Bifunctional glutathione synthetase mutant, and nucleotide sequence, preparation method and application thereof
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[0090] The present invention also provides a method for preparing the aforementioned bifunctional glutathione synthase mutant, which includes the following steps:
[0091] The wild-type GshF nucleotide sequence of Streptococcus salivarius of SEQ ID NO: 1 is subjected to one or more of random mutation, saturation mutation and iteration to obtain a nucleotide sequence encoding the aforementioned GshF mutant.
[0092] Insert the nucleotide sequence of the mutant into the vector to obtain a recombinant expression vector.
[0093] The recombinant expression vector is transformed into an expression strain to obtain a recombinant genetically engineered bacteria.
[0094] The recombinant genetically engineered bacteria are fermented, broken, and isolated to obtain a bifunctional glutathione synthase mutant.
[0095] The wild-type GshF nucleotide sequence of Streptococcus salivarius with SEQ ID NO:1 in the sequence table is subjected to random mutation, saturation mutation, iterative saturation ...
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[0102] Example 1: Construction and expression of wild-type GshF recombinant genetically engineered bacteria and purification and immobilization of recombinant protein
[0103] 1-1 Acquisition of wild-type GshF gene
[0104] In order to obtain mutant GshF with high synthesis 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) ), optimize the coding gene by E. coli codon preference and perform full gene synthesis, and name the wild-type bifunctional glutathione synthetase GshF-WT, and name the coding gene gshF-wt, optimized 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 bacteria
[0106] Reference figure 1 , Th...
Example Embodiment
[0117] Example 2: Preparation of GshF mutant
[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 present inventors used the recombinant expression vector pET-gshF-wt as the DNA template, and the primers were T7 universal primers (SEQ ID NOs: 15 and 16), and constructed a random sequence by means of error-prone PCR. 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, which means that a mutant has One to three amino acids are mutated, and the specific process of constructing a mutant library is as follows.
[0121] Error-prone PCR reaction system:
[0122] 10×Buffer
[0123] The error-prone PCR reaction conditions are: first denaturation at 95°C for 5 minutes; then den...
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