RU2360967C1 - Immobilised bacteria cells biocatalyst for decomposition of methylphosphonic acid and its esters - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention represents a biocatalyst containing immobilised bacteria cells included in polyvinyl alcohol cryogel, used to decompose methylphosphonic acid and its esters. The biocatalyst contains the following components (wt %): bacterial cells biomass - 0.125÷0.725, polyvinyl alcohol cryogel - 7.6÷12.8, aqueous phase - up to 100. Polyvinyl alcohol cryogel is formed in air. As individual bacterial cultures, Pseudomonas species 78G, Escherichia coli BN5α/pTrcTE-OPH, Escherichia coli SG13009 [pREP4]/pTES-His-OPH are used as able to degrade organophosphorous compounds with C-P-bonding. Methylphosphonic acid decomposition rate is 7.3-18 mg/l/h, while its esters are decomposed at 9.6-21.4 mg/l/h throughout 245-310 days.

EFFECT: biocatalyst is characterised with high rate of methylphosphonic acid and its esters decomposition over a long period of time.

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Description

The invention relates to biotechnology, and is a biocatalyst based on immobilized bacterial cells included in a matrix of gel media, with the help of which microbiological decomposition of methylphosphonic acid and its esters is carried out.

Methylphosphonic acid (IFC) and its esters are products of chemical detoxification of organophosphorus toxic agents (FOB), namely sarin, soman and Vx, which belong to the group of organophosphorus chemical warfare agents of nerve-paralytic action [Committee on Review and Evaluation of International Technologies for the Destruction of Non-Stockpile Chemical Materiel, National Research Council, Review of International Technologies for Destruction of Recovered Chemical Warfare Materiel. Washington, DC: National Academies Press, 2006, 128 p.], Whose stocks must be destroyed in accordance with the requirements of the International Convention on the Prohibition of the Development, Production, Stockpiling and Use of Chemical Weapons (adopted by the State Duma on 10/31/1997). A specific feature of the elimination of stockpiles of toxic substances, in accordance with the requirements of the Convention, is the need for additional processing of primary detoxification products to a state that excludes the possibility of their use for re-synthesis of starting materials. Also, along with the FOB itself, the primary products of their detoxification (IFC esters and IFC itself) have a certain toxicity [Munro NB, Talmage SS, Griffin GD, Waters LC, Watson AP, King JF, Hauschild V. The sources, fate, and toxicity of chemical warfare agent degradation products.// Environ. Health Perspect., 1999, V.107 (12), p.933-974.], Which necessitates their decomposition.

When implementing chemical methods for the decomposition of IFC esters, alkaline hydrolysis is carried out using highly concentrated solutions of potassium or sodium hydroxides with the addition of solutions of various salts [RF Patent No. 97100230, 1999. Method for the disposal of a poisonous substance of type Vx].

The disadvantage of this method is the need to use corrosion-resistant equipment, high temperatures (80-400 ° C) and compliance with long time intervals of exposure of the reaction masses (up to 3 months).

It should be noted that the CP bond is characterized by extremely high resistance to thermal degradation and chemical decomposition, even in the presence of strong acids and bases, and therefore chemical hydrolysis does not lead to the destruction of the CP bond in the molecule of MPC itself. Moreover, the breakdown of the CP bond is the main indicator of the effective destruction of toxic substances, since as a result of the breakdown of the CP bond, the hydrolysis process becomes irreversible.

Today, biotechnological methods for the decomposition of esters of IFCs and IFC itself, based on the ability of microorganism cells to use the energy of various chemical bonds present in the structure of substances, including toxic ones, are attracting great attention [Attaway N., Nelson JO, Baya AM, Voll MJ, White WE, Grimes DJ, Colwell RR Bacterial detoxification of diisopropyl fluorophosphate.// Appl. Environ. Microb., 1987, V. 53 (7), p. 1685-1689; DeFrank J.J., Cheng T.C. Purification and properties of an organophosphorus acid anhydrase from a halophilic bacterial isolate. // J. Bacfer W., 1991, V. 173 (6), p. 1938-1943; Konsaku M.A. Microbial carbon-phosphorus bond cleavage enzyme requires two protein components for activity.//J Bacteriol., 1989, V. 171 (8), p. 4504-4506; Osamura N., Murata K., Kimura A. Cytosolic C-P bond cleavage activity in bacterial cells isolated from soil.//./ J. Ferment. Bioeng., 1991, V.71 (2), p. 128-130; Pipke R., Amrhein N. Degradation of the phosphonate herbicide glyphosate by Arthrobacter atrocyaneus ATCC 13752.// Appl. Environ. Microb., 1988, V. 54 (5), p. 1293-1296]. Biotechnological methods are environmentally friendly, based on the functioning of intracellular microbial enzymes acting as biological catalysts, and compares favorably with chemical methods of decomposition of toxic substances in that:

- provide a break in the chemically stable CP bond,

- are realized at mesophilic temperatures (ambient temperatures);

- do not require the use of concentrated alkaline solutions and equipment made of special corrosion-resistant material.

