RU2143002C1 - METHOD OF γ-AMINOBUTYRIC ACID PRODUCING - Google Patents

Abstract

FIELD: amino acids, biotechnology, microbiology. SUBSTANCE: invention proposes a method of producing γ-aminobutyric acid (GABA) by metabolism of L-glutamic acid or its salts in the presence of biomass of bacterium Escherichia coli VKPM B-7460 or Escherichia coli VKPM B-7452 followed by purification and isolation of the end product. Decarboxylase activity of cells is 5 mcM GABA/min/mg of crude biomass and specific consumption of cells in the process of metabolism is 40 mg of crude biomass/1 g of GABA. The yield of the end product from glutamic acid is 55.6% wt.-% and that from sodium glutamate - 40 wt.-%. The content of GABA in the dry preparation - 98.7%. EFFECT: improved method of producing, increased yield and content. 1 tbl, 2 ex

Description

 The invention relates to the microbiological and medical industries and for the production of γ-aminobutyric acid from L-glutamic acid or its salts using the biomass of Escherichia coli bacteria.

 γ-Aminobutyric acid (GABA), as an effective neurotransmitter, is used in medicine as part of a number of drugs (aminalon, gammalon, picamilon, pantogam) for the treatment of brain disorders (memory, speech, hemiplegia and hypertension).

 A known chemical method for producing GABA during the alkaline hydrolysis of pyrrolidonocarboxylic acid (Garmaise et al. Canad. J. Chemistry, 1956, 34, 743-748; A.S. USSR N 452196). The disadvantage of this method is the high cost of pyrrolidone, harsh synthesis conditions, as well as the difficulty of hydrolysis of residual pyrrolidone in the body.

Known methods for producing GABA by microbiological synthesis (Japanese Patents N 69 01197, N 70 15437). After 90 h of cultivation of strains of Bact. cadaveris ATCC 9760 and E. coli ATCC 9637 in the culture fluid accumulate up to 4 g / l GABA. The product from the culture fluid separated at pH 2.0 is isolated by ion exchange chromatography on a Diaion SKI apparatus (type H ⁺ ). The yield of crystalline product from the culture fluid is 35%. The disadvantage of this method is the low yield of GABA and the long process time.

 Known methods for producing GABA by enzymatic decarboxylation of L-glutamic acid, where microorganism cells containing L-glutamate decarboxylase (GAD, EC 4.1.1.15) are used as a reaction catalyst (Japanese Patent No. 69 01196; Gubarev E.M. et al. Biochemistry. 1960.Vol. 25, No. 2, pp. 261-263), as well as immobilized GAD (A.S. USSR N 799318). The disadvantage of this method is the low glutamate decarboxylase activity of the cells and an additional procedure for the isolation and purification of GAD for immobilization.

Closest to the proposed invention is a method for producing GABA by decarboxylation of L-glutamic acid with bacterial cells of Arthrobacter simplex ATCC 15799 (Japanese Patent 69 01196). According to this method, a bacterial culture is grown at 30 ^o C, pH 7.0 for 4 days on a medium containing 1% L-glutamic acid, 1% glucose, 0.05% disubstituted potassium phosphate, 0.025% magnesium sulfate seven-water, 0.2% ammonium sulfate , 0.3% yeast extract and 0.3% chalk. The biomass concentration in the culture fluid at the end of the process is 6 mg / ml. To obtain GABA, 3 g of dry acetone-treated biomass is added to an aqueous solution of L-glutamic acid (10 g / 300 ml) at pH 5.0 and a temperature of 37 ^o C. The decarboxylation process is carried out for 2 hours until the complete conversion of L-glutamine acid in GABA while maintaining the initial pH value by automatically feeding 3 N. of hydrochloric acid. At the end of the process, the reaction mixture is freed from biomass and GABA is isolated by adding ethanol. Thus, 7 g of GABA, i.e. 1 g of dry acetonated cell powder synthesizes 2.3 g of GABA.

 The disadvantages of this method are the low biomass accumulation, long growth period and high biomass consumption for GABA production (0.43 g dry biomass per 1 g of product) due to the low glutamate decarboxylase activity of the strain used.

 Our task was to reduce the biomass consumption for the synthesis of GABA and increase the growth of microorganisms, thereby reducing the specific consumption of nutrient medium components per product unit.

