Enzymatic method of producing electroconductive polymers

FIELD: chemistry.

SUBSTANCE: disclosed is an enzymatic method of producing electroconductive polymers, specifically polyaniline, polypyrrole, polythiophene and substituted derivatives thereof. The method involves use of a salt of a transition metal complex as an enzymatic reaction accelerator, having redox potential in the range of 0.35-0.95 V. The salt is selected from a group comprising cyanide complexes of molybdenum, osmium, ruthenium, tungsten and iron. The process is carried out at pH 2.5-5.5 and temperature 0-30°C and the oxidant used is molecular oxygen.

EFFECT: method enables to carry out high-speed oxidative polymerisation using low concentration of enzymatic reaction accelerator and an acid dopant.

3 dwg, 12 ex

 

The invention relates to biotechnology and has a wide scope. Electrically conductive polymers, which include polyaniline, polypyrrol, polythiophene and their substituted derivatives have a wide range of potential use in electronic and optoelectronic nanodevices, sensors, light-powered systems, energy conversion, corrosion protection, static electricity and electromagnetic radiation to create artificial muscles and interfaces between electronic and biological systems (.Wessling, Synthetic Metals 93 (1998) 143-154; L.Groenendaal, F.Jonas, D.Freitag, H.Pielartzik, J.R.Reynolds, Advanced materials, 12 (2000) 481-493; B.Ch. Liu, L.-P. Ma, H.-M. Cheng, Advanced materials, 22 (2010) E28-E62). Depending on the desired properties, such as conductivity, solubility, ability to plenkoobrazovatel, use the appropriate monomers having the structure of different functional groups in different positions.

Typically, conductive polymers are synthesized by chemical or electrochemical methods. Electrochemical polymerization of monomers occurs without the addition of chemical oxidants, but is limited by the presence of a conductive substrate and its limited size. Chemical oxidation is a fairly easy method of obtaining a conductive polymer using strong oxidizing agents, is aka as ammonium persulfate, salts of trivalent ions of iron, bichromates, permanganates in quantities that equal or exceed the concentration of the used monomers. The reduction products of these compounds need to be processed, which creates additional costs when exercising the synthesis of conductive polymers. In addition, synthesis of some conducting polymers (such as polyaniline) should be carried out in strongly acidic medium (1M HCl or 1M H2SO4to ensure linear conductive structure of polyaniline formed according to the type of "head to tail" (J.-C. Chiang, A.G. Mac Diarmid, Synthetic Metals 13 (1986) 193-205; Y. Cao, P. Smith, A.J. Heeger, Synthetic Metals 32 (1989) 263-281). This requires corrosion-resistant equipment.

Thus obtained electroconductive polymers have very low solubility in most common organic and inorganic solvents and poor performance. To overcome this drawback using a variety of approaches, such as synthesis in micellar solutions, interfacial polymerization, neutral soluble polymeric stabilizers and polysulphonate. Despite the great attraction of the practical use of electrically conductive polymers used in the methods of chemical synthesis, as noted above, are Neue is overvoltage. In addition, synthesized by the above methods, conducting polymers have volatile composition, which may vary with small changes in the synthesis conditions (J.S.Stejskal, I.Sapurina, M.Troehova, E.N.Konyushenko, Macromolecules 41 (2008) 3500-3536).

The conductivity of the polymer is achieved, as a rule, acid doped. While the anions of the acids are the charge compensating anions in the main circuit conductive polymers.