A number of methods are known for biodegradation of alkaline hydrolysis products of sarin, soman and Vx, based on the use of complex compositional artificial multicomponent free cell consortia of various microorganisms [US Patent No. 6599733 B1 (2003); U.S. Patent No. 7,001,758 B1 (2006) Microbial biodegradation of phosphonates. C12N 1/20, B09B 3/00], providing decomposition of the esters of IFC or IFC itself. The composition of these consortia includes various bacterial cultures represented by many different strains (Methylobacterium radiotolerans strain GB21, Agrobacterium tumefaciens strain GB2GA, Klebsiella oxytoca strain GB2CS and GB272, Aureobacterium sp. Strains GB2, GB23, GB272 and 29. Before using them for the direct decomposition of methylphosphonic acid isopropyl ester (IPEMPK) at a concentration above 5 mM, the preliminary adaptation of the consortium cells under aerobic conditions is carried out with constant stirring (150 rpm) for 12 days at 28-29 ° C in a phosphate-free medium, prepared on the basis of 50 mM HEPES buffer (pH 7.2) and containing 2.7 mM (329.4 mg / l) IPEMPK, 0.5 g / l KCl, 0.5 g / l (NH ₄ ) ₂ SO ₄ , 10 ml / l of a standard solution of trace elements Wolin Salts (Sigma, USA). Next, the cells are transferred to a medium of a similar composition containing 2.4-6.0 mM IPEMPA, and the conversion of the MPA ester to MPA is carried out by 100% for 14 days. To decompose accumulated MFCs (230.4 mg / l) and to utilize the decomposition product of MFCs - phosphate ions - a carbon source (20 g / l corn syrup) is introduced into the medium, and after 5 days of cultivation of the consortium under the same conditions as above (pH, temperature, medium stirring), a 90-92% degree of decomposition of a broadcast with a CP bond is achieved. The functioning time of such a biocatalyst is 14 days.

This biocatalyst, which is a consortium of free bacterial cells, designed for the decomposition of MPC and its isopropyl ether, has a number of significant disadvantages, which are that:

- it provides low decomposition rates of IPEMPK and IFC, which, according to the developers of the biocatalyst, are 0.87 mg / l / h and 1.77 mg / l / h, respectively;

- it is necessary to control the constancy of the complex composition of the microbial consortium in order to ensure high efficiency of its action, despite the fact that the exact composition of the biocatalyst (concentration of various cells) is not indicated by the developers;

- Preliminary long-term adaptation of cells to toxic substrates is required (12 days);

- it is necessary to utilize spent bacterial biomass and cultivate cells in order to accumulate biomass and prepare on its basis a new portion of the biocatalyst in the form of a consortium after the completion of each cycle of the IFC decomposition process.

The use of immobilized cells in the implementation of many biotechnological processes makes it possible to use the same cells of microorganisms many times and, thus, to avoid the need to solve the problem of continuous utilization of accumulated biomass. In addition, the use of cells in an immobilized form allows to increase their resistance to various adverse factors (high concentrations of toxic substrate, changes in pH, etc.) and to ensure a longer and more efficient process with their participation [Nedovic V., Willaert R. Fundamentals of cell immobilization biotechnology // Kluwer Academic Publishers, 2004, 555 p.].

The main characteristics of any immobilized biocatalyst used to decompose the primary products of FOB hydrolysis are:

- the rate of decomposition of the toxic substrate, which determines the total duration of the process;

- the degree of decomposition of the toxic substrate (%);

- the presence / absence of the need to prepare the biocatalyst for its use for the decomposition of toxic substrates and the duration of such preparation;

- the duration of the possible effective use of the biocatalyst in the target process.

It is advisable to use these basic characteristics for a comparative analysis of known biocatalysts developed on the basis of immobilized cells of various microorganisms for the decomposition of MPA and its esters.

Known immobilized biocatalyst for the decomposition of IFC esters, which is activated sludge, namely, a collection of self-selected self-immobilized cells of various micro and macroorganisms, which folds up in river water treatment plants [Harvey S.P., Carey L.F., Haley M.V., Bossle PC, Gillitt N.D. Bunt Sequencing batch reactor biodegradation of hydrolyzed sarin as sole carbon source // Bioremed J., 2003, V.7 (3-4), p.179-185]. The composition of such an immobilized biocatalyst is not known.

To activate the immobilized biocatalyst in the form of activated sludge and use it for the decomposition of IPEMPK and IFC in alkaline hydrolysates of sarin, its preliminary adaptation is carried out for 74 days with a gradual increase in the concentration of IPEMPK and IFC in the medium to 2.8 g / l and 250.4 mg / g respectively. The use of an immobilized biocatalyst in the form of adapted activated sludge provides 99.6% decomposition of the MPA ester to its residual concentration of 10 mg / l in the medium for 15 days, that is, the IPEMPA decomposition rate is 7.75 mg / l / h, but the biocatalyst it is not capable of decomposing MPA, and an increase in the concentration of this compound in the medium is proportional to the amount of hydrolyzed ether (i.e., tens of times). The biocatalyst can be used to decompose the MPA ester for 200 days in a batch reactor with a 15-day cyclic retention of the medium in the reactor.

