Part:BBa_K4724012

IsPETaseM157S-6xHis Tag

IsPETase is a hydrolytic enzyme produced by Ideonella sakaiensis that can degrade PET, and M157S is a mutation from a bulky amino acid to a bulky amino acid, which shifts the active site and allows PET to bind better to the active pocket of the enzyme.

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 Concentrations of the products TPA and MHET of 500 nM WT and M157S 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

The M157S mutant showed reduced degradation compared to WT at 30°C, 37°C and 45°C.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 56
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 56
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 790
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 56
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 56
  Illegal AgeI site found at 546
  
• 1000
  COMPATIBLE WITH RFC[1000]