 The problem is solved by the creation of new recombinant strains of bacteria Escherichia coli GAD1 and Escherichia coli GADK10, with increased activity of glutamate decarboxylase and intensive growth in a nutrient medium.

 In our proposed method for producing GABA, the biomass consumption is 0.04 - 0.05 g of raw biomass for obtaining 1 g of GABA.

The activity of the strains is 4 to 5 u / mg of raw biomass. (For the unit of activity, take such an amount of enzyme that forms 1 μM GABA in 1 min at 37 ^o C).

 The productivity of the process of cultivation of microorganisms is 35 - 40 mg of crude biomass / ml, the duration of cultivation is 12 - 13 hours

 Strains of E. coli GAD1 and E. coli GADK10 were obtained by genetic engineering and contain plasmids that carry the glutamate decarboxylase gene and antibiotic resistance determinants. The strains are deposited in the All-Russian collection of industrial microorganisms and have registration numbers VKPM B-7460 and B-7452, respectively.

 Strains have the following cultural-morphological and physiological-biochemical characteristics.

 Cell morphology.

 Gram-negative, slightly mobile sticks with rounded ends, 1.5-2.0 microns in length.

 Cultural signs.

1. Meat peptone agar (MPA). After 24 hours of growth at 37 ^o C forms round whitish translucent colonies with a diameter of 1.5 - 3.0 mm; the surface of the colonies is smooth, the edges are even or slightly wavy, the center of the colonies is raised, the structure is homogeneous, the consistency is pasty, easily emulsified.

2. Agar Lowry. After 24 hours of growth at 37 ^o C forms round whitish translucent colonies with a diameter of 1.5 - 3.0 mm; the surface of the colonies is smooth, the edges are even, the structure is homogeneous, the consistency is pasty, easily emulsified.

3. Agar medium Adams. After 36 - 48 h of growth at 37 ^o C forms colonies with a diameter of 0.5 - 1.5 mm; grayish-white, translucent, slightly convex with a shiny surface.

4. Growth in meat and peptone broth. The specific growth rate at 37 ^o C is 1.2 h ^-1 ; after 24 hours of growth - a strong uniform turbidity, a characteristic odor.

 Physiological and biochemical characteristics.

 IPA injection growth: good growth throughout the injection. The microorganism is an optional anaerobic.

 It does not dilute gelatin.

 Growth in milk is good with coagulation of milk.

 Indole does not form.

Attitude to temperature. It grows on meat and peptone broth at 43 ^o C and below. The optimum temperature is 37 ^o C.

 Relation to pH. It grows on liquid media with a pH of 6.0 to 8.0. The optimum pH value is 7.0.

 Relation to carbon sources. It grows well on glucose, fructose, lactose, mannose, xylose, glycerin, mannitol.

 Attitude to nitrogen sources. Absorbs nitrogen in the form of ammonium, nitrates, and also nitrogen of some organic compounds.

 The strain E. coli GAD1 is resistant to ampicillin.

 The strain E. coli GADK10 is resistant to ampicillin and kanamycin.

 The content of plasmids. E. coli GAD1 cells contain a multi-copy hybrid plasmid pGAD1 (4.5 kb), which provides ampicillin resistance and carries the gadA gene.

 E. coli GADK10 cells contain a multi-copy hybrid plasmid pGADK10 (10.4 kb), which provides resistance to ampicillin and kanamycin and carries the gadA gene.

The method of obtaining GABA is as follows. Cultivation of Escherichia coli is carried out using nutrient media containing a source of carbon, nitrogen and mineral salts. As a carbon source, glucose, fructose, sucrose, glycerin, acetate or substrates containing them are used. Ammonium salts, urea, protein hydrolysates, etc. are used as a nitrogen source. To accelerate cell growth and increase their number, additives such as peptone, corn extract, and other components of culture media accepted in microbiology can be added to the nutrient medium. To increase GAD activity, its coenzyme, pyridoxalphosphate, can be added to the composition of the media. The cultivation of cells of Escherichia coli strains is carried out at a temperature of 24 - 40 ^o C, mainly 36 - 38 ^o C for 12-13 hours. The pH of the medium is maintained in the range 5.5 - 8.0, mainly 6.8 - 7.0. After growing, the cells are separated from the culture fluid and used as a catalyst in the reaction for producing GABA from L-glutamic acid or its salts. To increase the efficiency of the transformation process, cells can be subjected to heat treatment (freezing, heating) or treatment with some solvents (toluene, ethyl acetate and others), and can also be destroyed by sonication or other mechanical methods.

