The method of obtaining gamma-aminobutyric acid (gaba)

 

(57) Abstract:

The invention relates to biotechnology and is a method of obtaining gamma-aminobutyric acid (GABA) using auxotrophic for L-isoleucine bacteria Escherichia coli. Found the bacterium is grown in a nutrient medium containing at least 100 mg/l L-isoleucine, and then emit gamma-aminobutyric acid from the culture fluid. This invention allows to increase the release of GABA. 2 C.p. f-crystals, 2 PL.

The technical field

The present invention relates to the microbiological industry and medicine and relates to a method of producing gamma-aminobutyric acid (GABA) using auxotrophic for L-isoleucine bacteria belonging to the genus Escherichia.

Prior art

Gamma-aminobutyric acid (GABA) is a major inhibitory amino acid neurotransmitters nerve impulses in the brain of mammals. Being widely (albeit unevenly) widespread in the mammalian brain, GABA is supposed mediator priblizitelno 30% of the synapses in the brain. GABA mediates in many functions of synapses by forming complexes with proteins (R is happening by changing the conductivity chlorides, which usually, though not necessarily, leads to hyperpolarization of the cell (WO 0200221 A1). Because GABA is an effective neurotransmitter, it is used in medicine for the treatment of brain disorders such as amnesia, paraphasia (speech), hemiplegia (paralysis of half the body), hypertension and others. Also GABA can be used as carcinostatic agent (JP 60139622 A2).

Known chemical methods for the synthesis of GABA (Garmaise et al, Canad. J. Chemistry, 1956, 34, 742-748; USSR Author's certificate No. 452196). For medical purposes can also be ispolzovanie food enriched with GABA. There are various products derived from plants, enriched with GABA. These include tea leaves (tiled Japan's bid 9-205989), leaves of coffee (tiled Japan's bid 8-173111), plants of the family Cruciferae and their juice (European application EP 1082911 A2), filamentous fungi (JP 2000060536 A2), powder or juice of plants Brassicaceous species (JP 2001136929 A2) and others.

Described microbiological methods of obtaining GABA using the bacteria E. coli VKPM B-7460 and PMBC V containing a plasmid with the gene of glutamatdekarboksilazy (RF patent No. 2143002), Bad. cadaveris ATCC 9760 (JP69-01197), E. coli ATCC 9637 (JP70-15437) and Arthrobacter simplex ATCC 15799 (JP69-01196).

But so far the control of Escherichia, can produce GABA in the conditions when these bacteria are grown in a nutrient medium containing significant amounts of L-isoleucine.

Description of the invention

The object of the present invention is a method for gamma-aminobutyric acid (GABA), including the stage of growth auxotrophic for L-isoleucine bacteria belonging to the genus Escherichia, in a nutrient medium containing significant amounts of L-isoleucine.

The goal was achieved by establishing the fact that auxotrophic for L-isoleucine bacteria belonging to the genus Escherichia, in the course of cultivation in a nutrient medium containing L-isoleucine in quantities greater than necessary to complementaly auxotrophy of L-isoleucine, can produce GABA.

Thus was accomplished the present invention.

Thus, the present invention includes the following.

1) a method of obtaining a gamma-aminobutyric acid (GABA), which includes stages:

- cultivation of auxotrophic for L-isoleucine bacteria belonging to the genus Escherichia, in a nutrient medium containing at least 100 mg/l L-isoleucine, with the aim of production and accumulation of gamma-aminobutyric acid (GABA), and

3) the Method according to 1), which uses the bacterium Escherichia coli VL334thrC+or Escherichia coli 702ilvA (VKPM B-8012).

Hereinafter the present invention will be described in more detail. The method according to the present invention is a method for gamma-aminobutyric acid (GABA), which includes stages:

- cultivation of auxotrophic for L-isoleucine bacteria belonging to the genus Escherichia, in a nutrient medium containing at least 100 mg/l L-isoleucine, with the aim of production and accumulation of gamma-aminobutyric acid (GABA), and

- selection of gamma-aminobutyric acid (GABA) from the culture fluid. The term “auxotrophic for L-isoleucine bacterium” means that the bacterium requires L-isoleucine in the medium for their growth. In this case, “auxotrophic for L-isoleucine bacterium is a bacterium, which requires the presence in the medium of at least 10 mg/l of L-isoleucine. Therefore, the concentration of L-isoleucine in the medium for auxotrophic for L-isoleucine bacteria is usually not less than 10 mg/L. Methods of obtaining auxotrophic for L-isoleucine bacteria to the genus Escherichia” means, the bacterium belongs to the genus Escherichia according to the classification known to a specialist in the field of Microbiology. As examples of the microorganism belonging to the genus Escherichia, used in the present invention, may be mentioned the bacterium Escherichia coli (E. coif).

Examples of auxotrophic for L-isoleucine bacteria belonging to the genus Escherichia are strains deficient in the gene ilvA, such as strains of E. coli B7ILE (VKPM B-8013), VL334thrC+702ilvA (VKPM B-8012) and 237 (VKPM B-7925), described in European patent EP 1172433.

