Part:BBa_K4724015
IsPETaseA240_S242del-6xHis Tag
IsPETase is a hydrolase produced by Ideonella sakaiensis that can degrade PET. A240_S242del is because asparagine is a polar amino acid, and the deletion of the polar amino acid reduces the polarity of the protein so that the protein binds better to the hydrophobic surface of PET.
Constraction
The plasmid was PCR with pET-22b(+) as the vector, and the PCR bands were verified by nucleic acid gel electrophoresis after mutation. The plasmid with the correct bands was transformed into E.coliBL21(DE3) sensory state.
Fig.1 Nucleic acid gel electrophoresis for PCR of plasmid
Characterization
After protein purification, enzymatic reactions were performed to measure enzyme activity. The substrate used was PET powder, which was broken down into TPA and MHET in the presence of an IsPETase mutant, and the volume of purified enzyme solution required for a 500 μL reaction system was determined based on protein concentration. The reaction was carried out at 30°C, 37°C and 45°C for 48 h. After the reaction, the reaction solution was analyzed by high performance liquid chromatography (HPLC), and the liquid phase result at 6 min corresponded to TPA and the liquid phase result at 8 min corresponded to MHET. The peak areas of the products outputted from the liquid chromatography were converted into the product concentrations by standard curves, as shown in Fig.2
Fig.2 Concentrations of the products TPA and MHET of 500 nM WT and A240_S242del reacted with PET powder at different temperatures for 48h. (A) and (B) are the concentrations of the products obtained at a reaction temperature of 30°C; (C) and (D) are the concentrations of the products obtained at a reaction temperature of 37°C; (E) and (F) are the concentrations of the products obtained at a reaction temperature of 45°C. The concentrations of TPA and MHET were obtained at different temperatures.
Conclusion
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 56
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 637
Illegal PstI site found at 56 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 787
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 56
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 56
Illegal AgeI site found at 546 - 1000COMPATIBLE WITH RFC[1000]
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