Prepared, as indicated above, the cells are placed in a solution of glutamic acid for biotransformation. Biotransformation is carried out at pH 3.5 - 5.5, mainly 4.5 - 5.0, and a temperature of 30 - 50 ^o C, optimally 37 - 40 ^o C. During the process, the concentration of substrate and products is controlled by thin layer chromatography on paper or plates, Warburg method or by the amount consumed titrant (Salvadory C. et al., Ital. J. Biochem., 1970, V. 19, N 3, 1). The process is stopped when the substrate is completely processed into GABA. The reaction mixture is separated, the spent biomass is discarded, and the native solution is transferred to the stage of isolation and purification of GABA.

Example 1. Construction of plasmids pGAD1 and pGAD10 containing the gadA gene, and the creation of strains of E. coli GAD1 and E. coli GADK10
The construction of plasmids is as follows.

 1. At the first stage, the E. coli gadA gene is cloned using the polymerase chain reaction (PCR) method based on the known nucleotide sequence (Smith et al., J. Bacteriology, 1992, 174, 5820). For amplification of the gene using a pair of primers GAD5 and GAD3, flanking the coding part of the gadA gene from 5 'and 3'-ends, respectively.

GAD5: 5'-TTCCATATGGACCAGAAGCTGTTAACGG-3 '
GAD3: 5'-CAAGAGCTCTTATCAGGTGTGTTTAAAGCTGTTC-3 '
The nucleotide sequence of the primers includes the restriction sites necessary for subsequent cloning in the expression vector: the GAD5 oligonucleotide, contains the NdeI restriction site, and GAD3 the SacI.

 The chromosomal DNA of E. coli K12 TG1 strain (Maniatis T. et al., 1989, Molecular Cloning, A Laboratory Manual Cold Spring Harbor Laboratory, N. Y.) is used as a matrix.

The amplification cycle includes: denaturation of the DNA at 96 ^° C for 1 minute, annealing of the primer at 60 ^° C for 1 minute, DNA synthesis at 74 ^° C for 1.5 minutes. A total of 35 cycles are performed.

 DNA fragments synthesized during PCR are fractionated in a 1% agarose gel. DNA with mobility corresponding to the expected size of the gadA gene (≈ 1.4 kb) is isolated from agarose, treated with T4 phage polynucleotide kinase and subcloned into pUC19 vector (Stratagene) at the SmaI site.

 Plasmids containing the desired insert are selected by restriction analysis, and then the DNA of these plasmids is sequenced by the Sanger method (Sanger et al., Proc. Natl. Acad. Sci. USA, 1977, 74, 5463).

 2. At the second stage of operation, the DNA fragment with the glutamate decarboxylase gene is inserted into the pGEMEX1 vector.

 The expression vector pGEMEX1 (Promega) provides ampicillin resistance and carries the full-sized gene 10 of the T7 bacteriophage. A polylinker with a number of unique restriction sites, including the SacI site, is built directly between the stop codon and terminator of gene 10. At the site of the initiating codon, including it, there is an NdeI restriction site. Substitution of a part of gene 10 located between the restriction sites NdeI and SacI with the gadA gene makes it possible to use all the necessary regulatory elements of transcription and translation of gene 10 for expression of the latter.

a) Selected at the first stage, plasmids with the gadA gene are hydrolyzed by restriction enzymes NdeI and SacI, then a 1.4 kb DNA fragment containing the indicated gene is isolated;
b) the vector pGEMEX1 DNA is restricted with restriction enzymes AatII and SacI. The smaller of the 1063 kbp DNA fragments isolated from agarose;
c) DNA of the vector pGEMEX1 is hydrolyzed by restriction enzymes AatII and NdeI. The larger of the 2098 kb DNA fragments isolated from agarose.