As acidic deruosi agents are used as low-molecular-weight strong acids (sulfuric, hydrochloric, sulfokamforna, toluensulfonate and others), and polysulfonamide (poly(2-acrylamide-2-methyl-1-propanesulfonate), from sulphonated polystyrene and other) (US Patent, 7462298; Eur. Patent 686662; US Patent 5792558) and micelle-forming hydrophobic sulfonic acids or their salts (dodecylsulfonate sodium, dodecylbenzensulfonate acid) (M.G.Han, S.H.Foulger, Small, 2 (2006) 1164-1169; V.Rumbau, J.A.Pomposo, J.A.Alduncin, H.Grande, D.Mecerreyes, E.Ochoteco, Enzyme and microbial technology 40 (2007) 1412-1421; US Patent 7230071). Polysulphonate and micelle-forming hydrophobic sulfonic acids or their salts used in the matrix synthesis of conductive polymers to obtain aqueous dispersions of nanoparticles of polymers. Sometimes as dopants used as the Lewis acid (W.Baik, W.Luan, R.H.Zhao, S.Koo, K.-S.Kim, Synthetic Metals 159 (2009) 1244-1246).

The use of biocatalysts d is I the synthesis of conductive polymers is an alternative to chemical synthesis and is of great interest, because it allows the process of oxidative polymerization in a clean and relatively mild conditions (in aqueous solutions at room temperature, atmospheric pressure and slightly acidic pH values of the reaction medium), without the formation of a large number of toxic by-products and to obtain a polymer that is not contaminated with decomposition products of the oxidant. Thus, this approach is largely meets the requirements of green chemistry.

However, despite the attractiveness of using enzymes for the synthesis of conductive polymers in the scientific and patent literature describes only a limited number of examples of biocatalytic obtain polyaniline, polypyrrole, polythiophene with them (US Patent 5420237; US Patent 6569651; R.Cruz-Silva, E.Amaro, A.Escamilla, M.E.Nicho, S.Sepulveda-Guzman, L.Arizmendi, J.Romero-Garcia, F.F.Castillon-Barraza, M.H.Farias, J.Colloid and Interface Science 328 (2008) 263-269; Karamyshev A.V., Shieev S.V., Koroleva O.V., Yaropolov, A.I., Sakharov I.Yu., Enzyme and microbial technology. 33 (2000) 556-564).

The most common use biocatalysts for the synthesis of conducting polyaniline.

Enzymatic synthesis of polyaniline to a mixture of freshly under vacuum aniline and acid dopant was added an aqueous solution of the biocatalyst. As a last used oxidoreductase (peroxidase and laccase), isolated from various objects. For peroxidase reaction initiation p is ocess polymerization of aniline was performed, adding in portions to the reaction solution of hydrogen peroxide, with constant stirring. When using laccase as a catalyst for the reaction of oxidative polymerization of aniline oxidant was molecular oxygen, forming as product water recovery.

Biocatalytic synthesis of conductive polyaniline, despite the environmental attractiveness, also has several disadvantages. First, most of peroxidases from various sources are colorability and can be used in the synthesis only in the narrow range of pH values, the reaction solution near pH 4. At higher pH values, the reaction mixture is formed elektroprovodyashchie the branched polymer structure. At more acidic pH values peroxidase dissociate into heme and apparment, losing their catalytic activity. The acidic pH of the reaction medium is necessary to ensure the conductivity of polyaniline. Secondly, the reaction mixture is added in portions of the second pre-diluted peroxidase substrate is hydrogen peroxide, so that its concentration in the synthesis conditions did not exceed 1 mm. At higher concentrations of hydrogen peroxide peroxidase is inactivated, forming hydrogen peroxide inactive connection. Thirdly, peroxidases is from the roots of horseradish and other peroxidases are too expensive for industrial use in the synthesis of conductive polymers. Laccase compared with peroxidases have advantages. The recombinant enzyme is commercially produced by firm "NOVOZYMES" (http://www.novozymes.com/). and in the Russian Federation developed the experimental-industrial technology for the production of laccase from the basidiomycete Trametes hirsuta. Fungal laccase are kislotostabilen enzymes and as an oxidizer use oxygen. However, compared to the peroxidase-catalyzed oxidation of the monomer, laccase-catalyzed polymerization of aniline is slower and with a lower yield of the final product. Enzymatic oxidation of pyrrole, thiophene, its derivative 3,4-ethylenedioxythiophene (EDOT) with the participation of peroxidases and lackas does not occur or occurs very slowly. Thus, during the synthesis and expenses biocatalysts increase, which increases the cost of the final product.

