RU2399672C1 - Biocatalytic method of n-substituted aliphatic acrylamide synthesis and rhodococcus erythropolis bacteria strain for implementation thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method provides the use of said strain as a biocatalyst for N-substituted aliphatic acrylamide synthesis of acrylamide and primary aliphatic amines. The invention allows producing high-yield N-substituted aliphatic acrylamides. The yield of N-iaopropylacrylamide is 11 g/l, of N-dimethylaminopropiylacrylamide - 5 g/l.

EFFECT: Rhodococcus erythropolis bacteria strain, 37 RNCIM No As-1793 exhibit acylating activity and is capable to synthesise N-substituted aliphatic acrylamides.

2 cl, 5 ex

Description

The present invention relates to biotechnology and relates to the synthesis of N-substituted aliphatic acrylamides, in particular N-isopropyl acrylamide and N-dimethylaminopropyl acrylamide, by biocatalytic acylation of primary aliphatic amines with acrylamide using a bacterium strain Rhodococcus erythropolis with acylation activity as a biocatalyst.

N-substituted aliphatic acrylamides are widely used as monomers to obtain water-soluble polymers used in the production of various flocculants, adsorbents, and structure-forming agents used in industry and agriculture (Polyacrylamide. - M., 1992.).

Traditionally, N-substituted aliphatic acrylamides are obtained by chemical methods. The most universal of them is the acylation of aliphatic amines with acid chlorides of acrylic and methacrylic acids (MacWilliams D. S. Functional monomers. Their preparation, polymerization, and application. New York: M. Decker, 1973). Common disadvantages of chemical synthesis methods are the need to obtain and use highly toxic acid chlorides and the use of toxic organic solvents.

An alternative approach to the synthesis of chemical compounds is biocatalytic synthesis. When carrying out biocatalytic methods for the synthesis of chemicals, enzymes or cells of microorganisms are used as catalysts, and the synthesis process is carried out at low temperatures and pressure in aqueous media (RF 2053300).

The mentioned differences of biocatalytic methods from chemical ones are in many cases their advantages. The possibility of synthesizing in aqueous media leads to lower toxicity of production, the possibility of carrying out processes at low temperatures and pressures reduces its cost, more pronounced selectivity of enzyme-based catalysts allows to obtain cleaner substances. Additional advantages of biocatalysts over chemical catalysts are simpler methods for producing biocatalysts, as well as the possibility of modifying biocatalysts in a given direction for the synthesis of various structural variants of compounds.

To date, no biocatalytic methods for the synthesis of N-substituted aliphatic acrylamides are known.

The present invention is the development of a biocatalytic method for the synthesis of N-substituted aliphatic acrylamides.

The problem is solved by:

- obtaining strain Rhodococcus erythropolis 37, having acylating activity, deposited in the All-Russian collection of industrial microorganisms (VKPM) under the number VKPM As-1793 and

- development of a method for the synthesis of N-substituted aliphatic acrylamides from acrylamide and primary aliphatic amines using Rhodococcus erythropolis 37 as a bacterial biocatalyst.

The method proposed in the present invention is based on the acylation of primary aliphatic amines in an aqueous medium with amides of unsaturated carboxylic acids, in particular acrylamide. As a biocatalyst in the present method using a strain of Rhodococcus erythropolis VKPM Ac-1793, with acylating activity.

The inventive strain has an acylating activity with unique substrate specificity and is capable of synthesizing N-substituted aliphatic acrylamides, in particular N-isopropyl acrylamide, N-dimethylaminopropyl acrylamide.

The strain Rhodococcus erythropolis 37 VKPM Ac-1793 is isolated from the soil in a minimal medium with acetonitrile.

Characterization of the claimed strain

Morphological properties. The cells of the strain Rhodococcus erythropolis 37 VKPM Ac-1793 are motionless and stained according to Gram. They do not form a dispute, non-acid resistant. After 18-20 hours of growth at a temperature of 30 ° C on meat-peptone agar - MPA (Manual for practical studies in microbiology, Moscow State University, 1983, p. 93), the cells form weakly branching filaments, which after 48-72 hours break up into rod-shaped and cocciform elements. In old cultures, pleomorphic cells are observed in the form of flasks, pears, clubs. Cell division occurs according to the splitting and bending types.

On solid nutrient media (agar Luria Bertani, meat-peptone agar, Hotinger agar) after 48 hours of growth at a temperature of 30 ° C, the claimed strain forms round smooth colonies with a diameter of 1 mm, painted in pink.

