Method of immobilisation of chymotrypsin on nanoparticles of selenium or silver |
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IPC classes for russian patent Method of immobilisation of chymotrypsin on nanoparticles of selenium or silver (RU 2551317):
 
Method of stabilising nanoparticles of biogenic elements with enzymes / 2504582
Method involves synthesis of nanoparticles through a redox reaction while adding an enzyme for stabilising the formed nanoparticles directly into the reaction. The enzyme solution - chymotrypsin, concentration of which is selected from 0.02%, is mixed with a precursor solution of 0.013 M selenous acid or silver nitrate - 1%, 0.01%, 0.001%; the solutions are stirred and held at room temperature of 20°C or low temperature of 0°C for 30-60 minutes. A reducing agent - ascorbic acid or sodium borohydride - is then added to the reaction flask and the solutions are stirred and settled to complete the reaction.
 Tripsin synthesis method / 2403285
Invention relates to biotechnology. Prepared pancreatic gland of slaughter animals is ground to particle size of 7-11 mm and the extracted in acidified distilled water at 7-15°C while periodically stirring. The suspension is separated on a settling and filtering centrifuge with automatic unloading of the precipitate using a filter element with pore size of 1-5 mm. Separation of fractions after salting out ballast substances and tripsin is carried out using a filter element with pore size of 16-20 mcm with subsequent drying of the desired product by spraying or sublimation. Desalination is carried out through diafiltration on ultrafiltration apparatus using hollow fibres.
Method of stable cell cultures manufacture / 2392318
Embryonic tissue is flushed with enzyme solution at 25-37°C temperature. For enzyme neutralisation is used medium, made of Eagle medium with lactalbumine hydrolysate or bovine serum.
 Method of preparing cholesterol-esterase, tripsin, deoxyribonuclease, and ribonuclease from bovine pancreas / 2311455
Invention aims at biotechnological preparation of following medicaments: pancreatic ribonuclease, pancreatic deoxyribonuclease, tripsin, and cholesterolesterase. Method envisages grinding bovine pancreas and homogenation thereof. Resulting homogenate is extracted with cooled ethanol at constant stirring, centrifuged, and subjected to second extraction, now with sulfuric acid containing magnesium sulfate, at constant stirring. Extract containing desired enzymes is separated from precipitate via centrifugation. Ballast proteins are precipitated by ammonium sulfate at 30-35% saturation and removed from supernatant via centrifugation. Cholesterolesterase is precipitated by ammonium sulfate at 45-50% saturation of extract, tripsin at 60-65% saturation, and DNA-ase at 95-98% saturation followed by dialysis and chromatographic purification on DEAE-cellulose column.
Method for preparing trypsin / 2265053
Invention relates to a method for preparing proteolytic enzymes and can be used medicine, veterinary science and other branches. Method involves milling (homogenization) of pancreas tissue in tap water without using buffer solutions, extraction of product, separation of inert proteins and lipids. Separation of inert proteins and lipids is carried out by addition of 1% acid solution of chitosan to homogenate, stirring and settling. Prepared mixture is filtered through belting-fabric, trypsin-containing filtrate is concentrated by ultrafiltration through hollow fibers and subjected for drying. Invention provides increasing yield of trypsin, to enhance its specific activity, to simplify the technological process in preparing trypsin and to reduce duration of the process, to enhance safety and ecological purity of manufacturing process.
Nanosized sorbent for sorption of strains of aerobic microorganisms micrococcus albus and pseudomonas putida / 2545393
Invention proposes a nanosized sorbent for sorption of strains of anaerobic microorganisms Micrococcus albus and Pseudomonas putida. A sorbent represents nanosized water-suspended particles of nonstoichiometric cubic ferrite spinels with a general formula of MxFeyO4, where M is chosen from the following group: Co, Mn, Ni, x=0.50-0.96, x+y=3.
 Biocatalyst, method of its preparation and method of reetherification of vegetable oils using this biocatalyst / 2539101
Biocatalyst for reetherification of vegetable oils is offered which contains as enzymatic active substation the partially destructed cells or cell lysates of the recombinant producer strain rE. coli/lip, carrier consisting of silicon dioxide and of nanostructured carbon, and maltodextrin or high molecular weight polysaccharide as the water-retaining agent. A biocatalyst is prepared by mixing of cells or cell lysates of the recombinant producer strain rE. coli/lip with the carrier and water-retaining agent of carbon nature with the subsequent drying and fractionating. The method of reetherification of vegetable oils is performed both in a periodic mode in a mixing reactor, and in a continuous mode in a flowing reactor with the fixed layer of a biocatalyst at the temperature 60-80°C.
