SU1395673A1 - Method of producing immobilized luminescent bacteria photobacterium fischeri strain 6 - Google Patents

Abstract

The invention relates to the field of polymer chemistry and biotechnology and can be used in analytical chemistry to determine the acceptance of organic compounds in solutions. The purpose of the invention is to increase the duration of use of ani immobilized cells by increasing the time of their luminescence. luminescent bacteria Photo-bacterium fisheri strain 6 grafted with a copolymer of a synthetic polymer and (meth) acrylic acid chloride containing 50-170 mCa% of grafted oligochlorine with molol 1000 1600, at the rate of 1 ml of suspension containing 10 to 10 cells per 1-2 cm of graft copolymera. The luminescence time of the target product is 30-40 h 1 tabLo i SL

Description

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The invention relates to the chemistry of polymers and biotechnology, in particular, to the method of covalent immobilization of cells, and can be used in the S technique, in particular to create enhanced de-wipe sensors for determining various impurities in solutions based on immobilized marine luminescent cells. tsentih bacteria PhotobacteriuM fishe ri (CMPB) about

The purpose of the invention is to increase the duration of the use of immobilized cells by increasing the luminescence ipx time.

I The method consists in covalent immoisation of cells by treating their aqueous suspension with an acylating agent VOM; cells of marine luminescent bacteria Photobacterium flsheri strain b are used as cells; as an acylorrheic agent, an organic polymeric polymer and an acid (meth) acrylic acid acid copolymer (meth) acrylic acid copolymer is synthesized with a synthetic polymer and an acid (meth) acrylic acid acid copolymer (meth) acrylic acid; -170 wt.% Grafted oligochloride hydride with a molecular mass of 1000-1600 and the cell suspension formed from isoTKy are 1 ml of the suspension containing 10-10 cells 1-2 cm over the top of the graft copolymer; The preparation of immobilized JTOK (luminescent polymer material) is carried out in two stages:

In the first stage, a copolymer of a synthetic copolymer and an acid chloride of (meth) acrylic 1 DISK is obtained under the action of γ irradiation from the gas phase with a dose of 2.0-5.0 Mrado

In the second stage, a second) grafted copolymer is used: aqueous suspension of cells with a concentration of 10–10 cells / ml of suspension. The choice of this concentration range of cell suspension is due to the number of cells injected onto the surface (and, does not change at a higher concentration in the suspension; when the concentration of the cell suspension is less than iO cells / mp, the surface of the graft copolymer is not saturated with the immobilized (patches,

The grafting of the graft copolymer with CMPB suspension is carried out by immersing the graft material in a cell suspension in 0.05 M phosphate buffer.

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containing 0.3 M sodium chloride (pH 7.8) at 16–20 h at the rate of 1 ml of cell suspension per 1 t 2.

z cm graft copolymer, so that its entire surface is immersed in a cell suspension

The following classes of polymers can be used as starting polymers: polyolefins, polyesters, polyamides, polyacrylates, etc. Any form of polymers can be used; films, fibers, granules, complex profiles and polymer blocks, powders

The method allows to increase the duration of use of immobilized cells by increasing their luminescence time to 30-40 hours (instead of 8 hours for intact cells and less than an hour for immobilized in a known manner)

Example 1o A defatted ethanol film of polyethylene (PE) with an area of 100 cm is placed in a special vial with an overflow, in the lower part of which is poured 1 ml of methacrylic acid chloride (MAX) so that the liquid does not come into contact with the polymer. The ampoule is evacuated to a residual torr pressure, sealed and irradiated with Jf radiation (° C radiation source, GURH type installation) with a dose rate of 0.5 Mrad / h to a total dose of 2.5 Mrad During irradiation, the lower part of the ampoule is shielded with lead protection to prevent homopolymerization acid chloride

After irradiation, the ampules are opened, the film is washed with absolute ether to remove unreacted chporanhydride and dried. The amount of grafted MAX with an average chain length of 16 monomer units (molecular weight 1600) is 4.40 10 g / cm of the film surface