The disadvantages of this biocatalyst are:

- the inability of the biocatalyst to decompose MPC, which is a key substance with CP bond, the destruction of which ensures the irreversibility of the decomposition of FOV;

- the need for preliminary long-term adaptation of the biocatalyst (within 74 days) to a toxic substrate,

- the final composition of the immobilized biocatalyst in the form of adapted activated sludge is not known, which makes it impossible to analyze and maintain a stable microbial composition of the biocatalyst and, therefore, the reproducible level of biodegradation activity of the biocatalyst.

Known immobilized biocatalyst based on the artificial microbial bacterial consortium GB2 [DeFrank J.J., Guelta M., Harvey S., Fry I.J., Earley J.P., Lupton F.S. Biodegradation of hydrolyzed chemical warfare agents by bacterial consortia // Netherlands: Kluwer Academic Publishers. In: B. Zwanenburg et al. (Eds.), Enzymes in Action: Green Solution for Chemical Problems, 2000, p. 193-209]. The developed biocatalyst is designed for the decomposition of MPC, IPEMPK and methylphosphonic acid ethyl ester (EEMPK), formed, respectively, during alkaline hydrolysis of sarin and substance Vx, adopted by the US Army.

To obtain an immobilized biocatalyst, blocks of a foamed polyurethane carrier are placed in the reactor (1 l), which are held in the reactor by means of polyurethane hollow cylinders. Next, the reactor is filled with a medium containing 3.8 vol.% Alkaline hydrolyzate of FOV with one of the MPA esters as a source of phosphorus, as well as 1.2 g / l NH ₄ CI, 0.35 g / l KN ₂ PO ₄ and 10 ml / l standard solution of trace elements Wolin Salts, so that the total filling volume is 600-800 ml. Next, a mixture of cells (Methylobacterium radiotolerans strain GB21, Agrobacterium tumefaciens strain GB2GA, Klebsiella oxytoca strain GB2CS and GB272, Aureobacterium sp. Strains GB2, GB23, GB272 and Gb292), which make up the consortium, is introduced into the reactor. Adhesive immobilization of consortium cells on a porous carrier occurs.

Before using the immobilized biocatalyst for targeted decomposition of IFC esters, it is adapted to toxic substrates (the conditions and timing of the adaptation are not indicated by the developers).

Further, an adapted immobilized biocatalyst is used for the decomposition of IFC esters. To do this, alkaline hydrolysates of FOV containing MFK esters diluted 1000 times are introduced into the medium for culturing immobilized cells so that the initial concentration of MFK esters in the biocatalyst reactor is 500 mg / L (~ 4 mM). Additionally, a carbon source is introduced into the medium: glucose, isopropanol, glycerin, molasses or glycerin mixed with molasses (exact concentrations of substances are not indicated).

The maximum degree of decomposition of the esters of IFCs and IFCs under the action of an immobilized biocatalyst is 40% in 15 days, regardless of the concentration and source of carbon introduced into the medium. The biocatalyst can function for 40 days under periodic conditions.

The main disadvantages of this immobilized biocatalyst are:

- a fairly low rate of decomposition of compounds with CP bond under the action of an immobilized biocatalyst, which, according to the developers, is 0.56 mg / l / h;

- the need to use a multicomponent consortium of microorganisms to achieve the indicated maximum degree of degradation and the need to control and maintain a constant complex microbial composition of the immobilized biocatalyst to ensure its effective action;

- the need for adaptation of the immobilized biocatalyst to toxic substrates before use;

- the use to obtain an immobilized biocatalyst of the method of adhesion of the cells that make up the microbial consortium on the surface of the carrier leads to heterogeneity and heterogeneity of the composition of the biocatalyst, since cells of different bacterial strains exhibit, due to the different structure of the cell walls, different adhesive ability to the same material, which affects the efficiency of their retention on the surface of the carrier. Also, with the selected method of immobilization, serious diffusion problems arise for mass transfer processes carried out by adherent cells located in layers located in close proximity to the surface of the carrier.

Another immobilized biocatalyst is known, which is a direct improvement of the above-described biocatalyst based on an immobilized consortium [US Patent No. 6080906 (2000) Demilitarization of chemical munitions, A62D 3/00]. The method for its preparation and the composition of the used GB2 microbial consortium are the same as described above, but cubic granules of expanded polyurethane with a linear size of 1.25 cm containing activated carbon particles included in the polyurethane matrix are used as a carrier for adhesive immobilization of cells. The carrier is also held in the reactor using polyurethane hollow cylinders. The exact composition of the immobilized biocatalyst is not known.