As a prototype of the selected method of enzymatic synthesis of conducting polypyrrole using laccase and organic amplifier the reaction rate of the polymerization of pyrrole (N.-.Song, G..R.Palmore, J.Phys.Chem.B, 2005, 109. 19278-19287.), includes the following sequence of operations:

- preparation of a mixture of pyrrole and 2,2'-Azino-bis(3-ethyl-benzothiazoline-6-sulfonate) diammonium salt (ABTS) in acetate buffer with pH 4,0;

- initiating the reaction by addition of a solution l is ccasi from the fungus Trametes versicolor.

The reaction was carried out in Petri dishes at room temperature. The concentration ratio of monomer (pyrrole) and accelerator enzymatic reaction (ABTS) was 8:1. In the absence of ABCS the oxidation reaction of pyrrole was very slow. The disadvantages of this method of carrying out the oxidative polymerization of pyrrole are the high cost of the accelerator enzymatic reaction - ABTS and the necessity of its use for the polymerization at high concentrations. In addition, resulting from fermentative reactions of cation radicals ABTS can be combined with pyrrole radicals, leading to modifications of the synthesized polypyrrole, impairing its properties.

The objective of the invention is to develop a simple, environmentally friendly method of producing conductive polymers in water-insoluble and colloidal forms with a reduced time of synthesis, reduced the concentration of the redox mediator in the reaction medium and the absence of modification of the redox mediator of the obtained polymers. The problem is solved by the proposed method comprising carrying out the oxidative polymerization of the monomers in the presence of copper-containing oxidase (lakkis and bilirubinemia) and redox-mediators of these enzymes related to salts of transition metal complexes, accelerating enzyme is active the oxidation reaction of the monomers.

Laccase (p-diphenol:oxygen oxidoreductase, EC 1.10.3.2) and bilirubinaemia (bilirubin:oxygen oxidoreductase, EC 1.3.3.5) catalyze the oxidation of a wide range of organic and inorganic compounds by molecular oxygen with the simultaneous restore it to the water. These oxidases are commercially available or can be obtained by standard methods. In principle producers of these enzymes can be as natural organisms and organisms modified by genetic engineering methods. To obtain a particularly preferred lackas apply mushrooms such as Pleurotus, Phlebia, Trametes, Cerrena, Panus {Trametes hirsuta, Trametes pubescens, Trametes ochracea, Cerrena maxima, Coriolopsis fulvocinerea, Trametes versicolor, Panus tigrinus), producers of bilirubinemia was fungi Myrothecium verrucaria, Trachyderma tsunodae.

Laccase and bilirubinaemia can directly oxidize compounds potentials are below or slightly above the redox potential of the T1 copper center oxidase. When exceeding the capacity of the oxidation of substrates oxidase by more than 200 mV compared to the T1 center of the enzyme (the primary electron acceptor in the active centre oxidase), the efficiency of enzymatic catalysis by these enzymes is reduced, or the reaction does not proceed at all. The potentials of the oxidation of aniline, pyrrole, thiophene and 3,4 ethylenedioxythiophene have high the e values. Therefore, the rate of oxidation of aniline with participation of lackas relatively low, and the oxidation reaction ADOT did not leak at all.

Redox mediators called low molecular weight compounds that are substrates of lakas or bilirubinemia that as a result of enzymatic oxidation sustainable form highly reactive products. The latest in a diffusion-controlled regime can enter into chemical (non-enzymatic) reactions with other compounds that do not undergo oxidation involving only copper-containing oxidase. While the oxidized mediator is restored oxidizable compound to its original shape and thus, creating a vicious cycle. Formed during the enzymatic reaction of the oxidized form of the mediator may further oxidize compounds with ionization potentials greater than the redox potential of the T1 center of lakas and to accelerate the oxidation reaction of these compounds (SCHEME 1). Laccase-mediatory system used for the degradation of xenobiotics, for delignification of paper pulp, bleaching of fabrics (US Patent 5,965,510; WO 200,302,3133; WO 2003023043; WO 03016615; JP 2001037465).

