RU2539033C1 - RECOMBINANT BACTERIUM STRAIN Rhodococcus rhodochrous HAVING CONSTITUTIVE ACYLATING ACTIVITY, AND METHOD OF SYNTHESIS OF N-REPLACED ACRYLAMIDES USING THIS STRAIN AS BIOCATALYST - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention relates to recombinant bacterium strain Rhodococcus rhodochrous RNCIM Ac1960, which has a constitutive acylating activity and is obtained by replacement in a chromosome of strain Rhodococcus rhodochrous RCM Ac-1515D of a gene coding nitril hydrase with a gene coding acylamidase from strain Rhodococcus erythropolis 37 RNCIM Ac-1793, as well as to a synthesis method of N-replaced acrylamides from acrylamide and amines using it as a biocatalyst.

EFFECT: invention allows efficient production of concentrated solutions of N-replaced acrylamides.

2 cl, 1 dwg, 1 tbl, 5 ex

Description

The claimed group of inventions relates to biotechnology and relates to a recombinant strain of Rhodococcus rhodochrous, as well as a method for the synthesis of N-substituted acrylamides, in particular N-isopropylacrylamide or N-dimethylaminopropylacrylamide using cells of this strain as a biocatalyst.

N-substituted aliphatic acrylamides are derivatives of acrylamide - a monomer for the synthesis of a wide range of polymers, which are used in almost all industries. The development of polymer production technology in recent years has led to the development of new types of polymers, which along with the main monomer - acrylamide - also include various derivatives, including N-substituted acrylamides. These monomers play the role of modifiers of the physicochemical properties of polymers, significantly expanding the scope of their application. One of the significant applications of N-substituted acrylamides is the synthesis of water-soluble polymers used as adsorbents, flocculants and structure-forming agents in many industries and agriculture (Polyacrylamide. - M., 1992).

To date, N-substituted acrylamides are produced industrially in the traditional way — by chemical synthesis based on the acylation of aliphatic amines with acid chlorides of acrylic and methacrylic acids (MacWilliams DS Functional monomers. Their preparation, polymerization, and application. New York: M. Decker, 1973) . The reagents used for this synthesis are extremely toxic, and, given the increasing demand for N-substituted acrylamides, production by traditional technology is becoming increasingly dangerous for the environment and personnel.

A fundamentally different way to obtain N-substituted acrylamides is biocatalytic synthesis carried out in an aqueous medium.

A known method of biocatalytic synthesis of N-substituted acrylamides using a biocatalyst - cells of the strain Rhodococcus erythropolis VKPM Ac-1793, having acylating activity (RU2399672), due to the presence of the acylamidase enzyme in them.

The strain Rhodococcus erythropolis VKPM Ac-1793 (RU2399672) will be considered as the closest analogue of the claimed strain, and the method of biocatalytic synthesis of N-substituted acrylamides using the Rhodococcus erythropolis VKPM Ac-1793 strain strain (RU2399672) as the closest analogue as the biocatalyst.

The disadvantage of the strain Rhodococcus erythropolis VKPM Ac-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 the Rhodococcus erythropolis VKPM Ac-1793 strain as a biocatalyst.

The task of the claimed group of inventions is to expand the arsenal of methods for biocatalytic synthesis of N-substituted acrylamides.

The problem is solved by:

- designing a recombinant strain of bacteria Rhodococcus rhodochrous VKPM Ac-1960, which has constitutive acylating activity and obtained by substituting on the chromosome of the strain Rhodococcus rhodochrous VKM AC-1515D (RU 2053300) a gene encoding the structural part of the nitrile hydratase enzyme to the gene encoding the structural part of Rhodococcus erythropolis VKPM Ac-1793 while maintaining the promoter of the nitrile hydratase gene as a promoter of the acylamidase gene in the claimed strain;

- development of a method for the synthesis of N-substituted acrylamides using the inventive bacterial strain Rhodococcus rhodochrous VKPM Ac-1960 as a biocatalyst.

The strain Rhodococcus rhodochrous VKPM Ac-1960, into the chromosome of which the acylamidase gene from Rhodococcus erythropolis VKPM Ac-1793 is integrated, 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 rhodochrous VKPM Ac-1960 cells (used as a biocatalyst), N-substituted acrylamides, in particular, N-isopropyl acrylamide (or N-dimethylaminopropyl acrylamide), are synthesized.

Characterization of the claimed strain of Rhodococcus rhodochrous VKPM Ac-1960

Morphological properties. The cells of the strain are motionless and Gram stained. They do not form a dispute, non-acid resistant. At the age of 18-20 hours, the cells form long (up to 20 microns) weakly branching filaments, which after 48-72 hours break up into rod-shaped and coccoid 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.

Cultural properties. On solid nutrient media (MPA, Hottinger) after 48 h of growth, the strain forms round smooth colonies with a diameter of 1 mm, colored from pale pink to pink-orange. With growth, a film and a precipitate form on the meat-peptone broth. Litmus milk does not change.

