RU2844007C2 - Improved biocatalyst recombinant escherichia coli bacteria strain which synthesises nitrilase enzyme from achromobacter denitrificans - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is a recombinant Escherichia coli bacteria strain VKPM V-14588 – a biocatalyst with nitrilase activity, which produces the nitrilase enzyme, obtained by the transformation of the strain Escherichia coli VKPM V-10189 with the plasmid pNit31, shown in the drawing and bearing the nitrilase gene with the sequence SEQ ID 01.

EFFECT: invention provides production of nitrilase with high activity.

1 cl, 1 dwg, 3 ex

Description

Field of technology

The invention relates to biotechnology and concerns a recombinant strain of Escherichia coli , which is a biocatalyst strain producing a nitrilase enzyme that catalyzes the direct hydrolysis of carboxylic acid nitriles into corresponding acids, in particular acrylic acid in the form of ammonium acrylate.

State of the art

In recent years, the development of polymer production technology has led to a sharp increase in the demand for monomers, including acrylic acid and its derivatives. A well-known method for producing acrylic acid is vapor-phase oxidation of propylene. The disadvantages of this method include harsh process conditions (high temperature and pressure), the use of explosive raw materials (propylene), the tendency of products to spontaneous explosive polymerization and, as a result, the high cost of transportation in special containers, as well as the need to add polymerization inhibitors to the product. Concentrated solutions of acrylic acid are highly toxic to humans, and for use in the preparation of water-soluble polymers, additional dilution operations with water are necessary, associated with a hazard to personnel. An alternative to this method is the use of microorganisms to prepare aqueous solutions of acrylate. One option is the production of acrylates by sugar fermentation (US 2016017382), and the other is the production of acrylates by biocatalysis (RU 2731289). The advantage of these methods is the mild conditions of the processes carried out at room temperature, atmospheric pressure, and in an aqueous environment. The disadvantage of the fermentation method is the low concentration of the resulting acrylate solutions and the high degree of their contamination with the products of the vital activity of microorganisms. The biocatalytic method has the following advantages: it makes it possible to obtain aqueous solutions of sufficient concentration (20-30%) for direct use in the production of water-soluble polymers and ensures the purity of the resulting product.

As a known analogue of the claimed strain, one can consider the recombinant biocatalyst strain Rhodococcus rhodochrous VKPM AC-2111, which synthesizes the nitrilase enzyme, with a nitrilase activity of 6.5 units/mg d/w (units/mg dry weight) of cells (RU 2731289).

Disclosure of the essence of the invention

The technical result of the claimed invention is the development of a biocatalyst strain that produces nitrilase, which is distinguished by increased nitrilase activity compared to the known analogue.

The problem is solved by:

- construction of a recombinant strain of Escherichia coli bacteria VKPM B-14588, which has increased nitrilase activity compared to the known analogue, obtained by introducing into the Escherichia coli strain BL21(DE3) B-10189 a plasmid containing the Nit gene from Achromobacter denitrificans C-32 VKPM B-13889 under the control of the T7 bacteriophage promoter.

The claimed strain was obtained by cloning the nitrilase gene from the Achromobacter denitrificans C-32 VKPM B-13889 strain as part of the pLATE31 plasmid, under the control of the inducible promoter of the T7 bacteriophage; it is a biocatalyst strain with increased nitrilase activity compared to the known analogue.

The claimed strain is deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) and has the registration number Escherichia coli VKPM B-14588.

The strain Escherichia coli VKPM B-10189, into which the plasmid pNit31 with the nitrilase gene from Achromobacter denitrificans C-32 VKPM B-13889 is transformed, acquires nitrilase activity. When aqueous solutions of nitriles are added to the suspension of Escherichia coli VKPM B-14588 cells, organic acids are synthesized in the form of ammonium salts, in particular, acrylic acid in the form of ammonium acrylate.

Brief description of the drawings

The drawing shows the structure of the pNit31 plasmid used to obtain the claimed strain. Legend: Nit - Nit nitrilase gene; T7 promoter - phage T7 promoter; T7 terminator - phage T7 transcription terminator; lad - Lac repressor gene; ori - plasmid replicative genes; AmpR - ampicillin resistance gene.

Detailed explanations on obtaining and using the plasmid are given in the examples.

