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Glutamic acid decarboxylase recombinant plasmid as well as construction method and application thereof

A technology of glutamic acid decarboxylase and recombinant plasmid, which is applied in the field of genetic engineering, can solve the problems of low GABA yield, low activity, and low expression of recombinant glutamic acid decarboxylase, etc., and achieve reduced production costs, high safety, and suitable effect on large-scale industrial production

Active Publication Date: 2014-10-15
CATCH BIO SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The first technical problem to be solved by the present invention is the problem that the recombinant glutamic acid decarboxylase in the prior art has less expression and low activity in the host bacterium Corynebacterium glutamicum, and then provides a high-efficiency expression of GAD Glutamic acid decarboxylase recombinant plasmid and the construction method of the recombinant plasmid
[0006] The second technical problem to be solved by the present invention is the low yield of GABA produced by recombinant coryneform bacteria in the prior art, and a method for efficiently producing GABA by recombinant coryneform bacteria is provided

Method used

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  • Glutamic acid decarboxylase recombinant plasmid as well as construction method and application thereof
  • Glutamic acid decarboxylase recombinant plasmid as well as construction method and application thereof
  • Glutamic acid decarboxylase recombinant plasmid as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Construction of recombinant pEKEx2-gadB expression vector

[0047] The recombinant pEKEx2-gadB expression vector constructed in this example has a Ptac promoter and is an IPTG-inducible expression vector. The construction method is as follows: according to the amino acid sequence of glutamic acid decarboxylase gadB derived from Escherichia coli and the multiple cloning site of the shuttle expression vector pEKEx2, the following two primers are designed and synthesized for PCR amplification, in which E.coli K-12 genomic DNA is used as template.

[0048] YW1: 5'-GGA GTC GAC AAGGAGATATAGAT ATGGATAAGA AGCAAGTAAC G

[0049] Sal I

[0050] YW2:

[0051] 5'-5'-GGA GAGCTC TCACTTATCGTCGTCATCCTTGTAATCGGTATGTTTAAAAGCTGTTC-3’

[0052] SacI

[0053] Set up the following reaction system in a 50 μl PCR reaction tube:

[0054]

[0055]

[0056] The PCR reaction conditions are as follows:

[0057]

[0058] After the reaction, 10 μl was taken for 1.0% Agar...

Embodiment 2

[0060] Example 2 Construction of recombinant pEKEx2-pamyE-gadB expression vector:

[0061] The recombinant pEKEx2-pamyE-gadB expression vector constructed in this example has a maltose promoter and is a maltose-inducible expression vector. The construction method is as follows: according to the multiple cloning site of the shuttle expression vector pEKEx2 and the Ptac promoter sequence, the pEKEx2 vector is singly cut with PvuII restriction endonuclease, recovered and connected independently, and the transformed pEKEx2 vector without the Ptac promoter is obtained . Subsequently, according to the amino acid sequence of glutamic acid decarboxylase gadB derived from Escherichia coli, the sequence of the maltose promoter pamyE in the genome of Corynebacterium glutamicum ATCC13032, and the multiple cloning site of pEKEx2, the following four primers were designed and synthesized. The genome and E.coli K-12 genome DNA were used as templates, and the pamyE PCR products of YW3 and YW4...

Embodiment 3

[0074] Embodiment 3 Construction of recombinant bacteria:

[0075] The construction method of the recombinant bacterium of the present embodiment comprises the following steps:

[0076] (1) pick a single colony of Corynebacterium glutamicum in LBG liquid medium, and cultivate overnight at 30°C and 200rpm for 16h;

[0077] (2) Measure its OD 600 value, transfer the bacterial solution into a 250ml Erlenmeyer flask containing 30ml competent medium, the final OD of the bacterial solution 600 Value is 0.2;

[0078] (3) Continue culturing at 30°C, 200rpm for 3-5h to make OD 600 Around 0.9;

[0079] (4) Place the culture medium on ice for 15 minutes;

[0080] (5) Add the bacterial solution into a pre-cooled 50ml centrifuge tube, and centrifuge at 4°C and 4000rpm for 10min;

[0081] (6) Discard the supernatant, add 10% pre-cooled glycerol to the centrifuge tube, fully suspend the bacteria, and centrifuge at 4°C and 4000rpm for 10min;

[0082] (7) Discard the supernatant, and re...

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Abstract

The invention relates to a glutamic acid decarboxylase recombinant plasmid. A glutamic acid decarboxylase gene gadB is spliced with maltose promoter pamyE and then inserted into a shuttle expression vector pEKEx2 to form the recombinant plasmid pEKEx-pamyE-gadB. The invention further provides a construction method and an application of the recombinant plasmid. According to the recombinant plasmid, the expression quantity of recombinant glutamic acid decarboxylase is greatly improved, decarboxylation of glutamic acid is promoted, the yield of gamma-aminobutyric acid is further improved, the production cycle is shortened, the glutamic acid decarboxylase recombinant plasmid is more suitable for large-scale industrial production, the safety is higher, limitation to the product application area due to the safety problem is avoided, and the recombinant plasmid can be used in the fields of food and medicine.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a recombinant plasmid of glutamic acid decarboxylase and its construction method and application. Background technique [0002] γ-aminobutyric acid (γ-aminobutyric acid, GABA) is a non-protein amino acid that exists in a free state and is an inhibitory transmitter in the mammalian central nervous system. GABA has a variety of physiological functions, such as lowering blood pressure, preventing epilepsy, improving sleep, antidepressant, calming the nerves, promoting hormone secretion, protecting the liver and kidney, etc. Therefore, it has received widespread attention. At present, the preparation of GABA at home and abroad mainly adopts chemical synthesis or microbial fermentation. Due to the complex toxic components that need to be removed from the product and the poor safety of the product when the chemical synthesis method is used to prepare GABA, the microbial ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/77C12N15/66C12N1/21C12P13/00C12R1/15
Inventor 殷志敏王期赵娇娇温尧林
Owner CATCH BIO SCI & TECH
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