Part:BBa_K2110800

PETase

This codon optimized sequence codes the wild type protein called PETase. PETase is a Poly(ethylene terephthalate) hydrolase, which was found in Ideonella sakaiensis (strain 201-F6) by Japanese scientists. It allows Ideonella sakaiensis to degrade PET. If properly transferred into engineered organisms as E.Coli or yeast, secreted PETase can catalyze the hydrolysis of PET to produce mono(2-hydroxyethyl) terephthalate (MHET) as the major product. Optimum temperature is 40 degrees Celsius for PET film hydrolysis and optimum pH is 9.

Usage and Biology

In order to build a PET degradation device, we had to increase the secretion of PET degradation enzymes. So we searched for a novel signal peptide candidate through literature search and determined the cleavage site using computational analysis.

In order to release PETase (BBa_K2110800) from E. coli, the endogenous signal peptide was removed and a new signal peptide (NSP4) was attached. As a result, it can be seen that NSP4-PETase was secreted more into the media than PETase (BBa_K2110800).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 79
  Illegal BglII site found at 289
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Activity Assay
We detect the absorption peak of culture medium after culturing with PET film added for 2d. The absorption degree-wavelength curve is as follows.

References

Han, S., Machhi, S., Berge, M., Xi, G., Linke, T., & Schoner, R. (2017). Novel signal peptides improve the secretion of recombinant Staphylococcus aureus Alpha toxin H35L in Escherichia coli. AMB Express, 7(1), 93.