Physiological properties. Obligate aerob. Reduces nitrates. Test with methyl red, the Voges-Proskauer reaction is negative. Forms hydrogen sulfide. The oxidase-negative strain, catalase- and phosphatase-positive. Starch and cellulose do not hydrolyze; twins 60 and 80 hydrolyze. Adenine is not disposed of. Cells cannot withstand heat in skim milk at 72 ° C for 15 minutes. The strain grows at a pH of 6-9, a temperature of 5-45 ° C. The acid is formed from the following sugars and alcohols: glucose, fructose, maltose, sucrose, sorbitol, mannitol and glycerol. No gas formation was detected on any sugar. Ammonium compounds, nitrates and urea are used as the sole source of nitrogen. It uses maltose, beckoning, sorbitol, glucose, glycerin, lactate, pyruvate, benzoate, n- and m-hydroxybenzoate, tyrosine as the sole carbon source; does not use rhamnose, galactose, inositol, a-ketoglutarate. The cell wall of the strain Rhodococcus rhodochrous VKPM Ac-1960 contains meso-diaminopimelic acid, arabinose and galactose, which is typical for cinnamiform bacteria with type IV cell wall. Cells also contain lipid A (LCN), characteristic of Rhodococcus.

A distinctive feature of the claimed strain is a high level of stability of acylating activity in non-selective conditions, i.e. in the absence of antibiotics in the environment. The level of acylamidase gene loss after growth under non-selective conditions for the strain Rhodococcus rhodochrous VKPM Ac-1960 is less than 0.1%.

Obtaining a biocatalyst.

To obtain cells of the inventive strain Rhodococcus rhodochrous VKPM Ac-1960 in large quantities and with high acylamidase activity, the strain is grown at 25-30 ° C for 48-72 hours on synthetic media containing sugars or alcohols in a concentration of 0.1-4 as a carbon source %, and as a source of nitrogen, ammonium compounds, nitrates or urea in concentrations of 0.4-2%. The resulting cells are separated from the culture fluid by centrifugation at 5-16 thousand rpm, resuspended in 10 mm phosphate buffer pH 7.0-8.0 to a concentration of 20-40 g dry weight / l and stored in this form at 4 ° C.

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.

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 (from 1 to 10 g dry weight per 1 l of the reaction mixture) in a thermostatic vessel (at a temperature of from 20 to 40 ° C, preferably from 25 to 30 ° C) and subsequent stirring (from 100 to 250 rpm) for 1-48 hours. The pH value ranges from 8.5 to 11, more preferably 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 30 g / l of N-substituted acrylamides.

The invention is illustrated by the following figures.

Figure 1. The structure of the plasmid pRY1-H1-aam-H2. H1 — fragment of the promoter region of the cluster of nitrile hydratase genes from Rhodococcus rhodochrous VKM AC-1515D; aam — gene encoding acylamidase from Rhodococcus erythropolis VKPM Ac-1793; H2 — DNA fragment with the nhmG gene from Rhodococcus rhodochrous VKM AC-1515D; bla - a gene encoding beta-lactamase (a marker of resistance to ampicillin); tsr is a gene encoding 23 S rRNA methyltransferase (a marker of resistance to thiostrepton); E. coli oriR — region responsible for the replication of pUC19 plasmid in E. coli; oriT is the region responsible for conjugative plasmid transfer.

Example 1. Construction of an integration cassette for the replacement of nitrile hydratase genes on the Rhodococcus rhodochrous BKM AC-1515D chromosome with the Rhodococcus erythropolis VKPM Ac-1793 acylamidase gene

The integration cassette consists of the acylamidase gene from Rhodococcus erythropolis VKPM Ac-1793 flanked by fragments of the chromosome from the strain Rhodococcus rhodochrous BKM AC-1515D, designated H1 and H2. Fragment H1 contains a DNA region homologous to the regulatory region of nitrile hydratase on the chromosome Rhodococcus rhodochrous BKM AC-1515D. Fragment H2 contains a DNA region homologous to the nhmG gene on the Rhodococcus rhodochrous BKM AC-1515D chromosome. The recombinant plasmid for replacing the nitrile hydratase genes on the chromosome consists of an integration cassette cloned on the plasmid pRY1 (Ryabchenko L.E., Polyakova I.N., Yanenko A.S. Mobilizable plasmid vectors capable of conjugative transfer between E. coli and Rhodococcus cells , and their use for the construction of strains of Rhodococcus. Biotechnology, 2005, N5, s.6-13). This plasmid is not able to autonomously be maintained in the strain Rhodococcus rhodochrous BKM AC-1515D. If a plasmid contains a region homologous to the host chromosome, the plasmid is able to integrate into the chromosome through homologous regions.

The acylamidase gene and fragments H1 and H2 were obtained using the polymerase chain reaction (PCR), using the appropriate primers and DNA matrices listed in table 1.