The graft copolymer is incubated for 20 hours at 4 ° C in a suspension of CMPP of Photobacterium fisheri strain 6 in 0.05 M phosphate buffer containing 0.3 M sodium chloride (pH 7.8) at a concentration of 10 cells / mp of suspension. Grafted 1 cm films take 1 ml of cell suspension so that the entire surface of the film is covered with liquid

after immobilization, the grafted film is washed with M phosphate buffer, also containing 0.3 M sodium chloride (pH 7.8), until the washings of the wash water stop, which is determined on the photometer by the luminescence intensity of non-immobilized washed cells, placed in a thermostatted cell (24 C) constant mixing and measuring the luminescence intensity on a photometer with a photomultiplier IQ calibrated in the wavelength range of 480-500 nMo The time for which the luminescence intensity decreases by 50% (the luminescence time of the luminescent material) was 38 hours. Time 15.0 grams of the grafted oligomer per cm of luminescence of the intact control suspension (or 8.80 g per 2 cm). Time of life

film minescence instantly drops to oh

Example 12о All operations were carried out as in Example 11, adding maso% acrylamide. The intensity of the luminescence instantly drops to O

Example 13 All operations were carried out as in Example 1, using a cell suspension containing 10 million cells / ml, taken in an amount of 50 m for a total film area of 100 cm, is 1 ml of the suspension per 2 cm of the film surface. The film contains

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g

, 0 g grafted oligomer per cm (or 8.80 g per 2 cm). Time of life

film minescence instantly drops to oh

Example 12о All operations were carried out as in Example 11 by adding maso% acrylamide. The intensity of the luminescence instantly drops to O

Example 13 All operations are carried out as in Example 1, using a cell suspension containing 10 ppm cells / ml, taken in an amount of 50 ml per 100 film total area, 1 ml of suspension per 2 cm film surface. The film contains

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cells under the same conditions - 8 h.

Examples 2-8 All operations are carried out analogously to Example 1 o.

The data of the experiments on the examples are given in the table.

PRI me R 9o The PE film not subjected to the graft copolymerization with MAX was incubated for 20 hours at 4 ° C in suspension of MPCB (conditions are similar to Example 25) and the luminescence time of the sorbed cells is determined. The luminescence time is 9 hours, after which almost all of the immobilized cells, non-covalently (sorbed), were washed from the surface of the polymeric material.

Example 10 (comparative prototype). To suspension 10 ml KMLB

39 Cho cells

Example 14. All operations were carried out as in Example 1, using a suspension containing 10 million cells / ml in an amount of 50 ml per 100 cm of surface (or 1 ml per 2 cm). Cell life time 32 hours

Example 15 (control) All operations were carried out as in example 1, using 4 ml of cell suspension with a cell content of 10 mln / ml per 2 cm of the film surface. The cell lifetime is 40 hours. Toe does not increase with an increase in the ratio of the volume of cell suspension: i - 2 cm of surface.

Example 16 (control). All operations are similar to those of the example.

in 0.05 M phosphate buffer containing 35 °, a suspension with a concentration of 10 mln cells / 2 cm of surface in an amount of 1 ml is used. Candle time is 12 hours

0.03 m of sodium chloride (pH 7.8) containing 10 cells / ml was added with continuous stirring at 4 ° C with 100 µl (0.01 g) MAX, the mixture was brought to room temperature, added 1% g AA, 0.1 g BIS and polymerized under the action of the system potassium persulfate - potassium metabisulfite for 1 hour. A gel with 45 co-folded cells is obtained in the form of a covalently immobilized cell 45 KMLB in the form of a film. The intensity of the luminescence of a polymeric material with a covalently immobilized by this method KMLB is O, the Drug does not possess the properties of a luminescent polymeric material, therefore its luminescence time cannot be determined.

Example 11, A film with immobilized gg of CMLP is placed in a thermostated cell (temperature 24 ° C) and 10 mol of dioxane is added to the phosphate buffer. Luni cell intensity

Example 17 (control) All operations are carried out as in Example 1, using a ratio of 0.5 ml of suspension with a concentration of 10 mln / ml per 1 cm of surface. The luminescence time is 40 h, t, e, increasing the concentration of the suspension above 10 mln cells / ml does not lead to an increase in lifetime.

The average value of the surface area of the films varies from 1 to 100. Large areas cannot be placed in an irradiation unit of GURH type. The surface area of the polyamide powder in Example 6-10.