 The DNA fragments obtained in steps a, b, and c are mixed, treated with T4 DNA ligase, and the E. coli K-12 XL-1 Blue strain (Stratagene) is transformed. Using restriction analysis, clones with a recombinant plasmid containing the gadA gene insert were selected among transformants. The resulting plasmid (4.5 kb) was called pGAD1.

 To express the glutamate decarboxylase gene, the recombinant plasmid pGAD1 is transferred to a special E. coli strain BL21 (DE3) (Novagen) containing a chromosomal copy of the T7 phage RNA polymerase gene. In this way, a recombinant E. coli strain BL-21 (DE3) [pGAD1], hereinafter referred to as ampicillin resistant E. coli GAD1, is obtained.

 The strain activity is 4.0 - 4.5 u / mg of raw biomass.

3. At this stage of the work, a recombinant plasmid is obtained containing the gadA gene and carrying the determinants of resistance to two antibiotics - ampicillin and kanamycin for the convenience of further work with GABA producer strains. For this:
a) plasmid pGAD1 is digested with BamHI restrictase.

 b) the pUC4K vector containing the neo gene, which provides resistance to kanamycin and neomycin (Pharmacia), is hydrolyzed with the same restriction enzyme. A smaller 1.3 kb fragment. isolated from agarose.

 The DNA fragments obtained in steps a and b are mixed, treated with T4 DNA ligase, and the E. coli K12 XL-Blue strain is transformed with the resulting ligation mixture. The selection of transformants is carried out on the basis of resistance to ampicillin and kanamycin.

 Restriction analysis showed that two antibiotic-resistant transformants carry recombinant plasmids containing the original plasmid pGAD1 and the BamHI - BamHI fragment of the pUC4K vector with the neo gene in different combinations. So, there were plasmids with one neo fragment inserted into the pGAD1 plasmid in different orientations, plasmids containing two BamHI - BamHI fragments with the neo gene and one copy of pGAD1, as well as a structure with two sequentially located plasmids pGAD1 and one fragment with the neo gene. After introducing the obtained plasmids into the expression strain of E. coli B BL-21 (DE3) and evaluating the level of GABA production, a strain carrying the plasmid pGADK10 (10.4 kb) is selected with a tandem repeat of the original plasmid pGAD1 and one BamHI - BamHI fragment, containing the neo gene, as providing the highest level of production. In this way a recombinant E. coli strain BL-21 (DE3) [pGADK10], hereinafter referred to as E. coli GADK10, resistant to ampicillin and kanamycin, is obtained.

 The strain activity is 4.5 - 5.0 u / mg of raw biomass.

 Example 2. The process of obtaining γ-aminobutyric acid.

 1. Obtaining cell biomass of E. coli GAD1 and E. coli GADK10.

One-day cell colonies of E. coli GAD1 and E. coli GADK10, grown separately on MPA, are sterilized in Erlenmeyer flasks containing 100 ml of culture medium (10 flasks for each culture). An antibiotic-added L-broth is used as a nutrient medium: 0.01 g / l ampicillin for E. coli GAD1 and 0.005 g / l kanamycin for E. coli GADK10. Seeds are grown for 6 hours on a laboratory shaker at an initial pH of 7.3, a temperature of 37 ^° C and a rotation speed of 200 rpm. Then the culture contribute in an amount of 5 vol.% In the fermenters of the company "Marubishi" with a volume of 30 l, containing 20 l of culture medium of the following composition, g / l:
1. glucose - 1.00;
2. potassium phosphate monosubstituted - 0.20;
3. amino acid hydrolyzate - 1.50;
4. magnesium sulfate - 0.05;
5. ammonium chloride - 0.10;
6. peptone - 0.50;
7. pyridoxalphosphate - 0.0025;
8. tap water - up to 20 liters.

 In addition, 0.01 g / L ampicillin enters the fermentation medium for E. coli GAD1 strain, and 0.005 g / L kanamycin enters the medium for E. coli GADK10 strain.

 Amino acid hydrolyzate, potassium phosphate and peptone are sterilized in a fermenter at 0.8 ati for 45 minutes; other components are sterilized separately.

 Glucose, glucose-ammonia mixture, magnesium sulfate and ammonium chloride are sterilized in an autoclave at 0.5 ati for 30 minutes. Pyridoxalphosphate is dissolved in 100 ml of water and sterilizing filtration is carried out. Antibiotics are dissolved in sterile water. The components are introduced into the nutrient medium immediately before sowing.

