SU1698286A1 - Culture medium for cultivation of pseudomonas stutzeri: vkpm v-4724 strain producing restrictase psu1 - Google Patents

Description

one

(21) 4724816/13

(22) 07.27.89

(46) 12/15/91. Bul №46

(71) Scientific and Production Association Biolar and Scientific and Production Association Vector

(72) Yu.N.Boykov, V.E. Repin, S.H. Degt roar and N.V. Boikova

(53) 577.15 (088.8)

(56) Arkadieva Z.A., Trifonova T.V. / Agapova N.S. and Kozlova E.I. - Scientific. report high school biol. Science, 1976, №7, p. 113-115.

Gingeros T.R., Greenough L, Lehildkraut I., Roberts RJ. Two new restriction endonu- cleases from Protew vulgaris. - J. Nucleic Acids Res., 1981, vol. 9, No. 18, p. 4525-4536.

(54) NUTRIENT FOR CULTIVATING STRAIN PSEUDOMONAS STUTZERI VKPM B-4727 - RYSTRIKTASE PSU PRODUCT

(57) The invention relates to microbiology and concerns the composition of nutrient media.

intended for cultivation of the Psu I restriction enzyme producers. In order to increase the yield of the enzyme and its activity, it is proposed to use for the cultivation of the Psu I restriction enzyme Pseudomonas stutzeri VKPM-4727 a nutrient medium containing, as a nutrient base and source of amino nitrogen, pancreatic renal hydrolyzate with% content: total nitrogen 11.2; amine nitrogen 6.7; ash 9.7; moisture content 4.1, in the following ratio of components, g / l: pancreatic renal hydrolyzate 15.0– 20.0; ammonium sulfate 1.0-2.0; sodium citrate 0.5-0.7; ferric chloride 0.001-0.002; water up to 1 l. At the same time, the biomass yield reaches 12.5–13.0 g / l of culture liquid, the yield of restriction enzyme is 10,000–15,000 units / g of biomass, and the activity increases to 125,000–195,000 units / l of culture fluid, which is 2 -5 times higher than in a known environment. 1 tab.

(L

WITH

This invention relates to microbiology, in particular, nutrient media compositions for the cultivation of restriction enzyme producers PSU I.

The purpose of the invention is to increase the yield of the enzyme and its activity.

The essence of the invention lies in the fact that the nutrient medium for the cultivation of the producer of restrictase - Pseudomonas stutzeri strain VKPM 8-4727 contains pancreatic renal hydrolyzate and mineral salts as the nutritional basis and source of amino nitrogen.

o you

Pancreatic renal hydrolyzate is produced by enzymatic hydrolysis of waste (in this case, animal kidney), followed by drying in a spray dryer.

The pancreatic renal hydrolyzate is a light brown powder. It is rich in essential vital elements for normal growth, development of bacteria, manifestation of biosynthetic activity.

Renal hydrolyzate contains,% total nitrogen 11.2; amine nitrogen 6.7 ash 9.7; moisture content 4.1. Also, since the kidneys

TO)

with about

animals contain other biologically active substances (vitamins, microelements, etc.), then when preparing a hydrolyzate, it is enriched with these substances.

Introduction of pancreatic renal hydrolyzate contributes to activating growth, stimulating the viability of bacteria and the accumulation of restriction enzyme activity.

The introduction of ammonium sulfate contributes to the additional nutrition of bacteria with amino nitrogen and sulfur. As a source of carbon nutrition, sodium citrate is introduced into the nutrient medium. As a result of the joint use of sodium citrate and ammonium sulfate, the pH is maintained during the growth of the culture in the optimal range (7.2). Sodium ions, together with iron ions that make up iron chloride, act on the respiratory centers of bacteria.

When a renal hydrolyzate is administered in amounts less than 15.0 g / l (for example, 12.5 g / l), the yield of biomass and enzyme decreases, since there is not enough amine nitrogen for the growth and development of bacteria Pseudomonas stutzeri and, accordingly, for the biosynthesis of Psu I restriction enzymes. When pancreatic renal hydrolyzate is introduced in amounts of more than 20.0 g / l (for example, 2.5 g / l), the yield of biomass and enzyme decreases, since bacteria are repressed and Psu i restriction biosynthesis is produced.