The obtained immobilized biocatalyst (680 ml) provides at 20-25 ° C and pH 6.0-9.0 under aerobic conditions with a liquid volume in the reactor equal to 740 ml, 75% decomposition of organophosphorus compounds (EEMPK and IFC) for 15 days. Thus, this result is achieved at a concentration of immobilized biocatalyst in the environment of 920 g / l. The rate of decomposition of the esters of IFCs and IFCs under the action of this biocatalyst is not known, since the authors of this development do not indicate the initial concentrations of substances necessary to achieve 75% decomposition of substances in the indicated time.

The biocatalyst can be used to treat Vx hydrolysates containing EEMPA and MPA under periodic conditions of reactor operation for 8 working cycles of 15 days, which is 120 days in total.

This biocatalyst has all the main disadvantages indicated for the biocatalyst described above, and yet another one, which consists in the fact that activated carbon particles introduced into the carrier, according to the data of the biocatalysis developers, sorb IFC ether from the medium. As a result, a significant amount thereof remains bound in the carrier and is not degradable.

A known biocatalyst based on a bacterial consortium immobilized in polyvinyl alcohol (PVA) cryogel (Zhang Y., Autenrieth RL, Bonner JS, Harvey SP, Wild JR Biodegradation of neutralized sarin // Biotechnol. Bioeng., 1999, V.64 (2) , p.221-231), intended for the decomposition of IPEMPK, which is a product of alkaline hydrolysis of sarin. To obtain a biocatalyst, a consortium of bacterial cultures isolated from water samples collected at the landfills of the Edgewood Chemical and Biological Center of the US Army is used. The exact microbial composition of the consortium is not known.

Before immobilization, the cells of the consortium for a long time (5 cycles of 7 days) are adapted to media containing a carbon source (glucose, glycerin or sodium succinate), 1.5 g / l NH ₄ Cl, 0.3 g / l KH ₂ PO ₄ , 10 ml / l of a standard solution of trace elements Wolin Salts, 2 mg / l CoCl ₂ , 1 mg / l ZnCl ₂ and IEMPK, while from cycle to cycle the concentration of IEMPK in the medium is increased to a final concentration of 0.35 g / l.

Immobilization of cells adapted to IEMPK is carried out by incorporating bacterial biomass into PVA cryogel granules. For this, the cells in the stationary growth phase are separated by centrifugation from the medium used to adapt them and mixed with the PVA solution so that their concentration in the mixture is 10%. The cell suspension is instilled into a cooled solution of hexane, which leads to rapid cooling of the droplets and the formation of granules with a diameter of 2 mm The obtained biocatalyst granules are thawed and thoroughly washed with 15 mM HEPES buffer from a hydrophobic solvent.

The immobilized biocatalyst provides 85% decomposition of 350 mg / L of IEMPK for 400 h (~ 17 days), as well as the decomposition of 297.5 mg / L of MPC accumulated in the medium over the same period of time by 84.5%. An increase in the processing time does not lead to an increase in the degree of destruction of IEMPK and a decrease in the concentration of MPA in the system. The functioning time of the biocatalyst is 17 days.

The main disadvantages of this biocatalyst are:

- the use of a bacterial consortium of unknown composition, the control and maintenance of which is impossible to maintain the long and effective action of the biocatalyst;

- requires a preliminary long-term adaptation of cells (within 35 days) to toxic substrates before their immobilization,

- the method of immobilization involves the use of hexane as a hydrophobic medium for the formation of biocatalyst granules based on a hydrophilic carrier, which requires a subsequent mandatory step of thorough removal of toxic and hydrophobic solvent from the surface of the granules, since its presence leads to the formation of a hydrophobic film, significantly reducing the availability of dissolved in the medium substances, including oxygen, for cells inside the granules. As a result, immobilized cells quickly lose their viability, and, as a consequence of this, the effectiveness of the biocatalyst is rapidly reduced. The use of hexane, which is highly flammable and toxic to humans, and not just for microbial cells, to obtain an immobilized biocatalyst requires special safety measures when working with it (transportation, storage, use), as well as the need for special treatment of water containing hexane and obtained after washing the granules of the biocatalyst from a hydrophobic solvent;

- a rather low rate of decomposition of compounds with CP bond under the influence of an immobilized biocatalyst, which, according to the developers, is 0.74 mg / l / h for both IEMPK and IFC.

This technical solution, as the closest to the claimed type of carrier used (PVA cryogel), as well as the method of cell immobilization (inclusion in the gel) used to obtain the biocatalyst, is taken as a prototype.

The objective of the invention is to obtain a highly active biocatalyst for the decomposition of MPA and its esters, which are products of chemical detoxification of FOV, namely sarin, soman and Vx, based on cells of individual bacterial cultures immobilized in polyvinyl alcohol cryogel and capable of providing a high rate of decomposition of toxic substrates throughout a long time.

The problem is solved by creating a biocatalyst that contains cells of individual bacterial cultures of Pseudomonas species 78G, Escherichia coli DH5α / pTrcTE-OPH, Escherichia coli SG13009 [pREP4] / pTES-His-OPH capable of degrading organophosphorus compounds with a C-P bond and polyvinyl alcohol included in the cryogel, the formation of which occurs in air, and has the following component composition (wt.%): bacterial cell biomass - 0.125 ÷ 0.725, polyvinyl alcohol cryogel - 7.6 ÷ 12.8, aqueous phase - up to 100.

