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Bifunctional glutathione synthetase mutant, and nucleotide sequence, preparation method and application thereof

Active Publication Date: 2017-10-20
HUNAN FLAG BIOTECHNOLOGY CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] The present invention provides a bifunctional glutathione synthetase mutant, nucleotide sequence and its preparation method and application to solve the technical problems of low synthetic activity of the mutant enzyme and low batches of immobilized enzyme

Method used

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  • Bifunctional glutathione synthetase mutant, and nucleotide sequence, preparation method and application thereof
  • Bifunctional glutathione synthetase mutant, and nucleotide sequence, preparation method and application thereof
  • Bifunctional glutathione synthetase mutant, and nucleotide sequence, preparation method and application thereof

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preparation example Construction

[0090] The present invention also provides a method for preparing the above-mentioned bifunctional glutathione synthetase mutant, comprising the following steps:

[0091] One or more of random mutation, saturation mutation and iterative saturation is performed on the wild-type GshF nucleotide sequence of Streptococcus salivarius of SEQ ID NO: 1 to obtain the nucleotide sequence encoding the mutant GshF as before.

[0092] The nucleotide sequence of the mutant is inserted into the vector to obtain a recombinant expression vector.

[0093] The recombinant expression vector is transformed into the expression strain to obtain the recombinant genetically engineered bacteria.

[0094] The recombinant genetically engineered bacteria are induced by fermentation, crushed, and isolated to obtain a bifunctional glutathione synthetase mutant.

[0095] The wild-type GshF nucleotide sequence of Streptococcus salivarius with SEQ ID NO: 1 in the sequence listing is subjected to steps such as...

Embodiment 1

[0102] Example 1: Construction and expression of wild-type GshF recombinant genetically engineered bacteria and purification and immobilization of recombinant proteins

[0103] 1-1 Obtaining of wild-type GshF gene

[0104] In order to obtain a mutant GshF with high synthetic activity, weak product inhibition, strong heat resistance and good operational stability, the wild-type GshF gene and amino acid sequence used in the present invention are derived from Streptococcus salivarius (GeneBank accession number: WP_038676473 ), the coding gene was optimized through Escherichia coli codon bias and the whole gene synthesis was carried out, and the wild-type bifunctional glutathione synthetase was named GshF-WT, and its coding gene was named gshF-wt. The following nucleotide sequence and amino acid sequence are shown in SEQ ID NO:1 and SEQ ID NO:2.

[0105] 1-2 Construction of wild-type GshF prokaryotic expression vector and construction of recombinant genetically engineered bacteri...

Embodiment 2

[0117] Embodiment 2: Preparation of GshF mutants

[0118] 2-1 Preparation of GshF mutant with high synthetic activity

[0119] Construction of 2-1-1GshF mutant library

[0120] In order to improve the synthetic activity of wild-type GshF, the inventors used the recombinant expression vector pET-gshF-wt as a DNA template, and the primers were T7 universal primers (SEQ ID NO: 15 and 16), and constructed a random GshF by error-prone PCR method. Mutant library, and by adjusting the concentration of Mg2+ and Mn2+ and the concentration of dCTP and dTTP oligonucleotides in the error-prone PCR reaction system, the base mismatch rate of the mutant library is five per thousand, that is, to ensure that a mutant has 1 to 3 amino acids are mutated, and the specific process of constructing the mutant library is as follows.

[0121] Error-prone PCR reaction system:

[0122] 10×Buffer

5μL

2mmol / L dNTPS

5μL

100mmol / L dCTP

0.5μL

100mmol / L dTTP

0....

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Abstract

The invention discloses a bifunctional glutathione synthetase mutant, and a nucleotide sequence, a preparation method and an application thereof. At least one of sites in the third position, the 123rd position, the 161st position, the 194th position, 382nd position and 390th position of the amino acid sequence of a GshF mutant is different from the corresponding sites of the wild GshF amino acid sequence of Streptococcus salivarius in amino acids in order to obtain the GshF mutant having a high synthesis activity, a fast reaction rate and a good conversion efficiency. The synthesis vitality of a GshF-4 mutant is improved 12.8 times, the specific vitality is improved 11 times, the optimum temperature is increased by 20 DEG C, the vitality keeps at 91.3% after standing at 55 DEG C for 60 min, an immobilized enzyme of the GshF-4 mutant allows the concentration of GSH to reach 21 g / L when participating in the catalytic synthesis of the GSH at 50 DEG C under a pH value of 8.0 for 60 min, can be continuously used for 300 times or more, and has very good operating stability.

Description

technical field [0001] The invention relates to the field of bifunctional glutathione synthetase, in particular to a bifunctional glutathione synthetase mutant. In addition, the present invention also relates to a nucleotide sequence comprising the above-mentioned bifunctional glutathione synthetase mutant and its preparation method and application. Background technique [0002] Glutathione (GSH for short) is formed by condensation of glutamic acid, cysteine ​​and glycine through peptide bonds, and is an active tripeptide with various important physiological functions. In the natural environment, there are two forms of GSH, namely the reduced form and the oxidized form. Among them, the reduced form of GSH is the main reducing substance in cells, which plays a vital role in maintaining a suitable redox environment in organisms and can protect cells. Protected from oxidizing forms, toxic compounds and radiation. At the same time, GSH molecules are small, easily absorbed by t...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/52C12N15/70C12P21/02
CPCC12N9/93C12N15/70C12P21/02C12Y603/01
Inventor 黄斌周晶辉赵强熊孟玲刘婧莹曾红宇许岗
Owner HUNAN FLAG BIOTECHNOLOGY CO LTD
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