SU1532583A1 - Strain of paracoccus denitrificans bacteria as producer of endonuclease of restriction pde 12 i - Google Patents

Abstract

This invention relates to biotechnology. The aim of the invention is to identify the producer strain PARACOCCUS DENITRIFICANS VKPM B-4152, which provides the restriction endonuclease PDE 12 1, which is an isoschizomer of the restriction enzyme SAU 96 1 and capable of recognizing and cleaving the GGNCC nucleotide sequence. The strain was identified as a result of searching for strain-producing restriction enzymes among microorganisms living in Lake Balkhash, non-pathogenic for humans, undemanding to nutrient media, easily destroyed by ultrasound, provides an increased yield of the target product, which is 3000 u / g of biomass. The PARACOCCUS strain DENITRIFICANS B - 4152 produces a restriction endonuclease PDE 12 1, and has a GGNCC recognition site identical to the SAU 96 1 recognition site.

Description

The invention relates to biotechnology, in particular, to the production of restriction endonucleases capable of recognizing and cleaving the GGNCC nucleotide sequence.

The aim of the invention is to find out a new strain - producer.

providing a higher yield of the target product, non-pathogenic for humans and undemanding to nutrient media.

The bacterial strain Paracoccus denitrif leans VKPM B-415 2 was selected during the search for producer strains.

Rostriktaz among microorganisms, about (5thuchushchikh in the lake Balkhash, .-,

The strain Paracoccus denitrifleans B-4152 is characterized by the following morphological, cultural and physiological features: cells with (Phenic 1-1.1 µm in diameter, are found singly, in pairs or a: -regates. In young cultures, short rod-shaped cells can be found. Stationary They accumulate poly B-oxybutyra GramotriceTeline. Respiratory metabolism. Reactions to C-daza and catalase are positive. Not G1LOFILA, no need for growth. Gh factors, gelatin is not diluted. t -20 ° C.

On fish peptone agar, forms colonies grayish-white, shiny 8–9 mm in diameter with uneven edges, flat, do not grow into the agar. Growth is abundant. The RPB forms a uniform turbidity, sediment. Good growth on synthetic medium with mineral nitrogen.

Produced by the alleged strain; strictase is characterized by the following properties: it recognizes and specifically splits the sequence of nucleotides of the GGNCC; the optimum pH for restriction enzyme action is 7.5-9.0; optimal temperature for restriction enzyme activity, Mo ions are required, with an optimum concentration of 5-15 mM.

For the cultivation of Par.den B-4152, a nutrient medium of the following composition is used, g / l distilled

ry; Trypton 10, yeast extract

Cultivation was carried out at 4 with good aeration for 2 days. The output of biomass is 2-3 g / l of culturedness. The output of restrictase Pde 12 1: 45 u / g biomass.

Example. Par cells. den.,: (stored in 50% glycerol in L-bulone at t -20 C are seeded on a plate with L A. After 2 days of incubation | 1 or the cells are collected in a loop and added to 100 ml B and grown to Density is 1.5 at. Then the culture is introduced into 2 liters of B. grown to a density of 2.5 at. After cooling, the cells are harvested by centrifugation. Biomass yield 2.3 g wet weight b 1 liter.

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4 g of cells are suspended in 20 ml of 0.02 M Tris-HC1 buffer pH 7.5, containing 0.001 m / 2 | -mercaptoethanol and 0.1% Triton X-100, and are destroyed in an ultrasonic disintegrator. Cell debris is removed by centrifugation (18000 rpm, 30 min at) and the supernatant is applied to 30 ml of Whatman phosphocellulose P-11 (equilibrated with buffer A (0.02 M potassium phosphate buffer, pH 7.5; 0.001 M, 6-mercaptoethanol, 5 EDTA), then the column is rinsed with buffer A until the UV absorbing material disappears from the eluate.

The adsorbed material was eluted with a solution of 1 M NaCl in buffer A. Then, 60 ml of the resulting protein washes with a colony were dialyzed overnight against 2 liters of buffer L and applied to 10 ml of DEAE-cellulose (, TSE-52, Whatman, England), balanced with buffer A and elute with 100 ml gradient of 0-1.0 M NaCl in buffer A. The volume of each fraction is 5 ml. Aliquats from each fraction (1 μl) are incubated for 1 h with 1 μl of phage C1857 DNA and analyzed in agarose gel. The fractions cleaving the DNA of phage / (7-9) are pooled, dialized for 3 hours against 2 liters of buffer A and applied to 4 ml of P-11 phosphocellulose (Whatman, England) and eluted with 40 ml of 0-0.1 M gradient NaCl in buffer A. The volume of each fraction is 2 ml. The fractions containing Pde 12 I are determined as well as by chromatography on DEAE-cellulose. Fractions 14, 15 containing Pde 12 I are combined and dialyzed against buffer A containing 0.1 M NaCl and 50% glycerol. The resulting enzyme preparation (1 ml) has a specific activity of 12000 units / ml. The minimum amount of enzyme, neg., Required for complete cleavage of 1 µl of phage L DNA for 1 hour at 37 ° C is taken as a unit of activity. The enzyme preparation is stored. at. From 1 g of biomass receive 3000 units. restrictase Pde 12 I.

To determine the sequence of nucleotides recognized by the Pde 12 I restriction enzyme, the dac phase and pBR 322 restriction enzymes Pde 12 I and Sau 96 1 are separately hydrolyzed, as well as the co-hydrolysis with these restrictases. A complete picture of the fragments obtained during DNA hydrolysis with Pde 12 I and Sau 96 I was observed.

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515325836

Claims (1)

Separately and jointly, indicating the yield of the target enzyme indicates that the Pde 12 I restriction enzyme, which is 3000 U / g of biomass (12 times more than the restriction enzyme Sau 96 I, is more than in the prototype), recognizes and splits Sequence - The formula of the invention of nucleotides GGNCC. The resulting strain is non-pathogenic for the human Paracoccus denitrium bacterial strain; ultra-ficans VKPM B-4152 is easily destroyed - producing by endozvuk, and also provides the increased restriction nuclease Pde 12 I.