A method for producing gamma-aminobutyric acid

A technology of arginine and double arginine, which is applied in the field of fermentation engineering, can solve problems such as the difficulty of correct folding and achieve the effect of reducing the cost of separation and purification and simplifying the separation and purification procedure

Active Publication Date: 2018-06-05
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Glutamate decarboxylase is a homohexameric protein. Previous research in the laboratory has shown that the Sec system signal peptide PelB cannot secrete active glutamate decarboxylase outside the cell, possibly due to correct folding in the periplasmic space. Difficult to carry out effectively

Method used

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Experimental program
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Embodiment 1

[0021] The construction of embodiment 1 recombinant bacteria

[0022] 1) Genomic DNA was extracted from Escherichia coli E.coliw3110 as a template for PCR amplification. The primers for amplifying the signal peptide torA gene were: 5'-CGCCATATGATGAACAATAACGATCTCTTTCAGGC-3' and 5'-CATCCATGGCCGCTTGCGCCGCAGTC-3' to amplify glutamic acid The primers of decarboxylase gene gadB are: 5'-CATCCATGGATAAGAAGCAAGTAACG-3' and 5'-CCCTCGAGTCAGGTATGTTTAAAGCTGTT-3'.

[0023] 2) Digest the PCR product of the torA gene with restriction endonucleases NdeI and NcoI, digest the PCR product of the gadB gene with restriction endonucleases NcoI and XhoI, and treat the vector pET20b(+) with NdeI and XhoI. After the digestion product was purified, T4 ligase was ligated overnight at 22 degrees Celsius, transformed into E.coli JM109, and the correct transformant was screened. For the obtained correct transformant, extract the plasmid and transform E.coli BL21(DE3)

Embodiment 2

[0024] Example 2 The extracellular glutamic acid decarboxylase SDS-PAGE and glutamic acid decarboxylase enzyme activity detection of recombinant bacteria at shake flask level

[0025] 1) Shake flask horizontal fermentation of recombinant bacteria

[0026] a) Medium: Seed medium is LB medium (1L): tryptone 10g, yeast extract 5g, NaCl 10g, pH adjusted to 7.0; fermentation medium is TB medium (1L): tryptone 12g, yeast Extract 24g, Glycerin 5g, K 2 HPO 4 14.6g, NaH 2 PO 4 2H 2 o 3 .6g, glycine 7.5g, NaCl 5g.

[0027] b) Cultivation method: The seeds cultured overnight at 37° C. and 200 rpm were transferred to TB medium at an inoculum size of 5%, and cultivated at 37° C. and 200 rpm for 36 hours.

[0028] c) Induction condition: Induce OD 600 The value is 1.0 and the IPTG concentration is 0.7 mM.

[0029] 2) SDS-PAGE checks the expression of glutamic acid decarboxylase in the cell and outside the cell, with empty load as the control ( figure 1 Among them, 1 and 2 are empt...

Embodiment 3

[0031] Embodiment 3 Recombinant bacteria fermenter horizontal fermentation

[0032]1) The seed medium is LB medium (1L): tryptone 10g, yeast extract 5g, NaCl10g, pH adjusted to 7.0; fermentation medium is TB medium (1L): tryptone 30g, yeast extract 20g , glycerol 8g, Na2SO4 2.0g, (NH4)2SO4 2.5g, (NH4)2-H-citrate1.0g, K2HPO4 14.6g, NaH2PO4 2H2O3.6g, MgSO4 7H2O2.0g, thiamine100mg; feed medium (1L ): yeast extract 50g, glycerol 500g, MgSO4·7H2O3.4g; induction (1L): lactose 200g.

[0033] 2) Culture method: when the initial glycerol in the medium is exhausted, that is, when the dissolved oxygen in the fermenter rises, grow at a specific growth rate of 0.12h -1 Exponential flow plus feed medium; to be OD 600 When it reached 50, the feeding medium was changed from exponential feeding to constant feeding, with a flow acceleration rate of 12mL / h, and lactose was fed at a speed of 0.8g / L / h for induction, and the induction lasted for 24h.

[0034] 3) After 42 hours of fermentation, t...

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Abstract

The invention discloses a method for efficiently producing γ-aminobutyric acid, which belongs to the field of fermentation engineering. In the present invention, a double arginine signal peptide gene (torA) is fused with a glutamic acid decarboxylase gene (gadB), and transformed into an Escherichia coli host (Escherichia coli BL21 (DE3)) to obtain a protein that can secrete and express glutamic acid decarboxylase Recombinant bacteria pET20b(+)-torA-gadB / E.coli BL21(DE3). The extracellular glutamic acid decarboxylase activity of the bacteria can reach 5.11U / mL in shake flask and 15.82U / mL in 3L fermenter. Using this system, the yield of γ-aminobutyric acid can reach 203.7g / L with monohydrate and sodium glutamate as substrates.

Description

technical field [0001] The invention relates to a method for producing gamma-aminobutyric acid, in particular to a method for producing gamma-aminobutyric acid by using a genetically engineered bacterium that secretes and expresses glutamic acid decarboxylase, and belongs to the field of fermentation engineering. Background technique [0002] γ-aminobutyric acid (GABA for short), also known as 4-aminobutyric acid and aminobutyric acid, is a non-protein amino acid that exists widely in nature and has important physiological functions. In the human body, GABA is one of the three major inhibitory neurotransmitters in the central nervous system. It has physiological effects such as relieving anxiety, lowering blood pressure, and calming sleep, so it is widely used in medical care. The production of GABA by fermentation mainly uses the glutamate decarboxylase (glutamate decarboxylase, GAD, EC 4.1.1.15) of the microorganism itself to catalyze the decarboxylation of glutamic acid o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12N9/88C12P13/00C12R1/19
CPCC12N1/20C12N9/88C12N15/70C12P13/005C12Y401/01015C12N1/205C12R2001/19
Inventor 王小元赵安琪胡晓清李烨
Owner JIANGNAN UNIV
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