When ammonium sulphate is added in amounts less than 1.0 g / l (e.g., 0.5 g / l), the yield of biomass and restriction enzyme Psu decreases, since the amount of ammonium ions is not sufficient to alkalify the medium and maintain pH. With the introduction of ammonium sulphate in amounts of more than 2.0 g / l (for example, 2.5 g / l), the yield of biomass and Psul restriction enzyme decreases, since bacteria growth and enzyme biosynthesis are inhibited.

When sodium citrate is added in amounts less than 0.5 g / l (for example, 0.4 g / l), the biomass yield of bacteria and restriction enzyme Psu I decreases due to the lack of sodium ions in the medium to activate the cellular respiration of bacteria. The introduction of sodium citrate, in quantities of more than 0.7 g / l (for example, 0.8 g / l) causes a decrease in the biomass yield of bacteria and the restriction enzyme Psu gak as an inhibitory effect on the respiratory centers of cells occurs.

The introduction of ferric chloride in amounts less than 0.001 g / l (e.g., 0.0005 g / l) leads to a decrease in biomass yield and restriction enzyme Psu I, since there are not enough iron ions in the medium to activate cellular respiration of bacteria and chlorine ionosis to nourish the culture. With the introduction of ferric chloride in amounts of more than 0.002 g / l (for example, 0.0025 g / l), the yield of bacterial biomass and restriction enzyme Psu I decreases due to the fact that repressive effects on the respiratory centers occur and an excess of chlorine ions inhibits growth, development and biosynthetic activity of culture,

PRI me R 1. Nutrient medium contains, g / l:

Pancreatic renal

hydrolyzate 20.0

0 ammonium sulphate 2.0

Sodium citrate0.7

Ferric chloride0,002

VodaDo 1.0 l

The revitalization of the Pseudomonas 5 stutzeri 131 VKPM B-4727 strain was carried out on dry nutrient agar (dry nutrient agar 35.0 g / l; water 1.0 l) in petri dishes. Incubated in a heat chamber at 28 ° C for 24 h,

0 From the surface of the agar in Petri dishes the culture was transferred with a bacteriological loop on the reclining flasks with the nutrient medium of the given composition, pH 7.2.

Grow seed with 5 28 ° C for 18 h on the rocking chair.

Fermentation of the culture is carried out on a nutrient medium of this composition at a pH of 7.2, a temperature of 28 ° C, stirring at 200 rpm, air supply: 2 volumes of air 0 per volume of nutrient medium, grown until the optical density of the bacterial suspension 1 is reached, 7 opt. (FEC, filter number b, cuvette 0.5 cm).

Biomass yield 13.0 g / l of culture 5 liquid

The output of restrictase Psu 15000 units, / g biomass; 195000 units / g of culture fluid.

Gel electrophoresis is used to determine enzyme activity.

Enzyme activity is determined in

coarse bacterial biomass extract

Pseudomonas stutzeri 131 VKPM B-4727 and

at the end of the selection (after chromatography on

55 column with phosphoclusus).

Per unit of PsuI restrictase activity is taken as the minimum amount of enzyme required to fully digest 1 µg of phage A DNA for 1 hour at 37 ° C.

Enzyme extraction is carried out as follows.

50 g of biomass of Pseudomonas stutzeri bacteria are suspended with 100 ml of 10 mM Tris-HCl buffer, pH 7.2, containing 10 mM 2-mercapto ethanol, 1 mM sodium azide, 1 mM Trilon B (buffer A), and made up with the same buffer to the volume of 150 ml. 1.5 ml of 10% Triton X-100 and 150 mgl of lysozyme are added to the suspension. The lysis is carried out for 1 hour with constant stirring. Further fractionation of the lysatz is carried out in a two-phase separation system.

The resulting lysate is added to a mixture consisting of 37.0 ml of a 15% dextran T-500 aqueous solution, 56 ml of a 30% aqueous solution, polyethylene glycol (PEG) and 35 ml of 4.0 M sodium chloride (pH of the mixture 6.5). Mix thoroughly for 15 minutes and centrifuge in a Hlqh speed centrifuge (20000 g for 20 minutes). The top PEG-phase with a volume of 120 ml containing the enzyme is taken, diluted with twice the volume of buffer A, triton X-100 (0.1%) is added and applied to a phosphocellulose column equilibrated with buffer A containing 0.1 % Triton X- 100 (Buffer B). The enzyme is eluted with a 0-1.0 M linear gradient of sodium chloride concentration in buffer B. The fractions containing the restriction enzyme Psu i are concentrated by dialysis against buffer B containing 55% glycerol.

