RU2340663C1 - STRAIN OF BACTERIUM Paenibacillus sp, PRODUCER OF RESTRICTION ENDONUCLEASE Psp 1009I - Google Patents

Abstract

FIELD: chemistry; bio-technology.

SUBSTANCE: new strain Paenibacillus sp. has been obtained, which produces site specific restriction endonuclease Psp 1009I, capable of recognising and splitting the sequence of nucleotides 5'-GCCNNNNNGGC-3'. The strain may be used for the production of the highly active endonuclease Psp 1009I.

EFFECT: new connections contain useful biological properties.

1 cl, 3 dwg, 3 ex

Description

The invention relates to the search for new strains producing biologically active substances that are promising for the microbiological industry and genetic engineering, and for the production of a new strain producing a site-specific endonuclease capable of recognizing and cleaving the 5'-GCCNNNNNGGC-3 'nucleotide sequence.

In the process of analysis of soil samples of the Geyser Valley (Kamchatka), a bacterium of the genus Paenibacillus was isolated, which is a producer of a restriction endonuclease called Psp 1009l according to the generally accepted nomenclature [1]. The strain is cultivated at a temperature of 28-30 ° C and is a producer of endonuclease, which has high activity at a temperature of 37 ° C. The enzyme is an isoshizomer of the known BglI enzyme, which was first isolated in 1976 from a strain of Bacillus globigii [2].

Other prototypes of this enzyme are known: Bpu 86I from a strain of Bacillus pumilis 86 and Bse 631I from a strain of B. species 631 [3]. BsoJI from a strain of Bacillus stearothermophilus Azores9582A_ [4]. Tsp 219I from strain Thermus species_ [5]. Tsp 8EI from Thermus species 8E strain [6]. VanI from Vibrio anguillarum strain [7].

However, in strains of the genus Paenibacillus, restriction endonuclease with such specificity was isolated for the first time.

This strain contains only one enzyme, which is its advantage over Bacillus species 6-3-1 [3] and Thermus species 8E [6], which contain two endonucleases of different specificity.

The strain grows well on simple nutrient media and does not apply to pathogenic strains. The biomass yield is 5.5 ± 0.5 g / l L-broth. And the yield of the enzyme is 30,000 U / g.

A restriction endonuclease of this specificity is used to study the primary structure of DNA, physical mapping of various genomes and has not been previously detected in strains of the genus Paenibacillus.

The literature does not provide data on the output of enzymes for most prototypes of Psp1009I.

The most accessible and close to the claimed strain is a strain of Bacillus globigii, producing the restriction endonuclease BglI [3, 5], the prototype. The enzyme retains activity from two to six months of storage at -20 ° C. No data were found on the yield of the enzyme.

An object of the invention is to obtain a strain capable of growing on simple nutrient media producing a restrictase, recognizing and cleaving the nucleotide sequence of 5'-GCCNNNN / NGGC-3 ', with a high yield of the enzyme that is stable during storage and capable of being active under low-temperature reaction conditions.

The stated technical problem is solved by the identification and use of a producer strain of site-specific restriction endonuclease Psp1009I.

In the process of studying the properties of this strain, it was found that it belongs to the genus Paenibacillus.

Identification is carried out by conventional methods [9-10]. The strain has the following properties:

Gram-negative, mesophilic, forms endospores. On dense nutrient medium forms sprawling, flat colonies of irregular shape, with a complex surface structure. The cells of the strain Paenibacillus species Dg-1009 are aerobic, gram-negative, motile regular bacilli that reproduce by binary division; form elliptical swollen endospores located terminally. The cell sizes of the strain are in the range of 0.4-0.6 × 2-3.5 μm. Strain P.species Dg-1009 positive in tests for oxidase, catalase, esculin, in the reactions of VP and MR, on H ₂ S, reduces nitrates; does not have lecithinase and lipase, phosphatase, does not hydrolyze casein, is negative in tests for phenylalanine deaminase, arginine decarboxylase; does not utilize citrate, does not form indole; hydrolyzes esculin, arabinose, xylose, glucose, sucrose, lactose and maltose, does not utilize rhamnose and mannitol. The nucleotide composition of chromosomal DNA is 49.6 mol.% HZ.

Based on the data obtained, the isolated strain belongs to the genus Paenibacillus.

The resulting strain was deposited in the Museum of Cultures of the Federal State Institution of Science and Science of the VB "Vector" of Rospotrebnadzor under registration number B-1092, and the restriction endonuclease produced by it was named Psp1009I.

