RU2304165C1 - Method for production of acrylic monomers and bacterium strain rhodococcus rhodochrous producing the same - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: Specific bacterium strain Rhodococcus rhodochrous has ferment system including nitrilehydrase and amidase enzymes for hydrolysis of nitriles and amines. Claimed method includes application of said strain as biocatalyst in hydrolysis process both acrylonitrile and acrylamide. Method of present invention makes it possible to produce mixed solutions of acrylamide and ammonium acrylate monomers in industrial scale.

EFFECT: improved method for large scale production of acrylamide and ammonium acrylate monomers.

2 cl, 1 tbl, 6 ex

Description

The invention relates to biotechnology and relates to a biocatalytic method for producing acrylic monomers, namely, acrylamide and ammonium acrylate, as well as a strain of bacteria Rhodococcus rhodochrous, used as a biocatalyst in the implementation of this method.

Acrylamide, acrylic acid and its salts are valuable monomers for the preparation of water-soluble polymers. A significant proportion of them are copolymers of acrylamide and acrylic acid (or its salts), which are widely used as flocculants, reagents for oil production, dispersants, etc.

Traditionally, acrylamide and ammonium acrylate are obtained by hydrolysis of acrylonitrile in the presence of sulfuric acid or a copper catalyst (Plate and Slivinsky. Fundamentals of chemistry and technology of monomers. M: Nauka, 2002, p. 241). The main disadvantages of these methods: low concentration of monomers in the final solution (no more than 10%), as well as the formation of by-products and a large amount of waste.

An alternative method for producing acrylic monomers is the biocatalytic hydrolysis of acrylonitrile. In this case, microbial enzymes or microbial cells with nitrile hydrolytic activity are used as catalysts in the hydrolysis of acrylonitrile.

Known methods for producing acrylamide, in which various microbial strains containing the nitrile hydratase enzyme are used as a biocatalyst for the acrylonitrile hydrolysis, namely, the strain Rhodococcus rhodochrous VKPM S-926 (RF 1731814), its mutant version is the strain Rhodococcus rhodochrous VKPM RF-15151515 VKPM Ac331515 strain Rhodococcus rhodochrous J1 (JP 4234597/87, EP 0307926) and other strains, while the final product is 38-50% solutions of one monomer - acrylamide. Currently, these methods due to their high efficiency are used for the industrial production of acrylamide.

Known methods for producing another acrylic monomer - ammonium acrylate by hydrolysis of acrylonitrile in the presence of biocatalysts containing the nitrilase enzyme, based on strains of Rhodococcus sp. (US 5135858, US 6361981, US 5998180) and Alcaligenes denitrificans (RF 2177034). According to these methods, it is possible to obtain 30% and even 40% solutions of ammonium acrylate. The main problems of these processes remain the low level of activity of the biocatalysts used and their rapid inactivation.

A known method of producing ammonium acrylate using a biocatalyst Comamonas testesteroni 5-MGAM-4D (US 6670158) containing the enzyme system nitrile hydratase / amidase. Unlike nitrilase-based catalysts that convert acrylonitrile to ammonium acrylate in one step, a biocatalyst based on the nitrile hydratase / amidase system converts acrylonitrile to ammonium acrylate in two stages: in the first step, nitrile hydratase converts acrylonitrile to acrylamide, which is subsequently converted to acrylamide ammonium According to this method, the end product of the process is solutions of one monomer - ammonium acrylate. The effectiveness of this method remains low, due to the weak activity of amidase.

If the biocatalytic method for producing acrylamide has already been implemented on an industrial scale, the development of processes for producing ammonium acrylate is still at the stage of pilot plants. The reason for this is the lack of effective catalysts for the conversion of acrylonitrile to ammonium acrylate.

The objective of the present invention was to expand the arsenal of methods for producing acrylic monomers.

The problem is solved by:

- obtaining a strain of Rhodococcus rhodochrous M20 having a unique enzyme system for the hydrolysis of nitriles and amides, including nitrile hydratase and amidase enzymes, deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) under VKPM As-1728, and

- development of a method for producing acrylic monomers using this strain as a biocatalyst.

Due to the characteristics of the catalytic system of the claimed strain, mixed solutions of acrylamide monomers and ammonium acrylate in various ratios are obtained in the presence of hydrolysis of either acrylonitrile or acrylamide in its presence. These mixed solutions are of interest in the preparation of various types of copolymers, both directly and after their conversion into a solution of sodium acrylamide / acrylate.

The strain Rhodococcus rhodochrous VKPM Ac-1728 was obtained on selective media with chloroacetamide and acetamide and is a mutant of the strain Rhodococcus rhodochrous M8 - VKPM S-926 (RF 1731814).

Characterization of the claimed strain.

Taxonomic properties.

