Polyethylene terephthalate (PET), one of the most widely used plastics in the world, is currently causing serious environmental pollution. In recent years, more and more researchers have focused on the enzymatic degradation of PET, which is a more environmentally friendly degradation and recycling method than the traditional chemical and physical recycling methods. In this study, our team established an efficient secretion system of PET degradation enzyme SbPETase in E. coli BL21(DE3) using the signal peptide PelB and the colistin-releasing protein Kil, which not only improved the enzyme yield but also greatly simplified the purification process of PET degradation enzyme (Figure 1A). This provided a more convenient tool for further study of the enzyme. In addition, our team rationalized the PET hydrolase SbPETase from Schlegelella brevitalea sp. nov. and used the established secretion system to rapidly screen for triple mutants with significantly increased activity (Figure 1B).

The DNA sequence of SbPETase was inserted into the BamHI and XhoI sites of the pET22b vector (Figure 2A). The gene Kil was amplified by PCR from the plasmid containing the Kil gene and cloned into the NcoI and XhoI sites of the pRSFduet1 vector (Figure 2B).

We amplify SbPETase was amplified from the genome of S. brevitalea sp. nov (Figure3A.) and ligated it to the double-enzyme digestion pET22b vector, and We amplify Kil by PCR from the plasmid containing the Kil gene (Figure3B.), ligated to the double-enzyme digestion pRSFduet1 carrier.
To verify if the plasmid is correct, we digest plasmid pET22b-PelB-SbPETase with ApaLI and pRSFdeut-1-Kil with AseI (Figure 3C, E). We send the constructed recombinant plasmid to a sequencing company for sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region (Figure 3D, F.), and the plasmids were successfully constructed. So far, we have successfully obtained the recombinant plasmids.

We transferred the plasmid pET22b-pelB-SbPETase into E. coli BL21(DE3), expanded the culture in the LB medium and added IPTG to induce protein expression when the OD600 reached 0.4. After overnight induction and culture, we collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins. Next, we used nickel column (Ni-NTA) purification to purify the protein SbPETase.

In order to test if Kil signal peptide can improve the yield of SbPETase proteins in the culture medium, we also co-transformed the recombinant plasmids pET22b-PelB-SbPETase and pRSFDeut1-Kil into E. coli BL21(DE3), expanded the culture in the LB medium and added IPTG to induce protein expression when the OD600 reached 0.4. After overnight induction and culture, we purified the protein SbPETase we wanted and collected the data (Figure 5). In Figure5, when co_transformed pET22b-PelB-SbPETase and pRSFDeut1-Kil, the yield of protein SbPETase in the extracellular medium is higher than without it.

We set up an in vitro system and confirmed the ability to use the SbPETase to degrade PET materials. As shown in Figure 6, we demonstrated that SbPETase could degrade PET and BHET into MHET and a small quantity of TPA through HPLC (Figure 6A, B), the optimum conditions showed that at 30°C, pH 7.0 (BHET as substrate) or pH 8.0 (PET film as substrate), SbPETase showed the highest activity.We set up an in vitro system and confirmed the ability to use the SbPETase to degrade PET materials. As shown in Figure 6, we demonstrated that SbPETase could degrade PET and BHET into MHET and a small quantity of TPA through HPLC (Figure 6A, B), the optimum conditions showed that at 30°C, pH 7.0 (BHET as substrate) or pH 8.0 (PET film as substrate), SbPETase showed the highest activity.

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 7A we could find that there were two mutants (SbPETaseW132H, SbPETaseR259A) with improved catalytic efficiency of degrading PET (Figure 7A). 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 7B)