RU2522804C2 - METHOD OF SYNTHESIS OF N-SUBSTITUTED ACRYLAMIDES AND RECOMBINANT STRAIN Rhodococcus erythropolis FOR ITS IMPLEMENTATION (VERSIONS) - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: recombinant strains Rhodococcus erythropolis 37 p16-Ami RNCIM Ac-1937 and Rhodococcus erythropolis HX7 p16-Ami RNCIM Ac-1938 are constructed, constitutively producing the enzyme acylamidase with acylating activity. Also, the method of synthesis of N-substituted acrylamides is created, in particular N-isopropylacrylamide, and N-dimethylaminopropylacrylamide, using these strains as a biocatalyst.

EFFECT: invention enables to improve the efficiency of preparing N-substituted acrylamides.

3 cl, 1 tbl, 1 dwg, 9 ex

Description

The claimed group of inventions relates to biotechnology and relates to recombinant strains of Rhodococcus erythropolis, as well as to a method for the synthesis of N-substituted acrylamides, in particular N-isopropyl acrylamide or N-dimethylaminopropyl acrylamide, using cells of these strains as a biocatalyst.

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

The traditional method of industrial production of N-substituted acrylamides is carried out by chemical synthesis based on 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). The disadvantage of this method is the use of highly toxic acid chlorides, as well as toxic organic solvents.

An alternative approach to the synthesis of N-substituted acrylamides is biocatalytic synthesis occurring in an aqueous medium. A known method of biocatalytic synthesis of N-substituted acrylamides using a biocatalyst - cells of the strain Rhodococcus erythropolis 37 VKPM Ac-1793, having acylating activity (RU 2399672), due to the presence of the acylamidase enzyme in them.

The strain Rhodococcus erythropolis 37 VKPM Ac-1793 (RU 2399672) will be considered as the closest analogue of the claimed strain, and the method of biocatalytic synthesis of N-substituted acrylamides using as a biocatalyst cell strain Rhodococcus erythropolis 37 VKPM Ac-1793 (RU 2399672) - analog of the proposed method.

The disadvantage of the strain Rhodococcus erythropolis 37 VKPM As-1793 is that its acylating activity is inducible, that is, it requires the presence of an expensive inducer, acetanilide, in the culture medium. This increases the cost and reduces the competitiveness of the method - the closest analogue based on the use of strain VKPM As-1793 as a biocatalyst.

RU 2439154 provides the nucleotide sequence of a DAA37 DNA fragment from Rhodococcus erythropolis 37 VKPM Ac-1793 encoding the acylamidase enzyme. This information has opened up prospects for the design of more advanced biocatalysts with acylating activity by genetic engineering methods.

The task of the claimed group of inventions is the creation of recombinant strains of Rhodococcus erythropolis constitutively synthesizing acylamidase and the development of biocatalytic methods for the synthesis of N-substituted acrylamides using cells of these strains as a biocatalyst.

The problem is solved by:

- development of a method for the synthesis of N-substituted acrylamides using Rhodococcus erythropolis with constitutive acylating activity as a biocatalyst;

- designing a strain with constitutive acylating activity of Rhodococcus erythropolis 37 p16-Ami VKPM Ac-1937, by introducing a DAA37 DNA fragment of the hybrid plasmid p16-Ami in the recipient strain Rhodococcus erythropolis 37 VKPM Ac-1793 for the implementation of the proposed method;

- constructing a strain with constitutive acylating activity of Rhodococcus erythropolis HX7 p16-Ami VKPM As-1938, by introducing a DAA37 DNA fragment as part of the hybrid plasmid p16-Ami into the recipient strain Rhodococcus erythropolis HX7 VKPM As-1267 to implement the proposed method.

Strains of Rhodococcus erythropolis, into which a plasmid with a DAA37 DNA fragment from Rhodococcus erythropolis 37 VKPM As-1793 was introduced, acquires constitutive acylating activity and the ability to synthesize N-substituted aliphatic acrylamides. When aqueous solutions of acrylamide and amines are mixed in the presence of Rhodococcus erythropolis 37 p16-Ami VKPM Ac-1937 and Rhodococcus erythropolis HX7 p16-Ami VKPM Ac-1938 cells (used as a biocatalyst), N-substituted acrylamides, in particular N-isopropylacrylamide, are synthesized N-dimethylaminopropylacrylamide.

Characterization of the claimed strains.

