JPH0824571B2 - Preparation of thermostable restriction enzyme - Google Patents

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to a method for preparing a thermostable enzyme.

2. Description of the Related Art Various methods for preparing a restriction enzyme have been reported to date, but all of them require a complicated operation because the separation operation by ion exchange column chromatography or the like is performed twice or more. In addition, the final yield of enzyme was low. As described above, a restriction enzyme that can be separated only from a living organism in a trace amount, even if it is important in genetic engineering, cannot be commercialized because there is no economical separation / preparation method.

An object of the present invention relates to a method for preparing a thermostable restriction enzyme, and a novel preparation capable of obtaining the above enzyme having a high activity purity in a high yield by a simple operation. It is an attempt to provide the law.

The inventors of the present invention have, as a result of diligent research, conducted a heating for a time at which the contaminating endonuclease is substantially inactivated at the lowest temperature at which the heat-resistant restriction enzyme of interest begins to be inactivated. The present inventors have found that a thermostable restriction enzyme having a high activity purity can be prepared in a high yield by a simple operation by treatment, and have completed the present invention. That is, the present invention resides in a method for preparing a thermostable restriction enzyme, which comprises performing heat treatment at the lowest temperature at which the thermostable restriction enzyme begins to deactivate until the contaminating exonuclease is substantially deactivated. Thermostable cyanobacterium Synechococcus elongatus (Synechococc)
heat-resistant restriction enzyme Se derived from us elongatus)
l I is used. The heat treatment is performed at 60 ° C. for 10 minutes. Hereinafter, the present invention will be described in detail. The preparation method of the present invention can be applied to the preparation of various thermostable restriction enzymes such as Bst I and Spl I. In the following, the thermophilic cyanobacterium Synechococcus elongatus (Synechoco) will be described below.
ccus elongatus) (FERM P-12
393) the thermostable restriction enzyme Sel I present in the bacterial cells
The present invention will be described in detail by taking the preparation of This S
el I is a novel enzyme that the present inventors have succeeded in isolating for the first time, and has the enzymatic properties shown in Table 1 below. According to the present invention, in order to obtain the thermostable restriction enzyme Sel I, first, the cultured thermophilic cyanobacteria are collected, ultrasonically disrupted, and centrifuged at 30,000 G for 30 minutes to perform a restriction enzyme-containing soluble protein fraction. To get Then, a Q-Sepharose fast flow gel column is used as the anion exchange column, and the above-mentioned restriction enzyme-containing soluble protein fraction is adsorbed onto the column under 0 mM NaCl conditions.
Next, T with a concentration gradient from 0 mM to 100 mM was added.
E buffer (10 mM Tris-HCl, 1 mM ED
When the adsorbed substance was eluted with TA, pH 8.0, 0 mM-100 mM NaCl), TE with a NaCl concentration of 85 mM was obtained.
The restriction enzyme Sel I is eluted with the buffer. Exonuclease is slightly mixed in this solution. Therefore, as a means of removing this exonuclease, Sel
Heat treatment is performed at the lowest temperature at which I starts to be inactivated until the exonuclease is substantially inactivated. Specifically 6
Heat treatment at 0 ° C. for 10 minutes. In this way, SelI having high activity purity can be obtained. The features of the preparation method of the present invention are described below. Separation operation using a column takes time and effort, and repeating this as in the conventional method increases the loss of the target substance. In the preparation method of the present invention, the separation operation by the column is used only once. As a result, the operation is simplified and the loss of the target restriction enzyme is extremely small. A small amount of exonuclease is mixed in the restriction enzyme sample separated by the anion exchange column. The heat treatment used to remove this characterizes the present invention. That is, the present invention is to obtain a Sel I solution having an activity purity equivalent to that of a purified product by combining separation with an anion exchange column and heat treatment.
The specific heating temperature of 60 ° C. in the preparation of Sel I described above is the lowest temperature at which Sel I starts deactivating. The same applies to exonucleases. Therefore, if the sample is heat-treated at this temperature for an appropriate time, the exonuclease is inactivated, and
The deactivation of Sel I can be minimized. That time is 10 minutes. This means that the inactivation rates of Sel I and exonuclease at 60 ° C are equal,
Assuming that Sel I and exonuclease are present at a ratio of 9: 1, if Sel I is reduced by 1, exonuclease is also reduced by 1, and at this time, Sel I activity is purified. Actual sample (column fractionated)
Among them, Sel I is predominantly present over exonuclease. This is DNA for 1 hour, 4 hours, over night using Sel I sample.
This was confirmed by the fact that no difference was observed in the agarose gel electrophoresis pattern when digested. Experimentally examining the purification effect of Sel I activity by heat, 60
Exonuclease was able to reduce the activity to a negligible level by heat treatment at 10 ° C. for 10 minutes or more. As this qualitative evaluation method, Cutting-Ligation
-The Recutting method was used. As the quantification method, a method was used in which the unit was calculated from the change in OD260 nm generated when λ-DNA was digested with Sel I under the conditions of a reaction solution volume of 50 μL, a reaction temperature of 50 ° C., and a reaction time of 1 hour. The formula is ΔOD 260 nm × 50 μg [U: unit] (1 U: activity just enough to completely digest 1 μg of λDNA in 1 hour). The Sel I sample thus separated had an activity purity equivalent to that of the Sel I sample purified by the three-step column. That is, the DNA cleaved by the Sel I sample isolated by the method we invented could be religated by ligase, and the ligated DNA could also be cleaved by Sel I. This is a general method for qualitatively evaluating the activity purity of restriction enzymes. As described above, regarding the present invention, the heat-resistant cyanobacteria Synechococcus elongatus ( Synecho-c
Occurrence of Sel I from occus elongatus ) has been described as an example. It can also be applied to the preparation of various thermostable restriction enzymes with differences.

