Difference between revisions of "Part:BBa K5023002"
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[[Image:Bielefeld2012-Immobilized_proteins.jpg|500px|left|thumb|'''Figure 20''': The percentage of laccases immobilized to CPC-Beads. 99 % of ECOL, 97 % of BPUL and 79 % of BHAL and TTHL laccases were bound to the beads.]]
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== PHL7 ==
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[[Image:https://static.igem.wiki/teams/5023/wiki/design-page/design-page/design-page/phl7-parts-mhetygua-lucas-daniel-ovelar-vargas.png|500px|left|thumb|'''Figure 20''': The percentage of laccases immobilized to CPC-Beads. 99 % of ECOL, 97 % of BPUL and 79 % of BHAL and TTHL laccases were bound to the beads.]]
  
 
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Revision as of 00:55, 10 October 2023

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


PHL7

File:Https://static.igem.wiki/teams/5023/wiki/design-page/design-page/design-page/phl7-parts-mhetygua-lucas-daniel-ovelar-vargas.png
Figure 20: The percentage of laccases immobilized to CPC-Beads. 99 % of ECOL, 97 % of BPUL and 79 % of BHAL and TTHL laccases were bound to the beads.

Figure 20 shows the percentage of laccases bound after incubation with CPC-beads, relative to the original concentration. The concentration of laccases in the supernatant after incubation was measured using Roti®-Nanoquant. The results showed that only 1% of ECOL laccases was still present in the supernatant. This illustrates that ECOL was successfully immobilized on the CPC-beads.


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