Fermentation is carried out at 37 ^o C, pH 7.0 ± 0.1, gauge pressure 0.3 ati and aeration with an air flow rate of 1 l / (l x min) for 13 hours. The pH is automatically maintained by supplying a sterile glucose-ammonia mixture of the following composition:
1. glucose - 450 g / l;
2. ammonia - 62.5 g / l;
3. tap water - up to 4 liters.

 At the end of the process, the concentration of crude biomass of E. coli GAD1 is 35 g / l, E. coli GADK10 - 38 g / l.

The culture liquids are cooled to 15 ^o C and centrifuged on a milk separator ASG-3M at 6000 rpm The fresh cell mass of both strains is 700 ± 50 g. The resulting biomass is distributed into 100 g plastic bags and frozen at -20 ^o C in a Minsk-17 freezer with a holding time of 24 hours.

 2. Preparation of biomass.

 In order to increase the enzymatic activity of biomass, it is thermally activated.

Glutamate decarboxylase activity is determined by the amount of GABA formed after a 15-minute incubation of cells with a concentration of 0.25 mg of crude biomass / ml in 1 ml of a 0.01% solution of L-glutamic acid at 37 ^° C and pH 4.6. The reaction is stopped with 30 μl of a 30% sodium hydroxide solution.

 The concentration of the formed GABA is analyzed by thin-layer chromatography on paper FN-11 (Germany) using the system butanol - glacial acetic acid - distilled water (4: 1: 2).

The elution is carried out for 15 hours in a chromatographic chamber, after which the paper is treated with a 0.5% solution of ninhydrin in acetone and placed in a thermostat for 30 minutes at 60 ^o C.

The developed chromatographic spot is cut out and placed in 5 ml of a 0.5% solution of cadmium chloride for 1 hour at 60 ^° C. The optical density of the solution is measured on an SF-26 spectrophotometer at a wavelength of 540 nm. The concentration of GABA is calculated as the ratio of the optical density of the sample to the optical density of a standard 0.1% GABA solution.

 Glutamate decarboxylase activity in E. coli GAD1 strain was 4 u / mg crude biomass, and in E. coli GADK10 strain 5 u / mg crude biomass.

12.5 g of frozen E. coli GAD1 biomass and 10 g of E. coli GADK10 are diluted separately in two portions of 60 ml of 0.9% sodium chloride solution and thermostatted at 53 ^° C for 40 min in two 500 ml Bioflo reactors. Then the temperature is reduced to 37 ^o C and maintain this value throughout the process of biotransformation.

 3. Biotransformation.

 The process of obtaining GABA is carried out using sodium glutamate or L-glutamic acid as a raw material.

The theoretical GABA yield upon complete conversion of the substrate is:
- from sodium glutamate - 55 wt.%;
- from L-glutamic acid - 70 wt.%.

 The practical output of GABA at the stage of biotransformation in both cases is very close to theoretical. The residual concentration of the substrate does not exceed 0.5 g / l.

 When using sodium glutamate, a preliminary modification of the raw material is carried out by converting sodium glutamate to L-glutamic acid in order to prevent the formation of the sodium salt of GABA during the transformation reaction. When using L-glutamic acid as a raw material, this stage is excluded from the technological scheme.

 500 g of sodium glutamate are dissolved in 1500 ml of tap water (pH 7.7) and 160 ml of glacial acetic acid are added with vigorous stirring. A thick white suspension of L-glutamic acid is formed (pH 4.5). The reaction is represented by equation 1.

Далее суспензию фильтруют через фильтровальную бумагу, раствор сливают, а осадок L-глутаминовой кислоты передают на стадию биотрансформации. На стадии модификации сырья получают 470 г осадка L-глутаминовой кислоты с влажностью 30% (или 360 г L-глутаминовой кислоты). Потери L-глутаминовой кислоты в фильтрате определяются ее растворимостью и составляют 30 г. Процесс подготовки субстрата ведут аналогично для двух штаммов.