 Method of biodiesel production / 2533419
Method of biodiesel production is realised by the re-etherification in mixing natural oil, alcohol and a catalyst and following separation of the target product. The method is characterised by the fact that at the first stage of the re-etherification iron sulphate (II) is applied as the catalyst, after which iron sulphate and precipitated glycerol are separated and the mixture of alcohol, oil and ethers of fatty acids are supplied to the second stage of the re-etherification, at which as the catalyst used is an enzyme - lipase, immobilised on the surface, after which glycerol and the enzyme catalyst are separated and the mixture of alcohol and biodiesel is directed to a stage of the target product separation.
Bacterial system for normalising microbiocenosis of human and animal organism / 2530174
Invention relates to biotechnology. The bacterial system is intended for normalising microbiocenosis of a human or animal organism. The system comprises a support which is a carbon-mineral enterosorbent (enterumin), eubiotic bacteria (bifidobacteria) immobilised thereon and nutrient components and a protective medium. The carbon-mineral enterosorbent is modified by oxidative treatment (with hydrogen peroxide) and the bacteria titre in the finished preparation is 1 x 107 KOE/g of the bacterial system.
Biocatalyst for re-esterification of fats and method for production thereof / 2528778
Group of inventions relates to biotechnology and food industry. Disclosed is a method of producing a biocatalyst for re-esterification of fats. The method includes amination of granular silica gel or silicon dioxide with particle size of 0.3-1.0 mm with aminopropyltriethoxysilane. The obtained aminated support is treated with aqueous solution of glutaric aldehyde or glyoxal with concentration of 2.0 wt % or 5.0 wt % for 2 hours. The catalyst is immobilised on the treated catalyst by recycling through said support a solution of thermally stable lipase of Geobacillus lituanicus bacteria in a phosphate buffer at temperature of 0°C or 4°C at pH 6.5 for 12 hours. The obtained biocatalyst is then washed with aqueous solution of tris(hydroxymethyl)aminomethane hydrochloride. Also disclosed is a biocatalyst for re-esterification of fats obtained using said method.
 Photobiocatalyst for hydrogen formation and photocatalytic method of obtaining hydrogen / 2511053
Group of inventions relates to biotechnology. Claimed is photobiocatalyst, which includes hydrogenase, immobilised in amount not less than 0.1 nmol per cm2 on nanostructured mesoporous TiO2 film. Mesoporous film is prepared from TiO2 nanocrystals with size from 15 to 25 nm with specific surface 50-100 m2/g. Together with hydrogenase enzyme on titanium dioxide fim mobilized is photosensibiliser FS1 - pigment-protein complex of photosystem 1 in amount 0.01-0.04 nmol FS per 1 cm2. Obtained TiO2 film is 4-8 mcm thick, has specific surface 50-65 m2/g, pores with average radius 11.5 nm and specific volume 0.50-0.65 cm3/g. Also claimed is photocatalytic method of obtaining hydrogen in anaerobic conditions with application of claimed photobiocatalyst with illumination with light with λ=490-750 nm in presence of organic electron donor and electron carrier.
Method for immobilisation of l-phenylalanine-ammonium-lyase on magnetic nanoparticles / 2460790
What is presented is a method for immobilisation of L-phenylalanine-ammonium-lyase on magnetic nanoparticles of metal oxides in the presence of the condensing agent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.
Biocatalyst, method of preparing said biocatalyst and method of obtaining invert syrup using said catalyst / 2451546
Invention is meant for use in food industry and specifically relates to the technology of obtaining syrups containing glucose and fructose, used in confectionary and baking industry to make confectionary products and candy. Disclosed is a biocatalyst for producing invert syrup, a method of producing said biocatalyst and a method of producing invert syrup - an inversion of saccharose using the prepared biocatalysts. The biocatalyst for producing invert syrup contains the following in wt % on dry substances: yeast autolysate as enzyme-active biomass 30-50, nanocarbon component 5-15 and filler consisting of silicon dioxide - up to 100. A nanocarbon component with a nanofibre structure or a carbon nanotube structure or a nanodiamond structure or a bulbous nanocarbon structure is used. The method of obtaining invert syrup is realised in a reactor with a fixed bed of the biocatalyst described above at temperature not higher than 50°C.
