JPH03244381A - New restriction enzyme and its production - Google Patents

Abstract

PURPOSE:To enable industrial production of a new restriction enzyme CcoI having a wide range of temperature stability, pH stability and specific various enzymatic chemical properties by culturing a microorganism belonging to the genus Clostridium in a nutrient culture medium. CONSTITUTION:Clostridium coccoides B-2 (FERM P-11238) belonging to the genus Clostridium is cultured in a normal culture medium containing glucose as a carbon source, peptone as a nitrogen source, etc. The cultured microbial cells are subsequently collected according to a conventional method and the microbial cells are crushed by a method for ultrasonic treatment, etc. A cell-free extract solution obtained by a method such as centrifugation is then subjected to an ion exchange chromatographic method to afford a restriction enzyme CcoI, having functions to recognize and cleave a specific base sequence in double-stranded deoxyribonucleic acid, substrate specificity and various specified enzymatic chemical properties such as stable pH 5.0-10.0 and stable temperature at 70 deg.C. Thereby, industrial production of the restriction enzyme having the pH and temperature stability can be carried out.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a novel restriction enzyme and a method for producing the same.

[Prior Art] Restriction enzymes are endonucleases that recognize a specific base sequence on deoxyribonucleic acid (DNA) and cleave double strands within or near this sequence.

This restriction enzyme has high substrate specificity and reproducibility in its enzymatic activity, and is also useful for genetic manipulation such as gene isolation, base sequence analysis, protein structure analysis, and mass production of genetic materials. It is an essential enzyme.

It is also a reagent that will have important industrial significance in future elucidation and treatment of genetic diseases, artificial gene conversion, etc.

Restriction enzymes have been isolated from various microorganisms, and about 100 types are known to date, depending on their recognition base sequences and cleavage modes. only been discovered.

[Object of the present 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, bacteria belonging to the genus Clostridium were found to be different from the existing actinomycete Nocardia aero-colonigen.
It was discovered that isosisomers of the restriction enzyme Nael derived from es were produced.

It was discovered that this bacterium has a high productivity of restriction enzymes even when cultured for a short period of time, and that the restriction enzyme Cco I produced by this bacterium is highly stable against heat and pH, leading to the completion of the present invention. . That is, the present invention provides a restriction enzyme CcoI which has a wider range of temperature stability and p)I stability than the conventionally used restriction enzyme Nael, and an industrial production method thereof.

Means for Solving Problem E] The first invention of the present invention relates to a novel restriction enzyme CcoI having the following enzymatic chemical properties.

(a) Action and substrate specificity Restriction enzyme Cco I is ↓ in 2Kibutsu deoxyribonucleic acid ↑ It is an enzyme that recognizes this base sequence and cuts it with an arrow.

To determine the recognition site of the restriction enzyme CcoI, E. coli phage DNA, animal virus Ad2 IINA, E. coli phage φX174 RF DNA, E. coli phage 813 mp18 RF DNA, animal virus SV40 DNA, E. coli plasmid FpBR322D
The number of cleavages of each DNA in NA was determined.

Sono result, input DNA, SV40 DNA, M2OD DNA
1 full site, 13 sites of Ad2 DNA, pBR32
2 DNA was cut at 4 locations.

This is fur, Kusuno data (Gene, Vol, 10
゜P-3? I, 1980), wood enzymes were shown to recognize and cleave 5'-GCCGGC-3'. This is the restriction enzyme Nael (Kes
sler, [1:, and Holtke, Hl, ,
Gene, Vol. 4? , P-1-153, 1988).

So-C1SV40 []NAwo use, control F[[elementary Gc
oL! = When we tried tabultization with the restriction enzyme Nael, there was no change in the cut fragment, and the restriction enzyme GcoI
was confirmed to be an isosisomer of the restriction enzyme Nael.

The νj cutoff point was determined by the following method.

First, extract Escherichia coli phage M13 with restriction enzyme (:col).
The mp+a RF DNA was cut, and the phosphate at the cut end was removed with alkaline phosphatase.

Then, using polynucleotide kinase and [γ-32P]adenosine triphosphate, IINA
Radioactive phosphate was added to the 5' end of the fragment.

