Part:BBa_K4724028

IsPETaseQ119F/W159H-Linker-CBM11-6xHis Tag

IsPETase is a hydrolase produced by Ideonella sakaiensis that degrades PET. Q119F and W159H double mutants have the highest PET degradation activity among double mutants in our experiment, and CBM11 is the most hydrophobic domain in our experiment, so plasmids containing this composite element are constructed. Compared to the old part BBa K4724028, we designed a new part BBa_K2010999.

Characterization

1. HPLC

After protein purification, an enzymatic reaction is performed to measure the activity of the enzyme. The substrates used are PET film, PET powder, and filter cloth, under the action of IsPETase, PET powder is broken down into TPA and MHET. Determine the volume of purified enzyme solution required for 500 μL of the reaction system based on protein concentration. At 37 °C, it reacted with PET film, PET powder, and filter cloth for 48h, and after the end, the reaction solution was analyzed by high performance liquid chromatography, the liquid phase result of 6min corresponds to TPA, and the liquid phase result of 8min corresponds to MHET. Through the standard curve, the peak area of the product output by the liquid phase instrument is converted into the product concentration, such as in Fig.1.

Fig.1 Concentrations of the products TPA, MHET of the reaction of 500nM IsPETase^{Q119F/ W159H} - CBM11 with PET powder, PET film, and filter cloth at 37°C for 48h.

2. SEM After 48h degradation, we observed the changes on the surface of the PET film under an electron microscope.

Fig.2 (A) SEM images of PET films degraded by the enzyme-free system for 48h; (B) SEM images of PET films degraded by IsPETase for 48h; (C) SEM images of PET films degraded by IsPETase^Q119F/W159H - CBM11 for 48h.

By Fig.2, the degree of degradation of the PET material can be reflected by the degree of surface depression in the SEM image. Since (A) was only immersed in buffer solution for 48h without degradation by enzyme addition solution, it presents a smooth surface in the SEM image. (B) is the effect after 48h of WT degradation, a slight depression can be seen on the surface, but the degree of depression is not obvious. (C) is the effect of IsPETase^Q119F/W159H - CBM11 after 48h of degradation, it can be seen that some areas of depression are more obvious than the effect of WT.

Fig.3 (A) SEM images of the degradation of industrial filter cloth by the enzyme-free system for 48 h; (B) SEM images of the degradation of industrial filter cloth by IsPETase for 48 h; (C) SEM images of the degradation of industrial filter cloth by IsPETase^Q119F/W159H - CBM11 for 48 h. The degradation degree of the PET material can be reflected by the roughness of the surface in the SEM images.

2.Conclusion

IsPETase^{Q119F/ W159H}- CBM11 degraded PET powder at about 6 times the product concentration of IsPETase(BBa_K2010999), degraded PET film at about 5 times the product concentration of BBa_K2010999, and degraded filter cloth at about 6 times the product concentration of BBa_K2010999. However, it is clear that IsPETase^{Q119F/ W159H}- CBM11 is much more capable of degrading PET powder than PET film and filter cloth.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 56
  Illegal PstI site found at 913
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 56
  Illegal PstI site found at 913
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1134
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 56
  Illegal PstI site found at 913
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 56
  Illegal PstI site found at 913
  Illegal AgeI site found at 546
  Illegal AgeI site found at 926
  
• 1000
  COMPATIBLE WITH RFC[1000]