HU202273B - Process for producing new bfli restriction endonukleaze from bacillus cereus - Google Patents

Abstract

A subspecies of fluorescent MIMNGp 001018 strain of bacillus cereus (its mutants and variants) which produce a wood restrictive endonuclease enzyme coded BFIL are prepd. by aerobic submerged culture using a medium of assimilateable sources of carbon and nitrogen and mineral salts. - The BFIL enzyme is obtd. from this culture

Description

Scope of the description: 4 pages, 2 drawings, Figure 3

EN 202273 Β

Restriction endonucleases are those enzymes that recognize certain sequences specific to them in double-stranded DNA and then cleave both chains endonucleolytically.

To our knowledge, restriction endonucleases are produced only in prokaryotic organisms, ie bacteria and blue algae, but are quite common here. The number of restriction endonucleases known so far exceeds 400. Enzymes of different origins and structures can perform the same function, the so-called enzyme. isosisomeric enzymes. Recognition specificity is less than 100 in addition to the large number of enzymes.

In one group of enzymatic cleavage, cleavage is cut at the same site against the two chains, resulting in a blunt end. Another type of cleavage cuts the two chains asymmetrically, resulting in a 3 'or 5' overhang. Restriction endonuclease recognition sequences are often referred to as " palindrom, that is, a DNA section that is read-symmetric, the base sequence of one strand being read backward on the corresponding piece of the complementary strand,

CTGCAG eg: GACGTC *

Extending the recognition site or splitter to our versions is very important. The use and restriction of restriction endonucleases is now widespread and diverse. In all tasks, it is important, in some tasks, to determine that these enzymes define a specific DNA structure at the specified site. Expanding the number of configurable sites and structures will, of course, expand the application and response capabilities.

Highlighting a wide range of applications:

- Physical mapping of DNA

- Modification of DNA by recombinant DNA technology (production of mutants, breeding)

- Isolation of DNA by hybridization probes

- diagnosis based on DNA identification

- diagnosis based on DNA mutation confirmation

- confirmation of species, breed, strain identity, or difference based on DNA structure

- Determining the location of DNA methylation in the higher organism based on the splice, etc.

An interesting subgroup of restriction endonucleases is the type II S enzyme. They are cleaved outside the recognition site, not specifically linked to the palindrom recognition sequence, but also specifically (Gene 47, 1-154, 1986).

In our research, we managed to isolate a bacterial strain from which a type II S enzyme with a specificity not known to date can be obtained. The strain Bacillus cereus subsp. fluorescens by Industry and Industry

The National Collection of Agricultural Microorganisms was deposited under the number NCAIM Β (P) 001018.

The BflI enzyme prepared according to the invention has been subjected to assays to determine the recognition site and cleavage site. We found that the enzyme recognition site is:

ACGGC (N) iz

TGCCG (N) 13 * (cleavage from the recognition site at 12 or 13 nucleotides)

According to the invention, the preparation of the BflI enzyme is carried out by the method of Bacillus cereus subsp. Fluorescent NCAIM Β (P) 001018 was cultured in a well-ventilated medium containing assimilable carbon, nitrogen and mineral salts, and the resulting enzyme was recovered from the cell mass.

The invention is further illustrated by the following examples.

Example 1

Bacillus cereus subsp. Fluorescent NCAIM (P) 001018 was maintained on YTA agar by inoculation for 2-3 months at 37 ° C for 16 hours in an agitated refrigerator at + 4 ° C.

To prepare the enzyme, a plate of the same medium is poured into a Petri dish and the strain is plated. Some of the grown colonies were suspended in 10 ml of YTB medium with suckling and propagated overnight. 100 ml of YTB medium were inoculated with 1 ml of the .overnight suspension in a shake flask and incubated for 6 hours on a shaking table (180 rpm) at 37 ° C and used as a seed culture. For production fermentation, 1 L of YTB medium was sterilized in a 3 L Erlenmeyer flask, which was inoculated with 10 ml of seed culture. The propagation is carried out on a shaking table (180 rpm) at 37 ° C for 8 hours. When the fermentation was stopped, the fermentation broth was centrifuged (4000 rpm, 30 min), 50 g of fuganedves were obtained from 1 l of fermentation broth. Cells were suspended in double 0.01 M 2-mercapto-ethanol containing 0.01 M pH 7.5 Triz-HCl buffer, and the cells were ultrasound scanned using the SONIFER CELL DISRUPTOR for 10 minutes. Cell debris was removed by 60 min ultracentrifugation (35,000 rpm). NaCl was added to the supernatant to a concentration of 1 M and the cell extract was chromatographed on a 4 x 90 cm Bio-Gel A-0.5 m column. The eluent is 1 M NaCl, 0.01 M pH 7 = 7.5 Tris-HCl, 0.01 M 2-mercaptoethanol solution. 6 ml fractions were collected for restriction enzyme activity. The method is as follows: 0.01 M pH 7.8 Tris-HCl, 3 mM MgCl 2, 6 mM 2-mercaptoethanol, 0.1 mg / ml gelatin medium in 25 µl final volume of 1 µg lambda phage DNA and 2 µl sample is added. Incubation

