reaction was stopped by adding an enzyme reaction stop solution consisting of 0.1$BPB (Pro-F: Funol Blue) to the solution.Escherichia coli in the solution Phage λ DNA at 0.5 pg/m
The DNA was separated by electrophoresis on a 1% agarose gel containing 1 ml of ethidium bromite, and the end point was when the number and amount of DNA hands did not change after UV irradiation.

In the above reaction l! Enzyme activity that completely cleaves DNA was defined as 1 unit.

C Example j / Hetateroides pulcatus S-15 (Feikoken Bacterial Serial No. 11125) was cultured using steel shear and casamino acid liquid medium (Table 1). Preculture was carried out at 37°C for 24 hours, and main culture was carried out at 37°C for 48 hours by inoculating the preculture solution in an amount of 1/+00 of the main culture solution. When bacterial cells were collected by far-Q separation, approximately 12 g of wet bacterial cells were obtained. Cysteine Tomb #0. 4 g
Buffer A (10 layer N Tris-HCl,
pH7.5. 10 rmH 2-mercaptoethanol and 7 M magnesium chloride were added to the cod, treated with ultrasound, and centrifuged to obtain a cell-free extract. Purification was performed using the following high performance liquid chromatography (manufactured by Tosoh Corporation). The resulting cell-free extract was passed through a 0.45 m filter, and then DEA equilibrated with buffer B (17.5.7+sM 2-mercabutoethanol, ?+++H magnesium chloride).
E- It was adsorbed on Toyopearl Pack 850M (ion exchange chromatography).

0~. Elution was performed with buffer B with a linear concentration gradient of 40 hM sodium chloride to obtain a restriction enzyme fraction with a concentration of 60-120+sM sodium chloride.

The obtained restriction enzyme fraction was dialyzed overnight against buffer B containing 10% (V/V) glycerol. This dialyzed restriction enzyme fraction was equilibrated with buffer B containing 10$ (V/V) glycerol using TSKgel DEAE-5PW Glass.
After adsorption on (ion exchange chromatography), the enzyme was eluted with #bar B having a linear sodium chloride concentration gradient from O to 250 layer X to obtain a restriction enzyme fraction with a sodium chloride concentration of 100 to 130 mM.

This restriction enzyme fraction was concentrated 3 times and purified with TSKgel G3000SW GlaSs (
Gel filtration) with a retention time of 14.0~l
A restriction enzyme fraction was obtained at 5.0 minutes.

The obtained restriction enzyme fraction was dialyzed overnight against buffer B containing 50% (V/V) glycerol to obtain the final enzyme preparation (1,
200 units). No nonspecific DNA degrading enzymes were observed in the gel filtration fraction. [Effect] According to the present invention described above, the conventionally used restriction enzyme S
A simple industrial production method has become possible for a new restriction enzyme that has a wider range of salt stability than pl+.

Claims (3)

[Claims] (1) A novel restriction enzyme Bv having the following enzymatic chemical properties
uI. (a) Action and substrate specificity It recognizes the base sequence [there is a gene sequence] in double-stranded deoxyribonucleic acid and cleaves it at the position of the arrow (in the formula, A
is adenosine, G is guanosine, T is thymidine, and C is cytidine). (b) Optimum pH 8.0 (c) Stable pH 7.0 to 9.5 (d) Optimum temperature 37°C (e) Stable temperature 50°C However, by heating for 5 minutes. (f) Stable salt concentration 50 to 100mM, however, based on sodium chloride. (g) Molecular weight: 47,000 However, by gel filtration method. (2) A method for producing the restriction enzyme BvuI, which comprises culturing a restriction enzyme BvuI-producing bacterium belonging to the genus Bacteroides in a nutrient medium, and collecting the restriction enzyme BvuI from the culture. (3) The production method according to claim 2, wherein the restriction enzyme BvuI-producing bacterium belonging to the genus Bacteroides is Bacteroides vulgatus S-15.