The strain is stored in a nutrient medium with glycerin.

The following composition (g / l) is used for cultivation: bacto-tryptone (Difco) - 10.0, yeast extract (Difco) - 5.0, NaCl - 5.0, pH 7.0-7.2.

To obtain an enzyme preparation, the strain is grown in half-liter flasks in a shaker at 200 rpm and a temperature of 30 ° C for 24-48 hours. Cells are precipitated by centrifugation at 5000 rpm and a temperature of 4-5 ° C. Isolation is carried out by chromatographic purification on P-11 phosphocellulose (Whatman, England). All operations for the isolation of Psp1009I are carried out at a temperature of 4-5 ° C. Cells are suspended in buffer A (10 mM potassium phosphate buffer pH 7.5, 7 mM β - mercaptoethanol, 0.1 mM EDTA) at a rate of 4 ml per 1 g of biomass and destroyed by ultrasound disintegrator ("MSE", England), then clarified by centrifugation in a centrifuge J-21B (Bekman, USA) at a speed of 18,000 rpm for 30 minutes The extract was applied to a column (1.6 × 14 cm) of P-11 phosphocellulose (Whatman, England), washed with buffer A, and the enzyme was washed off in a NaCl concentration gradient (0.0-0.8 M). The fractions containing restrictase are dialyzed against buffer B (10 mM Tris-HCl pH 7.5, 1 mM β-mercaptoethanol, 0.1 mM EDTA. Fractions with the enzyme are dialyzed against a 50% glycerol solution in buffer A.

For electrophoresis use agarose company ("Sigma", USA). Restriction analysis is carried out in 20 μl of the reaction mixture containing 10 mm Tris pH 7.5, 10 mm MgCl ₂ , 10 mm NaCl, 1 mm dithiothreitol (DTT) and 1 μg of λсI857 DNA at a temperature of 25-30 ° C.

The output of the enzyme from 1 g of cells is 30,000 units. The unit of activity (E) is the minimum amount of the enzyme, which for 1 h at a temperature of 30 ° C under optimal conditions completely hydrolyzes 1 μg of DNA of phage λсI857.

The strain is unique not only in the presence of this restriction endonuclease, but also has additional properties. It belongs to gram-negative bacilli.

The defining difference of the proposed strain from the strain of the prototype is:

- the strain belongs to the genus Paenibacillus;

- growth on simple nutrient media with the addition of yeast extract;

- the presence of one enzyme Psp1009I.

As a result of screening, the restriction endonuclease Psp1009I was detected in the strain Paenibacillus sp.Dg1009.

To determine the recognizable nucleotide sequence of the restriction endonuclease Psp1009I, the experimentally obtained by agarose gel electrophoresis of the fragments formed during the hydrolysis of λcI857, T7, pBR322, pUC19 DNA is compared with the control ones (Figs. 1, 2). The results of the comparative analysis allow us to conclude that this restriction enzyme recognizes the nucleotide sequence (5'-GCCN ₅ GGC-3 '). Figure 3 presents the electrophoregram of the products of DNA hydrolysis of phage λсI857 with the enzyme Bsp1009I dor. 1), BglI and Psp1009I (dor. 2) and BglI, recognition site (5'-GCCN ₅ GGC-3 '), (dor.3). As can be seen from the figure, the identity of the splitting bands in parallel and joint hydrolysis confirms their isoshisomerism.

To determine the recognizable sequence of nucleotides by the restriction endonuclease Psp1009I (Fig. 1), the fragments experimentally obtained by electrophoresis in a 1% agarose gel are formed by hydrolysis of DNA of phage λсI857. The results of the comparative analysis allow us to conclude that this restriction endonuclease recognizes the nucleotide sequence (5'-GCCNNNNNGGC-3 '). As can be seen from the presented figure 3, the identity of the splitting bands in parallel and joint hydrolysis confirms their isoshisomerism.

Interpretation of the obtained experimental data suggests that the studied restriction endonuclease is an isochisomer of BglI restriction enzyme.

Thus, a highly active restriction endonuclease that recognizes and cleaves the 5'-GCCNNNNNGGC-3 'nucleotide sequence from a strain belonging to the genus Paenibacillus having an optimal restriction temperature of 20-30 ° C was first isolated.

Since the proposed strain was obtained for the first time and was never used as a producer of restriction endonuclease, we can conclude that the proposed strain meets the criteria of the invention of "novelty" and "inventive step".

Description of figures:

Figure 1 - identification of restriction endonuclease from strain P. species. Dg1009.

