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Glutamic acid decarboxylase mutant and preparation method thereof and application

A glutamic acid decarboxylase and mutant technology, applied in the field of molecular biology, can solve problems such as unfavorable GAD application, further improvement of heat resistance stability, etc., to reduce screening workload, improve experimental efficiency and feasibility, The effect of increasing the probability of positive mutation

Inactive Publication Date: 2016-04-06
NINGBO INST OF TECH ZHEJIANG UNIV ZHEJIANG
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, experiments have shown that the half-life of glutamic acid decarboxylase at 55°C is only 30.9 minutes, which is very unfavorable for the application of GAD in the biological preparation of GABA, and its heat resistance and stability need to be further improved.

Method used

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  • Glutamic acid decarboxylase mutant and preparation method thereof and application
  • Glutamic acid decarboxylase mutant and preparation method thereof and application
  • Glutamic acid decarboxylase mutant and preparation method thereof and application

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Embodiment 1

[0039] 1. Construction of mutants

[0040] According to the glutamate decarboxylase gene design of Lactobacillus brevis (Lactobacillus brevis) GAD1407 13 pairs of site-directed mutagenesis primers, wherein the primers of mutant G364P are as follows:

[0041] G364P-F: 5'-ATCGCTCACTAAATTACCGGGCTTTTCCCTCATTAAT-3';

[0042] G364P-F: 5'-TAATGAGGGAAAAGCCCGGTAATTTAGTGAGCGATTTC-3';

[0043] With pET28a(+)-GAD1407 plasmid ( figure 2 ) as a template for site-directed PCR amplification ( image 3 ). The PCR amplification system is 50 μL, including: PrimeSTARMax DNA Polymerase 25 μL, 1 μL upstream primer (10 μM), 1 μL downstream primer (10 μM), 1 μL plasmid template (100 ng / μL), and high-temperature sterilized ultrapure water to make up to a total volume of 50 μL.

[0044] The PCR amplification program was as follows: denaturation at 98°C for 1 min, followed by an amplification cycle, that is, denaturation at 98°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 3 min, a t...

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Abstract

The invention discloses a glutamic acid decarboxylase mutant and a preparation method thereof and application. An amino acid sequence of the mutant is as shown in SEQ ID No. 2, and a nucleotide sequence is as shown in SEQ ID No. 1. The invention further discloses an expression unit containing genes for encoding the glutamic acid decarboxylase mutant, a recombinant plasmid and a transformant. According to information of comparison with a thermococcus kodakarensis glutamic acid decarboxylase (GAD) amino acid sequence, a method of site-directed mutagenesis is adopted to introduce proline residue at an amino acid locus corresponding to lactobacillus brevis GAD, and the thermal stability of the GAD is improved through rational design. The mutant has better thermal stability in the process of catalyzing L-glutamic acid or sodium salts thereof to generate gamma-aminobutyric acid and is favorable for industrial production of gamma amino acid butyric acid (GABA).

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a glutamic acid decarboxylase mutant and its preparation method and application. Background technique [0002] Glutamate decarboxylase (GAD for short; EC4.1.1.15) uses pyridoxal phosphate (PLP) as a coenzyme to specifically catalyze the decarboxylation of L-glutamic acid to synthesize γ-aminobutyric acid (γ-aminobutyrate , GABA) enzymes. GABA is widely found in animals, plants and microorganisms. It is a naturally occurring non-protein amino acid. It has physiological functions such as inhibiting excessive excitement and relieving nervous tension in the mammalian central nervous system. In addition, GABA has many important physiological functions such as preventing vascular sclerosis, delaying aging, repairing skin, lowering blood pressure, calming the nerves, improving sleep and memory, and is widely used in industries such as medicine and health, functional food and ...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12P13/00
CPCC12N9/88C12P13/00C12Y401/01015
Inventor 胡升黄俊方文姬梅乐和赵伟睿吕常江
Owner NINGBO INST OF TECH ZHEJIANG UNIV ZHEJIANG
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