This DNA fragment with radioactive phosphate is digested with restriction enzyme B.
Among the newly generated fragments cut with be I, 388b
p (base pair) was separated and fractionated by polyacrylamide gel electrophoresis.

When this fragment was examined for its nucleotide sequence from the 5' end using the Maxam-Kirnoheit method, the restriction enzyme CcoI obtained from these experiments recognized the nucleotide sequence ↓ ↑ and cut it at the position indicated by the arrow. It turned out to be.

(b) Optimal conditions for enzyme reaction and optimal pH for enzyme stability:
The optimum pH of the restriction enzyme Cco I was 8.0.

Stable pH: Restriction enzyme CcoI maintained 100% activity at pH 5.0 to 1000 during treatment at 4°C for 24 hours.

Optimal temperature The optimum temperature for the double restriction enzyme CcoI was 45°C.

100% activity was maintained even after heating for 5 minutes at a stable temperature of 70°C.

Stable salt concentration: Sodium chloride concentration 50mM to loomM
showed stable activity. The optimal salt concentration was 50mM.

(c) Molecular tic: TSKgel G3000S
62,000 by the gel filtration method using W Glass (manufactured by Tosoh).The second invention of the present invention is to culture a restriction enzyme CcoI-producing bacterium belonging to the genus Clostridium in a nutrient medium, and to obtain restriction enzymes from the culture. This method is characterized by collecting the enzyme CcoI.

The microorganism used in the present invention may be any CcoI-producing bacterium belonging to the genus Clostridium, but for example, Clostridium coccoides B- isolated from human feces may be used.
2 (Feiko Kenboku Joyori No. 11238) is suitable.

The wood fungus had the following physiological properties.

1. Obligate anaerobes 2. Obligate spore-forming bacteria +, Durham-positive short rods (0.6-1
．． OXo, 8-3.0141) 3 Motile nitrate production Indole production Gelatin liquefaction Lecithinase activity Lipase activity Hemolytic reaction 4 Succinic acid/acetic acid fermentation Peptone, yeast extract, Fildes' solution, glucose medium 5 Carbon sources Assimilable Arabinose + Xylose + Rhamnose
+ Nluportose + Repose + Glucose + Mannose + Fructose + Galactose + Sucrose + Mannitol +
Cellobiose + lactose +, trehalose
+ melibiose +, raffinose + meletitose +, nluchtol + inositol +, glycerol mannitol +, erythritol Based on the above physiological properties, this bacterium was classified as Clostridium in “The World of Intestinal Bacteria: Isolation and Identification of Anaerobic Bacteria” (written by Tomozoku Mitsuoka).・Clostridium coc
coides).

There are no restrictions on the culture method, and any culture method that can be grown using a commonly used culture method for Clostridium bacteria may be used.

For example, sugars such as glucose and euclose 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 NTn.

Growth is also improved by adding a few percent of blood components.

Extraction and purification of wood enzymes can be carried out according to general restriction enzyme purification methods.

That is, cultured bacterial cells are collected according to a conventional method, and the bacterial cells are disrupted by a method such as ultrasonication.

After disruption, a cell-free extract is obtained by a method such as centrifugation.

This extract is purified by a combination of chromatography methods such as ion exchange chromatography, gel filtration, and affinity chromatography to obtain the restriction enzyme CcoI.

The activity of CcOI was measured as follows.

10mM Tris-HCl, pH 7,5,7mM 2-
Mercaptoethanol, 71M magnesium chloride, 50
The enzyme was added to a reaction system consisting of mM sodium chloride and 1 µg of DNA to bring the total volume to 50 ρ°C, and the reaction was carried out at 37°C for 1 hour.

Add 1$ S[]S (sodium dodecyl sulfate) to the reaction solution.
, 50%G IJ -1= O-L, 0.1$BPB
The reaction was stopped by adding 511β of an enzyme reaction stop solution consisting of (bromophenol blue).

0.51.1g of Escherichia coli phage DNA in the reaction solution
It was separated by tX agarose gel electrophoresis containing 1 m4 of ethidium bromide, and the end point was when the number and amount of /Hend of the υV heat-irradiated DNA stopped changing.

