Part:BBa_K3787002
S245I PETase-MHETase
A coding sequence of the S245I 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, S245I PETase mutant 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.
Design of S245I PETase mutant
The mutant makes the substrate binding site, subunit II more cutinase-like and increases the hydrophobic property of the enzyme. It was created by our team in iGEM 2019.
Link: https://parts.igem.org/wiki/index.php?title=Part:BBa_K2982005
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 6× 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 S-M protein was expressed (Figure 1, lane 5). Clear bands with correct sizes were observed in the western blot of all purified proteins (Figure 2, lane 5).
These results demonstrated that our protein, S245I PETase-MHETase chimera, was successfully expressed and purified.
Scanning Electron Microscope (SEM)
(i) PET film incubated with S245I PETase-MHETase chimera
(ii) PET film incubated with buffer only
The pitting of PET film surface resulting from the digestion of S245I 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).
SEM image of PET film digested with S245I PETase-MHETase exhibited PET depolymerization activity.
HPLC
Products released from PET films after digestion with S245I PETase-MHETase were detected and quantified by HPLC.
(i) TPA Standard
Maximum intensity: 2650 cps at 4.64 min
(ii) Products released from the PET film incubated with S245I PETase-MHETase
Maximum intensity: 1160 cps at 4.66 min
Maximum intensity: 1080 cps at 4.67 min
(iii) Products released from the PET film incubated with buffer only
Maximum intensity: 700 cps at 4.69 min
Quantification of TPA released from PET film digestion with S245I PETase-MHETase
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 chimera proteins 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 S245I PETase-MHETase, suggesting that chimeras exhibit PET depolymerization activity (Table 1). Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 348
Illegal PstI site found at 1889
Illegal PstI site found at 1918 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 304
Illegal NheI site found at 1038
Illegal PstI site found at 1889
Illegal PstI site found at 1918
Illegal NotI site found at 1586 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 348
Illegal PstI site found at 1889
Illegal PstI site found at 1918 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 348
Illegal PstI site found at 1889
Illegal PstI site found at 1918
Illegal NgoMIV site found at 1513
Illegal AgeI site found at 627 - 1000COMPATIBLE WITH RFC[1000]
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