Part:BBa_K4390117
N-terminal L2NC-tagged MHETase
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.
The fusion protein with N-terminal L2NC silica tag and MHETase for MHET hydrolysis.
Usage and Biology
We designed the C-terminal Car9-tagged MHETase to make the construct functional for both MHET hydrolysis and silica immobilisation.
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).
L2NC is a truncated version of the L2 ribosomal protein from E. coli, designed for fusion to C-terminal of a protein using JUMP assembly. This tag contains just the N and C-terminal regions of L2 which were shown to have silica binding capacity in previous experiments, therefore allowing the use of a smaller tag without compromising on binding affinity. The attachment of L2NC silica tag on the C-terminus of the functional enzyme would result in the 14.19 kDa increasement in weight. From literature, the dissociation constant between L2NC silica tag and silica beads is 1.7nM. Therefore, this tag facilitates immobilisation to silica surfaces, enabling enzyme immobilisation or purification using silica-based spin columns (Kim et al., 2020).
Design
C-terminal N-terminal L2NC-tagged MHETase was assembled by JUMP assembly with: T7 promoter (P part)-B0034 RBS (R part)-L2NC(N part)-[FAST-PETase] (O part)-L1U1H08 (CT part). All the codon is optimized for BioBrick and JUMP assembly.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 110
- 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.
Kim S, Joo K, Jo B, Cha H. Stability-Controllable Self-Immobilization of Carbonic Anhydrase Fused with a Silica-Binding Tag onto Diatom Biosilica for Enzymatic CO2 Capture and Utilization. ACS Applied Materials & Interfaces. 2020;12(24):27055-27063.
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