Glutamic acid decarboxylase mutant with enhanced pH stability and application thereof

A glutamic acid decarboxylase and mutant technology, applied in the field of mutants, can solve the problems of inactivation of enzyme molecules, increased corrosion of production equipment, accelerated equipment aging, etc., and achieve the effect of improving the stability of enzyme activity

Active Publication Date: 2016-02-03
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the optimal pH of Escherichia coli glutamic acid decarboxylase (GAD) is too low, which is 3.8-4.5, and the enzyme molecule is prone to depolymerization and inactivation when the pH is higher than 6.0, which limits its industrial application.
Since the substrate used in the industrial production of GABA is L-sodium glutamate, which is neutral and slightly alkaline, in order to achieve the optimum pH condition of L-glutamic acid decarboxylase

Method used

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  • Glutamic acid decarboxylase mutant with enhanced pH stability and application thereof
  • Glutamic acid decarboxylase mutant with enhanced pH stability and application thereof

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Experimental program
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Effect test

Embodiment 1

[0024] The construction of embodiment 1 mutant expression plasmid and the acquisition of recombinant strain

[0025] According to the lpgad gene sequence in the 3254376bp whole genome nucleic acid sequence of Lactobacillus plantarum GB01-21 in NCBI, the two primers of the glutamic acid decarboxylase coding gene were designed, and then the PCR point mutation intermediate was designed according to the amino acid site to be mutated Primers:

[0026] The mutant gene was amplified in vitro by overlap extension PCR.

[0027] The primers used for site-directed mutagenesis were:

[0028]P1: 5'-GAC(GGATCC)ATGGCAATGTTATACGGTAA-3'(BamHI)

[0029] P2: 5'-GGC(GCGGCCGC)TCAGTGTGTGAATAGGTATT-3'(NotI)

[0030] gadE91Rprimer1: 5'-CGACAAATCTCGGTACCCCCCGCACGGCCGA-3'

[0031] gadE91Rprimer2: 5'-TCGGCCGTGCGGGGGTACCGAGATTTGTCG-3'

[0032] gadE91Aprimer1: 5'-CGACAAATCTGCGTACCCCCCGCACGGCCGA-3'

[0033] gadE91Aprimer2: 5'-TCGGCCGTGCGGGGGTACGCAGATTTGTCG-3'

[0034] Extract the chromosome of Lacto...

Embodiment 2

[0038] Embodiment 2 Expression of mutant glutamate decarboxylase and Ni-NTA purification

[0039] Inoculate the recombinants stored in cryopreserved tubes into LB medium containing kanamycin (final concentration: 50 μg / mL), culture with shaking at 37°C overnight, transfer to fermentation medium at 1% inoculum the next day, and culture at 37°C To OD about 0.6-0.8, add human IPTG to a final concentration of 0.5mmol / L, and induce expression overnight at 16°C. The cells induced by IPTG were ultrasonically disrupted, and the supernatant was analyzed by SDS-PAGE. A specific band with a molecular weight of about 53kDa was detected, and the specific enzyme activity of the supernatant was measured. Centrifuge the overnight induced expression bacterial solution at 10000r / min, 4°C for 15min, collect the bacterial cells, suspend the bacterial cells with pH 7.4 PBS buffer solution, ultrasonically break the cells, and then filter through a 0.45 μm filter membrane to select the expression ve...

Embodiment 3

[0040] The determination of the stability of embodiment 3 wild enzyme and mutant enzyme under different pH

[0041] Dilute the pure enzyme solution obtained in the previous step to a concentration of 2.5mg / mL, and then carry out enzymatic reactions respectively. The total volume of the enzymatic reaction is 500 μL, take 490 μL of 0.2M buffer solution with different pH (3.0-7.0), which contains 0.01 mMPLP, 100 mML-sodium glutamate, add 10 μL of enzyme solution, and separate the buffer solution and enzyme before the reaction The solution was preheated at 40°C for 5 minutes, then mixed, reacted at 40°C for 30 minutes, and finally boiled quickly to terminate the reaction, centrifuged, diluted 5 times with 5% trichloroacetic acid (TCA), and precipitated protein in a refrigerator at 4°C for about 3 hours. It is then detected by HPLC.

[0042] The results showed that the relative enzymatic activity of the glutamic acid decarboxylase mutant was increased from 38% to 72% or 84% at pH ...

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Abstract

The invention discloses an acid-stable glutamic acid decarboxylase (GAD) mutant with pH stability migrating to neutral range, and belongs to the field of biological engineering. The encoding gene of glutamic acid decarboxylase derived from actobacillus plantarum GB01-21 glutamic acid decarboxylase is subjected to site-specific mutagenesis, and the glutamate E on the 89th site is mutated to arginine R or alanine A. The glutamic acid decarboxylase mutant has relative enzyme in pH value of 6.5 increased from the original 38% up to 72% or 84%, and the enzyme activity stability in the neutral pH range is increased significantly. For the transformation with a glutamic acid decarboxylase mutant synthesized by recombinant escherichia coli, the GABA yield reaches 260 g/L, and the yield is 99.6%; and for the transformation with a glutamic acid decarboxylase mutant synthesized by recombinant Corynebacterium glutamicum, the GABA yield reaches 116 g/L, and the yield is 99.5%. The invention reduces the fermentation equipment loss under acidic conditions, and lays foundation for the efficient synthesis of gamma-aminobutyric acid.

Description

technical field [0001] The invention discloses a mutant whose pH stability of acid-stable glutamate decarboxylase (GAD) shifts to a neutral range, and belongs to the field of bioengineering. Background technique [0002] γ-Aminobutyric acid (GABA) is a functional non-protein natural amino acid widely found in animals, plants and microorganisms. It is an important inhibitory neurotransmitter in the central nervous system of vertebrates and has sedative and anticonvulsant properties , increase appetite, promote digestion, anti-oxidation and other functions, and have a significant impact on animal heat stress resistance, reproductive performance, hormone secretion, and carcass quality. With the gradual deepening of the research on GABA, its function has been widely used in various fields, especially in food and animal husbandry, the development of food and feed rich in GABA and its application in animal production have become domestic and foreign. Therefore, the research on GA...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N5/10C12N15/70C12P13/00
CPCC12N9/88C12P13/005C12Y401/01015
Inventor 饶志明徐美娟杨套伟张显
Owner JIANGNAN UNIV
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