1-BglI / λ, 3-BglII / λ5-HindIII / λ, 7-BssT1I / λ; 2, 4, 6 - different variants of the cell extract of strain P. species. Dg1009.

Figure 2 - electrophoregram of hydrolysis of various DNA with a cell extract of the strain Paenibacillus sp.Dg-1009:

1 - DNA λ, 2 - λ + Psp1009I, 3 - T7 + Psp1009I, 4 - T7 DNA, 5 - pUC19, 6 - pUC19 + Psp1009I, 7 - pBR322, 8 - pBR322 + Psp1009I.

Figure 3 is an electrophoregram of the products of DNA hydrolysis of phage λсI857 with the enzyme Psp1009I (d.1), BglI (recognition site 5'-GCCNNNNNGGC-3 ') (d.2). Psp1009I and BglI (dor. 3).

The invention is illustrated by the following examples of specific performance.

Example 1. Analysis of the strain for the presence of restriction endonucleases. Separate colonies were taken from a solid medium and added to 200 μl of TEN buffer (0.1 M Tris pH 7.5, 0.01 M EDTA, 0.05 M NaCl). For destruction, lysozyme and triton X-100 are used. The cell suspension is centrifuged, aliquots are taken and introduced in a ratio of 1:10 in the incubation mixture containing 1 μg of DNA. The selected enzyme was used to hydrolyze several substrates: DNA of phages λ and T7 and plasmid DNA pBR322 pUC19. The data are presented in figure 2.

Example 2. Obtaining biomass and isolation of the enzyme. Before starting work, the cells of the producer strain are subcultured with a bacteriological loop on an agar medium with yeast extract or RPA (fish nutrient agar). Weeds are placed in a thermostat at a temperature of 28 ± 2 ° C. After 18 hours of incubation, the grown cells are transplanted into flasks with fresh nutrient medium,

Cultivation is carried out on a rocking chair at 28 ± 2 ° C with aeration, with stirring - 200 rpm for 24 hours. Cells are harvested by centrifugation on a J-21 centrifuge at 5,000 rpm and a temperature of 4-5 ° C. The biomass yield is 5 g / l of medium.

Disintegration, isolation and purification of the enzyme is carried out according to the method described above.

Example 3. Determination of the specificity of the enzyme. The specificity of the enzyme is determined by the picture of parallel and joint hydrolysis of DNA of phage lambda (figure 3). The identity of the hydrolysis patterns in lanes 1-3 indicates that these enzymes recognize and cleave the same nucleotide sequence 5'-GCCNNNNNGGC-3 '.

List of references

1. Smith, N.O., Nathans, D. // J. Mol. Biol. 1973. V.81. R. 419-423.

2. Pirrotta V. Two restriction endonucleases from Bacillus globigii .. // Nucleic Acids Res. 3: 1747-1760 (1976).

3. V.E. Repin, L. R. Lebedev, I. S. Andreeva, L. I. Puchkova, Yu. P. Zernov, G. D. Serov, T. A. Tereshchenko, G. N. Afinogenova, N.M. Pustoshilova. Producers of restriction endonucleases among natural microbial isolates and the development on their basis of technologies for producing enzyme preparations. // Biotechnology, 1998, No. 2, S.18-27.

4. Jones, S., Hill, V.J., Vincent, S., Clark, D.R. Unpublished observations.

5. Korpela, J., Hjorleifsdottir, S. Unpublished observations.

6. Welch, S.G., Williams, R.A.D. Two thermostable type II restriction endonucleases from Icelandic strains of the genus Thermus: Tsp4C I (ACN / GT), a novel type II restriction endonuclease, and Tsp8E I, an isoschizomer of the mesophilic enzyme Bgll (GCCNNNN / NGGC). // Biochem. J. 309: 595-599 (1995).

7. Wood, Unpublished observations.

8. Roberts, R. J., Macelis, D. // Nucleic Acids Res. - 2000. - V.28. - P.306-307.

9. Puchkova L.I., Ushakova T.A., V.E. Repin. Testing and isolation of highly purified restriction endonucleases. // Applied biochemistry and microbiol., 2002, T. 38, No. 1, p.20-24.

10. Maniatis T., Fritsch E., Sambrook J. Methods of genetic engineering. Molecular Cloning M.: Mir, 1984.480 s.

Claims (1)

The bacterial strain Paenibacillus sp., Deposited in the Collection of microorganism cultures of the Federal State Institution of Science "State Scientific Center for Virology and Biotechnology" Vector "under No. B-1135, producer of restriction endonuclease Psp 1009I.

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