Next, the suspension is filtered through filter paper, the solution is drained, and the precipitate of L-glutamic acid is transferred to the biotransformation stage. At the stage of raw material modification, 470 g of L-glutamic acid precipitate with a moisture content of 30% (or 360 g of L-glutamic acid) is obtained. Losses of L-glutamic acid in the filtrate are determined by its solubility and amount to 30 g. The preparation of the substrate is carried out similarly for the two strains.

Biotransformation is carried out in two Multigen reactors with a volume of 2 l each at a pH of 4.6 ± 0.1, a temperature of 37 ^o C and minimal stirring. The pH value is maintained by glacial acetic acid.

 The reaction is represented by equation 2.

В реакторы загружают по 500 мл водопроводной воды и по 230 г полученной на предыдущей стадии сырой L-глутаминовой кислоты (первая порция). Вносят по 10 мл клеточных суспензий, приготовленных на стадии 2. В дальнейшем, начиная со второго часа, суспензии биомассы подают перистальтическими насосами фирмы "Verder" со скоростью 5 мл/час в течение процесса. Через 5 часов вносят еще по 230 г L-глутаминовой кислоты.

500 ml of tap water and 230 g of the crude L-glutamic acid obtained in the previous step (the first portion) are loaded into the reactors. 10 ml of cell suspensions prepared in step 2 are introduced. Subsequently, starting from the second hour, biomass suspensions are supplied by Verder peristaltic pumps at a rate of 5 ml / hour during the process. After 5 hours, another 230 g of L-glutamic acid are added.

 The completeness of processing the substrate is determined by stabilizing the pH of the reaction mixture and by the results of thin-layer chromatography. The final reaction volumes are 1000 ml, pH 5.0. After 15 hours of the process, 260 g / l GABA is accumulated in the reaction mixtures. The residual concentration of L-glutamic acid in solutions is 0.5 g / l.

 Thus, 260 g of GABA are obtained from 500 g of sodium glutamate at the biotransformation stage. That is, with respect to monosodium glutamate, the yield is 52% by weight. The biomass consumption of E. coli GAD1 is 0.05 g of crude biomass / g of GABA, E. coli GADK10 is 0.04 g of raw biomass / g of GABA.

 4. Isolation and purification of GABA.

The process of isolation and purification of GABA consists of the following steps:
1. microfiltration of the reaction mixture;
2. ultrafiltration of the GABA solution;
3. Evaporation of the GABA solution in a vacuum dryer;
4. crystallization of GABA in ethanol and drying of crystals.

 Microfiltration of the reaction mixture is carried out on a Vladipor membrane filter with a pore diameter of 0.45 μm.

Ultrafiltration of the solution is carried out on a Millipor Pellicon installation equipped with a 5.0 SQ.FT cartridge with a pore diameter of 10 kD, at a temperature of 10 ^° C and a pressure of 2 atm.

Evaporation of the GABA solution is carried out on a vacuum evaporator LPH 03125, manufactured in Hungary, at a residual pressure of 100 mbar and a temperature of 60 ^o C to a residual moisture content of 6%.

GABA crystallization is carried out in 94% ethanol. 300 g of one stripped off mixture are diluted in 1200 ml of ethanol at a temperature of 5 ^o C. After precipitation of the GABA crystals, the mother liquor is drained and ethanol is distilled off. GABA crystals are dried on an LPH 03125 vacuum evaporator at a residual pressure of 100 mbar and a temperature of 60 ^° C for 6 hours, weighed, and the resulting preparation is analyzed according to FS 42-1903-82.

 The results of the isolation and purification of GABA are shown in the table.

 Thus, 200 g of a dry preparation of GABA containing 98.7% of the basic substance was obtained. The specific consumption of crude biomass per unit of product is 0.05 g / g for E. coli GAD1 strain and 0.04 g / g for E. coli GADK10 strain. When using sodium glutamate as a raw material, the total GABA yield is 40 wt.%; when using L-glutamic acid as a raw material, 55.6 wt.%.

Claims (1)

 A method of producing γ-aminobutyric acid by biotransformation of L-glutamic acid or its salts in the presence of a biomass of bacteria having glutamate decarboxylase activity, followed by isolation and purification of the target product, characterized in that bacterial strains Escherichia coli VKPM B-7460 or Escherichia coli VKPM B- 7452.

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