The proposed method enables one stage: synthesis of conductive polymers (polyaniline, polypyrrole, polythiophene) and their substituted derivatives, in the process of oxidative polymerization of the corresponding monomial is the moat and in the presence of dissolved in the reaction mixture of the reaction accelerator, acid dopant and oxidant is molecular oxygen at a pH of 2.5 to 5.5, a temperature 0-55°C, catalyzed by glycerol phosphate oxidase. As solvents used are water or water-organic solutions.

As accelerator enzymatic reaction using salt cyanide complexes of transition metals, such as hexacyanoferrate(2+), occasionalpaper(4+), hexazinone(2+), hexacyanoruthenate(2+), occasionalist(4+), redox potential (E°) which are in the region of 0.35-0.95 In Rel. normal hydrogen electrode (NHE). As oxidant use molecular oxygen. As biocatalysts using fungal laccase and bilirubinaemia. As acid dopant use of strong inorganic acids, organic sulfonic acids, polysulphonate or their salts and acid Lewis.

The enzymatic method of obtaining a conductive polymer using low concentrations of amplifiers reactions of inorganic nature and acid dopant is environmentally friendly, single-stage, allows the process of oxidative polymerization of monomers with high speed and high output in the kinetically controlled regime, which reduces the final products - polyaniline, polythiophene, polypyrrol or their substituted derivatives.

The invention is explained in the following and examples.

Example No. 1. Matrix synthesis of polyaniline (PANI) using as dopant poly(2-acrylamide-2-methyl-1-propanesulfonate) (the PUMPS) in the presence of the accelerator enzymatic reaction occasionalist(4+).

10 ml 0,05 m Na-phosphate-citrate buffer solution, pH 3.5, containing fresh aniline at a concentration of 25 mm and poly(2-acrylamide-2-methyl-1-propanesulfonate) with molecular weight of 2000 kDa at a concentration of 25 mm calculated on the unit of the polymer, (the ratio of Monomeric aniline and link the PUMPS is 1:1) was stirred for 1.5 hours for linking and establishing the electrostatic balance between the positively charged molecules of aniline (pKa 4,63) and negatively charged sulfo-polymer at a temperature of 20°C. Then the resulting solution to acceleration of enzymatic polymerization reactions with constant stirring solution was added occasionalist(4+) potassium (E°=0,78) with the final concentration in the reaction medium is 0.05 mm (the ratio of the concentrations of the monomer and the accelerator 500:1) and continued to stir the solution for 10 minutes. The polymerization reaction was initiated by introducing a reaction solution was homogeneous according to the gradient electrophoresis of the drug laccase from the fungus Trametes hirsuta concentration in the reaction mixture of 2.0×10-7M (the activity of the enzyme preparation is left 80 µmol of pyrocatechin/ min-mg protein). The synthesis of the polymer was performed in air for 24 hours at 20°C with constant stirring. About the formation of interpolymer electrically conductive PANI complex and the PUMPS were judged visually by a colour change of the solution and the change in the spectrum in the region of wavelengths of 350-900 nm. After making the reaction mixture laccase solution quickly became blue, and then turned into a dark green, indicating the formation of emeraldine salt of polyaniline. The conductivity of the dried sample PANI/the PUMPS, the measured standard two-point method was equal to 10.2 MSM/see On UV-visible spectra of aqueous dispersions of nanoparticles of interpolymer complex PANI/the PUMPS have absorption bands in the region of 420 nm and 760 nm, which corresponds to emeraldine salt of polyaniline (figure 1).

Example No. 2. Synthesis of polyaniline on the matrix of the PUMPS was carried out similarly as described in example No. 1, but the reaction mixture was added occasionalist(4+) of potassium with the final concentration in the reaction medium is 0.01 mm (the ratio of the concentrations of the monomer and the accelerator 2500:1) the polymerization reaction, as measured by the increase in optical density of the solution at a wavelength of 750 nm was 1.2 times less than in example No. 1.

