Difference between revisions of "Part:BBa K3787001"

Latest revision as of 13:28, 20 October 2021

WT PETase-MHETase

A coding sequence of the Wild Type PETase-MHETase chimera.

The sequence is optimized for the expression of Escherichia coli, strains DH5α and C41(DE3).

Origin and Biology

This part is a chimeric protein used to depolymerize PET into its constituting monomers. It consists of two enzymes, Wild Type PETase and MHETase, both belong to the hydrolase superfamily. The PETase part degrades PET into bis(2-hydroxyethyl) terephthalic acid (BHET), mono(2-hydroxyethyl) terephthalic acid (MHET), and terephthalic acid (TPA), while the MHETase part degrades MHET into TPA and ethylene glycol (EG) by cleavage of the ester bond within the polymer.

The C-terminus of PETase is linked to the N-terminus of MHETase using a 12 amino acid serine-glycine linker.

These two enzymes were originally found in the bacteria Ideonella sakaiensis, which uses PET as a carbon source, and integrates the degradation products into its metabolic cycle.

Characterisation

In our experiment, this gene was inserted into an expression vector, PET-21b which is used due to its high copy number, the presence of T7 promoter and a lac operon. We use DH5ɑ as host cells due to its high insert stability. Then, extracted DNA is transformed into E. coli C41(DE3), which we use to perform the protein induction and purification.

After the protein was induced using 0.5mM IPTG, it was purified and extracted using a column with nickel resin due to the presence of a 6X His-Tag which was fused with C terminus of MHETase. After purification, SDS-PAGE and Western blot were performed to confirm the identity of expressed protein using His-Tag antibody.

Our SDS-PAGE results showed that the purified P-M protein was expressed (Figure 1, lane 4). Clear bands with correct sizes were observed in the western blot of all purified proteins (Figure 2, lane 4).

These results demonstrated that our protein, Wild Type PETase-MHETase chimera, was successfully expressed and purified.

Scanning Electron Microscope (SEM)

(i) PET film incubated with WT PETase-MHETase chimera

(ii) PET film incubated with buffer only

The pitting of PET film surface resulting from the digestion of WT PETase-MHETase was observed (Figure 3). No pitting of PET film surface was observed when PET film was incubated with buffer-only solution (Figure 4).

Products released from PET films after digestion with WT PETase-MHETase were detected and quantified by HPLC.

HPLC

(i) TPA Standard
Maximum intensity: 2600 cps at 4.64 min
Figure 5. HPLC profile of 0.397µM TPA standard

</center> (ii) Products released from the PET film incubated with WT PETase-MHETase
Maximum intensity: 680 cps at 4.69 min

Maximum intensity: 1340 cps at 4.67 min Figure 7. HPLC profile of products released from PET film digestion using 16µL WT PETase-MHETase

</center> (iii) Products released from the PET film incubated with buffer only
Maximum intensity: 990 cps at 4.65 min
Figure 8. HPLC profile of products released from PET film digestion with buffer only

</center> Quantification of TPA released from PET film digestion with WT PETase-MHETase Table 1. Calculated concentration of TPA released from PET film digestion with WT PETase-MHETase

</center> HPLC profiles demonstrated that the detection peak representing TPA monomer has a retention time at 4.64 minutes (Figure 5). 8μL and 16μL of extracted WT PETase-MHETase was used to digest PET film in order to investigate their PET depolymerization activity. HPLC data showing that trace amounts of TPA were detected in eluents of PET film digestion with WT PETase-MHETase, suggesting that the chimera exhibit PET depolymerization activity (Table 1). Sequence and Features