 Peroxidase enzymatic activity stabiliser / 2445271
Invention relates to biochemistry. Disclosed is a peroxidase enzymatic activity stabiliser. The stabiliser is in form of cobalt ferrospinel nanoparticles, components of which vary in the interval Co0.7±0.05Fe2.3±0.05O4-Co1±0.05Fe2±0.05O4, which are suspended in a sodium phosphate buffer solution with concentration of 0.01-10 mg/ml.
Method of producing surface-modified nanoparticles for immobilising biological substances / 2425879
Disclosed is a method of producing surface-modified nanoparticles for immobilising biological substances. The method involves modifying Fe3O4 nanoparticles containing ester groups. The obtained particles are then treated with aminopropyl triethoxy silane. Further, the suspension of nanoparticles is incubated with a condensing agent and the enzyme is immobilised.
 Method of obtaining silver nanoparticles / 2526390
Method of obtaining silver nanoparticles includes preparation of water solutions of silver nitrate with the concentration of 0.001÷0.02 M/l and L-cysteine with the concentration of 0.00125÷0.04 M/l. The obtained solutions are mixed with the molar ratio of silver nitrate and L-cysteine in the range of 1.25÷2.00 and kept at a temperature of 15÷55°C for 0.34÷48 hours in a protected from light place with obtaining a solution of a supramolecular polymer. The obtained solution of the supramolecular polymer is diluted with water in a volume ratio of 1:1. A water solution of sodium borohydride with the concentration of 0.003÷0.010 M/l is prepared and added to the solution of the supramolecular polymer with constant mixing.
Method of stabilising nanoparticles of biogenic elements with enzymes / 2504582
Method involves synthesis of nanoparticles through a redox reaction while adding an enzyme for stabilising the formed nanoparticles directly into the reaction. The enzyme solution - chymotrypsin, concentration of which is selected from 0.02%, is mixed with a precursor solution of 0.013 M selenous acid or silver nitrate - 1%, 0.01%, 0.001%; the solutions are stirred and held at room temperature of 20°C or low temperature of 0°C for 30-60 minutes. A reducing agent - ascorbic acid or sodium borohydride - is then added to the reaction flask and the solutions are stirred and settled to complete the reaction.
Method of silver extraction from sewages and technological solutions / 2497760
Invention relates to chemical industry and environment protection. Silver is extracted from water with application of compositional sorbent in quantity 50-200 mg/dm3 of water. Sorbent consists of cellulose fibres which contain, in wt %, not less than 94 % of fibres with length not more than 1.23 mm and not less than 54% of fibres with length not more than 0.63 mm, and immobilised on them lead sulphide in quantity 50-300 wt.f. per 100 wt.f. of fibres. Processed water is separated from sorbent by pressure flotation. Float-sludge or its part is returned into process of water processing. Float-sludge and processed water discharged from the process are utilised.
 Method of producing silver nanocompositions based on synthetic water-soluble polymers / 2485051
Invention relates to a method of producing compositions of silver nanoparticles based on water-soluble synthetic copolymers. The method involves reducing silver ions in the presence of a water-soluble polymer. The polymers used, which combine properties of a silver ion reducing agent and a stabiliser of the formed nanoparticles, are copolymers of 2-deoxy-2-methacrylamido-D-glucose with 2-dimethylamino-ethylmethacrylate or 2-diethylamino-ethylmethacrylate of general formula:
 Method for obtaining ionic silver solution / 2471018
Metallic silver is diluted in distilled water till electrolyte is formed. After electrolyte is formed as a result of anodic silver oxidation and self-dilution of oxide, dilution process is interrupted, electrolyte is drained and magnetised by passing it through a glass tube going through magnetic field of constant magnet. Then, at weak mixing of the solution, dilution process of metallic silver is continued till hardly transparent black suspension is formed; after that, the process is stopped. Settled concentrate is separated; in addition, clean electrolyte is magnetised and again brought into circulation, and deposit of crystalline hydrate of silver oxide (1) is used in order to obtain water solution of ionic silver, at which crystalline hydrate is diluted in water, magnetised in magnetic field, filtered and drained to glass bottles to be stored.
