Difference between revisions of "Part:BBa K2982003:Design"

 
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===Source===
 
===Source===
  
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Modified from wild type IsPETase sequence from 'Structural Insight into Molecular Mechanism of Poly (ethylene terephthalate) Degradation', Nature Communication, 2018.
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Modified from wild type IsPETase sequence from 'Characterization and engineering of a plastic-degrading aromatic polyesterase', PNAs, 2018.
  
 
===References===
 
===References===
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Austin, H. P., Allen, M. D., Donohoe, B. S., Rorrer, N. A., Kearns, F. L., Silveira, R. L., . . . Beckham, G. T. (2018). Characterization and engineering of a plastic-degrading aromatic polyesterase. Proceedings of the National Academy of Sciences, 115(19). doi:10.1073/pnas.1718804115

Latest revision as of 04:39, 7 August 2019


Coding sequence for W159H/S214H IsPETase double mutant


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 348
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 304
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 348
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 348
    Illegal AgeI site found at 627
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The mutation site, W159 locate in substrate binding site, subsite II where three MHET moieties are bound through hydrophobic interaction. Trp 159 in PETase has effect of extending the hydrophobic surface adjacent to the active site. Mutation, W159H results in more space on binding center. The mutation site, S214 locate in the edge of the substrate binding site have effect on substrate binding. Both single mutant of W159H and S214H show higher activity than that of the wild type enzyme. Histidine is often found in the active sites of enzymes, where its imidazole ring can readily switch between uncharged or positively charged to catalyze the making and breaking of bonds. In TfCut2, Histidine 169 and Histidine 224 are located at the corresponding positions of Trpytophan 159 and Serine 214 in subsite II of IsPETase. The resulting double mutant may have effect on substrate binding.


Source

Modified from wild type IsPETase sequence from 'Characterization and engineering of a plastic-degrading aromatic polyesterase', PNAs, 2018.

References

Austin, H. P., Allen, M. D., Donohoe, B. S., Rorrer, N. A., Kearns, F. L., Silveira, R. L., . . . Beckham, G. T. (2018). Characterization and engineering of a plastic-degrading aromatic polyesterase. Proceedings of the National Academy of Sciences, 115(19). doi:10.1073/pnas.1718804115