Table 1 List of primers and DNA templates used to generate fragments of the integration cassette H1, aam and H2. Fragment Primer Name Primer sequence DNA matrix H1 Straight 5'-GGGGGATATCCCCGGAGTCTTCATCCTGTTTTGGGATGCG-3 ' Chromosomal DNA from R. rhodochrous BKM AC-1515D Back 5'-GATCCATGGCCTCATTCCTTTCATCGGAGC aam Straight 5'-GTCCCATGGCCGAACAGAATCTGC Chromosomal DNA from R. erythropolis VKPM Ac-1793 Back 5'-GGGGAGCTCGATTCAGACGTCATCACAC

CAATCTAGCC-3 ' H2 Straight 5'-GGGGAGCTCGACTCGATGATCGGTGTCAGTAATGCG-3 ' Chromosomal DNA from R. rhodochrous BKM AC-1515D Back 5'-GGGGAATTCAGTACCCACCGAGCCGAATTAGGAACCACCG-3 '

The primer sequences contain restriction enzyme sites BamHI, Ncol, Sad, EcoRI, which are necessary for connecting fragments to each other and introducing the cassette into plasmid pRY1. The scheme of the plasmid containing the integration cassette is presented in figure 1.

Example 2. Construction of the claimed strain of Rhodococcus rhodochrous VKPM Ac1960

To construct the inventive strain, a plasmid with an integration cassette is introduced into the R. rhodochrous BKM AC-1515 Ac strain using conjugation. To transfer the plasmid to R. rhodochrous BKM AC-1515D, an auxiliary E. coli S17-1 strain is used, which is capable of providing conjugative transfer of plasmid derivatives pRY1 to Rhodococcus recipient strains. To carry out conjugative transfer, plasmid pRY1-H1-aam-H2 is introduced into E. coli S17-1 strain by chemical transformation of DNA. Conjugation crosses between E. coli S17-1 and R. rhodochrous BKM AC-1515D are carried out by mixing exponentially growing cultures of both strains, incubating them on solid medium in the absence of selective agents overnight, and then incubating for 4 days in the presence of thiostrepton antibiotics (selection against R. rhodochrous BKM AC-1515D cells that did not receive the plasmid) and nalidixic acid (selection against E. coli S17-1 cells). Thiostrepton-resistant clones of R. rhodochrous BKM AC-1515D contain the plasmid pRY1-H1-aam-H2 integrated into the chromosome. The exclusion of a plasmid from the chromosome in some cases leads to the replacement of the nitrile hydratase gene with the acylamidase gene. In this case, the cell acquires sensitivity to thiostrepton, but retains acylating activity. So, we obtained a strain containing the acylamidase gene on the chromosome, which was deposited in the All-Russian collection of industrial microorganisms of the Federal State Unitary Enterprise GosNIIgenetika as Rhodococcus rhodochrous VKPM Ac-1960.

Example 3. Obtaining a biocatalyst based on the inventive strain Rhodococcus rhodochrous VKPM Ac1960

In an Erlenmeyer flask (750 ml), containing 50 ml of a synthetic MS nutrient medium of the following composition, wt.%: Glucose - 2.5; urea - 0.06; MgSO ₄ - 0.05; CoCl ₂ - 0.004; FeSO ₄ - 0.0005; water - the rest, add 0.5 ml of culture (10 ⁹ cells / ml) of the inventive strain, previously grown on the same medium. The flask was then incubated with stirring (200 rpm) at 30 ° C for 72 hours. Thus obtained culture has an acylating activity of 25 mm IPAA / l * h The resulting biomass of the strain Rhodococcus rhodochrous VKPM Ac1960 is separated by centrifugation at 5 thousand rpm and stored at + 4 ° C.

Example 4. Synthesis of N-isopropylacrylamide using a biocatalyst based on the inventive strain Rhodococcus rhodochrous VKPM Ac1960

Aqueous solutions of acrylamide and isopropylamine (pH 10) are mixed, then a suspension of cells of the strain Rhodococcus rhodochrous Ac1960, obtained as in Example 3, 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 on a dry weight / L basis. 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 32 g / L.

Example 5. Synthesis of N-dimethylaminopropylacrylamide using a biocatalyst based on the inventive strain Rhodococcus rhodochrous VKPM Ac1960

Aqueous solutions of acrylamide and dimethylaminopropylamine (pH 10) are mixed, then a suspension of Rhodococcus rhodochrous Ac1960 strain obtained as in Example 3 is added to the reaction mixture as a biocatalyst. The concentrations of acrylamide and dimethylaminopropylamine in the mixture are 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 8 g / L.

In this way,

- a strain of Rhodococcus rhodochrous VKPM Ac1960 was constructed, which has constitutive acylating activity and, unlike the closest analogue strain, does not need an acylating activity inducer and is able to grow on a synthetic mineral medium;

- the claimed method for the synthesis of N-substituted acrylamides using the inventive strain as a biocatalyst allows to obtain concentrated (8-30 g / l) solutions of N-substituted acrylamides, which exceeds the method closest to 1.5 times.

Claims (2)

1. Recombinant bacterial strain Rhodococcus rhodochrous VKPM Ac1960, which has constitutive acylating activity and is obtained by substituting on the chromosome of the strain Rhodococcus rhodochrous VKM Ac-1515D a gene encoding nitrile hydratase with a gene encoding acylamidase from strain Rhodococcuscus erythrocytes 37. 2. A method for the synthesis of N-substituted acrylamides from acrylamide and amines using a bacterial strain as a biocatalyst according to claim 1.

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