The authors of the present invention found that auxotrophic for L-isoleucine bacteria belonging to the genus Escherichia, in the course of cultivation in a nutrient medium containing L-isoleucine in quantities greater than necessary to complementaly auxotrophy of L-isoleucine, can produce GABA. In other words, in conditions where the need for L-isoleucine is not a limiting factor to the growth of auxotrophic for L-isoleucine bacteria, cultivation of these bacteria leads to the accumulation of GABA in the culture fluid. The concentration of L-isoleucine should not be less than 100 mg/L. Detailed mechanism established fact remains unidentified.

In this image the Yong way, similar to traditional methods of fermentation, in which GABA is produced by using bacteria.

The nutrient medium used for cultivation, can be both synthetic and natural, provided that the medium contains sources of carbon, nitrogen, mineral supplements and the required amount of L-isoleucine, necessary for the growth of bacteria. The number of L-isoleucine, used in the method according to the present invention, should not be less than 100 mg/l To carbon sources include various carbohydrates such as glucose and sucrose, and various organic acids. Depending on the nature of the assimilation of the used microorganism can be used alcohols such as ethanol and glycerol. As the nitrogen source can be used various inorganic ammonium salts such as ammonia and ammonium sulfate, other nitrogen compounds such as amines, a natural nitrogen sources such as peptone, soybean hydrolysate, fermentolizat microorganisms. As mineral additives can be used potassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride and similar compounds. As vitamins Magny conditions, such as mixing the culture fluid on the rocking chair, stirring with aeration, at a temperature in the range from 20 to 40°C, preferably in the range from 30 to 38°C. the pH of the environment is maintained within the range of from 5 to 9, preferably from 6.5 to 7.2. pH can be adjusted by ammonia, calcium carbonate, various acids, bases and buffer solutions. Usually growing within 1 to 5 days, resulting in the accumulation of the target acid in the culture fluid.

After cultivation, solids such as cells can be removed from the culture fluid by centrifuging or by filtration through a membrane, and then GABA can be isolated and purified by methods of ion-exchange chromatography, concentration and crystallization.

The best way of carrying out the invention

In more detail the present invention will be explained below with reference to Examples.

Example 1. Production of GABA using scarce on ilvA gene of E. coli strain VL334thrC+.

Strain VL334 (VKPM B-1641) is auxotrophic for L-isoleucine and L-threonine with the strain containing mutations in genes thrC and ilvA (U.S. patent 4278765). Natural allele thrC gene was transferred by the method of General treatise was received auxotrophic for L-isoleucine strain VL334thrC+. The specified strain had the ability to production of L-glutamic acid (European patent EP 1172433).

The nutrient medium for fermentation contained 60 g/l glucose, 25 g/l ammonium sulfate, 2 g/l KN2PO41 g/l MgSO4, 0.1 mg/ml thiamine and 25 g/l of chalk (pH 7.2). L-isoleucine was added in various concentrations. Glucose and chalk were sterilized separately. 2 ml of culture medium were placed in test tubes and inoculated with one loop of the microorganism. The cultivation was carried out at 30°C biscuits 3 days with stirring.

GABA was determined using the Waters Accq-Tag Amino Acid Analysis Method” in accordance with the manufacturer's recommendations. The method consists in obtaining derivatives of peptides and amino acids from hydrolyzed proteins before applying to the column, followed by separation of the derivatives obtained using reverse-phase HPLC with fluorescence detection. Factor retention (RC) is defined as the ratio of the retention time of the peak of the substance (RT) to the retention time of L-phenylalanine (eluent pH 5.05). RC L-Proline = 0.695, RC GABA=0.701. Wavelength excitation fluorescence detector was 250 nm, the wavelength region of the emission was 320-560 nm. The maximum emission for L-Proline status is DAMI mass spectroscopy and NMR.

The number of GABA and L-glutamic acid was measured using paper chromatography (mobile phase: butanol acetic acid-water =4:1:1). Cultivation results are presented in table 1.

Example 2. Production of GABA using scarce on ilvA gene of E. coli strain 702ilvA.

The strain 702ilvA (VKPM B-8012) is a strain that is deficient in the gene ilvA, and has the ability to production of L-Proline (European patent EP 1172433). The strain 702ilvA were grown in the same conditions described in Example 1. Cultivation results presented in table 2.

1.The method of obtaining gamma-aminobutyric acid (GABA), including the stage of growth auxotrophic for L-isoleucine bacteria Escherichia coli in a nutrient medium containing at least 100 mg/l L-isoleucine, and excretion of gamma-aminobutyric acid (GABA) from the culture fluid.

2. The method according to p. 1, wherein the bacterium is modified to increase the expression of genes of the biosynthesis of L-glutamate.

3. The method according to p. 1, characterized in that use strain of the bacterium Escherichia coli VL334thrC+or Escherichia coli 702ilvA (VKPM B-8012).



 

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