Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Recombinant engineered bacteria expressing glutamic acid decarboxylase on the surface, its construction method and application

A technology of glutamic acid decarboxylase and recombinant engineering bacteria, applied in the biological field, can solve the problems of cumbersome steps, insufficient apparent catalytic activity, influence of mass transfer resistance, etc., and achieves the effects of simple equipment, easy industrial application and simplified operation.

Active Publication Date: 2019-12-27
EAST CHINA UNIV OF SCI & TECH
View PDF5 Cites 0 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Recombinant engineered bacteria expressing glutamic acid decarboxylase on the surface, its construction method and application
  • Recombinant engineered bacteria expressing glutamic acid decarboxylase on the surface, its construction method and application
  • Recombinant engineered bacteria expressing glutamic acid decarboxylase on the surface, its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1. Construction of recombinant plasmids for intracellular expression and surface display of glutamate decarboxylase in Escherichia coli

[0055] The construction process of the recombinant plasmid for intracellular expression and surface display of glutamate decarboxylase in E. coli is as follows figure 1 Shown. According to the β subtype gene sequence of E. coli GAD in GenBank (see SEQ ID No. 1 or GenBank No. EF551361.1), the primers gadB-F and gadB-R were designed and synthesized. The sequences are as follows:

[0056] gadB-F: 5’-AATTGGATCCATGGATAAGAAGCAAG-3’, the base corresponding to the underline indicates the BamHI restriction site;

[0057] gadB-R: 5'-AAGGCTCGAGGGTATGTTTAAAGCTG-3', the base corresponding to the underline indicates the XhoI restriction site.

[0058] Using the total DNA of the E. coli strain as a template, PCR amplification was performed using a pair of gadB-F and gadB-R primers. The results are shown in figure 2 , Where 1 is PCR amplified gadB g...

Embodiment 2

[0065] Example 2 Expression of E. coli recombinant intracellular GAD enzyme and recombinant surface display GAD enzyme

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

[0067] The cells were collected by centrifugation at 5000×g for 20 minutes at 4°C, wa...

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] Take 10OD of the bacteria obtained in Example 2 and collect the bacteria by centrifugation. Resuspend in 0.2MpH4.0 citric acid-disodium hydrogen phosphate buffer containing 0.1ML-sodium glutamate and 10mMPLP, and react for 15min at 30°C and 180rpm / min. Centrifuge at 12000rpm for 30min, take 10ul of supernatant, and measure the amount of GABA produced by Berthelot colorimetric method.

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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 particularly relates to a recombinant engineered bacteria expressing glutamate decarboxylase on the surface, and a construction method and application thereof. Background technique [0002] Gamma-aminobutyric acid (GABA) is widely found in animals, plants and microorganisms. It is a natural functional non-protein amino acid. It has the functions of lowering blood pressure, anti-convulsant, preventing epilepsy, improving sleep, anti-depression, improving brain cells, promoting hormone secretion and protecting liver and kidney. It can also be used as synthetic biodegradable material polyamide-4 (PA4) and environmentally friendly The plastic solvent N-pyrrolidone precursor substance. Therefore, GABA has a wide range of applications in medicine, food and health care, chemical industry, and agriculture. [0003] At present, the main preparation methods of GABA at home and abroad include chemical synthesis,...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com