Escherichia coli glucosamine synthase mutant and its application
A technology of glucosamine and Escherichia coli, applied in the field of genetic engineering and microbial fermentation, can solve problems such as corrosion of equipment, fishy smell of products, and allergic effects.
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Embodiment 1
[0037] The acquisition of embodiment 1 Escherichia coli glucosamine synthase gene
[0038] Using 1 μg of Escherichia coli genomic DNA as a PCR reaction template, design forward primer GlmS-F according to the sequence shown in SEQ ID NO.3: 5′-CGGATCCATGTGTGGAATTGTTGGCGC-3′, reverse primer GlmS–R: 5′ -CCTCGAGTTACTCAACCGTAACCGATTTTGCCA-3'; wherein, the parts in italics are restriction enzyme cutting sites BamHI and XhoI respectively. The PCR reaction was carried out in 50 μl total system, and the reaction conditions were: denaturation at 94°C for 5 min; denaturation at 94°C for 50 s, annealing at 58°C for 1 min, extension at 72°C for 2 min, a total of 30 cycles; extension at 72°C for 10 min. Take 3 μl of PCR amplification products for agarose gel electrophoresis verification, the results are as follows figure 1 shown. Take 100 μl of the PCR product for agarose gel electrophoresis, and recover the target fragment according to the instructions of the gel recovery kit.
Embodiment 2
[0039] Embodiment 2 Construction of wild-type glucosamine synthase gene expression vector
[0040] After the PCR product in Example 1 was digested with restriction endonucleases BamHI and XhoI, it was ligated with pET-24d(+) plasmid (purchased from Novagen) digested with BamHI and XhoI endonucleases, The constructed vector was named pET-GlmS, and then the pET-GlmS mixture was used to transform Escherichia coli DH5-α (purchased from Promega), and the sequence was determined to extract the plasmid of the transformed cell library and transform Escherichia coli BL21 (DE3) strains (purchased from promega company).
Embodiment 3
[0041] Example 3 Error-prone PCR amplification of Escherichia coli glucosamine synthase gene
[0042] Using the property that Taq DNA polymerase does not have 3′-5′ proofreading function, under high magnesium ion concentration (8mmol / L) and different concentrations of dNTP (where the concentration of dATP and dGTP is 1.5mmol / L, the concentration of dTTP and dCTP 3.0mmol / L) to control the frequency of random mutations, introduce random mutations into the target gene, and construct a mutation library. The template concentration A260 value is 1000ng / mL, the enzyme concentration is 5U / μL, and the primer concentration is 100μM. The optimal mutation rate in the experiment is about 0.6%.
[0043] Error-prone PCR reaction system (100μl):
[0044]
[0045]
[0046] The PCR program was: pre-denaturation at 96°C for 4 minutes; denaturation at 94°C for 1 minute, annealing at 56°C for 1 minute, extension at 75°C for 2 minutes, 45 cycles; and finally extension at 75°C for 15 minutes. ...
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