Morphological properties. The cells of the VKPM Ac-1728 strain are motionless and Gram stained. They do not form a dispute, non-acid resistant. At the age of 18-20 hours on meat-peptone agar - MPA (Manual for Practical Activities in Microbiology, Moscow State University, 1983, p. 93), the cells form long / up to 20 μm / 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, such as MPA, after 48 hours of growth, the inventive strain forms round smooth colonies with a diameter of 1 mm, colored in 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 strain is oxidase-negative, 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 pH 6-9, temperature 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 nitrogen source. It uses maltose, mannitosorbitol, glucose, glycerin, lactate, acetate, pyruvate, benzoate, p- and m-hydroxybenzoate, tyrosine as the sole carbon source; does not use rhamnose, galactose, inositol, a-ketoglutarate.

Biochemical analysis showed that the cell wall of the VKPM strain As-1728 contains mesodiaminopimelic acid, arabinose, and galactose, which is typical for nardi-like bacteria with the IY type of cell wall (Nesterenko et al., Nocard-like and coryne-like bacteria, Naukova Dumka, 1985, p. 90). Cells also contain lipid A (LCN), characteristic of Rhodococcus.

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

Based on the above and in accordance with Bergey’s guidelines (Bergey Bacterial Identifier, M .: Mir, 1997), the strain VKPM Ac-1728 is assigned to the genus Rhodococcus and the species rhodochrous.

Optimum cultivation conditions.

To obtain cells of the inventive strain in large quantities and with high activity of nitrile hydratase and amidase, the strain is grown at 25-30 ° C for 24-72 hours on synthetic media containing pyruvate, acetate or glucose as a carbon source in concentrations of 0.1-4%, and as a source of nitrogen, ammonium, nitrate salts or urea in concentrations of 0.4-2%. Expensive components such as vitamins, amino acids or extracts are not required for the growth of the strain.

A distinctive feature of the claimed strain is its ability to synthesize nitrile hydratase and amidase enzymes constitutively, i.e. in the absence of known inducers in the medium, such as nitriles, amides of organic acids (Table 1). In this case, the inventive strain VKPM As-1728 exhibits a higher activity of both of these enzymes than the parent strain VKPM S-926.

Table 1. The nitrile hydratase and amidase activities of the claimed strain VKPM As-1728 in comparison with the parent strain VKPM S-926. Strain The specific activity of nitrile hydratase, μm acrylamide / min · mg cells by dry weight The specific activity of amidase, μm acrylate / min · mg cells by dry weight Without inductor With inductor Without inductor With inductor VKPM As-1728 one hundred 120 2,3 2,5 VKPM S-926 6 67 0.05 0.1

To determine the activity of enzymes, the strains were grown at 30 ° C with intensive aeration (200 rpm) in Erlenmeyer flasks (750 ml in volume) containing 150 ml of culture medium of the following composition (wt.%): K ₂ HPO ₄ 3Н ₂ O - 0.05; KH ₂ PO ₄ - 0.05; MgSO ₄ 7H ₂ O - 0.05; FeSO ₄ 7H ₂ O - 0.0005; CoCl ₂ 6H ₂ O - 0.001; sodium pyruvate - 0.5; and NH ₄ Cl - 0.2; water is the rest. After 24 hours, the resulting cultures were divided into two parts and an inducer, acetamide (2 g / l), was added to one of them and cultivation continued for another 20 hours. Cells are separated by centrifugation and used to determine the activity of nitrile hydratase and amidase.

The enzymatic activity of the strain is determined by measuring the hydrolysis rate of a 1% solution of acrylonitrile (for nitrile hydratase) or acrylamide (for amidase) at 20 ° C. The unit of activity is the amount of enzymatic activity required to convert 1 μm of the substrate into a product in 1 min.

The concentrations of acrylonitrile, acrylamide and ammonium acrylate are determined by high pressure liquid chromatography (Moreau et al., Analyst, 1991, p. 1381-1383). The specific activity of nitrile hydratase is expressed in μM acrylamide / min · mg cells by dry weight. The specific activity of amidase is expressed in μM acrylate / min · mg cells by dry weight.

The method in General is as follows.

The method of obtaining mixed solutions of acrylamide and ammonium acrylate is carried out by mixing an aqueous solution of either acrylonitrile or acrylamide with an aqueous suspension of the biocatalyst in a thermostatic vessel (at a temperature of 4 to 55 ° C, preferably from 18 to 30 ° C), followed by stirring (from 100 to 250 rpm). The pH value varies from 4 to 10, preferably from 6 to 8. The concentration of the biocatalyst (calculated on dry weight) varies from 0.4 mg to 4 mg per 1 ml of the reaction mixture and determines the ratio of the components of the obtained mixed solutions of acrylamide and ammonium acrylate.

A biocatalyst having a specific nitrile hydratase activity of at least 60 u / mg and an amidase activity of at least 0.5 u / mg is used both as free cells of the inventive strain obtained by separation from the culture fluid by centrifugation and subsequent suspension in distilled water, and in immobilized form, for example in the form of cells included in the polyacrylamide gel.

Both acrylonitrile and acrylamide are introduced into the reactor either simultaneously, or in portions, or continuously. The concentration of acrylonitrile or acrylamide in the reaction medium is from 0.5 to 7%, preferably from 0.5 to 2%.