Example No. 3. Synthesis of polyaniline on the matrix of the PUMPS was carried out similarly as described in example No. 1, but when the temperature is e 30°C. The conductivity of the dried sample PANI/the PUMPS was 3.5 MSM/see

Example No. 4. Synthesis of polyaniline on the matrix of the PUMPS was carried out similarly as described in example No. 1, but at a temperature of 0°C. To this reaction mixture was placed in a vessel with ice. The conductivity of the dried sample complex PANI/the PUMPS amounted to 14.3 MS/see

Example No. 5. Synthesis of polyaniline on the matrix of the PUMPS was carried out similarly as described in example No. 1, but instead of homogeneous drug laccase used the cultural filtrate liquid basidiomycete Trametes hirsuta. The oxidase activity of the drug was 3 µmol of pyrocatechin/min per ml of culture fluid.

Example No. 6. For comparison, the synthesis of polyaniline on the matrix of the PUMPS was carried out similarly as described in example No. 1, but without adding to the reaction mixture accelerator enzymatic reaction. In the process of synthesis solution slowly changes color from blue to green and after 24 hours turns into dark green. UV-visible spectra of complex PANI/the PUMPS in the same way as in example No. 1, has an absorption band in the region of 420 nm and 760 nm, the absorbance at 760 nm 1.5 times less than in example No. 1. In the absence of an accelerator, a polymerization reaction, as measured by the increase in optical density at a wavelength of 760 nm, was 4-5 times lower than in example No. 1. Electropo Timoti dried sample was 2.4 MSM/see

Example No. 7. Non-matrix enzymatic synthesis of conducting polyaniline using as the acid dopant sulfocarbamoyl acid and occasionalist(4+) potassium as accelerator enzymatic polymerization reactions.

10 ml reaction mixture containing fresh aniline at a concentration of 0.15 M and (1S)-(+)-10-sulfocarbamoyl acid (CCM) at a concentration of 0,155 M in bidistilled water (pH of 2.8) was stirred on a magnetic stirrer for 30 minutes. Then in the resulting solution to accelerate enzymatic polymerization reactions with constant stirring solution was added occasionalist(4+) of potassium with the final concentration in the reaction medium is 0.1 mm, and continued to stir the solution for 10 minutes. Initiating the polymerization was carried out by adding a solution of a laccase from the fungus Trametes hirsuta (concentration in the reaction mixture to 3.0·10-7M). The synthesis was carried out at 4°C in air with constant stirring for 24 hours. After the reaction residue of the synthesized polymer was separated by centrifugation (12000 rpm, 10 min) and washed several times 0.05 mm solution of the CCM to remove excess reagents and the resulting oligomers. The precipitate was dried in vacuum. About the formation of synthesized optically active polyaniline was judged visually on izmeneniyami solution. The yield was 59%.

Example No. 8. Synthesis of PANI on the matrix of the PUMPS was carried out similarly as described in example No. 1, but as biocatalyst used laccase isolated from the basidiomycete fungus Cerrena maxima.

Example No. 9. Synthesis of conductive polyaniline in an aqueous solution of micelles dodecylbenzenesulfonate sodium (N) and accelerator enzymatic polymerization reactions.

A solution of micelles was obtained by dissolution of dodecylbenzenesulfonate sodium (10 mm) in 0.05 M Na-citrate-phosphate buffer (pH 3,8) with gentle stirring on a magnetic stirrer. In the original solution was added dropwise freshly aniline with a final concentration of 10 mm. Stirring was continued for 1 hour at room temperature (approximately 22°C). Then in the reagent solution was made solution occasionalist(4+) of potassium with the final concentration in the reaction medium is 0.05 mm. The reaction was initiated by introducing into the reaction mixture laccase (concentration in the reaction mixture - 2·10-7M). The reaction was conducted for 24 hours at room temperature and constant stirring on a magnetic stirrer under aerobic conditions. Complex formation of PANI/Na was measured spectrophotometrically at the wavelength interval 190-1000 nm. During the reaction solution at a white-turbid through blue-green became dark green color that the witness is only about education emeraldine PANI salt. Exit MRS. calculated by aniline was 67%. In the absence of accelerator enzymatic reaction output PANI was 31%.

