SU1402615A1 - Nutritive medium for cultivating klebsicella pneumonial bacteria vkpm v-1826 as producer of restrictase - Google Patents

Abstract

This invention relates to microbiology and can be used in the production of the restriction endonuclease Kpnl from biomass for work in biotechnology and biochemistry. The aim of the invention is to increase the biomass yield. The components of the nutrient medium were selected experimentally in two stages using a computer according to the fractional factor planning program for DFE-2. The concentrations of the medium components, in wt%, were proposed: glucose 0.6-0.65; peptone 1.6-1.7; yeast extract 1,2-. 1.3; disubstituted phosphoric acid sodium 0.85-0.87; ammonium chloride 0,37-0,40; magnesium sulfate salt 0.02; distilled water - the rest. The nutrient medium provides a high yield of raw biomass (L-ZO g / L) and enzyme (T-Yu units per 1 g of raw biomass). 2 tab. S (l

Description

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This invention relates to microbiology and can be used in the production of restriction endonuclease from biomass for biotechnical work and biochemistry.

The purpose of the invention is to increase the biomass yield.

Prepare a nutrient medium based on I l as follows. Peptone and yeast extract are weighed and dissolved in 200 ml of water, boiled for 15-20 minutes, cooled, the pH is adjusted to 7.0-7.2 and the precipitate is separated in a centrifuge at 6000 rpm for 20 minutes. The supernatant is drained; to the measuring vessel, add salt, glucose, water to the mark. The pH of the medium is checked, adjusted to 7.0-7.2 if necessary, and sterilized at a pressure of 1 atm for 30 minutes.

The results of cell culture in media 1-4 are presented in Table. one.

As can be seen from the data table. 1, after 5 cells grow as follows. In environments 2 and 3 with a lower concentration of components, growth is worse than the beginning of starvation of cells, and medium 4 with a higher concentration of components does not improve growth in relation to the growth of cells on medium 1. Consequently, the selected concentration of components of medium 1 is optimal.

; Example. To compare the output of the raw biomass, parallel cultivation of K.pneumoniae COK8) VKPM B-1823 is carried out in the proposed medium and control medium (known). The proposed environment, g / l: glucose 6.5; nienTOH 17; yeast extract 13; IfejHPO ,. 8.7; NH4C1 4.0; MgS04-7H, 0 0.02, distilled water to 1 l. Wednesday (control), g / l: glucose 1; NaCl 10.0; yeast extract (Difso) 5; bacto-peptone (Difeo) 10, distilled water to 1 l, pH of the medium 7.0. The cultivation is carried out in 750 ml Erlenmeyer flasks in 200 ml of medium at 3-7 seconds with a swing of 200250 rpm. The optical density of the cell suspension is expressed in optical units (i.e.) and measured on a KFK-2 photoelectric calorimeter instrument at a wavelength of 540 nm in a cuvette 3.05 mm thick. Samples are taken simultaneously.

The results are presented in table 2.

According to the results of table. 2 that cell growth on the proposed medium in

two times better than the famous. In addition, experiments have shown a direct correlation between cell growth and enzyme accumulation, so cells are precipitated from the culture fluid by the end of the logarithmic growth phase. The yield of raw biomass on the proposed medium is 3-5 times higher than on the known one. Consequently, the total yield of the enzyme. The concentration of the Kpnl restriction endonuclease in the cell extract load after the destruction of the cells by ultra sound is r l-18-l o sm, per 1 g of raw biomass. The minimum amount of enzyme required for complete hydrolysis of 1 μg of phage DNA at 37 ° C in 15 minutes was taken as a unit of activity. Restriction endonucleases derived from biomass are valuable drugs used for scientific research, they are subject to high requirements for activity and purity.

Thus, the nutrient medium includes the components used in the microbiological industry. The combination of the components in the proposed medium, selected according to the result of a two-stage fractional-factor experiment on a computer, makes it possible to obtain K.pneumoniae OK8 cells in an amount of 20-30 g per 1 l of medium.

Claims (1)

Invention Formula The nutrient medium for cultivating the bacteria Klebsiella pneumoniae VKPM B-1826 - a producer of the restriction enzyme Kpnl, containing glucose, peptone, yeast extract, a mineral source of sodium and chlorine, and distilled water, so that in order to increase the yield of biomass, the supplement also contains a syrme. magnesium, as the source of sodium - disubstituted sodium phosphate, and the source of chlorine - ammonium chloride, in the following ratio of components, wt,%: Glucose Pepton Yeast Extract Ammonium Chloride 0,60-0,65 1,6-1,7 1.2-1.3 0.37-0.40 0.85-0.87 0.015-0.020 Else Table 1 The results of the cultivation of K.pneumoniae OK8 Optical density, optical units, with cultivation time, h 10.28 0.7 2il 2.7 3.7 20.27 0.7 1.9 2.3 2.8 30.26 0.7 1.6 1.7 2.5 40.27 0.7 2.1 2.7 3.2 one . Table 2 The concentration of K.pneimoniae OK8 cells on the proposed medium and the known Time Optical Density; Output of Raw Bio Growth; Capacity of Mass Culture, g h (o.ei) in sredet j-precontracted contractive role-played role played 21,11,1 4,53,11,943 84,62,964 248,04,020-306-7 f Compiled by I. Privalov Editor N. Kishtulinets Tekhred A. Kravchuk Proofreader S. Shekmar Order 2826/18 Circulation 520 Subscription VNIIPI USSR State Committee for inventions and discoveries 113035, Moscow, Zh-35, Raushsk nab., 4/5 Production and printing company, Uzhgorod, st. Project, 4