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FIELD: biotechnology. SUBSTANCE: method of immobilisation of chymotrypsin on nanoparticles of selenium or silver is proposed. The solution of chymotrypsin at a concentration of from 1·10-3 to 1 wt % is added the solution of selenious acid in the concentration range of 1.3·10-4-1.5 wt % or silver nitrate in the concentration range of 1·10-4-1 wt %. Then ascorbic acid is added to the reaction medium in the concentration of 1·10-3-0.7 wt % or solution of sodium borohydride in a concentration of from 0.01 to 0.6 wt %. The solution is stirred and left to complete the reaction. The process is carried out at a temperature of from 0 to 50°C. EFFECT: method enables to obtain a stable enzyme-colloidal complex and maintain in it over 90 percent of chymotrypsin activity in a broad pH range, and to increase the catalytic activity of nanocomplexes in certain pH ranges higher than the activity of native chymotrypsin at optimum point. 3 tbl, 3 ex
The invention relates to the field of Bioorganic chemistry, biotechnology, and nanochemistry, specifically to develop methods of immobilization of enzymes, ensuring the continued stability of their molecules, as well as the increase of specific enzyme activity. Immobilized enzymes have several advantages over native: continuity of conducting enzymatic process for adjusting the speed of the catalyzed reaction and yield of product; the directed change of properties of the enzyme (specificity, dependence of catalytic activity on pH and other environmental parameters, stability to denaturing influences); the possibility of regulating the catalytic activity of immobilized enzymes by changing media properties. In this area a large number of technical solutions, among which the most widespread chemical and physical methods of immobilization. Chemical method of immobilization is to covalent binding of biomolecules with pre-activated carrier, modified with reactive functional groups (amino, azido-, carboxyl, hydroxyl, etc.). Physical immobilization method is the adsorption of the enzyme on a solid support (usually a polymer) due to the physical �Il (ion-ion, hydrophobic, hydrogen bonds, etc). Also known a method of producing immobilized proteolytic enzyme (RU # 1041567, IPC C12N 11/10, 15.09.1983) providing a solution containing the aldehyde group of the carrier in a buffer solution and the subsequent addition of a proteolytic enzyme. As the carrier used xiaorong dissolved in 0.1 M trioxymethylene buffer (pH 8.5), and the accession of the enzyme is carried out at a ratio of carrier-enzyme 1:1. The main disadvantages of this method include the following: a scarce and expensive media - xiaorong, low stability of the target product, the need for storage of the preparation at low temperature (0-4°C). Also known physical method of immobilization of enzymes (EN 2167197, IPC C12N 11/14, SR 19/02, 20.05.2001) describing the composite for the saccharification of starch comprising the enzyme glucoamylase and solid support on the surface of immobilized glucoamylase, the carrier is zauglerozhenny silicate, which has a specific surface area of not less than 2 m2/g and is made in the form of pellets, honeycomb monoliths or foam. The carrier is prepared in a way that allows for enhanced adsorption properties zauglerozhenny aluminosilicate. In the preparation of the carrier to the original silicate with a specific surface�of 0.1 to 24 m 2/g is applied Nickel. Then spend the pyrolysis of propane-butane mixture in the presence of the media, resulting in zauglerozhenny silicate, specific surface area of which exceeds several times the specific surface area of the aluminosilicate source. The thus prepared carrier has a structure containing a large amount of mesopores, suitable in size for the sorption of molecules of the enzyme. Immobilization of glucoamylase is to conduct the process of physical adsorption on the surface of the obtained carrier. Physical adsorption is carried out by immersing the carrier in an aqueous solution of the enzyme and soak it for 6 hours with occasional stirring. The disadvantages of this invention are associated with the multi-stage technological process, with the use of pyrolysis for the deposition of the layer of porous carbon on the carrier surface, which is associated with high energy costs and large volume organic chemicals (propane-butane). In addition, as a result of sorption of the immobilized enzyme has low activity-50-80%(of the activity of the free enzyme). The closest technical solution is a method of immobilization of L-phenylalanin-ammonium-lyase on magnetic nanoparticles (see us Pat. Of the Russian Federation №RU 2460790, IPC SR 