Part:BBa_K4724007
IsPETaseT116R-6xHis Tag
We mutate threonine into arginine. We chose this mutation because PET hydrophobic surfaces have a negative charge, arginine is a basic amino acid with a positive charge, and the PET surface attracts each other.
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.coli BL21(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 WT and T116R at 500 nM at different temperatures. Fig.2 Fig.2 Concentrations of TPA and MHET of 500nM WT and T116R reacted with PET powder at different temperatures for 48h. (A) and (B) are the product concentrations obtained at a reaction temperature of 30°C; (C) and (D) are the product concentrations obtained at a reaction temperature of 37°C; (E) and (F) are the product concentrations obtained at a reaction temperature of 45°C. The product peak areas of 500 nM WT and T116R were converted into product concentrations as shown in Fig.2.
Fig.2 Concentrations of the products TPA and MHET of 500 nM WT and T116R 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 PstI site found at 56
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 790
- 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]
| None |