The composition of the nutrient media (wt.%):

Agar Luria Bertani:

Tripton 0.5 Yeast extract 0.25 NaCl 0.5 Agar agar 2 Water Rest

Meat-peptone agar:

Agar Agar 2

Meat-peptone broth - the rest

Agar Khotinger:

Agar Agar 2

Hottinger Bouillon (Guide to Practical Activities in Microbiology, Moscow State University, 1983, p. 93) - the rest.

Physiological properties:

The inventive strain assimilates lactose, acetate, succinate, fumarate, glycerin, fructose, acetamide, acrylamide, butyramide, isobutyramide, propionamide, valeramide. The optimal pH of the strain is in the range of 6.5-8.5.

Strain storage:

Storage of the claimed strain is carried out in the form of lyophilized cells.

The nucleotide sequence of the gene of the ribosomal 16S RNA

The nucleotide sequence of the gene of the ribosomal 16S RNA of the claimed strain corresponds to the genus Rhodococcus, species erythropolis.

Based on the foregoing and in accordance with Bergey’s guidelines (Berji Bacterial Identifier, Moscow, Mir, 1997), strain VKPM Ac-1793 is assigned to the genus Rhodococcus and the species erythropolis.

Optimum cultivation conditions:

To obtain cells of the inventive strain having acylating activity, the strain is grown at 25-30 ° C for 24-36 hours on synthetic media containing acetanilide at a concentration of 0.2-0.4%, or together fructose 0, as a carbon source. 2-0.4% and acetanilide 0.4-0.6%, and as a source of nitrogen, ammonium or nitrate salts in concentrations of 0.4-2%.

The composition of the synthetic medium M3, wt.%

K ₂ HPO ₄ * 3H ₂ O 0.7 KH ₂ PO ₄ 0.3 MgSO ₄ 0.05 FeSO ₄ 0,0005 a mixture of vitamins 0.05 deionized water rest

The method in General is as follows:

The preparation of an aqueous solution of N-substituted aliphatic acrylamides, in particular N-isopropylacrylamide and N-dimethylaminopropylacrylamide, is carried out by mixing aqueous solutions of acrylamide and aliphatic primary amines, in particular isopropylamine and dimethylaminopropylamine, in concentrations of 1-10% with a suspension of biocatalyst temperature 20-40 and ° C and pH 9.5-11 for 1-48 hours. The concentration of the biocatalyst in a mixture of 2-16 g dry weight / l.

The biocatalyst is stored and used in the form of free cells of the inventive strain, obtained by separation from the culture fluid by centrifugation at 5-16 thousand rpm and subsequent suspension in 10 mm phosphate buffer pH 7.0-8.0 to a concentration of 20-40 g dry weight / l The concentration of the resulting N-substituted aliphatic acrylamide is determined by gas chromatography. Analysis of the reaction mixture is carried out on a gas chromatograph LXM-80 (Russia) with a flame ionization detector. The reaction products are separated using a quartz column 32 m long packed with FFAP sorbent at a constant temperature of 180 ° C. Helium is used as the carrier gas.

The resulting N-substituted aliphatic acrylamide is isolated from the solution by extraction with benzene, redissolution in diethyl ether, precipitation at a temperature of 20 ° C, and filtration.

The invention is illustrated by examples.

Example 1. Obtaining a biocatalyst

In an Erlenmeyer flask (750 ml) containing 100 ml of synthetic nutrient medium M3 containing acetanilide at a concentration of 0.4% and NH ₄ NO ₃ at a concentration of 0.2%, add 1 ml of culture (10 ⁹ cells / ml) of the claimed strain , previously grown on Luria Bertani's medium for 16 hours at 30 ° C with stirring (300 rpm). Then the flask was incubated with stirring (200 rpm) at 30 ° C for 36 hours. The biomass is separated by centrifugation at 5 thousand rpm, resuspended in 10 mm phosphate buffer pH 7.0 to a concentration of 20 g / l and stored at + 4 ° C. Thus obtained cells in an amount of 20 mg dry weight have an acylating activity of 4.4 units / g dry cell weight.

Acylating activity is determined by the rate of synthesis of N-isopropylacrylamide according to the following procedure:

Mix 300 μl of a 10% aqueous solution of acrylamide, 250 μl of a 10% aqueous solution of isopropylamine (pH 10), 450 μl of a suspension of cells containing 2.4 mg of cells. Incubated at 37 ° C for 2 hours, the cells are separated by centrifugation for 1 minute at 10 thousand rpm, the content of N-isopropylacrylamide is determined by gas chromatography.

