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A glutamic acid decarboxylase recombinant plasmid and its construction method and application

A technology of glutamic acid decarboxylase and recombinant plasmid, applied in the field of genetic engineering, can solve the problems of low GABA yield, low expression of recombinant glutamic acid decarboxylase, low activity, etc., to reduce production costs and be suitable for large-scale industrialization. High production and safety effects

Active Publication Date: 2017-01-18
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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  • A glutamic acid decarboxylase recombinant plasmid and its construction method and application
  • A glutamic acid decarboxylase recombinant plasmid and its construction method and application
  • A glutamic acid decarboxylase recombinant plasmid and its construction method and application

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 glutamate decarboxylase gadB from E. coli and the multiple cloning site of the shuttle expression vector pEKEx2, the following two primers are designed and synthesized for PCR amplification, among which E. coli K-12 genomic DNA is used as template.

[0048] YW1: 5’-GGA GTCGAC AAGGAGATATAGAT ATGGATAAGA AGCAAGTAAC G

[0049] Sal I

[0050] YW2:

[0051] 5’-5’-GGA GAGCTC TCACTTATCGTCGTCATCCTTGTAATCGGTATGTTTAAAGCTGTTC-3’

[0052] SacI

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

[0054]

[0055]

[0056] The PCR reaction conditions are as follows:

[0057]

[0058] After the reaction, take 10 μl for 1.0% Agarose electrophoresis identification. Test results such...

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 cut with PvuII restriction endonuclease, recovered and autonomously connected to obtain the modified pEKEx2 vector without Ptac promoter . Subsequently, according to the amino acid sequence of glutamate decarboxylase gadB from Escherichia coli, the pamyE sequence of the maltose promoter of Corynebacterium glutamicum ATCC13032, and the multiple cloning site of pEKEx2, the following four primers were designed and synthesized, respectively, using Corynebacterium glutamicum ATCC13032 Genomic and E. coli K-12 genomic DNA were used as templates, and the pamyE PCR products of YW3 an...

Embodiment 3

[0074] Example 3 Construction of recombinant bacteria:

[0075] The method for constructing recombinant bacteria of this embodiment includes the following steps:

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

[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 The value is 0.2;

[0078] (3) Continue to incubate at 30℃ and 200rpm for 3-5h to make the OD 600 Around 0.9;

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

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

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

[0082] (7) Discard the supernatant, wash it twice with 10% ...

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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 specifically relates to a glutamate decarboxylase recombinant plasmid and a construction method and application thereof. Background technique [0002] γ-aminobutyric acid (GABA) is a kind of non-protein amino acid in free state, and it is an inhibitory transmitter of mammalian central nervous system. GABA has a variety of physiological functions, such as lowering blood pressure, preventing epilepsy, improving sleep, anti-depression, calming nerves, promoting hormone secretion, and protecting the liver and kidneys. Therefore, it has received widespread attention. At present, the preparation of GABA at home and abroad mainly uses chemical synthesis or microbial fermentation. When the chemical synthesis method is used to prepare GABA, the toxic components that need to be removed from the product are complex and the product safety is poor. Therefore, compared with the chemical synthesis method, the...

Claims

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Application Information

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Patent Type & Authority Patents(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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