19/04, B01D 15/38, SC 31/10, 10.09.2012), sanlucas�ISA in use as a carrier for immobilization of magnetic nanoparticles, representing the metal oxides. Immobilization of the enzyme on the nanoparticles is realized through advanced modification of their surface. The first stage is receiving magnetic nanoparticles containing on the surface of the electrophilic ester group, by reacting polymethyl methacrylate, the corresponding metal chloride and diethylene glycol. The second stage consists in forming on the surface of the nanoparticles of the layer of aminopropyltriethoxysilane due to the reaction of aminolysis electrophilic fragments of the media. The obtained nanoparticles are used for the subsequent immobilization of enzymes using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. Significant and obvious disadvantages of the described method are multi-stage, the need to resuspension nanoparticles obtained in the first stage, the additional surface modification and subsequent chemical immobilization of the enzyme on the surface of nanoparticles, which involves the use of additional reagents and complicates the process. In addition, chemical immobilization significantly affects the conformation of the enzyme, which reduces its catalytic activity. Immobilized enzyme retained only from 64% to 75% of its activity in the free state in a narrow pH range from 7.5 to 90. Technical challenge and a positive result of the claimed invention is to develop a one-step method of immobilization of different enzymes on nanoparticles of different nature, the result of which is to obtain time-stable colloidal solution of nanocomplex, the enzymatic activity of which is comparable or greater activity than the free enzyme in a wide pH range and do not have the stated disadvantages of the prototype. The invention consists in developing a method of immobilization of enzymes (in particular, chymotrypsin) on the nanoparticles of biogenic elements (e.g. selenium and silver). The specified task and the result of the invention is achieved by a redox reaction in the presence of enzyme solution intended for immobilization, for example - chymotrypsin, which are formed nanoparticles with adsorbed enzyme. To a solution of the enzyme, the concentration of which can vary from 0.001 to 1 wt.% add a solution of an oxidant, for example, se acid (H2SeO3) in a concentration range of 1.3·10-4To 1.5 wt.% or silver nitrate (AgNO3) in a concentration range from 1·10-4-1 mass%. Then the reaction medium is introduced reductants, such as ascorbic acid (from 1·10-3A-0.7 wt.%) Il� solution of sodium borohydride (0.01 to 0.6 wt.%). The process is conducted at a temperature of 0 to 50°C. the Solutions were mixed and left to complete the reaction. At the end of the reaction obtained stable solutions of the nanoparticles immobilized on the surface of enzymes due to the forces of physical adsorption. Distinctive features of the proposed method are shown above; the proposed method of immobilization of enzymes has obvious advantages over the prototype. Analysis of prior art did not allow to find a published solution, which would be used by the totality of essential features of the claimed method. This demonstrates the method according to the invention the condition of patentability as "novelty" and "inventive step". When identifying the materiality of novelty signs was obtained as follows. The catalytic activity of the enzyme-colloidal complex is maintained almost at the level of the free enzyme or exceeds it. Immobilized in this way, the enzyme shows higher catalytic activity over a wide pH range, including in sub-optimal intervals for the native enzyme, unlike the prototype. The method allows to immobilize the enzyme on the nanoparticles of different nature, combining in a single drug as the catalytic properties Firmin�and, and its own biological activity of the matrix-carrier. The proposed method of immobilization of one - stage and is easy to implement in terms of technology. Method of immobilization of enzyme by physical nature and requires spending additional chemical agents or the pre-modification of the particle surface. Nanocomplexes exhibit stability up to 1 year. Selected intervals of the concentrations of precursors and reducing agents due to the fact that for large values of the concentrations of the reactants (more than 1.5 wt.% H2SeO3and about 1 wt.% AgNO3) are formed aggregative unstable solutions of nanocomplexes with low enzymatic activity. At low concentrations (less than 1.3·10-4wt.% H2SeO3and 1·10-4wt.