Rhodococcus erythropolis 37 p16-Ami VKPM Ac-193 7 is a derivative of the strain Rhodococcus erythropolis 37 VKPM Ac-1793, into which a plasmid with a DAA37 DNA fragment encoding acylamidase synthesis was introduced. The strain Rhodococcus erythropolis 37 p16-Ami VKPM Ac-1937 additionally acquired a marker of resistance to apramycin.

Rhodococcus erythropolis HX7 VKPM Ac-1938 is a derivative of the strain Rhodococcus erythropolis HX7 VKPM Ac-1267, into which a plasmid with a DAA37 DNA fragment encoding acylamidase synthesis was introduced. The strain Rhodococcus erythropolis HX7 p16-Ami VKPM Ac-1938 additionally acquired a marker of resistance to apramycin.

Optimum cultivation conditions.

To obtain the cells of the claimed strains in large quantities and with high acylamidase activity, the strains are grown at 25-30 ° C for 24-72 hours on synthetic media containing sucrose, acetate or molasses in concentrations of 0.1-4% as a carbon source, and in as a source of nitrogen, ammonium or nitrate salts in concentrations of 0.4-2%.

Obtaining a biocatalyst.

Cells of biocatalysts - recombinant strains of Rhodococcus erythropolis with constitutive acylating activity are obtained as follows.

The seed, representing a culture of bacteria Rhodococcus erythropolis, carrying the p16-Ami plasmid, is grown in Luria Bertani (LB) liquid medium having a composition in wt.%: Tryptone 0.5, yeast extract 0.25, NaCl 0.5, water - the rest) with apramycin 100-200 μg / ml at 20-30 ° C until an optical density of 4-5 units is reached at 540 nm. After incubation for 24-30 hours, the cells are precipitated, washed and used as a biocatalyst.

The obtained cells are stored and used in the form of an aqueous suspension obtained by separating the cells 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 acylating activity of the strains 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 culture. Incubated at 37 ° C for 2 hours, cells are separated by centrifugation for 1 minute at 10 thousand rpm, the content of N-isopropylacrylamide is determined by gas chromatography.

Culture activity is expressed in mm IPAA synthesized in 1 hour per liter of culture.

A distinctive feature of the claimed strains is their ability to synthesize the enzyme acylamidase constitutively, i.e. in the absence of known inducers in the medium, such as acetanilide (Table 1). Moreover, the recombinant strains of Rhodococcus erythropolis exhibit higher activity than the recipient strains of Rhodococcus erythropolis that do not contain the DAA37 DNA fragment encoding acylamidase synthesis in the recombinant plasmid p16-Ami.

Table 1 Acylating activity of recombinant and recipient strains of Rhodococcus erythropolis Strain Acylating activity, mm IPAA / l * h Without inductor With inductor Rhodococcus erythropolis 37 p16-Ami VKPM Ac-1937 * 5 5 Rhodococcus erythropolis HX7 p16-Ami VKPM Ac-1938 * 5 5 Rhodococcus erythropolis 37 VKPM
Ac-1793 ** 0 2 Rhodococcus erythropolis HX7 VKPM Ac-1267 ** 0 0

* grown as in example 4;

** grown on media of the following composition (wt.%):

medium with inducer: acetanilide-4, NH ₄ NO ₃ - 2, K ₂ HPO ₄ * 3H ₂ O - 7, KH ₂ PO ₄ - 3, MgSO ₄ - 0.5, FeSO ₄ - 0.005, deionized water - the rest;

medium without inducer: sucrose - 4, NH ₄ NO ₃ - 2, K ₂ HPO ₄ * 3H ₂ O - 7, KH ₂ PO ₄ - 3, MgSO ₄ - 0.5, FeSO ₄ - 0.005, deionized water - the rest.

The method of synthesis of N-substituted acrylamides in General

The synthesis of N-substituted acrylamides, in particular N-isopropylacrylamide or N-dimethylaminopropylacrylamide, is carried out by mixing aqueous solutions of acrylamide and aliphatic primary amines, in particular isopropylamine and dimethylaminopropylamine, in concentrations from 1% to 10% with a suspension of biocatalyst (g from 1 to 10% by dry weight per 1 liter of the reaction mixture) in a thermostatic vessel (at a temperature of 20 to 40 ° C, preferably 25 to 30 ° C) and subsequent stirring (100 to 250 rpm) for 1-48 hours. pH varies from 8.5 to 11, preferably more precisely from 9 to 11.

The content of the final product, N-substituted acrylamide, is determined by gas chromatography.

The inventive method allows to obtain solutions containing from 1 to 20 g / l of N-substituted acrylamides.

The invention is illustrated by the following examples.

Example 1. Construction of a recombinant plasmid p16-Ami.