Example No. 10. Matrix enzymatic synthesis of PANI using as dopant the PUMPS and hexacyanoferrate(2+) potassium (E°=0.43 In) as accelerator enzymatic reaction polymerization of aniline.

Synthesis of polyaniline on the matrix of the PUMPS was carried out similarly as described in example No. 1, but in the reaction mixture instead of occasionality(4+) was added potassium hexacyanoferrate(3+) potassium (concentration in the reaction mixture to 0.3 mm). The synthesis time was 24 hours. UV-visible spectrum of interpolymer complex PANI/the PUMPS is visible clearly pronounced absorption band in the region of 770 nm. The conductivity of the sample was 14 MSM/see

Example No. 11. Enzymatic synthesis of polyethyleneoxide (PEDOT) on the matrix of the PUMPS using laccase from the fungus Trametes hirsuta and accelerator enzymatic polymerization reactions occasionalist(4+) potassium.

10 ml of 0.05 M Na-citrate-phosphate buffer solution pH 3.5, containing EDOT at a concentration of 7 mm and the PUMPS at a concentration of 10 mm was stirred for 10 min in an ultrasonic bath, and then 30 min on a magnetic stirrer at room temperature (22°C). Then in the resulting solution to accelerate the enzymatic reaction was added occasionalist(4+) potassium con who offered a concentration in the reaction medium is 0.05 mm (the ratio of the concentrations of the monomer and the accelerator 140:1). The polymerization reaction ADOT initiated by introducing into the reaction medium of preparation of fungal laccase concentration of enzyme in the reaction mixture 3·10-7M. Synthesis was performed with stirring under aerobic conditions for 24 hours. The formation of polyethyleneoxide observed visually and by the change of the spectrum in the region of wavelengths 190-1000 nm. After carrying out the enzymatic polymerization solution was purchased dark blue color. Figure 2 illustrates the spectrum of the synthesized polymer. The conductivity of the synthesized complex PEDOT/the PUMPS was 25 MSM/see

Example No. 12. Enzymatic synthesis of polypyrrole (PP) on the matrix of the PUMPS using laccase from the fungus Trametes hirsuta and accelerator enzymatic polymerization reactions occasionalist(4+) potassium.

10 ml of 0.05 M Na-citrate-phosphate buffer solution pH 3.5, containing pyrrole at a concentration of 10 mm and the PUMPS at a concentration of 10 mm was stirred for 30 min on a magnetic stirrer at room temperature (25°C). Then in the resulting solution to accelerate the enzymatic reaction was added occasionalist(4+) of potassium with the final concentration in the reaction medium is 0.05 mm (the ratio of the concentrations of the monomer and the accelerator 200:1). The reaction of polymerization of pyrrole initiated by introducing into the reaction medium of preparation of fungal laccase concentration of enzyme in actionnow mixture 3·10 -7M. Synthesis was performed with stirring under aerobic conditions for 24 hours. The conductivity of the synthesized complex PP/the PUMPS was 8.2 MSM/see

The enzymatic method of obtaining a conductive polymer such as polyaniline, polypyrrole, polythiophene and their substituted derivatives, providing for the oxidative polymerization of the corresponding monomers in the presence of oxidase, acid dopant, oxidant, acceptable for oxidase, and accelerator enzymatic reaction, characterized in that as the accelerator enzymatic reaction using salt complex of a transition metal having a redox potential of from 0.35 to 0.95, selected from the group consisting of cyanide complexes of molybdenum, osmium, ruthenium, tungsten, iron, at this pH is 2.5 to 5.5 and a temperature of 0-30°C, and as oxidant use molecular oxygen.



 

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11 cl, 5 tbl

FIELD: food industry.