Implementation of the invention

Microbiological characteristics of the claimed strain of Escherichia coli VKPM B-14588

The Escherichia coli strain VKPM B-14588 has the following morphological and cultural properties. The culture consists of gram-negative, facultatively anaerobic rod-shaped motile cells, 0.4-0.8 × 1-3 μm in size. The Voges-Proskauer reaction is negative, the reaction with methyl red is positive. The culture does not form H ₂ S and does not hydrolyze urea. The strain is oxidase-negative and catalase-positive. The cells grow well on simple rich nutrient media, such as LB and MPA (F. Gerhardt et al. Methods of General Bacteriology in 3 volumes, Mir, 1984). The optimum growth temperature is 37°C, the optimum pH is 7.2. On dense nutrient media, such as MPA, LB (F. Gerhardt et al. Methods of General Bacteriology in 3 volumes, Mir, 1984) it forms whitish shiny colonies, 2-3 mm in size, with smooth edges, and does not form spores. The strain Escherichia coli VKPM B-14588 contains a marker of resistance to ampicillin.

Microbiological characteristics of the Escherichia coli strain VKPM B-10189 used to obtain the claimed strain

The Escherichia coli strain VKPM B-10189 has the following morphological and cultural properties. The culture consists of gram-negative, facultatively anaerobic rod-shaped motile cells, 0.4-0.8 × 1-3 μm in size. The Voges-Proskauer reaction is negative, the reaction with methyl red is positive. The culture does not form H ₂ S and does not hydrolyze urea. The strain is oxidase-negative and catalase-positive. The cells grow well on simple rich nutrient media, such as LB and MPA (F. Gerhardt et al. Methods of General Bacteriology in 3 volumes, Mir, 1984). The optimum growth temperature is 37°C, the optimum pH is 7.2. On dense nutrient media, such as MPA, LB (F. Gerhardt et al. Methods of General Bacteriology in 3 volumes, Mir, 1984) it forms whitish shiny colonies, 2-3 mm in size, with smooth edges, and does not form spores. The strain Escherichia coli VKPM B-10189 contains the gene for RNA polymerase from bacteriophage T7 under the control of the lac promoter. The strain Escherichia coli VKPM B-10189 is defective in the presence of Lon and OmpT proteases and Dcm methylase. The strain Escherichia coli VKPM B-10189 is a λDE3 lysogen. The strain Escherichia coli VKPM B-10189 does not carry markers of antibiotic resistance.

Microbiological characteristics of the strain Achromobacter denitrificans C-32 VKPM B-13889 used to obtain the claimed strain

The strain Achromobacter denitrificans C-32 VKPM B-13889 has the following morphological and cultural properties. The culture consists of single gram-negative, strictly aerobic rod-shaped motile cells with peritrichous flagella, 0.5-1×1-3 μm in size. Does not form indole. Does not hydrolyze gelatin. The strain is oxidase-positive and catalase-positive. The cells grow well on simple rich nutrient media, such as LB and MPA (F. Gerhardt et al. Methods of General Bacteriology in 3 volumes, Mir, 1984). Can use acetate as a carbon source for growth. The optimum growth temperature is 30°C, the optimum pH is 7.2. On dense nutrient media, such as MPA, LB (F. Gerhardt et al. Methods of General Bacteriology in 3 volumes, Mir, 1984) it forms whitish shiny colonies, 2-3 mm in size, with smooth edges, and does not form spores. The strain Achromobacter denitrificans C-32 VKPM B-13889 does not contain markers of resistance to antibiotics.

To obtain cells of the Escherichia coli VKPM B-14588 biocatalyst strain containing the nitrilase gene, the strain is grown at 37°C for 2-5 hours to an optical density OD ₆₀₀ of -0.6 on complete (LB) or synthetic media containing glucose or acetate at concentrations of 0.1-4% as a carbon source and ammonium compounds, nitrates, or yeast extract at concentrations of 0.4-2% as a nitrogen source. Upon reaching an optical density OD ₆₀₀ of -0.6, the inducer IPTG (isopropylthiogalactoside) is added to the medium to a final concentration of 2 mM and cultivation is continued at 18-22°C for 2-8 hours.

The obtained 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 or the cells are separated by centrifugation at 4-16 thousand rpm, the resulting cell sediment is stored as a paste at minus 18°C.

The enzymatic activity of cells is determined using acrylonitrile as a substrate, according to the following procedure: 1 ml of the cell suspension is mixed with acrylonitrile (to a final concentration of 20.0 g/l) in sealed plastic test tubes, the test tubes are placed in a water bath at a temperature of 20°C and thermostatted for 10 min. Transformation is carried out with constant stirring. The reaction is stopped by adding 0.05 ml of 6 N HCl to the reaction mixture. Bacterial cells are separated by centrifugation. The concentration of acrylic acid in the supernatant is calculated based on the amount of formed ammonium, which is determined spectrophotometrically using Nessler's reagent. The amount of enzyme catalyzing the formation of 1 μmol of ammonium acrylate per minute is taken as a unit of specific nitrilase activity.