The unit of activity is the amount of enzyme required for the synthesis of 1 mM N-isopropylacrylamide in 1 hour.

Example 2. Obtaining a biocatalyst with a higher yield

The biocatalyst is grown, as in example 1, but the M3 medium additionally contains 0.4% fructose. The biomass is also resuspended, but to a concentration of 32 g dry weight / L.

The result is biomass in an amount of 80 mg dry weight with an acylating activity of 2.2 units / g dry cell weight.

Example 3. Synthesis of N-isopropylacrylamide from acrylamide and isopropylamine

Aqueous solutions of acrylamide and isopropylamine (pH 10) are mixed, then a suspension of the cells of the inventive strain obtained as in Example 1 is added to the reaction mixture. The concentrations of acrylamide and isopropylamine in the mixture are equimolar and make up 3% (0.42 mmol) and 2.5% ( 0.42 mmol), respectively. The concentration of cells in the mixture is 8 g dry weight / L. The reaction mixture was incubated at 37 ° C for 6 hours. Then the cells are separated by centrifugation (5 thousand rpm) and the amount of product obtained is determined by gas chromatography. The concentration of the resulting solution of N-isopropylacrylamide is 11 g / L.

Example 4. Synthesis of N-dimethylaminopropylacrylamide from acrylamide and dimethylaminopropylamine

Aqueous solutions of acrylamide and dimethylaminopropylamine (pH 10) are mixed, then a suspension of the cells of the inventive strain obtained as in Example 1 is added to the reaction mixture. The concentrations of acrylamide and dimethylaminopropylamine in the mixture are equimolar and equal to 1% (0.14 mmol) and 2.5% ( 0.14 mmol), respectively. The concentration of cells in the mixture is 2.4 g dry weight / L. The reaction mixture was incubated at 37 ° C for 24 hours. Then the cells are separated by centrifugation (5 thousand rpm) and the amount of product obtained is determined by gas chromatography. The concentration of the resulting solution of N-dimethylaminopropylacrylamide is 5 g / L.

Example 5. Obtaining N-isopropylacrylamide

50 ml of a suspension of cells of the inventive strain obtained as in example 2, contribute to a vessel with a volume of 200 ml. Then, 25 ml of an 8% solution of acrylamide and 25 ml of an 8% solution of isopropylamine with a pH of 10 are added there. The process is carried out with stirring (200 rpm), with a pH control, at a temperature of 37 ° C. After 24 hours, the process is completed, the cells are separated by centrifugation (10 thousand rpm). The resulting supernatant is extracted 3 times with an equal volume of benzene. Then the benzene is evaporated on a vacuum rotary evaporator at 38-40 ° C, the resulting oily liquid is dissolved in 15 ml of diethyl ether. The solution was dehydrated with anhydrous sodium sulfate and concentrated on a vacuum rotary evaporator. When cooled to 20 ° C, a white and yellow crystalline precipitate precipitated with a characteristic odor.

The obtained crystalline precipitate is analyzed by determining the melting temperature and the content of elements using elemental analysis:

- the melting point of the obtained crystals 62-64 ° C corresponds to the known (Journal of American Chemical Society, v.73, 1951, p.4076) for N-isopropylacrylamide - 62-63 ° C.

-element analysis revealed the following content of elements in the obtained sample of the substance (%): C - 63.77; H, 9.68; and N, 11.52. The element contents calculated for N-isopropylacrylamide are the same as experimentally determined (%): C - 63.71; H, 9.73; and N, 12.38.

Based on the determination of the melting temperature and the correspondence between the calculated and real results of elemental analysis, a conclusion is drawn on the correspondence of the obtained substance to N-isopropyl acrylamide.

Thus, the obtained strain Rhodococcus erythropolis 37 VKPM Ac-1793, with acylating activity, capable of synthesizing N-substituted aliphatic acrylamides. Based on it, a biocatalytic method for the preparation of N-substituted aliphatic acrylamides, in particular N-isopropylacrylamide and N-dimethylaminopropylacrylamide using microorganism cells, was first developed.

Claims (2)

1. Biocatalytic method for the synthesis of N-substituted aliphatic acrylamides from acrylamide and primary aliphatic amines using the bacterial strain Rhodococcus erythropolis 37 VKPM Ac-1793, which has acylating activity and is capable of synthesizing N-substituted aliphatic acrylamides, as a biocatalyst. 2. The bacterial strain Rhodococcus erythropolis 37 VKPM Ac-1793, with acylating activity and capable of synthesizing N-substituted aliphatic acrylamides.