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Recombinant engineering bacterium with surface exhibiting and expressing glutamic acid decarboxylase as well as construction method and application of recombinant engineering bacterium

A technology of glutamic acid decarboxylase and recombinant engineering bacteria, which is applied in the biological field, can solve problems such as insufficient apparent catalytic activity, cumbersome steps, and the impact of mass transfer resistance, and achieve the effects of easy industrial application, simple equipment, and simplified operation

Active Publication Date: 2017-05-31
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to overcome the drawbacks that the intracellular enzyme of recombinant expression bacteria needs to be separated and purified after the cell is broken, and the steps are cumbersome; The influence of mass transfer resistance and insufficient apparent catalytic activity

Method used

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  • Recombinant engineering bacterium with surface exhibiting and expressing glutamic acid decarboxylase as well as construction method and application of recombinant engineering bacterium
  • Recombinant engineering bacterium with surface exhibiting and expressing glutamic acid decarboxylase as well as construction method and application of recombinant engineering bacterium
  • Recombinant engineering bacterium with surface exhibiting and expressing glutamic acid decarboxylase as well as construction method and application of recombinant engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 , Construction of Escherichia coli Intracellular Expression and Surface Display Glutamate Decarboxylase Recombinant Plasmid

[0055] The construction process of recombinant plasmids for intracellular expression and surface display of glutamic acid decarboxylase in Escherichia coli is as follows: figure 1 shown. According to the β subtype gene sequence of the GAD of Escherichia coli in GenBank (see SEQ ID No.1 for the specific sequence, or GenBank No.EF551361.1), design and synthesize primers gadB-F and gadB-R, and its sequence is as follows:

[0056] gadB-F: 5'-AATT GGATCC ATGGATAAGAAGCAAG-3', the base corresponding to the underline indicates the BamH I restriction site;

[0057] gadB-R: 5'-AAGG CTCGAG GGTATGTTTAAAGCTG-3', the base corresponding to the underline indicates the Xho I restriction site.

[0058] Using the total DNA of Escherichia coli strains as a template, PCR amplification was performed using the primer pair gadB-F and gadB-R, and the re...

Embodiment 2

[0065] Example 2 Expression of Recombinant Intracellular GAD Enzyme and Recombinant Surface Displayed GAD Enzyme in Escherichia coli

[0066] The plasmids pET28a-gad B, pET28a-INP171-gadB and pET28a-INP224-gadB were respectively transformed into E.coli BL21(DE3) competent cells, and the plates containing LB solid medium containing 50ug / mL kanamycin were coated, 37 Incubate overnight at ℃. Single clones were picked, inoculated into LB liquid medium containing 50ug / mL kanamycin, and cultured at 37°C in a constant temperature shaker at 180rpm for 10-12h. Take the culture solution and inoculate it into fresh LB medium containing 50ug / mL kanamycin with a 2% inoculum amount, shake it in a shaker at 37°C and 180rpm until the OD600 reaches 0.6-0.8, add a final concentration of 0.4mM IPTG, 16°C, 180rpm continued shaking culture for 14-16h to induce the expression of GAD enzyme.

[0067] Collect the cells by centrifugation at 5000×g at 4°C for 20 minutes, wash once with citric acid-...

Embodiment 3

[0069] Example 3 Analysis of the apparent enzyme activity of recombinant engineering bacteria containing intracellular expression of GAD enzyme and surface display expression of GAD enzyme

[0070] Get the thalli that 10OD embodiment 2 obtains, centrifuge and collect thalline. Resuspend in 0.2M pH4.0 citric acid-disodium hydrogen phosphate buffer containing 0.1M L-sodium glutamate and 10mMPLP, react at 30°C for 15min at 180rpm / min. Centrifuge at 12000rpm for 30min, take 10ul supernatant, and use Berthelot chromogenic method to measure the amount of GABA produced.

[0071] It has been determined that the apparent enzyme activity of the recombinant engineering bacteria expressing GAD enzyme on the surface is 200U / g, while the apparent enzyme activity of the recombinant engineering bacteria expressing GAD enzyme in the cells under the same culture conditions is 170U / g, the former is 17.65% higher than the latter .

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Abstract

The invention relates to a recombinant engineering bacterium with the surface exhibiting and expressing glutamic acid decarboxylase as well as a construction method and application of the recombinant engineering bacterium. The preparation method mainly comprises the steps of constructing ice nucleaiton protein-based recombinant engineering bacterium with the surface exhibiting glutamic acid decarboxylase, inoculating a glutamic acid decarboxylase recombinant engineering bacterium into a culture medium, carrying out shaking culture, adding IPTG or lactose to induce the expression of glutamic acid decarboxylase, and collecting recombinant bacteria with the surface exhibiting glutamic acid decarboxylase, wherein the recombinant bacteria with the surface exhibiting glutamic acid decarboxylase is used for preparation of gamma-aminobutyric acid from the substrate glutamic acid decarboxylation. According to the preparation method, a permeabilization treatment process required in whole-strain expression of conventionally expressed endoenzyme is avoided, recombinase is exhibited and expressed on the surface of the recombinant bacteria, and a substrate can be in contact with the recombinase to finish conversion without entering bacteria cells, so that the purpose of improving the expression activity of the recombinant bacteria is achieved. The preparation method is simple, convenient and low in cost, the operation is simple, and the expression activity of the recombinant bacteria can be efficiently improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant engineering bacterium displaying and expressing glutamic acid decarboxylase on the surface, a construction method and application thereof. Background technique [0002] γ-Aminobutyric acid (GABA) widely exists in animals, plants and microorganisms, and is a natural functional non-protein amino acid. It has the functions of lowering blood pressure, anti-convulsion, preventing epilepsy, improving sleep, anti-depression, improving brain cells, promoting hormone secretion, protecting liver and kidney, etc. It can also be used as a synthetic biodegradable material polyamide-4 (PA4) and environmentally friendly Precursor substance of the plastic solvent N-pyrrolidone. Therefore, GABA has a wide range of applications in medicine, food health care, chemical industry, agriculture and so on. [0003] At present, there are three main preparation methods of GABA at ho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N1/19C12N9/88C12P13/00C12R1/19C12R1/07C12R1/225C12R1/645
CPCC12N9/88C12P13/005C12Y401/01015
Inventor 赵黎明范立强李明伟秦臻陈启明邱勇隽
Owner EAST CHINA UNIV OF SCI & TECH
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