Recombinant plasmid p16-Ami is a derivative of plasmid pN30 containing a replicon for autonomous replication in Rhodococcus (Ryabchenko L.E., Novikov A.D., Golyshin P.N., Yanenko A.S. Determination of the nucleotide sequence and structural analysis of the cryptic plasmid pN30 from the oil oxidizing strain of Rhodococcus erythropolis, Genetics, 2005, Volume 41, N12, pp. 1725-1727), and the plasmid pBlueScript containing replicon for autonomous replication in E. coli (M.A. Alting-Mees, JA Sorge, JM Short. pBluescriptll: Multifunctional cloning and mapping vectors. Methods in Enzymology, Volume 216, 1992, Pages 483-495). Plasmid pl6-Ami also contains the acylamidase gene from Rhodococcus erythropolis 37 VKPM Ac-1793 and the regulatory region of the nitrile hydratase gene from the strain Rhodococcus rhodochrous M8 (Veiko V., Yanenko A., Alekseeva M., Sintin A., Gulko L., Ratmanova K. , Ovcharova I., Andreeva L., Astaurova O., Polyakova I., Spiders V., Voronin S, Debabov V. Cloning and determination of the nucleotide sequence of the nitrile hydratase gene from Rhodococcus rhodochrous M8. // Biotechnology. 1995. No. 5-6. S.3-5). DNA fragments containing the acylamidase gene and regulatory region are obtained by PCR amplification. To obtain the acylamidase gene, the DNA of the pET16b-ami plasmid is used as a matrix, in which this gene is cloned without flanks (RU 2439154), and oligonucleotides homologous to the ends of the acylamidase gene with the addition of NcoI and EcoRI endunuclease sites are used as primers. To obtain a DNA fragment containing the regulatory region of the nitrile hydratase gene, the chromosome of strain M8 is used as a matrix, and oligonucleotides homologous to the ends of the regulatory region of the nitrile hydratase gene with the addition of PstI and Ncol endunuclease sites are used as primers.

The DNA fragments thus amplified are inserted into the pRY16 plasmid at the PstI and EcoRI sites (FIG. 1) and used to transform the E. coli XL1 Blue recipient strain. The obtained transformants are resistant to ampicillin 100 μg / ml, contain the plasmid p16-Ami, including the acylamidase gene and the regulatory region of nitrile hydratase from Rhodococcus rhodochrous M8.

Example 2. Construction of the claimed strain of Rhodococcus erythropolis 37 p16-Ami VKPM Ac-1937

The plasmid p16-Ami, obtained as in example 1, was introduced into the strain Rhodococcus erythropolis 37 using electrotransformation. Transformants selected on LB medium with apramycin (100 μg / ml) after incubation of the plates for 48 h at 30 ° C are a strain of Rhodococcus erythropolis 37 containing the plasmid p16-Ami. Such a strain was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) as Rhodococcus erythropolis VKPM Ac-1937.

Example 3. Construction of the claimed strain of Rhodococcus erythropolis VKPM HX7 p16-Ami Ac-1938

Plasmid p16-Ami, obtained as in example 1, was introduced into the strain Rhodococcus erythropolis HX7 using electro-transformation. Transformants selected on LB medium with apramycin (100 μg / ml) after incubation of the plates for 48 h at 30 ° C are a strain of Rhodococcus erythropolis HX7 containing the plasmid p16-Ami. Such a strain was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) as Rhodococcus erythropolis VKPM Ac-1938.

Example 4. Obtaining a biocatalyst based on the inventive strain Rhodococcus erythropolis pl6-Ami VKPM Ac-1937

In an Erlenmeyer flask (750 ml) containing 100 ml of LB medium containing apramycin at a concentration of 100 μg / ml, add 1 ml of culture (109cl / ml) of the strain Rhodococcus erythropolis 37 p16-Ami VKPM Ac-1937, previously grown on the same medium for 16 hours at 30 ° C with stirring (300 rpm). The flask was then incubated with stirring (300 rpm) for 36 hours at 30 ° C. Thus obtained culture of the inventive strain Rhodococcus erythropolis 37 p16-Ami VKPM Ac-1937 has an acylating activity of 5 mm IPAA / ml * h. Cell biomass is separated by centrifugation at 5 thousand rpm and stored at + 4 ° C.

