Polyethylene terephthalate (PET) is the most widely produced polyester plastic and its accumulation in the environment has become a global concern, so it is really important to solve this problem.
In order to carry forward the spirit of iGEM, and inherit and spread the value of iGEM, we specially searched the iGEM Biological Parts library for related projects and picked BBa_K3997000, IsPETase. This is a biological part submitted by iGEM21_WFLA_YK_PAO in 2021, they found IsPETase has the activity of degrading PET materials, but didn’t totally achieve degrading PET materials. So it is really important to identify other candidates for PET materials degradation.
In this project, our team carried out an efficiency enzyme SbPETase for this part in the laboratory, adding data from PET materials degradation testing to dedicate protein function. What’s more, we also developed several mutants of SbPETase that can be used for both PET and BHET, which also provides more ideas for future iGEM teams to optimize PET materials degradation enzymes. This information can be a good reference for future iGEM teams working on PET materials degradation.
In order to verify if our new composite part SbPETase, especially SbPETase-mutants we developed, we transferred the recombinant plasmid into E. coli BL21(DE3) to purify the protein, mixed it with substrates and set up an in vitro PET degradation screen platform. After screening SbPETase and its mutants by detecting the compounds in the reaction mixture, we identified several more active mutants, thereby achieving PET and BHET materials degradation.

SbPETase is a novel enzyme for Poly (ethylene terephthalate) (PET) hydrolysis. This hydrolase can degrade PET into small fragments, then transport the degraded products for further "digestion", and finally convert them into two relatively simple organic compounds, ethylene glycol, and terephthalic acid.

In order to construct the recombinant plasmid, we amplify SbPETase was amplified from the genome of S. brevitalea sp. nov and ligated it to the double-enzyme digestion pET22b vector. After identifying the correct colony through enzyme digestion, we sent the corresponding plasmid to the company for Sanger sequencing.In order to construct the recombinant plasmid, we amplify SbPETase was amplified from the genome of S. brevitalea sp. nov and ligated it to the double-enzyme digestion pET22b vector. After identifying the correct colony through enzyme digestion, we sent the corresponding plasmid to the company for Sanger sequencing.

To screen more efficiently PETase, we did site-direct mutation of SbPETase. PCR was performed using the identified correct pET22b-pelB-SbPETase plasmid as a template with the corresponding site-directed mutant primers. The PCR amplicons were digested with DpnI for 2 h, and the digested products were transformed into E. coli DH5α competent cells and incubation at 37℃ overnight. We constructed 3 mutants: L61T, W132H, and R259A. After identification of the correct mutant SbPETase plasmids, we co-transformed plasmids pET22b-PelB-SbPETase-mutants and pRSFDeut1-Kil into E. coli BL21(DE3) and purified the corresponding mutant protein.To screen more efficiently PETase, we did site-direct mutation of SbPETase. PCR was performed using the identified correct pET22b-pelB-SbPETase plasmid as a template with the corresponding site-directed mutant primers. The PCR amplicons were digested with DpnI for 2 h, and the digested products were transformed into E. coli DH5α competent cells and incubation at 37℃ overnight. We constructed 3 mutants: L61T, W132H, and R259A. After identification of the correct mutant SbPETase plasmids, we co-transformed plasmids pET22b-PelB-SbPETase-mutants and pRSFDeut1-Kil into E. coli BL21(DE3) and purified the corresponding mutant protein.

We tested the activity of mutant SbPETase towards PET and BHET materials using the in vitro platform we set up. In Figure 2A we could find that there were two mutants (SbPETaseW132H, SbPETaseR259A) with improved catalytic efficiency of degrading PET (Figure 2A). The activity of another mutant SbPETaseL61T was only improved towards degrading BHET. We then combined these three mutants (SbPETaseW132H, SbPETaseR259A, SbPETaseL61T) to generate three double mutants and one triple mutant. An in vitro activity assay showed that the triple mutant had the highest catalytic efficiency towards PET and BHET degradation (Figure 2B)