GB2183657A - New restriction enzyme and process for producing the same - Google Patents

Abstract

The restriction enzyme is capable of recognizing the following base sequence in double-stranded DNA and cleaving the DNA chain at the positions marked with arrows: <IMAGE> wherein A is adenosine, G is guanosine,T is thymidine and C is cytidine. The optimum pH for the enzyme is approximately 8.5, the stable pH range is 6.0 to 9.5 and the optimum working temperature is 60 to 65 DEG C. A process for producing this enzyme comprises growing a microorganism of the genus Acinetobacter and which is capable of producing the enzyme in a culture medium and subsequently collecting the enzyme thus formed from the culture broth.

Description

SPECIFICATION New restriction enzyme and process for producing the same This invention relates to a new restriction enzyme and to a process for producing the same. More particularly, it relates to a new restriction enzyme produced by a bacterium belonging to the genus Acinetobacter, and to a process for producing the same.

Restriction enzymes are endonucleases that are capable of recognizing a specific sequence of bases on a deoxyribonucleic acid (DNA) molecule and of cleaving the double-stranded DNA chain at specific sities. As a result of recent progress in molecular genetics, biochemistry and related sciences, it is now clear that DNA is the carrier of genetic information, and restriction endonucleases have been extensively used for various purposes such as clarification of genetic diseases, mass production of genetic materials based on genetic engineering, etc.

About 100 kinds of endonucleases have so far been isolated from many microorganisms, each being identified by the specific base sequence it recognizes and by the cleavage pattern it exhibits. A larger number of restriction enzymes with diversified enzymatic characteristics are still necessary for successful gene manipulation.

To the best of our knowledge, however, no restriction enzyme has been discovered, which is capable of recognizing the base sequence in a double-stranded DNA molecule as shown below and of cleaving it at the arrow-marked sites, 5'--iiC C G G A--3' 3'-AG G CCtT-5' wherein A, G, T and C represent adenosine, guanosine, thymidine and cytidine, respectively.

Hence the object of this invention is to provide a new restriction enzyme that is capable of recognizing the base sequence in a double-stranded DNA molecule as shown above and of cleaving it at the arrow-marked sites.

One aspect of this invention relates to a new restriction enzyme having the following properties: (a) Action and substrate specificity: Recognizes the base sequence in a double-stranded DNA molecule as shown below, and cleaves it at the arrow-marked sites, 5'--T1C C G G A--3' 3'-AG G C CtT5' wherein A, G, T and C represent adenosine, guanosine, thymidine and cytidine, respectively.

(b) Optimal pH : approximately 8.5 (c) Stable pH range: 6.0 to 9.5 (d) Optimal working temperature : 60 to 65"C The new restriction enzyme of this invention cleaves double stranded A-DNA at 24 sites, pBR322 DNA at one site, and adenovirus type-2 DNA at eight sites, but exhibits no cleavage action upon vex174 RF DNA and SV40 DNA.

Another aspect of this invention relates to a process for producing said new restriction enzyme, which comprises growing a microorganism belonging to the genus Acinetobacter and capable of producing said restriction enzyme in a culture medium and collecting the enzyme thus formed from the culture liquid.

Any strains of Acinetobacter capable of producing said new restriction enzyme can be used for the purpose of this invention. A typical example is Acinetobacter calcoaceticus, which has been deposited at the Fermentation Research Institute, Agency of Industrial Science and Technology, under FERM BP-935. The bacteriological properties of this strain are described on page 437 of "Bergey's Mannual of Determinative Bacteriology" (8th edition).

Any nutrients that can be assimilated by the strains capable of producing said new restriction enzyme may be added to the culture medium. Glucose and others may be used as a carbon source, while yeast extract, peptone, Brainheart's infusion and others are suitable as a nitrogen source.

The yield of the restriction enzyme varies greatly depending on the culture conditions. Good results are generally obtained at a temperature in the range from 25 to 40"C and at a pH in the range from 4 to 8; the highest output is achieved in one or two days culturing with aeration and agitation. It is needless to say that optimal culture conditions should be selected case by case according to the strain used and the composition of culture medium. The restriction enzyme produced by the process of this invention is accumulated mainly inside the microbial cells.

The microbial cells containing the restriction enzyme can be separated from the culture liquid by, for example, centrifugation.

