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Method for producing gamma-amino butyric acid through continuous conversion of immobilized enzyme

A technology for immobilizing enzymes and aminobutyric acid, applied in biochemical equipment and methods, immobilized on/in organic carriers, botany equipment and methods, etc., can solve the problem of complex production process and γ-aminobutyric acid output The difference is large, the reaction efficiency needs to be improved, etc., to achieve the effect of simple separation of enzyme and product, simple and fast immobilization process, and good industrialization prospects

Inactive Publication Date: 2012-10-10
天津启仁医药科技有限公司
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Problems solved by technology

[0007] At present, the biosynthesis method is the mainstream production technology of GABA. A comprehensive comparison of domestic and foreign biosynthesis methods to produce GABA, different strains and different production methods, the yield of GABA varies greatly, and The fermentation cycle is long, and how to separate and purify the product from the complex fermentation broth after fermentation has become a complex, time-consuming and cost-increasing part of the production process
In recent years, with the development and application of enzyme immobilization technology in the pharmaceutical industry, research on the conversion of immobilized GAD to γ-aminobutyric acid has also been reported. Wu Guoqi et al. (Analytical Chemistry, 1999, 27 (7): 759-763 Carboxymethylated allyl dextran biochemical resin, Lin Shaoqin et al. (Pharmaceutical Biotechnology, 2005, 12 (2): 101-105) used chitosan material to immobilize GAD, Chen Weiqing et al. (Chinese Journal of Biochemical Medicine, 2007 , 28(4):224-227) was immobilized by the synergistic effect of sodium alginate and gelatin, with sodium alginate and gelatin as the carrier, hardened by calcium chloride and cross-linked by glutaraldehyde, the embedding method of Escherichia coli GAD Immobilization, and try to prepare γ-aminobutyric acid by continuous reaction in packed bed reactor, but the reaction efficiency needs to be improved

Method used

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  • Method for producing gamma-amino butyric acid through continuous conversion of immobilized enzyme
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  • Method for producing gamma-amino butyric acid through continuous conversion of immobilized enzyme

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Embodiment 1

[0029] Cloning and expression of embodiment 1 glutamic acid decarboxylase gene

[0030] According to the glutamic acid decarboxylase gene sequence of Escherichia coli BL21 included in the NCBI database, the upstream primer GAD1: 5'-CGC GGA TCC ATG GAT AAG AAG CAA G-3' and the downstream primer GAD2: 5'CCG CTC GAG CGG TCA GGT were designed ATG TTT AAA G-3'.

[0031] The glutamic acid decarboxylase gene was obtained by PCR amplification using the genome of Escherichia coli BL21 as a template. After the target band was recovered by 1% agarose gel electrophoresis, the pET21a(+) vector was connected to construct pET21a(+)-GAD, and transfection Inject BL21(DE3) to obtain genetically engineered bacteria BL21(DE3)-pET21a(+)-GAD.

[0032] Take a single colony of glutamic acid decarboxylase engineered bacteria and inoculate it in LB medium, cultivate overnight at 37°C to obtain a saturated culture, inoculate the saturated culture at 1% in LB medium containing Amp (100 μg / ml), 37 ℃Cont...

Embodiment 2

[0034] The high-performance liquid chromatography detection of embodiment 2γ-aminobutyric acid

[0035] The content of L-glutamic acid and γ-aminobutyric acid was determined by pre-column derivatization HPLC method, and the derivatization reagent was o-phthalaldehyde reagent. The pre-column derivatization method is as follows: accurately measure 100 μL of sample solution, add 50 μL of derivatization reagent, mix and react for 2 minutes, and take 20 μL of sample for analysis. Column: C 18 Chromatographic column (Inertsil ODS-SP5μ, 4.6×250mm); mobile phase: 0-11min, B phase 8%-100%; room temperature; flow rate 1mL / min; detection wavelength 340nm.

[0036] The reaction solution was centrifuged to get the supernatant, which was derivatized by the above method and then analyzed by HPLC. The results are shown in figure 1 , compared with the γ-aminobutyric acid standard curve to calculate the molar conversion rate of L-sodium glutamate in the substrate solution.

Embodiment 3

[0037] The immobilization of embodiment 3 glutamic acid decarboxylase

[0038] Take 10 mL of cellulose microspheres (see Chinese Patent, Application No. 200710057042.9) and add them to 50 mL of the cell lysate of the genetically engineered bacteria BL21(DE3)-pET35b-GAD obtained in Example 1, shake overnight at 4°C. After washing with sodium phosphate buffer, add 0.01% to 1% of different concentrations of glutaraldehyde for cross-linking, then block with 1M Gly for 10 h, and wash with sodium phosphate buffer to obtain immobilized glutamic acid decarboxylase, that is, immobilized enzyme. Store at 4°C for later use. Take 1 mL of cellulose microspheres immobilized under different immobilization conditions, add the same dose of L-sodium glutamate to investigate the immobilization efficiency, the results are as follows: figure 2 As shown, the optimal concentration of crosslinker glutaraldehyde is 0.1%.

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Abstract

The invention discloses a method for producing gamma-amino butyric acid through continuous conversion of an immobilized enzyme. The method comprises the following steps of: inducing and expressing an established glutamic acid decarboxylase gene engineering bacterium BL21 (DE3)-pET35b-GAD; performing cell disruption; collecting supernatant; and researching optimal reaction conditions and immobilizing conditions of a glutamic acid decarboxylase in fusion expression with a cellulose binding domain (CBD). Cellulose is specific adsorbed by a CBD label, and a cellulose carrier is added into cell lysate, so that the specific adsorption of the glutamic acid decarboxylase is realized, and the immobilized glutamate decarboxylase is crosslinked by using a crosslinking agent. The conversion conditions are optimized and the optimal catalytic reaction conditions are as follows: the pH is 5.5; the reaction time is 2 hours; the concentration of a substrate L-sodium glutamate is 40g / L; the concentration of the immobilized enzyme is 8mL / L; and the concentration of a coenzyme pyridoxal phosphate aldehyde is 0.1mM. Under the optimal reaction, continuous conversion is repeated for 10 times and the conversion rate of the substrate L-sodium glutamate is more than 93%.

Description

technical field [0001] The invention belongs to the technical field of amino acid production by biotechnology, and relates to a method for producing gamma-aminobutyric acid through an immobilized enzyme method. Background technique [0002] γ-aminobutyric acid is a natural amino acid with high physiological activity, which is widely distributed in animals and plants. It is an important inhibitory neurotransmitter that has been studied more deeply at present, and participates in various metabolic activities. According to the current research, it is found that the physiological activities of GABA are mainly manifested in the following aspects: (1) sedating nerves and anti-anxiety; (2) lowering blood pressure; (3) treating Parkinson's disease, epilepsy, senile dementia and other diseases (4) Reduce blood ammonia to solve ammonia poisoning, thereby improving liver function; (5) Improve brain vitality; (6) Promote ethanol metabolism; (7) Others such as preventing skin aging, eli...

Claims

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Application Information

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IPC IPC(8): C12P13/00C12N15/60C12N15/70C12N9/88C12N11/12
Inventor 高智慧任丽梅余琼林刘磊王文芳丁国钰
Owner 天津启仁医药科技有限公司
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