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Glutamate decarboxylase (GAD) thermally-stable variant G56P gene and application thereof

A glutamic acid decarboxylase, thermostable technology, applied in the field of molecular biology, can solve problems such as unfavorable enzyme application

Inactive Publication Date: 2013-04-10
NINGBO INST OF TECH ZHEJIANG UNIV ZHEJIANG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the GAD expressed by this gene is quickly inactivated in the environment of 60°C and above, which is not conducive to the application of this enzyme in the biological preparation of GABA

Method used

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  • Glutamate decarboxylase (GAD) thermally-stable variant G56P gene and application thereof
  • Glutamate decarboxylase (GAD) thermally-stable variant G56P gene and application thereof
  • Glutamate decarboxylase (GAD) thermally-stable variant G56P gene and application thereof

Examples

Experimental program
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Embodiment Construction

[0020] For further illustrating the present invention, specifically illustrate in conjunction with following examples:

[0021] 1. Construction of recombinant plasmids

[0022] Lactobacillus brevis (Lactobacillus brevis) CGMCC NO.1306 was cultivated to the mid-logarithmic growth phase, and 4.5 mL of the bacterial liquid was centrifuged at 10,000 rpm for one minute, then the supernatant was discarded, and genomic DNA was extracted using a kit (Shanghai Sangong) according to the instructions.

[0023] Design the following degenerate primers:

[0024] Upstream degenerate primer: 5′-cg ggatcc atggcWatgttRtaYggWaaac-3' (as shown in SEQ ID NO.5);

[0025] Downstream degenerate primer: 5′-gg gaattc ttagtgHgtgaaYccgtattt-3' (as shown in SEQ ID NO.6);

[0026] Wherein, "ggatcc" in the upstream primer is the BamH I restriction site, and "gaattc" in the downstream primer is the EcoR I restriction site.

[0027] Use upstream primers and downstream primers to pair up, select and opt...

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Abstract

The invention relates to a glutamate decarboxylase (GAD) thermally-stable variant G56P gene. Site-specific mutagenesis occurs at the 56th site of the nucleotide sequence of the GAD thermally-stable variant G56P gene. According to researches, the thermal stability of GAD coded through the variant gene obtained after mutagenesis at the site is enhanced. Through the site-specific mutagenesis on the basis of wild GAD gene coding, the thermal stability of the corresponding variant enzyme at 60-70DEG C is improved. With the improvement of the thermal stability of the enzyme, higher operating temperature can be adopted during industrial production to improve reaction speed and increase substrate solubility, and the production of GABA (gamma-aminobutyric acid) through biological transformation by using the enzyme is facilitated.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a glutamic acid decarboxylase variant G56P gene and its application. Background technique [0002] Site-directed mutagenesis (site-directed mutagenesis or site-specific mutagenesis) refers to the technique of introducing specific base pair changes at designated sites of the target DNA fragment. It is a powerful tool for studying the complex relationship between protein structure and function, and it is also a common means of modifying genes in the laboratory. Site-specific changes, deletions, or insertions of specific bases in a known gene can change the corresponding amino acid sequence and the structural and functional characteristics of the protein, and help to understand the relationship between protein structure and function. Especially in the rational design of enzymes, or the use of random mutation and other directed evolution techniques to screen out important ...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N9/88C12N15/63C12N1/21C12P13/00C12R1/24C12R1/19
Inventor 梅乐和郁凯胡升黄俊
Owner NINGBO INST OF TECH ZHEJIANG UNIV ZHEJIANG