This enzyme can be extracted and purified by using known techniques commonly employed for the purification of restriction endonucleases. Thus, for example, the collected microbial cells are dispersed in a suitable buffer, and then broken down by ultrasonic treatment to allow extraction of the enzyme by the buffer solution. After removal of the cell residue by ultracentrifugation, ammonium sulfate is added to the supernatant for salting out, and the precipitate which separates out is dissolved in a potassium phosphate buffer (pH: 7.5) and dialyzed against a buffer of the same composition. The dialyzate is purified by ion-exchange chromatography, molecular-sieve chromatography and affinity chromatography, giving the restriction enzyme of this invention.In one embodiment the dialyzate is adsorbed on phosphocellulose P11 (Whatman) packed in a column, and the adsorbed portion is eluted with 0 to 1 .OM potassium chloride solutions. The active fractions are again adsorbed on DEAE-cellulose DE52 column (Whatman), followed by elution with 0 to 1 .OM potassium chloride solutions. The active fractions collected are then adsorbed on heparin-Sepharose CL-6B (Pharmacia Fine Chemicals), foilowed by washing with 0.7M potassium chloride solution and elution with 1 .OM potassium chloride solution. The active fraction is further purified by adsorption on aminohexyl-agarose column (Bethesda Research Laboratories) and elution with 0 to 1.5M potassium chloride solutions, giving a standard sample of the restriction enzyme of this invention.

The activity of this enzyme was determined according to the method described below.

A substrate solution of the composition shown in Table 1 was prepared.

TABLE 1 10mM Tris-HC1, pH: 7.5 7mM MgC12 7mM 2-Mercaptoethanol 60mM NaCI 0.01% Bovine serum albumin 1.0jug A- D NA This substrate solution (501) was preheated to 37"C, a sample of the restriction enzyme to be tested was added to allow the enzymatic reaction to proceed at that temperature, and the reaction was stopped 10 minutes later by the addition of 5p1 of a terminator solution (1% SDS, 50% glycerol, 0.02% Bromophenol Blue). The reaction mixture was then applied to a 1% agarose slab gel, and electrophoresis was conducted at a constant voltage of 10 V/cm for one to two hours. The buffer solution used for the electrophoresis was 90mM Tris-borate buffer containing 2.5mM EDTA (pH: 8.3).

DNA bands can be detected by UV irradiation if 0.5 Ug/ml ethidium bromide is previously added to the gel.

Eiectrophoresis was regarded as complete when the number and intensity of the bands for DNA fragments no longer changed.

The enzyme activity which ensures complete decomposition of 1 pug yg A- DNA after one hour's reaction at 37"C was defined as one unit.

The new restriction enzyme obtained by the process of this invention has the properties as described below.

(1) Action and substrate specificity: This enzyme is capable of recognizing the base sequence on a double stranded DNA molecule as shown below and cleaving it at the arrow-marked sites, 5'-T C G G A-3' 3'-AG G C 5' wherein A, T, C and G are as defined before.

The recognition base sequence of this enzyme was determined as described below.

The enzyme cleaved adenovirus type-2 DNA at eight sites, but failed to cleave vex174 RF DNA and SV40 DNA. In addition, it cleaved both Dam+ A-DNA and Dam A-DNA at more than 20 sites, the number of resulting fragments being smaller by 3 in the former than in the latter. Furthermore, the enzyme cleaved Dam- pBR322 DNA at one site, but showed no action upon Dam+ pBR322 DNA. These facts suggested that this enzyme would be able to recognize part of 5'-GATC-3'- a sequence which can be recognized by E. coli dam gene and undergoes methylation at A. Double digestion tests of Dam pBR322 using the restriction enzyme of this invention together with Hind Ill, Sal I, Hinc II and Pst I each revealed that the enzyme of this invention recognizes a site near at 170 bp on pBR322 DNA molecule.We searched sequence GATC in the vicinity of 1700 bp to find sequence TCCGGATC in the region from 1664 to 1671 bp. This indicates that the enzyme of this invention recognizes sequence TCCGGA. The number of sequences TCCGGA in adenovirus type-2 DNA, vex174 RF DNA, SV40 DNA and A-DNA was found to be 8, 0, 0 and 24, respectively, and this is in good agreement with the experimental data obtained above.

The sites of cleavage by the restriction enzyme of this invention were determined as described below.

Palindromic oligonucleotide d (GTTCCGGAAC) having the recognition site by the enzyme of this invention in the middle was synthesized by the solid-phase process and phosphorylated at the 5'-terminal end with polynucleotide kinase and [y~32p] adenosine triphosphate. The 32P-labeled molecule thus obtained was then annealed to give a double-stranded DNA, which was digested with the enzyme of this invention. The reaction mixture was analyzed on a DEAF-cellulose TLC plate (Masherey & Nagel Co.), giving a labeled spot of trinucleotide (5'-pGTT).It was thus concluded that this enzyme is capable of recognizing the base sequence as shown below and cleaving it at the arrow-marked sites, 5'TtC C G G A-3' 3'--AG G C CTT-5' (2) Optimal conditions for enzymatic activity: a) Optimal working temperature: The optimal working temperature of this restriction enzyme was 60 to 65"C.

b) Optimal pH: The optimal pH of this restriction enzyme was about 8.5.

c) KCI and NaCI concentration: The optimal salt concentration for this restriction enzyme was 150mM for both KCI and NaCI.

d) Stable pH range: The stable pH range for this restriction enzyme was 6.0 to 9.5.