%) AgNO3remains a large number of the free enzyme, and increasing the enzymatic activity is not observed. By changing the ratio of the concentration of the enzyme and the nanoparticles can be adjusted to size and enzymatic activity. Varying the amount of reducing agent can change the size of the resulting complexes. The resulting nanocomplexes are combined in one preparation of various properties associated with nature as nanoparticles (antimicrobial, antioxidant, anti-inflammatory, Antica�ceregene, detoxifying) and with the properties of enzymes (catalytic activity, substrate specificity, coupled enzymatic reaction). Immobilized enzyme shows a higher proteolytic activity, or in the entire pH range or within separate intervals pH profile. To prove compliance of the claimed solution the condition of patentability "industrial applicability" and for a better understanding of the essence of the claimed invention provides examples of specific performance for chymotrypsin on the selenium nanoparticles and silver. Example 1. For the immobilization of chymotrypsin on the selenium nanoparticles In flat-bottomed flask, 30 ml, was placed 0.5 ml of 0.2% solution of chymotrypsin (XT), add 7.5 ml of distilled water, then with stirring on a magnetic stirrer was added 1 ml 0,013 M se acid (H2SeO3) and after stirring for 10 min was added as a reducing agent ascorbic acid (C6H8O6) 1 ml of 0.025 M. Continue to mix for another 5 min and then leave before the completion of the reaction (24 hours) at room temperature. The obtained molecular solution has a characteristic reddish-orange color, the solution pH 3,2. Solution is stable up to 1 year. Nanocomplexes selenium-chymotrypsin shows a higher proteolytic activity compared with a sheer enzyme in a wide pH range.
Example 2. For the immobilization of chymotrypsin on silver nanoparticles In flat-bottomed flask, 30 ml, was placed a suspension of sodium borohydride (NaBH4) 0,011 g, placed the flask and add 10 ml of distilled water. Dissolved NaBH4aged 30-40 min at a time in a flask with 50 ml injected 5 ml of a 0.1% solution of XT and 1 ml of 0.02% solution of silver nitrate (AgNO3). The mixture was stirred in a circular motion bulb in the mold with ice and leave it there for 30-40 minutes. After equalization of the temperature in the flask with XT and with silver nitrate injected 4 ml of the solution prepared. Begins a violent reaction, the reaction flask remains in ice for 1 hour and then placed in the refrigerator for 18-24 hours. The reaction turns the solution black color, the pH of which is 10.4. The solution is stable from 1 month to 1 year.
As a result of application of the method get drugs that effectively maintain the catalytic properties of the enzyme more than 90% of them or significantly exceed compared with the free enzyme. Enzyme-colloidal complex shows high activity in a wide pH range. The method allows for the immobilization of the nanoparticles, with its own biological activity, and various in nature. The whole process is one�tadinya and is easy to implement in terms of technology. Example For the immobilization of chymotrypsin on the selenium nanoparticles at 50°C in flat-bottomed flask, 30 ml, was placed 0.5 ml of 0.2% solution of chymotrypsin (XT), add 7.5 ml of distilled water, then with stirring on a magnetic stirrer was added 1 ml 0,013 M se acid (H2SeO3) and after stirring for 10 min the flask was placed in a thermostat heated to 50°C, or in a water bath at the same temperature. After keeping the reaction flask in an incubator for 30 to 45 minutes (when the temperature becomes equal to 50°C) was added as a reducing agent ascorbic acid (C6H8O6) 1 ml of 0.025 M. Continue to mix for another 5 min and then leave before the completion of the reaction (24 hours) in an incubator at a temperature of 50°C. the Obtained molecular solution has a characteristic red color, the solution pH 3,2. Solution is stable up to 1 year. Nanocomplexes selenium-chymotrypsin shows a higher proteolytic activity compared with the pure enzyme in a wide pH range.
Method of immobilization of chymotrypsin on the selenium nanoparticles or silver, characterized by the fact that the formation of complexes of the redox reaction in the presence of the enzyme solution as follows: to a solution of the enzyme a - chymotrypsin with a concentration of 1·10-3/sup> up to 1 wt.% added a solution se acid (H2SeO3) in a concentration range of 1.3·10-4- 1.5 wt.% or silver nitrate (AgNO3) in a concentration range from 1·10-4- 1 wt.%; then the reaction medium is introduced reducing agents: ascorbic acid in a concentration of from 1·10-3To 0.7 wt.% or a solution of sodium borohydride at a concentration of from 0.01 to 0.6 wt.%; the process is conducted at a temperature of 0 to 50°C, the solution was stirred and left to complete the reaction.
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