The decomposition of IFC and its esters using the proposed biocatalyst based on immobilized cells is carried out when it is introduced into a nutrient medium containing the target substance, as well as a carbon source.

The proposed biocatalyst is characterized by a long period of functioning (up to 310 days), and is also capable of decomposing the esters of MPA and MPA at a rate of up to 21.4 mg / l / h and 18 mg / l / h, respectively.

Unlike analogs and prototypes, the cells of individual bacterial cultures are used to obtain the inventive biocatalyst, rather than bacterial consortia, which can significantly simplify the process of producing a biocatalyst in large quantities with well reproducible properties and monitor its condition during operation, as well as easily establish / lack of contamination of the biocatalyst by extraneous microflora.

As individual cultures that form the basis of the claimed immobilized biocatalyst, bacterial cells are used that are highly capable of destroying organophosphorus compounds, for example, Pseudomonas species 78G cells [RF Patent No. 2154103 (2000) Strain Pseudomonas species 78G intended for degradation of degradation products of organophosphorus poisonous substances . C12N 1/20; 9/88], Escherichia coli cells DH5α / pTrcTE-OPH [RF Patent No. 2232807 (2002) Recombinant plasmid DNA pTrcTE-ORN and organophosphate hydrolase enzyme producer, C12N 1/21, 15/52, 15/70], Escherichia coli cells SG13009 [pREP4] / pTES-His-OPH [RF Patent No. 2255975 (2003) Recombinant plasmid DNA pTES-His-OPH and producer of oligo-histidine-containing organophosphate hydrolase, C12N 1/21, 15/52, 15/70]. These cells are producers of intracellular highly active enzymes that catalyze the cleavage of O-P, S-P, F-P, and C-P bonds. Their use in an immobilized form makes it possible to break the target bond, and also to utilize the reaction products formed during this break from the reaction medium. Thus, when using such cells, reaction products are sequentially removed from the reaction medium (upon decomposition of the MPA ester, MPA is formed, which decomposes to phosphoric acid and is absorbed by the cells). When using consortia, all these successive transformations are carried out by different cells of microorganisms, while the vital products of some cells are substrates for others. The diffusion of the metabolites of one cell to other cells as substrates, the bioavailability of such substrates for spatially separated cells significantly affect the rate of decomposition of the main substances as a whole. In the case of highly active monocultures, all these processes are carried out by the same cells, which as a whole allows us to get rid of the mass transfer problems present in consortia and significantly increase the rate of conversion of substances.

To obtain an immobilized biocatalyst, monocultures of bacterial cells are grown on rich nutrient media known for these strains in order to accumulate metabolically active biomass, which is concentrated (concentrated) before incorporation into the matrix of a polymer carrier. This concentration is carried out by known methods, for example by centrifugation.

The use of polyvinyl alcohol (PVA) cryogel cells as a carrier, formed upon freezing and thawing of a polymer solution, is due to the high porosity of the gel matrix, which provides easy diffusion of substrates and products, respectively, to and from immobilized cells, as well as good performance characteristics of PVA-based cryogel [Lozinsky V.I. Cryogels based on natural and synthetic polymers: production, properties and applications // Uspekhi Khimii, 2002, vol. 71 (6), p. 559-585]. Moreover, in the claimed technical solution, biomass immobilization is carried out by suspending it in a PVA solution followed by cryogenic treatment (freezing, keeping in a frozen state and thawing) in an atmosphere of air, rather than a hydrophobic liquid, as in the prototype, under certain conditions, which leads to the formation of durable polymer cryogel containing cells included in its matrix. This completely eliminates all technological and environmental problems, as well as the problems of the safe organization of production, existing when implementing the method for producing a biocatalyst using the prototype using hexane. In addition, during the cryogenic treatment, the resulting immobilized biocatalyst, unlike the prototype, can be given any necessary shape, in particular, the shape of spherical granules, irregularly shaped particles, gel blocks, discs, sheets, tubes, etc. For this, the biomass suspension in the gel solution is frozen in an appropriate form.

The present invention provides for the formation of a PVA cryogel with simultaneous immobilization of cells. The composition of the inventive biocatalyst and the ratio of the components were selected based on the experiments and are unique, providing outstanding characteristics of the biocatalyst.

The actual decomposition of IFC and its esters using a biocatalyst based on immobilized cells consists in the fact that the biocatalyst is introduced into the reactor, where at a pH of 7.5-9.0 the microbial decomposition of toxic substrates is carried out and they are disposed of as a source of phosphorus in the presence of a source carbon, which can be used commonly used in microbiological production of glucose, glycerin, molasses, etc.

