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Glutathione synthetase mutant, encoding gene and application

A glutathione and synthetase technology, applied in the direction of peptides, enzymes, ligases, etc., can solve the problems of high production cost, low concentration of glutathione product, low catalytic activity of glutathione synthase, etc., to achieve The effect of reducing production costs and improving market competitiveness

Active Publication Date: 2015-02-04
BONTAC BIO ENG SHENZHEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a glutathione synthetase mutant, coding gene and application, aiming at solving the low catalytic activity of glutathione synthase in the existing glutathione production process , low concentration of glutathione product and high production cost

Method used

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  • Glutathione synthetase mutant, encoding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Construction of parental glutathione synthase gene vector plasmid:

[0037] Primers MP-F and MP-R were designed according to the gene sequence of GenBank (GenBank NC_015516). The gene encoding glutathione synthase was amplified from Melissococcus plutonius ATCC 35311 using the primer pair MP-F and MP-R.

[0038] Amplification conditions were: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1% Triton X-100, 50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP, 400 nM primer MP-F, 400 nM primer MP-R, 1.0 U Pfu DNA polymerase (Promega, USA), Pick a little Melissococcus plutonius ATCC 35311 bacteria with an inoculation loop, and adjust the reaction volume to 50 ml with sterile water.

[0039] The PCR amplification reaction program was: 95°C for 3 minutes, 40 cycles: 95°C for 50 seconds, 50°C for 30 seconds and 72°C for 1 minute, and finally 72°C for 10 minutes. The amplified product was digested with restriction endonucleases NdeI and AscI and lig...

Embodiment 2

[0043] Site-directed mutagenesis of glutathione synthase site 128

[0044] In order to mutate glutamine (Q) at position 128 in the parental amino acid sequence to arginine (R) to obtain mutant Q128R, the plasmid pRSET-MP (see Example 1) was used as a template to design primer pairs 128RF and 128RR ( See Table 1).

[0045] Use the primer pair MP-F and 128RR to amplify the F-RR fragment, and the primer pair 128RF and MP-R to amplify the RF-R fragment. The specific sequences of primers MP-F and MP-R are shown in Table 1.

[0046] The above amplification reaction conditions are: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4) 2 SO 4 , 2 mM MgSO 4 , 0.1% Triton X-100, 50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP, 400 nM primer MP-F and 400 nM primer 128RR, or 400 nM primer 128RF and 400 nM primer MP-R, 1.5 U Pfu DNA polymerase (Promega, USA), 20 ng pRSET-MP, and the reaction volume was adjusted to 50 microliters with sterile water.

[0047] The PCR amplification reaction pr...

Embodiment 3

[0053] Site-directed mutagenesis at position 256 of glutathione synthase

[0054] In order to mutate the Ala (A) at the 256th position in the parental amino acid sequence to Ser (S) to obtain the mutant A256S, the plasmid pRSET-MP in Example 1 was used as a template to design primer pairs 256SF and 256SR (see Table 1). Show).

[0055] The primer pair MP-F and 256SR was used to amplify the F-SR fragment, and the primer pair 256SF and MP-R was used to amplify the SF-R fragment. The specific sequences of primers MP-F and MP-R are shown in Table 1.

[0056] The above amplification reaction conditions are: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1% Triton X-100, 50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP, 400 nM primer MP-F and 400 nM primer 256SR, or 400 nM primer 256SF and 400 nM primer MP-R, 1.5 U Pfu DNA polymerase (Promega, USA), 20 ng pRSET-MP, and the reaction volume was adjusted to 50 microliters with sterile water.

[0057] The...

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Abstract

The invention discloses a glutathione synthetase mutant, an encoding gene and an application. The glutathione synthetase mutant is obtained through point mutation of the sequence 2 in a sequence table, wherein the point mutation is at least one mutation at the 128th position, the 256th position and the 320th position. By means of mutation of the glutathione synthetase gene sequence, the glutathione synthetase mutant having high catalytic activity is finally obtained and is higher than a parent by at least 50% in glutathione synthetase catalytic activity with disodium adenosine triphosphate, L-sodium glutamate, L-cysteine and glycine being a substrate. When immobilized glutathione synthetase is employed for preparing glutathione, the concentration of the prepared glutathione is higher than 50 mM, so that the glutathione synthetase mutant is available in industrialized production of the glutathione with production cost being reduced and market competitiveness of related products being enhanced.

Description

technical field [0001] The invention relates to the fields of molecular biology and biotechnology, in particular to a glutathione synthetase mutant, coding gene and application. Background technique [0002] Glutathione, namely γ-L-glutamyl-L-cysteinyl glycine (glutathione, GSH), is formed by condensation of L-glutamic acid, L-cysteine ​​and glycine through peptide bonds A biologically active tripeptide compound with both γ-glutamyl and sulfhydryl groups. [0003] There are two forms of glutathione: reduced GSH and oxidized GSSG. Only GSH is active, and GSSG in organisms needs to be reduced to play its important physiological functions. GSH is widely distributed in animal, plant and microbial cells in nature. GSH has a variety of important physiological functions in organisms, especially plays a vital role in maintaining a suitable redox environment in organisms. GSH also has unique physiological functions, known as longevity factor and anti-aging factor. Because GSH ha...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/52C12P21/02
CPCC07K5/0215C12N9/93C12Y603/02003
Inventor 傅荣昭
Owner BONTAC BIO ENG SHENZHEN
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