SUBSTANCE: pasteurised food product containing a proline-specific protease has water activity equal to at least 0.85. Used as the enzyme is protease extracted from Aspergillus or belonging to the S28 serine proteases family. The optimal activity of the said protease is at a pH value from 1 to 7, preferably - at a pH value from 2 to 6. Additionally proposed is a food product containing less than 1 wt % of protein or peptides. The said food products are produced by way of addition of a proline-specific protease to them.

EFFECT: such products consumption ensures gluten peptides splitting and is recommended to patients suffering from gluten intolerance.

17 cl, 5 dwg, 2 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: method of obtaining a yeast extract involves treating Saccharomyces cerevisiae yeast cells with purified phospholipase A and separating the yeast extract from treated yeast cells. Yeast cells undergo proteolysis during treatment with phospholipase. Treatment with phospholipase is carried out at pH 2-10, and the amount of phospholipase ranges from 0.001 to 1 mg enzymatic protein/g dry substance. Yeast extract output is equal to 68.8%, protein output is equal to 67.4% and degree of hydrolysis is equal to 71.8%.

EFFECT: method enables to obtain an end product with high output, protein content and degree of hydrolysis.

6 cl, 3 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to a method for producing a product of reaction catalysed by a protein showing 2-oxoglutarate-dependent enzyme activity such as (2S,3R,4S)-4-hydroxy-b-isoleucine or its salt and 4-hydroxy-b-proline or its salt with using a bacterium of a genus selected from a group consisting of Escherichia, Corynebacterium, Arthrobacter, Aspergillus and Bacillus, transformed by a DNA fragment containing a gene coding the protein showing 2-oxoglutarate-dependent enzyme activity with said bacterium modified so that: the gene coding 2-oxoglutarate dehydrogenase is inactivated, or the gene coding 2-oxoglutarate dehydrogenase, and the gene coding aminotransferase with branched-chain amino acid are inactivated.

EFFECT: invention enables high-effective production of said compounds and their salts.

14 cl, 6 dwg, 9 tbl, 9 ex

FIELD: medical technology.
SUBSTANCE: invention relates to the area of biotechnology, namely to the method for detection and typing of human papillomavirus (HPV) in a sample, as is a reaction vessel. The invention also relates to apparatus for use in the kit and the HPV detection and typing method. The assay comprises: performing a nucleic acid amplification reaction on a sample, the amplification reaction being intended to amplify an HPV target sequence in a non-type specific manner; obtaining single stranded oligonucleotides from any amplification products; allowing single stranded oligonucleotides to hybridise where possible with the HPV type-specific probes chosen from the group containing SEQ ID NO:1 - SEQ ID NO:133 and chosen according to their type, as given in table 1, provided on a solid support, the support being located within a reaction vessel suitable for containing the sample; and detecting hybridised oligonucleotides if HPV is present in the sample.

EFFECT: specific identification of the HPV types if HPV is present in a sample.

63 cl, 7 dwg, 2 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: method involves preparation of plant oil with heating to 80°C, alkaline ethanolysis using potassium hydroxide in ethanol with molar concentration of 2 mol/dm3 to obtain an ether-glycerine mixture which is separated to form two fractions - glycerine and a mixture of ethers. The mixture of ethers (biodiesel fuel) undergoes filtration, sorption purification and dehydration. The obtained biodiesel fuel is stored. Preparation of the plant oil is carried out such that before heating, the plant oil is mixed with 1% aqueous solution of an Ecofriend enzyme-probiotic preparation and the obtained mixture is held for 24 hours at temperature 23-27°C. After holding for 24 hours, the mixture of plant oil and this preparation is heated to said heating temperature. The products are taken in the following ratio, pts.wt: plant oil 5; 1% aqueous solution of Ecofriend enzyme-probiotic preparation 5; potassium hydroxide in ethanol 5.

EFFECT: method increases the amount of the obtained biodiesel fuel.