A significant difference of the claimed invention is a new, previously unknown composition of the biocatalyst and the combination of its components, namely the type of bacterial monocultures used to create the biocatalyst, the ratio of bacterial biomass and PVA cryogel formed in air, rather than a hydrophobic solvent, in the presence of cells upon receipt of the biocatalyst for the decomposition of IFC and its esters, which were previously not known.

A significant technical result of the claimed invention is the ability of the biocatalyst to decompose MPC and its esters at high speeds with a long life.

The following are specific examples illustrating the claimed technical solution.

Example 1. Biocatalyst based on immobilized cells of Pseudomonas sp. 78G included in the polyvinyl alcohol cryogel for decomposition of MPC.

8.4 g of the biomass of bacterial cells of Pseudomonas sp. 78 G, obtained after separation from the growth medium, initially containing 20 g / l glucose, 4.0 g / l yeast extract, 3.0 g / l (NH ₄ ) ₂ SO ₄ , 5 mg / l CoCl ₂ × 6H ₂ O , 5 mg / l ZnSO ₄ × 4H ₂ O (pH 6.8-7.0), centrifuged (20 min, 8000 rpm), mixed with 167.6 g of an 8% solution of polyvinyl alcohol prepared on the basis of saline (0.9% NaCl), at room temperature until a homogeneous mass. This suspension is then dispensed using a metering device (syringe, dispenser, etc.) into 0.1 ml 96-well immunological plates, which are placed in a freezer at -20 ° C, kept frozen for 17 hours, thawed at + 8 ° C. Get immobilized biocatalyst in the form of granules of a cylindrical shape, having the following composition (wt.%): Biomass of bacterial cells - 0.48 (by dry weight); PVA cryogel - 7.6; aqueous phase up to 100.

The obtained immobilized biocatalyst without any adaptation to a toxic substrate provides 100% decomposition of 485 mg / L of MPA in a 1 L aerobic reactor with a periodic cultivation at 28 ° C with continuous stirring of the medium (180 rpm) for 27 hours upon application in a minimal cultivation medium containing 0.1 g / l MgSO ₄ × 7H ₂ O, 0.5 g / l NaCl, 1 g / l NH ₄ Cl, 1 g / l NaHCO ₃ (pH 8.0), 15 g / l glucose as a carbon source. Thus, the IFC decomposition rate is 18 mg / l / h. The resulting biocatalyst is able to function in such a system for up to 250 days.

Example 2. Biocatalyst based on E. coli cells DH5α / pTrcTE-OPH included in the PVA cryogel for decomposition of MPC.

4.3 g of the biomass of bacterial cells of E. coli DH5α / pTrcTE-OPH obtained after separation from the growth medium, initially containing 10 g / l of tryptone, 5 g / l of yeast extract, 5 g / l of NaCl (pH 6.8), 47.7 mg / l of isopropylthiogalactoside, centrifuged (10 min, 10,000 rpm), is mixed with 171.7 g of a 10% solution of polyvinyl alcohol, prepared on the basis of 50 mm Na-carbonate buffer (pH 10), at room temperature until smooth. The resulting mixture was poured in an even layer on a baking sheet with sides 3 cm high to obtain gel blocks 0.5 cm high, frozen at -15 ° C, kept frozen for 15 hours, thawed at + 10 ° C and, thus, obtain a biocatalyst, having the following composition (wt.%): biomass of bacterial cells - 0.24 (by dry weight); PVA cryogel - 9.76; aqueous phase up to 100.

The obtained immobilized biocatalyst without any adaptation to a toxic substrate provides 100% decomposition of 480 mg / l of MPA in a 1 l aerobic reactor with a periodic cultivation mode at 28 ° C with continuous stirring of the medium (200 rpm) for 45 hours upon application to a minimal culture medium containing 0.1 g / l MgSO ₄ × 7H ₂ O, 0.5 g / l NaCl, 1 g / l NH ₄ CI, 1 g / l NaHCO ₃ (pH 8.0), 5 g / l glucose as a carbon source. Thus, the IFC decomposition rate is 10.7 mg / l / h. The resulting biocatalyst is able to function in such a system for up to 280 days.

Example 3. Biocatalyst based on cells of Pseudomonas sp. 78G included in the PVA cryogel for the decomposition of IFC and IFC isobutyl ether.

8.4 g of the biomass of bacterial cells of Pseudomonas sp. 78 G, obtained after separation from the growth medium, initially containing 20 r / l glucose, 4.0 g / l yeast extract, 3.0 g / l (NH ₄ ) ₂ SO ₄ , 5 mg / l CoCl ₂ × 6H ₂ O , 5 mg / l ZnSO ₄ × 4H ₂ O (pH 6.8-7.0), centrifuged (30 min, 6000 rpm), mixed with 167.6 g of a 10% solution of polyvinyl alcohol prepared on the basis of saline (0.9% NaCl), at room temperature until a homogeneous mass. This suspension is then dispensed using a metering device (syringe, dispenser, etc.) into 0.15 ml 96-well immunological plates, which are placed in a freezer at -10 ° C, kept frozen for 24 hours, thawing is carried out at + 8 ° C. Get immobilized biocatalyst in the form of granules of a cylindrical shape, having the following composition (wt.%): Biomass of bacterial cells - 0.45 (by dry weight); PVA cryogel - 9.5; aqueous phase up to 100.