HU 202273 ° C for 60 minutes. The reaction was stopped with 3 mL of reagent, consisting of 0.2 M EDTA, 50% sucrose, 0.1% bromophenol blue. The mixture was electrophoresed on a 1.4% agarose gel.

The electrophoresis buffer was 0.05 M Tris acetate pH 8.05, 0.02 M sodium acetate, mM EDTA. 200 V, 2 hours. DNA was detected by staining with ethidium bromide (see Biochemistry 12, 3055 (1973)). Fractions showing enzyme activity were pooled and dialyzed against PC buffer. For the next purification, a Whatx DE52 DEAE-cellulose column equilibrated with a PC buffer was used at 3 x 25 cm, the separation was carried out by linear gradient elution with 0-0.5 M NaCl in 1000 ml of PC buffer. Fractions eluted at 0.15-0.20 M NaCl were collected and pooled as described above for PC buffer.

Finally, on a 1.5 x 15 cm Whatman P 11 phospho-cellulose column, the enzyme-active proteins eluting with the 0.50-0.7 M NaCl content were separated by dialysis against 200 ml of PC buffer.

The resulting enzyme yields 50 units / g wet cell. Sign: BflI. The enzyme does not require Na- or K-, Mg ² * needs approx. 3 mM, pH optimum 7.8, heat optimum 37 ° C.

Applied in the examples media and buffer composition: YTA medium: Bacto tripton 10 g. Bacto yeast extract 5 g, NaCl 5 g> Bacto agar 15 g, tap water ad 1 1 Sterilization: 120 ° C for 30 minutes YTB medium: Bacto tripton 10 Ski Bacto yeast extract 5 g> NaCl 5 g. tap water ad 1 1 Sterilization: at 120 ° C for 30 minutes. PC Buffer: 0.01 M pH 7.4, potassium phosphate buffer,

Example 2

Characterization of the BflI enzyme

a) Proof of recognition

Double-cleavage of the pBR 322 plasmid with the new enzyme and with and with the following enzymes is known in the known art: Bspl, Hpall, Alul, MboII, Avall, Sali, HindIII, BamHI, Ball, PstI, BglI, Foki. Enzymes are Biolabs and REANAL products. Analysis of the resulting fragments shows that the new enzyme cleaves the plasmid pBR322 at three positions between the base pairs 600-620, 1150-1170 and 2950-2980. There are two homologous regions on the three sections above: CCTAA and ACGGC. A similar study with Lambda bacteriophage excludes the CCTAA recognition site and confirms the ACGGC construct; since its incidence is proportional to the number of detectable breakdown fragments. This recognition site is a Type II S enzyme, indicating that the cleavage site is outside the recognition sequence.

b) Determination of the cleavage site

In this assay, pKLT1 plasmid or other low molecular weight DNA was used to cleave the HindIII-Hinfl section of plasmid pKLT1. The base sequence of the desired DNA fragment is shown in Figure 1. The fragment is labeled at the 5 'end with polynucleotide kinase in the presence of gamma- ³² P-ATP. [Description of the known method: Maniatis Foot. Manual, Cold Spring Harbor Leg., NY (1982)]. The preparations were digested with the test enzyme in the following medium: 1.01 M pH = 7.8 Tris-HCl buffer, 0.003 M MgCl 2, 100 Mg / mL gelatin, 1 mL of the enzyme solution of Example 1, 1 Mg DNA fragment; A total volume of 50 m1 was incubated at 37 ° C for 1 hour. During digestion, approx. A HindIII-BflI fragment of 30 bp is generated. This is investigated by the Maxam-Gilbert method. Methods Enzymol. 5: 499-560 (1980)]. According to the results of the gel analysis, the cleavage site is ACGGC (N) iz and TGCCG (N) n (see Figure 2).

0.1 mM EDTA,

0.01 M 2-mercapto-ethanol.

Storage Buffer:

0.05 M KCl

0.01 M pH = 7.5 Tris-HCl buffer

0.1 mM EDTA

1.0 mM dithio-treitol

200 Mg / ml of bovine serum albumin 50% glycerol.

Example 3

From the fermentation broth prepared in Example 1, the cell mass was recovered. For 26 g of wet cell material, 40 ml of extraction buffer (0.01 M pH 7.5 tris-HCl containing 0.01 M 2-mercaptoethanol) was added. Ultrasonic treatment (50% working time, 10 min duration), the debris removed by centrifugation (35,000 rpm, 60 min).