2 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: described is oligonucleotide probe with complementary end sequences by type of "molecular beacon", ensuring fluorescent detection in identification of causative agents of glanders and melioidosis B. pseudomallei and B. Mallei by method of polymerase chain reaction with oligonucleotide primers BTTS-sl/BTTS-as3, complementary to fragment of gene 23S pRNA B. pseudomallei and B. mallei: 5'-(FAM)-CGCGCACCGGCAGTGATGAGCCACGCGCG-(RTQ1)-3', where FAM is carboxyfluorescein, fluorescent dye, whose absorption wavelength constitutes 492 nm, and fluorescence wavelength is 520 nm, RTQ1 is fluorescence quencher with range of quenching 470-570 nm.

EFFECT: application of hybridisation probe makes it possible to identify causative agents of glanders and melioidosis in short term with high sensitivity and specificity in biological material and environment objects.

3 ex

FIELD: chemistry.

SUBSTANCE: invention discloses acylamidase enzyme AA37 from Rhodococcus erythropolis 37 All-Russian collection of industrial microorganisms Ac-1793, with a sequence given in the description. A nucleotide sequence which codes this enzyme is defined. The invention describes a method for synthesis of N-substituted acrylamides in aqueous medium from acrylamide and amines in the presence of an acylamidase biocatalyst in isolated state or in a composition with E.coli cells.

EFFECT: invention enables to obtain N-substituted aliphatic acrylamides from acrylamide and primary aliphatic amines in an aqueous medium.

3 cl, 14 ex

FIELD: medicine.

SUBSTANCE: what is offered is a method for target nucleic acid (NA) detection providing a) NA sample enrichment by target sequences ensured by DNA-RNA hybrid formation between the NA-target and a probe complementary at least to its site, linkage of the produced hybrids with a solid carrier and removal of the NA both/either non-hybridised and/or non-linked with a substrate; b) amplification of the target NA enriched samples and target NA detection in the produced set with the help of an oligonucleotide complementary to a first target NA site, and a probe complementary to a second target NA site where an oligonucleotide sequence and a probe sequence form with the target NA the DNA-RNA hybrid detected by any of a set of common methods.

EFFECT: new method is high-sensitive and high-specific and enables to distinguish between high-homologous NA sequences.

22 cl, 19 tbl, 17 ex

FIELD: medicine.

SUBSTANCE: method for producing an Escherichia coli cell fraction showing protease inhibiting activity provides bacteria cells preservation in the presence of buffered 80-90% glycerine, processing by 3% triton X-100 for removing a cell membrane. Produced cytoplasm proteins are extracted by the increasing salt concentration, namely 0.14 M, 0.35 M; 2 M NaCl, 6 M guanidine hydrochloride with 0.1% β-mercaptoethanole. It is followed by affine sepharose 4B chromatography with immobilised trypsin and estimation of protease inhibiting activity in eluates.

EFFECT: invention can be used in analysis of molecular-genetic mechanisms of procaryote cell structure formation and roles of protein components in their organisation, and also genome remodelling nature that is necessary for disclosing of regulation pathways of mechanisms of macro-and microorganism action, and also search of new drug targets and development of ecologically safe new drugs.

3 tbl, 4 dwg

FIELD: chemistry.

SUBSTANCE: antistatic coating composition, containing an acrylate- and polyaniline-based resin modified with cyanate-acrylate, which is capable of chemically bonding with acrylate-based resin. The antistatic coating composition can additionally contain a plasticiser, a photosensitiser and organic solvents. The antistatic coating composition, where the cyanate-acrylate-modified polyaniline is obtained using a two-step method: 1) obtaining cyanate-acrylate through a reaction of hydroxyacrylate (formula (1)): O=C=N-R1-N=C=O, where R1 denotes alkylene or arylene containing 10 or less carbon atoms) and diisocyanate (formula (2)): where R2 denotes alkylene or arylene containing 10 or less carbon atoms) and 2) covalent bonding of cyanate-acrylate to the polyaniline backbone chain to form a polyaniline-acrylate copolymer.

EFFECT: formation of a film with excellent adhesion properties and surface tension with UV curing.

13 cl, 8 ex

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