The obtained immobilized biocatalyst without any adaptation to toxic substrates provides 100% decomposition of 380 mg / l of MPA isobutyl ether (IBEMPA) in a 1 L aerobic reactor with a continuous cultivation mode at 30 ° С while stirring the medium with bubbling for 31 h when introduced into minimum cultivation medium containing 0.1 g / l MgSO ₄ × 7H ₂ O, 0.5 g / l NaCl, 1 g / l NH ₄ Cl, 1 g / l NaHCO ₃ (pH 8.0), 20 g / l of glycerol as a carbon source. Thus, the decomposition rate of IFC isobutyl ether is 12.3 mg / l / h.

268 mg / l of the MPA produced by the decomposition of IBEMPA is also decomposed by an immobilized biocatalyst and at the same time (31 h) in the same reaction medium in the presence of IBEMPK it decomposes by 95%, i.e., the decomposition rate of MPC is 8.2 mg / l / h The resulting biocatalyst is able to function in such a system for up to 300 days.

Example 4. A biocatalyst based on E. coli SG13009 [pREP4] / pTES-His-OPH cells included in PVA cryogel for decomposition of isopropyl ether of MPA and MKF.

11 g of biomass of bacterial cells of E. coli SG13009 [pREP4] / pTES-His-OPH obtained after separation from the culture medium, initially containing 12 g / l of tryptone, 24 g / l of yeast extract, 4 g / l of glycerol, 6.95 g / l KN ₂ PO ₄ , 12.54 g / l KN ₂ PO ₄ × 3H ₂ O, 2.37 mg / l CoCl ₂ (pH 6.8-7.0), 23.8 mg / l isopropylthiogalactoside, by centrifugation (15 min, 8500 rpm), mixed with 869 g of a 13% PVA solution prepared on the basis of sterile tap water at room temperature until a homogeneous mass is obtained. The resulting suspension is distributed into the wells of immunological 96-well tablets with a spherical bottom of 0.2 ml. Freezing is carried out at -20 ° C, the duration of aging in a frozen state is 28 hours, thawing at + 4 ° C. Get a biocatalyst with a hemispherical form of granules having the following composition (wt.%): Biomass of bacterial cells - 0.125 (by dry weight); PVA cryogel - 12.8; aqueous phase up to 100.

The obtained immobilized biocatalyst without any adaptation to toxic substrates provides 95% decomposition of 300 mg / L of MPA isopropyl ether (IPEMPK) in a 1 L aerobic reactor with a periodic regime of cultivation at 26 ° C with continuous stirring of the medium (180 rpm) for for 20 hours when introduced into a minimal culture medium containing 0.1 g / l MgSO ₄ × 7H ₂ O, 0.5 g / l NaCl, 1 g / l NH ₄ Cl, 2 g / l NaHCO ₃ (pH 9, 2), 15 g / l molasses as a carbon source. Thus, the decomposition rate of IPEMPA is 14.25 mg / l / h.

217 mg / l of MPA produced by the decomposition of IPEMPA is also decomposed by an immobilized biocatalyst and at the same time (20 hours) in the same reaction medium in the presence of IPEMPK it decomposes by 84%, that is, the decomposition rate of MPC is 9.1 mg / l / h The resulting biocatalyst is able to function in such a system for up to 310 days.

Example 5. Biocatalyst based on E. coli SG13009 [pREP4] / pTES-His-OPH cells included in PVA cryogel for decomposition of isobutyl ether of MPA and MPA in the reaction masses obtained as a result of chemical destruction of substance Vx according to the RD-4M formulation .

23.2 g of the biomass of bacterial cells of E. coli SG13009 [pREP4] / pTES-His-OPH obtained after separation from the medium, as described in Example No. 4, is mixed with 456.8 g of an 8% solution of polyvinyl alcohol prepared based on 50 mm Na-phosphate-carbonate buffer (pH 8.0) at room temperature until a homogeneous mass. The resulting suspension is distributed in a rectangular metal form with sides 3 cm high to obtain gel blocks 0.3 cm high. Freezing is carried out at -20 ° C, the duration of aging in the frozen state is 16 hours, thawing at + 4 ° C. The thawed biocatalyst in the form of a gel layer is cut and pellets in the form of a parallelepiped with a size of 0.3 × 0.3 × 0.3 cm are obtained. The obtained biocatalyst has the following composition (wt.%): The biomass of bacterial cells is 0.725 (dry. mass); PVA cryogel - 7.6; aqueous phase up to 100.

