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
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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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