In the above reaction, the enzyme activity that completely cleaves 114% DNA was defined as one unit.

[Example] Clostridium coccoides B-2 (Feikoken Bibori No. 11238) was cultured anaerobically using a steel jar and a 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 1/100 of the main culture solution.

When the bacterial cells were collected by centrifugation, about 12 g of wet bacterial cells were obtained.

Table 1 Casamino acid liquid medium, pH 7.0 (NaOH) 1
1 Casamino acids 4.0g Yeast extract 3.0g Polypeptone
3.0g glucose
10. Og Sodium Sodium
1.58 Cysteine hydrochloric acid 0.4g
Obtained bacterial cells7. Buffer A (10mM) Lis-HCl, pH 7,5, 10mM 2-mercaptoethanol.

7IIM magnesium chloride) 50IIQ was added, the cells were crushed with a French press, and a cell-free extract was obtained by centrifugation.

Purification was performed using the following high performance liquid chromatography (manufactured by Tosoh Corporation).

After passing the obtained cell-free extract through a 0.45 μm filter, buffer B (10 mM) was added to Lis-HCl.

pH 7,5,7 M2-mercaptoethanol, 7m
It was adsorbed onto DEAE-Toyopearlvac 850M (ion exchange chromatography) equilibrated with Mgnesium chloride). Elution was performed with buffer MB having a linear concentration gradient of 0 to 400 mM sodium chloride to obtain a restriction enzyme fraction at a concentration of 300 mM sodium chloride.

The obtained restriction enzyme fraction was dialyzed overnight against buffer B containing 10% (V/V) glycerol. This restriction enzyme fraction is
It was adsorbed on TSKgel DEAE-5PW Glass (ion exchange chromatography) equilibrated with buffer B containing 02 (V/V) glycerol.
Buffer B with a linear sodium chloride concentration gradient of 0 H
A restriction enzyme fraction with a sodium chloride concentration of 120 to 180 mM was obtained.

The obtained restriction enzyme fraction was dialyzed overnight against buffer B containing 10% (V/V) glycerol. This restriction enzyme fraction
TSKgel Heparin-5PW Glass (
Affinity chromatography).

Elution was performed with buffer B with a linear sodium chloride concentration gradient of 0 to 250+*M to obtain a restriction enzyme fraction with a sodium chloride concentration of 40 to 80 mM.

This restriction enzyme fraction was concentrated 18 times and transferred to TSKgel G3000SW Glas in buffer B containing 200+*M sodium chloride and 10X (V/V) glycerol.
s (gel filtration).

A restriction enzyme fraction was obtained at a retention time of 12.0 to 16.0 minutes to obtain a final enzyme preparation (960 units).

Nonspecific NA degrading enzyme and phosphatase were not observed in the gel filtration fraction.

[Effects] According to the present invention described above, a simple industrial production method of a new restriction enzyme having a wider range of pH stability and temperature stability than conventionally used restriction enzymes has become possible.

Claims (3)

[Claims] (1) A novel restriction enzyme Cc having the following enzymatic chemical properties
oI. (a) Action and substrate specificity Recognizes the base sequence 5'-GCCGGC-3'3'-CGGCCG-5' in deoxyribonucleic acid and cleaves it at the position of the arrow (in the formula, G
indicates guanosine and C indicates cytidine). (b) Optimum pH 8.0 (c) Stable pH 5.0 to 10.0 (d) Optimum temperature 45°C (e) Stable temperature 70°C However, by heating for 5 minutes. (f) Stable salt concentration 50 to 100mM, however, based on sodium chloride. (g) Molecular weight: 62,000 However, by gel filtration method. (2) Cultivate restriction enzyme CcoI-producing bacteria belonging to the genus Clostridium in a nutrient medium, and extract the restriction enzyme CcoI from the culture.
A method for producing a restriction enzyme CcoI, which comprises collecting the restriction enzyme CcoI. (3) The production method according to claim 2, wherein the restriction enzyme CcoI-producing bacterium belonging to the genus Clostridium is Clostridium coccoides B-2.