Part:BBa_K4390032
MHETase
Cleaves MHET, the degradation product of PET. Used in plastic degradation
This part is not compatible with BioBrick RFC10 assembly but is compatible with the iGEM Type IIS Part standard which is also accepted by iGEM.
Usage and Biology
MHETase is an enzyme discovered in Ideonella sakaiensis at the same time as PETase (Yoshida, 2016). MHETase can hydrolyse Mono-(2-hydroxyethyl) terephthalic acid (MHET), the major product of PETase, further to terephthalic acid (TPA) and ethylene glycerol (EG). An advantage of using MHETase is that PETase degrades PET into MHET majorly, and MHET had a higher inhibitory effect on the overall hydrolysis of PET since it also originated from the other reaction product, that is, from BHET. Since TPA and EG did not affect the enzyme hydrolysis of PET, the observed inhibition caused by MHET and BHET is probably due to their ester bonds that occupy the TfCut2 substrate binding site (Pirillo, V, et al., 2021). From literature, there is a ∼60 Å long intrinsically disordered tether structure (residues 1–25) at the N-terminus of the MHETase (Pinto et al., 2021).
Design
The MHETase (BBa_K4390032) encode the peptide sequence of MHETase with additional 2 base pairs to fit in JUMP assembly O part design. The codon is optimized for BioBrick and JUMP assembly.
Characterization
The MHETase (BBa_K4390032) were used for different Lv.1 JUMP assembly with various silica tags at N- and C-terminus.
| Untagged MHETase | K4390091 |
| N-terminal L2NC-tagged MHETase | K4390117 |
| N-terminal L2NC-linker-tagged MHETase | K4390118 |
| N-terminal Car9-tagged MHETase | K4390087 |
| C-terminal L2NC-tagged MHETase | K4390079 |
| C-terminal L2NC-linker-tagged MHETase | K4390080 |
| C-terminal Car9-tagged MHETase | K4390078 |
The [N-terminal L2NC-tagged MHETase] plasmid was failed to construct since we didn’t obtain the Lv.0 plasmid of N-terminal L2NC silica tag. And we failed to construct all three C-terminal tagged MHETase construct since there was a stop codon on the MHETase DNA we ordered.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Reference
Yoshida S, Hiraga K, Takehana T, Taniguchi I, Yamaji H, Maeda Y et al. A bacterium that degrades and assimilates poly (ethylene terephthalate). Science. 2016;351(6278):1196-1199.
Pirillo V, Pollegioni L, Molla G. Analytical methods for the investigation of enzyme‐catalyzed degradation of polyethylene terephthalate. The FEBS Journal. 2021;288(16):4730-4745.
Pinto A, Ferreira P, Neves R, Fernandes P, Ramos M, Magalhães A. Reaction Mechanism of MHETase, a PET Degrading Enzyme. ACS Catalysis. 2021;11(16):10416-10428.
| None |