The inventive method allows to obtain mixed solutions of acrylamide / ammonium acrylate in ratios from 0.5: 99.5 to 99.5: 0.5.

Example 1

5 ml of a culture of the strain Rhodococcus rhodochrous VKPM Ac-1728, previously grown for 2 days at 30 ° C on a medium of the following composition (wt.%): K ₂ HPO ₄ · 3H ₂ O - 0.05; KH ₂ PO ₄ - 0.05; MgSO ₄ · 7H ₂ O - 0.05; FeSO ₄ · 7H ₂ O - 0.0005; CoCl ₂ · 6H ₂ O - 0.001; glucose - 4; urea — 0.2, water — the rest, introduced into an Erlenmeyer flask containing 150 ml of culture medium of the same composition, and incubated for 72 hours at 30 ° C and stirring at a speed of 200 rpm. Cells are separated by centrifugation (10,000 rpm). Some of the cells are stored frozen at -20 ° C, and the remaining part is suspended in 10 mM phosphate buffer (pH 7.0) (Rabinovich and Khavin. Brief chemical reference book, Moscow: Chemistry, 1991, p. 280) to a concentration of 12.0 mg cells / ml and stored at + 4 ° C. The cells thus obtained have a specific nitrile hydratase activity of 120 u / mg and an amidase activity of 2.5 u / mg.

Example 2

2.1 ml of a cell suspension of the inventive strain obtained as in example 1, is introduced into a 100 ml glass vessel with a magnetic stirrer and a temperature control system. Then 17.7 ml of distilled water are added there. Using a peristaltic pump at a constant speed of 1.06 ml / h, pure acrylonitrile was added to the reaction mixture. The process is carried out with stirring at a speed of 200 rpm and a temperature of 18-20 ° C. After 5 hours, the process is completed, the cells are separated by centrifugation (10,000 rpm), and the concentrations of acrylamide and ammonium acrylate, which are equal to 20% and 10%, are determined in the obtained supernatant.

Example 3

The process is carried out as in example 2, but a suspension of the inventive strain is added in an amount of 4.2 ml, distilled water is added in an amount of 15.6 ml. The result is a solution containing 15.3% acrylamide and 16% ammonium acrylate.

Example 4

The process is carried out as in example 2, but a suspension of the inventive strain is added in an amount of 6.7 ml, distilled water is added in an amount of 13.1 ml. The result is a solution containing 10.6% acrylamide and 19.6% ammonium acrylate.

Example 5

In a 1000 ml glass vessel with a magnetic stirrer and a temperature control system, 238 ml of a 40% solution of acrylamide obtained from acrylonitrile using biocatalyst M33 (RF patent 2053300), 95.2 ml of a cell suspension of the inventive strain obtained as in example 1, and 23.8 ml of distilled water. The process is carried out at 18-20 ° C and constant stirring (200 rpm). After five hours, the reaction was stopped by separating the cells by centrifugation (10,000 rpm). The resulting solution contains 15.2% acrylamide and 15.5% ammonium acrylate.

Example 6

To obtain an immobilized form of the biocatalyst, to 2.2 ml of a solution containing 20% acrylamide and 0.8% N, N-methylenebisacrylamide is added to 2 ml of a cell suspension (40 mg of cells by dry weight / ml) obtained as in Example 1. 250 μl of a 1.6% potassium persulfate solution and 25 μl of N-tetramethylethylenediamine (TEMED) are added. The gelation process is carried out at 4 ° C for 60 minutes The gel block is mechanically fragmented using a sieve with a hole diameter of 0.25 mm.

4.5 g of the obtained granules are introduced into a 100 ml glass vessel with a magnetic stirrer and a temperature control system. Then, 20.0 ml of distilled water is added to the vessel. Pure acrylonitrile is added to the reaction mixture using a peristaltic pump at a constant speed of 1.0 ml / h at a temperature of 18-20 ° C. 5 hours after separation of the granules by centrifugation (5000 rpm), a solution containing 9.4% acrylamide and 11.5% ammonium acrylate is obtained.

Thus, a strain of Rhodococcus rhodochrous VKPM Ac-1728 was obtained, which has a unique enzyme system for the hydrolysis of nitriles and amides, including nitrile hydratase and amidase enzymes. Based on it, a method for producing acrylic monomers was developed, which allows one to obtain mixed solutions of two monomers - acrylamide and ammonium acrylate in various ratios.

The strain Rhodococcus rhodochrous VKPM Ac-1728 is recommended for industrial production of acrylic monomers.

Claims (2)

1. A method of obtaining a mixed solution of acrylamide and ammonium acrylate by hydrolysis of either acrylonitrile or acrylamide when using the bacterial strain Rhodococcus rhodochrous VKPM Ac-1728 as a biocatalyst. 2. The bacterial strain Rhodococcus rhodochrous VKPM As-1728, having an enzyme system for the hydrolysis of nitriles and amides, including the enzymes nitrile hydratase and amidase.