INDUSTRIAL APPLICABILITY When Sel I is prepared by the conventional restriction enzyme separation method, wet cell 2
The amount of restriction enzyme obtained from 0 g was about 100 units. It is considered that this method resulted in a large loss of the enzyme because the separation operation using the ion exchange column was repeated three times. On the other hand, according to the preparation of the present invention, since the separation by the column is performed only once, the operation is simple, and the yield is 10 times or more that of the conventional method. Therefore, the preparation method of the present invention is extremely useful as a means for providing a thermostable restriction enzyme that occupies an important position in gene manipulation technology.

[Example] Thermophilic cyanobacteria Synechococcus elongatus
(Synechococcus elongatus)
0.01 g of (FERM P-12393) cells was inoculated into the medium shown in Table 2 below, and cultured at a temperature of 50 ° C. for 168 hours. Obtained Synechococcus elongatus (Synech
ococcus elongatus) wet cells 20 g
Was suspended in 40 ml of TE buffer, ultrasonically disrupted,
Centrifugation was performed at 000 G for 30 minutes, and the supernatant was used as a restriction enzyme-containing soluble protein fraction (1). Next, inner diameter 2
The above fraction (1) was adsorbed on a Q-Sepharose fast flow gel packed in a column of 0 mm and a height of 60 mm. After washing off the non-adsorbed components with TE buffer (0 mM NaCl), the flow rate was 2.5 ml / min for 2 hours, 0 mM.
The adsorbed components were made to flow out with TE buffer having a NaCl concentration gradient from 1 to 200 mM. As a result, the restriction enzyme Sel I was eluted at a NaCl concentration of 60 mM to 80 mM. A small amount of exonuclease was contained in 15 ml of the enzyme solution (2) obtained here. Therefore, heat treatment was performed to inactivate this. Enzyme solution (2) 1
A test tube containing 5 ml was immersed in hot water at 60 ° C. for 10 minutes.
The amount of restriction enzyme in the obtained enzyme solution (3) was 1000 uni.
It was ts or more. However, 1 unit means the amount of enzyme that completely decomposes 1 μg of λDNA in 50 μl of the enzyme reaction solution at 50 ° C. for 1 hour. Also, λDNA, p
BR322, φ × 174 was digested with this enzyme solution (3) and electrophoresed, and the obtained band pattern was examined.
Acinetobacter sp
It coincided with the derived restriction enzyme AccII (optimal pH 7.5, optimal temperature 37 ° C./60 mM NaCl).

Claims (1)

[Claims] 1. A thermophilic cyanobacterium, Synechococcus elongatu.
s) is cultivated, cells are collected, ultrasonically disrupted, and then centrifuged.
Adsorb the obtained protein fraction to an anion exchange column,
Restriction enzyme eluted with TE buffer with NaCl concentration of 85 mM
SelI is heat-treated at 60 ° C. for 10 minutes , which is a thermostable restriction enzyme S having the following enzymatic properties.
High-yield preparation of elI. Action: Cleavage 5'side of the base sequence CGCG of DNA Substrate specificity: CGCG optimum pH: pH 6.5 optimum temperature: 50-55 ° C Metal ion requirement: Mg ²⁺ only required deactivation temperature: 70 ° C, 10 Minute