The obtained immobilized biocatalyst without any adaptation to toxic substrates provides 65% decomposition of 2370 mg / l of IFC isobutyl ether (IBEMPA) in a 1 L aerobic reactor under continuous cultivation at 30 ° C while stirring the medium with bubbling for 72 hours and when using as a culture medium for the reaction mass obtained as a result of chemical destruction of substance Vx according to the formulation RD-4M, adopted in the Russian Federation for the destruction of organophosphorus toxic substances [Shkodich P.E., Zheltobryukhov V.F., Kla Uchev V.V. Environmental and hygienic aspects of the problem of the destruction of chemical weapons. // Volgograd: VolSU Publishing House, 2004, 236 s], diluted 100 times with the minimum medium described in the above Examples, with the additional introduction of 25 g / l glucose as a carbon source. The reaction mass obtained after chemical destruction of Vx has the following composition: 23.7% IBEMPK, 29% diisobutylmethylphosphonate, 36.9% N-methylpyrrolidone, 1.2% caprolactam, and 8.9% bis- [2- (N, N -diisopropylamino) ethyl] disulfide, 0.3% water. Thus, the decomposition rate of IBEMPA is 21.4 mg / l / h.

1087 mg / l (or 11.3 mmol) of the MPA produced by the decomposition of IBEMPA is also decomposed by an immobilized biocatalyst and, at the same time (72 h), in the same reaction medium in the presence of IBEMPK and other components of the reaction mixture decomposes into 70 %, that is, the IFC decomposition rate is 15.1 mg / l / h. The resulting biocatalyst is able to function in such a system for up to 250 days.

Example 6. Cell-based biocatalyst Pseudomonas sp. 78G included in the PVA cryogel for the decomposition of isopropyl ether IFC and IFC as a part of the reaction masses obtained as a result of chemical destruction of sarin by chemical hydrolysis.

An immobilized biocatalyst obtained as described in Example 3, when introduced into a medium containing reaction masses obtained after chemical destruction of sarin by treatment with a 10 M NaOH solution at 90 ° C and diluted 1000 times with a minimum medium containing 0.1 g / l MgSO ₄ × 7H ₂ O, 0.5 g / l NaCl, 1 g / l NH ₄ Cl, 8 g / l NaHCO ₃ (pH 9.5) and 25 g / l glucose as a carbon source, without which or adaptation to toxic substrates 100% decomposition of 250 mg / l of MFK isopropyl ether (IPEMFK) in an aerobic reactor with a volume of 1 l does not discontinuous cultivation at 28 ° C while stirring the medium with bubbling for 26 hours. Thus, the decomposition rate of the isofutyl ether of MPA is 9.6 mg / l / h.

190 mg / l of the MPA produced by the decomposition of IPEMPA is also decomposed by the immobilized biocatalyst and at the same time (26 h) in the same reaction medium in the presence of IPEMPK it decomposes 100%, i.e., the decomposition rate of MPC is 7.3 mg / l / h The resulting biocatalyst is able to function in such a system for up to 245 days.

Thus, such a previously unknown combination of biocatalyst components, that is, biomass of cells of individual bacterial cultures having high biodegradation activity with respect to MPA and its esters, and PVA cryogel formed in air, with the claimed ratio of the components of this biocatalyst and results in a biocatalyst having the following advantages compared to analogues and prototype.

1. The inventive biocatalyst has a significantly improved decomposition rate of both MPA and its esters, which reach, respectively, 18 mg / l / h (Example 1) and 21.4 mg / l / h (Example 5), which exceeds the same the performance of analogues and prototype is 2.8 times for IFC esters and 10 times for IFC. Moreover, the biocatalyst allows the decomposition of toxic substrates in a wide range of concentrations.

2. The inventive biocatalyst is characterized by an extended period of functioning, in particular up to 310 days (Example 4), which exceeds the duration of use of similar biocatalysts, presented as analogues and prototype, by more than 1.5-22 times.

3. The developed immobilized biocatalyst does not need adaptation before its use for the decomposition of MPC or its esters, taken for these purposes in the form of pure substances or as part of the reaction masses of a complex chemical composition.

4. The formation of a biocatalyst in the absence of hexane can significantly technically simplify the process of producing a biocatalyst based on polyvinyl alcohol cryogel, diversify the form of the resulting biocatalyst, and significantly improve its environmental, economic and social aspects, in terms of eliminating the negative effects of a toxic solvent on human health.

Claims (1)

A biocatalyst for the decomposition of methylphosphonic acid and its esters, which are the products of the chemical decomposition of organophosphorus toxic agents such as sarin, soman and Vx, containing immobilized biomass of bacterial cells and polyvinyl alcohol cryogel as a carrier, characterized in that the biocatalyst contains cells of individual bacterial cultures of Pseudomonas cultures 78 Escherichia coli DH5α / pTrcTE-OPH, Escherichia coli SG13009 [pREP4] / pTES-His-OPH capable of degradation of organophosphorus compounds with a C-P bond and incorporated into the polyvinyl cryogel about alcohol, the formation of which occurs in an air environment, and has the following component composition, wt.%:
Bacterial cell biomass 0.125 ÷ 0.725 Polyvinyl alcohol cryogel 7.6 ÷ 12.8 Water phase Up to 100