Plastics are widely used around the world because of their insulation, corrosion resistance, and low price. In 2016, plastic production reached 335 million tons per year, with Europe alone producing 60 million tons. Plastic production is expected to double in the next 20 years. As a result, a huge amount of plastic waste has accumulated in our environment. As of 2015, about 6,300 tons of plastic waste was generated, of which about 9% was recycled, 12% was incinerated, and 79% was deposited in landfills or the natural environment (Figure 1).
As one of the most widely used plastics in the world, polyethylene terephthalate (PET) has caused serious pollution to the environment. There are certain ways to degrade PET, however, the chemical degradation of PET needs to involve hydrolysis, thermal cracking such as strong acid and alkali, high temperature, and high pressure, then causing energy consumption and secondary pollution. PET can be recycled in various ways after being used, but the overall recycling rate is less than half, and the amount that can be reused after recycling is limited. Therefore, compared with chemical and physical recycling, enzymatic degradation of PET, as a way of biodegradation, is a more environmentally friendly degradation and recycling method.

Biological PET recycling is highly energy-intensive and the quality of the plastic decreases with each recycling cycle. Enzymes, on the other hand, only require an aqueous environment and a temperature of 65 to 70 degrees Celsius for their work. Another plus is the fact that they break down the PET into its components terephthalic acid and ethylene glycol, which can then be reused to produce new PET—resulting in a closed cycle. So far, however, biological PET recycling has only been tested by a pilot plant in France.

In this study, a high-efficiency secretion system of PET-degrading enzymes was established in Escherichia coli BL21(DE3) by 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-degrading enzymes. It provides a more convenient tool for the further study of the enzyme. In addition, we also rationally designed the PET hydrolase SbPETase from Schlegelella brevitalea sp. nov. and used the established secretion system to rapidly screen the triple mutants with significantly improved activity (Figure 2).

To construct a high-efficiency secretion system in E. coli, we developed a platform that combines signal peptide PelB (pectate lyase B signal peptide) and colicin release protein Kil. In this project, extracellular PETase was achieved by E. coli BL21(DE3) using a sec-dependent translocation signal peptide, pelB, for secretion, and the signal peptide is removed by a signal peptidase.
First, the gene SbPETase was amplified from the genome of S. brevitalea sp. nov. by PCR and cloned into the pET22b vector. The gene Kil was amplified by PCR and cloned into the pRSFduet1 vector.
Next, we extracted plasmids pET22b and pRSFduet1 from DH5α, linked the target gene and the vector into complete plasmids with homologous recombinant enzymes, and transferred the recombinant plasmids into DH5α. 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, and the plasmids were successfully constructed.
Then, we purified the SbPETase protein through Ni-NTA and set up an in vitro system to confirm if SbPETase can degrade PET and BHET materials.
Finally, after identifying that SbPETase protein works well in degrading PET and BHET, we performed the following site-directed mutagenesis: L61T, W132H, and R259A. All the SbPETase mutant proteins were obtained from the cultured medium directly and an in vitro activity assay was performed, which generated two mutants (SbPETaseW132H, SbPETaseR259A) with improved catalytic efficiency of degrading PET. 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 toward PET and BHET degradation.

1. Successfully construct pET22b-PelB-SbPETase and pRSFDeut1-Kil.
2. Expressed and purified SbPETase protein.
3. The SbPETase protein can degrade PET and BHET materials.
4. Detect the activity of SbPETase mutant proteins in degrading PET and BHET.