To the supernatant (48 ml) 1.25 g KCl and

3.36 ml of 10% Polymin P (Serva, Heidelberg) was added, the mixture was slowly stirred at melting ice for 30 min.

The pad is separated by centrifugation (20,000 rpm, 20 min). The crude protein-containing solution was dialyzed for 6 hours. As a first purification step, Heparin-Sepharose chromatography was performed on a 1.5 x 15 cm column at a development rate of 5 30 ml / h, using a PC buffer containing 0.0-0.6 M NaCl as eluent. The active fraction can be obtained at 0.06-0.15 M NaCl. The purification was carried out on a 1.5 x 5.0 cm aminopentyl chromatography column, eluting with 0.0-0.6 M NaCl PC buffer. The collected fractions are 0.12-0.18 M NaCl.

The solution is dialyzed against storage buffer. The activity of the resulting preparation corresponds to that of Example 1.

Claims (1)

PATIENT PERSONALITY A method for advancing BflI restriction endonuclease, characterized in that Bacillus cereus subsp. Fluorescens NCAIM Β (P) 001018 or its mutant or variant producing the above enzyme is grown in a submerged aerobic medium containing assimilable carbon, nitrogen source and mineral salt, and the BflI enzyme is recovered from the cell mass. Example 4 Bacillus cereus subsp. Fluorescent NCA1M Β (P) 001018 was cultured on YTA agar. Three days of sloping agar culture was washed with 5 ml of AGK medium, and 300 ml of AGK medium was inoculated with the suspension in a 1 L Erlenmeyer flask. The culture was continued at 37 ° C for 20 hours with shaking (rpm 350). The culture was centrifuged (8000 rpm, 30 min) to obtain 24 g fuganedge cell mass, which was processed as in Example 1. The resulting enzyme has the same cleavage as Bfll. The result of the digestion of known DNA molecules is described in Figure 3. Composition of AGK Medium: asparagine 1 g glucose 5 g corn mash powder 2 g potassium dihydrogen phosphate 0.05 g 40 magnesium sulfate 0.01 g tap water 1 1. Published by the National Bureau of Invention, Budapest Responsible for the publication: dr. Gabriella Szvoboda Head of Department R 4981 - KJK 91.3911.66-13-2 Alföldi Printing House Debrecen - Responsible leader: Viktor Szabó CEO -4Int Cl ⁵ C 12 N 9/22 C 12 N 1/20 pKLT1 fragment sequence 513 523 533 543 553 563 573 AAGCTTGCTT CTTTGCTGAC GAGTGGCGGA CGGGTGAGTA ATGTCTGGGA AACTGCCTGA TGGAGGGGGA 583 593 603 613 623 633 643 TAACTACTGG AAACGGTAGC TAATACCGCA TAACGTCGCA AGACCAAAGA GGGGGACCTT CGGGCCTCTT 653 663 673 683 693 703 713 3CCATCGGAT CGCGGTGG1C CCACCTGACC CCATGCCGAA CTCAGAAGTG AAACGCCGTA GCGCCGATGG 723 733 743 753 763 773 783 iCGGCAGTAG CGCGGTGGTC CCACCTGACC CCATGCCGAA CTCAGAAGTG AAACGCCGTA GCGCCGATGG 793 803 813 823 833 843 853 AGTGTGGGG TCTCCCCATG CGAGAGTAGG GAACTGCCAC CCATCAAATA AAACGAAAGG CTCAGTCGAA 863 873 883 893 903 913 923 .GACTGGGCC TTTCGTTTTA TCTGTTGTTT GTCGGTGAAC GCTCTCCTGA GTAGGACAAA TCCGCCGGGA 933 943 953 963 973 983 993 CGGATTTGA ACGTTGCGAA GCAACGGCCC GGAGGGTGGC GGGCAGGACG CCCGCCATAA ACTGCCAGGC 1003 1013 1023 1033 1043 1053 1063 TCAAATTAA GCAGAAGGCC ATCCTGAGGA TGGCCTTTTT GCGTTTCTAC AAACTCCCGG TACCGGCCGG 1073 ACCGGCCAA G Figure 1 Plasmid pKLT1 Hindii! - Hlnfl section -5 EN 202273 Β Int Cl ⁵ C 12 N 9/22 C 12 N 1/20 oh, yo.SlU-'BVJS