Example 5. Obtaining a biocatalyst based on the inventive strain Rhodococcus erythropolis HX7 p16-Ami VKPM Ac-1938

In an Erlenmeyer flask (750 ml) containing 100 ml of LB medium containing apramycin at a concentration of 100 μg / ml, 1 ml of culture (109 cells / ml) of Rhodococcus erythropolis strain HX7 p16-Ami VKPM Ac-1938, previously grown on the same medium for 16 hours at 30 ° C with stirring (300 rpm). The flask was then incubated with stirring (300 rpm) for 36 hours at 30 ° C. Thus obtained culture of the inventive strain Rhodococcus erythropolis HX7 p16-Ami VKPM Ac-1938 has an acylating activity of 5 mm IPAA / ml * h. Cell biomass is separated by centrifugation at 5 thousand rpm and stored at + 4 ° C.

Example 6. Synthesis of N-isopropylacrylamide using a biocatalyst based on the inventive strain Rhodococcus erythropolis 37 p16-Ami VKPM Ac-1937

Aqueous solutions of acrylamide and isopropylamine (pH 10) are mixed, then a suspension of Rhodococcus erythropolis 37 p16-Ami VKPM Ac-1937 strain obtained in Example 4 is added to the reaction mixture. The concentrations of acrylamide and isopropylamine in the mixture are 6% and 3%, respectively. The concentration of the biocatalyst in the mixture is 8 g dry weight / L. The reaction mixture was incubated at 37 ° C for 7 hours. Then the cells are separated by centrifugation (5 thousand rpm). The concentration of N-isopropylacrylamide in the reaction mixture was determined by gas chromatography. The concentration of N-isopropylacrylamide in the resulting sample is 10 g / L.

Example 7. Synthesis of N-isopropylacrylamide using a biocatalyst based on the inventive strain Rhodococcus erythropolis HX7 p16-Ami VKPM Ac-1938

The synthesis is carried out as in example 7, but as a catalyst, a suspension of cells of the strain Rhodococcus erythropolis HX7 p16-Ami VKPM Ac-1938, obtained as in example 5, is used. The concentration of the resulting solution of N-isopropylacrylamide is 10 g / L.

Example 8. Synthesis of N-dimethylaminopropylacrylamide using a biocatalyst based on the inventive strain Rhodococcus erythropolis 37 pl6-Ami VKPM Ac-1937

Aqueous solutions of acrylamide and dimethylaminopropylamine (pH 10) are mixed, then a suspension of Rhodococcus erythropolis 37 p16-Ami VKPM Ac-1937 strain obtained as in Example 4 is added to the reaction mixture as a biocatalyst. The concentration of acrylamide and dimethylaminopropylamine in the mixture is 6% and 2.5 % respectively. The concentration of cells in the mixture is 8 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 the final product is determined by gas chromatography. The concentration of the resulting solution of N-dimethylaminopropylacrylamide is 2 g / L.

Example 9. Synthesis of N-dimethylaminopropylacrylamide using a biocatalyst based on the inventive strain Rhodococcus erythropolis HX7 p16-Ami VKPM Ac-1938

The synthesis is carried out as in example 9, but as a biocatalyst, a suspension of cells of the strain Rhodococcus erythropolis HX7 p16-Ami VKPM Ac-1938, obtained as in example 5, is used. The concentration of the resulting solution of N-dimethylaminopropylacrylamide is 2 g / l.

Thus, the claimed method for the synthesis of N-substituted acrylamides is cheaper and more efficient than the closest analogue, as it is based on the use of recombinant bacterial strains of Rhodococcus erythropolis VKPM Ac-1937 or Rhodococcus erythropolis VKPM Ac-1938, which have constitutive acylating activity in 2, as a biocatalyst , 5 times higher than the activity level of the initial strain (which is the closest analogue strain) and does not require the presence of an expensive inducer, acetanilide, in the medium when grown.

Claims (3)

1. Recombinant strain of Rhodococcus erythropolis VKPM Ac-1937 with constitutive acylating activity and obtained by introducing an acylamidase-encoding DNA fragment of DAA37 from a strain of Rhodococcus erythropolis VKPM Ac-1 and the regulatory region of the gene of nitrile hydratase Rhodocococcoccus . 2. Recombinant strain of Rhodococcus erythropolis VKPM Ac-1938 with constitutive acylating activity and obtained by introducing an acylamidase-encoding DNA fragment of DAA37 from the Rhodococcus erythropolis strain VKPM Ac-1 and the regulatory region of the nitrile hydratase gene from the strain Rhodocococcyccus . 3. The method of synthesis of N-substituted acrylamides by mixing aqueous solutions of acrylamide and an aliphatic primary amine with the addition of Rhodococcus erythropolis bacteria having acylating activity as a biocatalyst, characterized in that cells of the recombinant strain according to claim 1 or 2 are used as a biocatalyst.

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