Preparation method of gamma-aminobutyric acid, whole-cell catalyst and application

A whole cell catalyst, aminobutyric acid technology, applied in the whole cell catalyst and application, the field of preparation of γ-aminobutyric acid, can solve the problems of low γ-aminobutyric acid content, unsatisfactory glutamic acid decarboxylase conversion rate and the like , to achieve the effect of being conducive to maintenance, activity and stability, and avoiding the formation of inclusion bodies

Pending Publication Date: 2022-06-24
绵阳晟氏健康科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is: the content of gamma-aminobutyric acid prepared by the microbial synthesis method is low, and the conversion rate of glutamic acid decarboxylase in the conversion process is not ideal

Method used

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  • Preparation method of gamma-aminobutyric acid, whole-cell catalyst and application
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  • Preparation method of gamma-aminobutyric acid, whole-cell catalyst and application

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preparation example Construction

[0043] The preparation method of a kind of γ-aminobutyric acid provided in the embodiment of the present invention comprises the following steps:

[0044]S1) Preparation of whole-cell catalyst: using the autotransporter AIDA-1 as a carrier protein, the expression of glutamate decarboxylase is displayed on the cell surface to obtain a whole-cell catalyst;

[0045] S2) Preparation of γ-aminobutyric acid: L-glutamic acid or its salt is used as the substrate, catalyzed and converted under the action of whole cell catalyst to generate γ-aminobutyric acid.

[0046] Using surface expression display technology, the expression of glutamate decarboxylase is displayed on the cell surface, so that in the process of catalytic conversion, the glutamate decarboxylase expressed on the cell surface can directly react with the substrate to avoid intracellular expression. Compared with intracellularly expressed enzyme proteins, the surface expression display method is more conducive to the foldi...

Embodiment 1

[0068] Example 1: Whole cell catalyst preparation

[0069] 1) Recombinant expression plasmid:

[0070] Primers were designed according to the gene sequence of glutamate decarboxylase gadB encoded by Escherichia coli str.K-12substr.MG1655 reported in GenBank.

[0071] Forward primer: 5'-CGGggtaccATGGATAAGAAGCAAGTAACGG-3'

[0072] Reverse primer: 5'-GCgagctcGGTATGTTTAAAGCTGTTCTGTTG-3'

[0073] The target gene was amplified by DNA polymerase Pfu using Escherichia coli genome as template, and the PCR product size was 1415bp. The target fragment was recovered and digested with the restriction enzymes KpnI and SacI together with the plasmid pAIDA1. The obtained target gene fragment was connected to the vector backbone overnight, and then transferred to Escherichia coli DH5α competent cells and coated on a chloramphenicol-resistant plate. , incubate overnight at 37°C upside down. Then single colonies were identified by colony PCR with forward and reverse primers, and double-enzym...

Embodiment 2

[0083] Example 2: Whole-cell catalytic preparation of γ-aminobutyric acid

[0084] In a 20L transformation tank, put 400g of the whole-cell catalyst obtained in Example 1, 374g of sodium glutamate monohydrate, and 164g of sodium acetate, add water to dissolve, adjust the pH value to 5.0 with acetic acid, set the volume to 10L, and heat up to 37 ℃, 2 g of magnesium chloride hexahydrate and 0.26 g of PLP (pyridoxal phosphate) were put in, and the conversion was started. During the biotransformation process, the temperature was controlled at 35 to 40°C, and the rotational speed was controlled at 250 rpm.

[0085] When the pH begins to rise, add glutamic acid to control the pH at 5.4 to 5.6, and control the content of L-glutamic acid to be below 5 g / L during the process; the pH rises slowly in the later stage of the reaction, and the foam gradually decreases, stop adding L-glutamic acid; then use a small amount of sulfuric acid to control the pH value, and the reaction ends when ...

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Abstract

The invention discloses a preparation method of gamma-aminobutyric acid, a whole-cell catalyst and application, the preparation method comprises the following steps: preparation of the whole-cell catalyst: expressing and displaying glutamate decarboxylase on the surface of a cell by using an autotransporter AIDA-1 as a carrier protein to obtain the whole-cell catalyst; the gamma-aminobutyric acid is prepared by taking L-glutamic acid or a salt thereof as a substrate, and carrying out catalytic conversion under the action of a whole-cell catalyst to generate the gamma-aminobutyric acid. Through a surface expression display technology, glutamate decarboxylase expression is displayed on the cell surface, and glutamate decarboxylase can directly react with a substrate in a catalytic conversion process, so that a cross reaction occurring during intracellular expression is avoided; compared with intracellular expression zymoprotein, the surface expression display method provided by the invention is more beneficial to folding, dissolving and soluble expression of glutamate decarboxylase, is more beneficial to maintenance of activity and stability of glutamate decarboxylase, and avoids formation of inclusion bodies.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a preparation method of γ-aminobutyric acid, a whole-cell catalyst and applications. Background technique [0002] Gamma-aminobutyric acid (4-aminobutyric acid) is a strong neuro-inhibitory amino acid with physiological effects of sedation, hypnosis, anticonvulsant and blood pressure lowering. It is a naturally occurring non-protein amino acid, which can promote the activation of the brain, strengthen the brain and improve intelligence, anti-epilepsy, promote sleep, beautify the skin, delay the aging function of the brain, and supplement the human body's inhibitory neurotransmitters. The blood pressure lowering effect can promote the improvement and protection of renal function. Daily supplementation of a small amount of gamma aminobutyric acid is beneficial to the relief of heart and brain blood pressure, and can also promote the balance of amino acid metabolism in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/00C12N15/70C12N15/66C12N15/60C12N1/21C12R1/19
CPCC12P13/005C12N15/70C12N9/88C12Y401/01015C12N1/20
Inventor 张瑞李晚军李倩刘鹏曾帅
Owner 绵阳晟氏健康科技有限公司
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