The invention is illustrated by the following examples.

Example 1. Construction of a plasmid containing the nitrilase gene from Achromobacter denitrificans C-32 under the control of a bacteriophage promoter

A hybrid plasmid containing the Nit nitrilase gene under the control of the T7 bacteriophage promoter was obtained by inserting fragments containing the Nit gene (1110 bp) in the correct orientation into the pLATE31 plasmid, according to generally accepted methods (J. Sambrook, EF Fritsch, T. Maniatis, Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Pr., 1989). 1147 bp. the fragment containing the Nit gene was obtained by PCR using the genomic DNA of the Achromobacter denitrificans strain C-32 VKPM AC-13889 as a template and primers F-nit (5'-gtggtggtgatggtgatggcc ctactttgctgggaccggttctt-3'), R-nit (5'-aagaaccggtcccagc aaagtagggccatcaccatcaccaccac-3'), the structure of which was calculated based on the sequence SEQ ID N1.

The hybrid plasmid of the target construct is selected in E. coli XL 1-Blue cells among clones. The plasmid structure is confirmed by sequencing. One of the hybrid plasmid variants, containing the nitrilase gene under the control of the T7 phage promoter, is designated as pNit31.

Example 2. Construction of the claimed strain

The claimed strain of Escherichia coli VKPM B-14588 was obtained by introducing DNA of plasmid p Nit31, isolated from E. coli XL 1-Blue cells (pNit31), into E. coli BL21 (DE3) cells using electroporation. Among the transformants grown on LB medium with ampicillin and IPTG (isopropylthiogalactoside), a clone was found that exhibited maximum nitrilase activity when cultivated in test tubes on a medium of the following composition (g/l deionized water): Na ₂ HPO ₄ *12H ₂ O - 15.0; KH ₂ PO ₄ - 2.0; CH ₃ COONa - 7; Yeast extract - 20; MgSO ₄ *7H ₂ O - 0.25; sodium fumarate - 3, pH - 6.5-7.0 (hereinafter referred to as the MFS medium).

This clone is deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) as the Escherichia coli strain VKPM B-14588.

Example 3. Production of biomass of the claimed strain and determination of activity

The biomass of Escherichia coli VKPM B-14588 is produced in 750 ml flasks containing 100 ml of MFS medium, into which 2 ml of the culture, previously grown on LB medium containing 100 mg/ml ampicillin, are added overnight at 37°C. The flasks are cultured at 37°C until the optical density of the culture is 0.6, IPTG (isopropylthiogalactoside) is added to 2 mM, then the temperature is reduced to 22°C and the cultivation is continued for 8 hours with constant stirring. Then the biomass of the Escherichia coli VKPM B-14588 strain is separated by centrifugation at 5 thousand rpm and the resulting cell suspension is stored at +4°C.

The enzymatic activity of cells is determined using acrylonitrile as a substrate. One milliliter of cell suspension is mixed with acrylonitrile (to a final acrylonitrile concentration of 20.0 g/l) in sealed plastic test tubes, the test tubes are placed in a water bath at 20°C and thermostatted for 10 min. Transformation is carried out with constant stirring. The reaction is stopped by adding 0.05 ml of 6 N HCl to the reaction mixture. Bacterial cells are separated by centrifugation. The concentration of acrylic acid in the supernatant is calculated based on the amount of formed ammonium, which is determined spectrophotometrically using Nessler's reagent. The amount of enzyme catalyzing the formation of 1 μmol of ammonium acrylate per minute is taken as a unit of specific nitrilase activity.

The nitrilase activity of Escherichia coli VKPM B-14588 cells is 30 units.

Thus, the claimed improved biocatalyst strain, developed using the claimed Escherichia coli strain VKPM B-14588, ensures more efficient production of carboxylic acids, in particular acrylic acid. This result is achieved due to the higher nitrilase activity of the claimed biocatalyst strain (30 units/mg d.v. cells) in contrast to the known strain (6.5 units/mg d.v. cells).

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Claims (1)

The recombinant strain of Escherichia coli bacteria VKPM B-14588 is a biocatalyst with nitrilase activity, producing the nitrilase enzyme obtained by transforming the strain of Escherichia coli VKPM B-10189 with the plasmid pNit31, shown in Fig. 1 and carrying the nitrilase gene with the sequence SEQ ID 01.