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ml

suspensions b

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containing 10 in 1 ml of intact cells, an aqueous solution of isopropanol is added and the change in the

39 Cho cells

Example 14. All operations were carried out as in Example 1, using a suspension containing 10 million cells / ml in an amount of 50 ml per 100 cm of surface (or 1 ml per 2 cm). Cell life time 32 hours

Example 15 (control) All operations were carried out as in example 1, using 4 ml of cell suspension with a cell content of 10 mln / ml per 2 cm of the film surface. The cell lifetime is 40 hours. Toe does not increase with an increase in the ratio of the volume of cell suspension: i - 2 cm of surface.

Example 16 (control). All operations are similar to those of the example.

 ° using a suspension with a concentration of 10 mlno cells / 2 cm surface in an amount of 1 ml. Time candle 12h,

no cells

Example 17 (control) All operations are carried out as in Example 1, using a ratio of 0.5 ml of suspension with a concentration of 10 mln / ml per 1 cm of surface. The luminescence time is 40 h, t, e, increasing the concentration of the suspension above 10 mln cells / ml does not lead to an increase in lifetime.

The average value of the surface area of the films varies from 1 to 100. Large areas cannot be placed in an irradiation unit of GURH type. The surface area of the polyamide powder in Example 6-10.

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e p

ml

suspensions b

18, K 1

containing 10 in 1 ml of intact cells, an aqueous solution of isopropanol is added and changes are recorded

luminescence intensity of cells depending on alcohol concentration. Full luminescence quenching is observed at a final inhibitor concentration of (159-0.1) 10 mol / l

Example 19. A 1 cm film (prepared according to Example 3) containing 10 immobilized cells was placed in a solution containing isopropanol and recorded a film glow depending on the alcohol concentration. A conical inhibition of luminescence was observed in the polymeric luminescence quenching / mol.

13956736

bilized CMLB with functional activity and an increased cell luminescence time (up to 30-40 h), the target product can be used to indicate organic impurities in solutions.

Analogous (2,140, O-10Ny cells are sensitive, obtained in examples 1-2, 4-8, therefore, the sensitivity of int ct and covalently immobilized

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Claims (1)

Invention Formula The method of obtaining immobilized luminescent bacteria Photobacterium um fischeri step 6, including treating the aqueous suspension of bacteria with an organic acylating agent, carrying out the immobilization using an organic polymer, separating the obtained cells from the reaction tichesically identical mixtures that differ from KJneTOK to hydrophobic molecules of the practical 20 in order to increase the duration of use of immobilized cells by increasing the time of their luminescence, immobilization is carried out by treatment with an acylating reagent, which uses a synthetic polymer and acid chloride (meth) as a polymer acrylic acid containing 50-170 masd by mass of grafted ; Thus, both native and AND: immobilized cells are about | synakno sensitive to hydrophobic compounds The yield of luminescent cells from immabilization by the proposed method is 60-70% of their initial amount, which is determined by the DZ by counting the cells in the initial and final suspensions. i Thus, the invention allows to obtain luminescent polymethyl materials with covalently immuno-oligochloride hydride, mol. 1000 - 1600 Dilton, and the processing of cell suspension is carried out at the rate of 1 ml of a suspension containing from 10 to 10 cells per 1-2 cm of the surface of the graft copolymer Polyethylene film Popietipenterefapat, film PP Nylon-6, EOLOKKO Polusmetip-force sa, MAX OH Kah MAX OH Poliemml t graiulk d "with an meter 0.1 MP AH 2.54.40501616001,01038 3.04.93781311001, 5.05.941701010001.01o 30 .3.05,02.831414000, 2.54.26561515000, 2.54,43611515000, Invention Formula The method of obtaining immobilized luminescent bacteria Photobacterium um fischeri step 6, including treating the aqueous suspension of bacteria with an organic acylating agent, carrying out the immobilization using an organic polymer, separating the obtained cells from the reaction oligochloride with mol.m. 1000 - 1600 Dilton, and the processing of cell suspension is carried out at the rate of 1 ml of a suspension containing from 10 to 10 cells per 1-2 cm of the surface of the graft copolymer The physicomechanical properties of ns-xo-nsk polymers and modified polymers are almost identical. OH M & X 2.0 2.93 Z5 6.0 7.35200 Mala efficiency imnob leasht. Destruction of the polymer. Polia (aa 9 cells sorption) Continuation 1800 2.5 8901.0 O1,0 ten 10 1-8 , 2-Jfr 9