Part:BBa_K5023002

Polyester Hydrolase PHL7

PHL7 is a recently discovered metagenomic-derived polyester hydrolase. This enzyme is capable of efficiently degrading amorphous polyethylene terephthalate (PET) found in post-consumer plastic waste. It Is stable in a temperature range suitable for PET hydrolysis between 65°C to 70°C. It hydrolyzes PET most effectively around 70°C, which is close to the glass transition temperature (Tg) of the polymer. PHL7 interacts with the terephthalic acid (TPA) moiety of its substrate in a lock-and-key mechanism, rather than an induced fit. This is similar to the enzyme LCC but different from IsPETase. The ligand-induced opening of the substrate-binding groove in PHL7 is restricted due to decreased flexibility of subsite I, which is influenced by the residue H185. This prevents the aromatic residue W156 from moving away from the active site. The aromatic π-stacking clamp, comprised of residues F63 and W156, is crucial for the binding and hydrolysis of BHET. The overall folds of PHL7 and its cocrystal structure with TPA (PHL7×TPA) are nearly identical, with minor deviations in the orientations of active site amino acids.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 307
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 307
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 778
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 307
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 307
  Illegal NgoMIV site found at 92
  Illegal NgoMIV site found at 193
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 543
  

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File:Https://static.igem.wiki/teams/5023/wiki/registry/phl7lucas.jpeg

Figure 22: Illustration of ABTS oxidation by ECOL with time compared to the negative control. The increase in ABTS oxidized proves laccase activity.

Figure 22 shows the illustration of ABTS oxidation by ECOL with time compared to the negative control. The increase in ABTS oxidized proves laccase activity even if a direct comparison with the original and not immobilized laccase solution was not possible due to the measuring methods.

Schematic of the Hifi cloning of D2

References

RICHTER, P. K. et al. Structure and function of the metagenomic plastic-degrading polyester hydrolase PHL7 bound to its product. Nature Communications, v. 14, n. 1, p. 1905, 5 abr. 2023. https://doi.org/10.1038/s41467-023-37415-x