(3) Molecular weight: 300,000 i 150,000 (by gel-filtration method with Sephadex G-200).

The following Example will further illustrate this invention but is not intended to limit its scope.

Example 1 Acinetobacter calcoaceticus (Ferm BP-935) was cultured in 160 liters of a medium having the composition shown below at 32 C for 10 hours with aeration and agitation, and the grown bacterial cells (about 1280 g on a wet basis) were collected from the culture broth 160 liters by means of a refrigerated centrifuge.

TABLE 2 Polypeptone 10 g Yeast extract 5 g Glucose 1 g NaCI 5g Deionized water 1 e pH 7.2 Three hundred and fifty grams of the bacterial cells obtained above were suspended in 700 ml of an extractive buffer solution (l0mM potassium phosphate buffer, 10mM 2-mercaptoethanol; pH: 7.5), the suspension was treated in an ultrasonic crusher to break down the cell walls, and the resulting mixture was centrifuged (100,000 x g, one hour) to remove the residue.

To the extract thus obtained, was added ammonium sulfate to 70% saturation, the precipitate which separated out was collected by centrifugation and dissolved in buffer solution A (1 OmM potassium phosphate buffer containing 1 0mM 2-mercaptoethanol and 5% glycerol; pH: 7.5), and the solution was dialyzed overnight against buffer solution A.

The dialyzate was adsorbed on phosphocellulose P11 (Whatman) packed in a 300-ml column and previously equilibrated with buffer solution A. After washing with buffer solution A, the adsorbed portion was eluted with 0 to 1.OM potassium chloride solutions (linear concentration gradient technique). The restriction enzyme activity was detected in fractions corresponding to 0.65 to 0.70 KCI concentrations.

These active fractions were collected and dialyzed overnight against buffer solution A, the dialyzate was adsorbed on DEAE-cellulose DE52 (Whatman) packed ina 10-mI column and previously equilibrated with buffersolutionA, and the adsorbed portion was eluted with 0 to 1 .0M potassium chloride solutions (linear concentration gradient technique). The restriction enzyme activity was detected in fractions corresponding to 0.20 to 0.25 KCI concentrations.

These active fractions were collected and dialyzed against buffer solution A, and the dialyzate was adsorbed on heparine-Sephrose CL-6B (Pharmacia Fine Chemicals) packed in a 4-ml column and previously equilibrated with buffer solution A. After washing with buffer solution A containing 0.7M potassium chloride, the adsorbed portion was eluted with buffer solution A containing 1.OM potassium chloride solution.

The active fraction thus obtained was again adsorbed on aminohexyl-agarose (Bethesda Research Laboratories) packed in a 4-ml column and previously equilibrated with buffer solution A. After washing with buffer solution A, the adsorbed portion was eluted with 0 to 1.5M potassium chloride solutions (linear concentration gradient technique). The restriction enzyme activity was detected in fractions corresponding to 0.32 to 0.65M potassium chloride solutions.

The active fractions were collected and concentrated by the addition of polyethyleneglycol, and an equal amount of glycerol was added to the concentrate, giving the final standard sample of the restriction enzyme of this invention.

This standard sample was free from any nonspecific DNase or phosphatase.

The purification method described above gave 3,000 units of the enzyme from 350 g of wet microbial cells.

As may be apparent from the foregoing, this invention provides a novel restriction enzyme having a unique substrate specificity that has never been known and a process for producing the same on an industrial basis.

Claims (3)

1. New restriction enzyme having the following properties: (a) Action and substrate specificity Recognizes the base sequence in a double-stranded deoxyribonucleic acid molecule as shown below, and cleaves it at the arrow-marked sites, 5'-T CG G A-3' 3'-AG G CCtT-5' (wherein A, G, T and C represent adenosine, guanosine, thymidine and cytidine, respectively).

(b) Optimal pH: approximately 8.5 (c) Stable pH range: 6.0 to 9.5 (d) Optimal working temperature: 60 to 65"C

2 A process for producing the restriction enzyme as defined in claim 1, which comprises growing a microorganism belonging to the genus Acinetobacter and capable of producing said new restriction enzyme in a culture medium and collecting the enzyme thus formed from the culture broth.

3. The process for producing the new restriction enzyme as defined in claim 2, wherein said microorganism belonging to the genus Acinetobacter is Acinetobacter calcoaceticus.