A purified Psu I enzyme is obtained in the form of a clear, colorless liquid.

Psu I restriction enzyme cleaves phage A DNA into 3 sites.

Content of nonspecific exo-and adonucleases: after isolation, none. Stored restrictase Psu i at -10 ° C for 1 year.

PRI mme R 2. Nutrient medium contains, g / l:

Pancreatic renal hydrolyzate17,5

Ammonium sulphate1,5

Sodium citrate0,6

Ferric chloride0.0015

VodaDo 1.0 l

Growing biomass: adapting the culture, inoculating the inoculum, fermenting the culture is carried out as in Example 1.

Biomass yield 12.75 g / l of culture fluid.

The output of restrictase Psul 12500 units / g of biomass; 160000 units / l of culture fluid. The activity determination, isolation of the enzyme Psu i, and storage is carried out analogously to example 1.

PRI me R 3 Nutrient medium contains, g / l:

Pancreatic renal hydrolkat15,0

5 ammonium sulphate 1.0

Lemon Sodium 0.5

Ferric chloride 0.001

VodaDo 1.0 l

Cultivation of biomass: adaptation of culture 10, inoculation of seed, culture fermentation is carried out analogously to example 1,

8b biomass stroke 12.5 g / l of culture liquid.

15 Output restriction Psu I 1COOO units / V biomass; 125,000 units / l of culture fluid.

Determination of activity, excretion; Enzyme Psu I and storage is carried out 0 as in Example 1.

EXAMPLE 4 Nutrient medium contains, g / l:

Pancreatic renal hydrolieat12,5

5 Ammonium sulphate 0.5

Lemon Sodium0.4

Ferric chloride0,0005

VodaDo 1.0 l

Growing the biomass: adapting the culture, inoculating the seed, fermenting the culture is carried out as in Example 1.

Biomass yield 5.1 g / l of culture liquid.

5 Exit restriction enzyme Psu I 2300 units / g bio-mass; 11730 units / l of culture fluid. The determination of the activity, isolation of the enzyme Psu I, and storage is carried out as in Example 1.

0P e m i r 5. The nutrient medium contains, g / l:

Pancreatic renal hydrolyzate22,5

Ammonium sulfate2.5

5Sodium citrate0.8

Ferric chloride0.0025

Water Up to 1.0 l

Growing biomass: adaptation. culture, inoculation of seed 0-a, fermentation of the culture is carried out as in example 1.

Biomass yield 5.7 g / l of culture liquid.

The output of restrictase PSU I 3100 units / g 5 biomass.

Activity determination, isolation of the Psu I enzyme and storage is carried out as in Example 1.

Example The table presents data on the comparative analysis of the cultivation of the producer strain Pseudomonas stutzert VKPM B-4727 on the known and proposed media.

When grown on the proposed medium of the restriction enzyme producer Psu I - Pseudomonas stutzeri VKPM B-4727, the biomass yield of bacteria increases 2 times, and the yield and activity of restriction enzymes increase 3-5 times

Claims (1)

Invention Nutrient medium for cultivation of the strain Pseudomonas stutzer VKPM B-4727, a producer of the restriction enzyme Psu I, including an amine nitrogen source, a nutrient medium Known Content, g / l Peptone 10.0 Sodium Chloride 5.0 M sleep water 1.0 l Biomass yield B, 3 g / l of culture fluid Restriction enzyme yield 6000 units / g of biomass; 37800 units / l of culture fluid 0 five non-saline salts and water, so that, in order to increase the yield of the enzyme and its activity, the medium contains pancreatic renal hydrolyzate as a source of amino nitrogen. and as mineral salts, ammonium sulphate, sodium citrate and iron chloride in the following ratio of components, in g / l: Pancreatic renal hydrolyzate15,0-20,0 Ammonium sulfate 1.0-2.0 Sodium citrate0,5-0,7 Ferric chloride 0.001-0.002 Water up to 1 l Offered Pancreatic renal hydrolyzate 20 Ammonium Sulphate 2.0 Lemon Sodium 0.7 Ferric chloride 0,002 Water 1.0 l 12.5-13.0 g / l of culture fluid 10,000-15,000 units / g of biomass; 125000-195000 units / l of culture fluid