Global plastic problem has been a widely discussed issue. After the invention of polyethylene terephthalate (PET) plastic bottles in 1973, the global PET plastic production has risen dramatically. PET bottles are known for their strength and for their durability. Unfortunately, the convenience of using PET bottles comes at a cost. More than 360 million tonnes of plastic waste is produced annually worldwide [1], and an estimated amount of 5.25 trillion pieces of plastic and microplastic are currently floating around the ocean [2]. By 2050, it is estimated that more plastic than fish will be filling up our oceans [3]. Our team, HK_GTC, notices the severity of plastic pollution, and is dedicated to solving the global problem of plastic pollution and arousing public awareness to this issue.
We believe that PET plastic is the main contributing factor of global plastic pollution. PET contributes 94% by weight in a plastic bottle [4], and it contributed to 20% of global plastic production in 2020 [5]. To ease the plastic pollution problem, our project focuses on completely depolymerizing PET plastics into its constituting monomers. In 2016, a species of PET-digesting bacteria, Ideonella sakaiensis, was discovered in a Japanese recycling plant located in Sakai. This bacteria was found to secrete PETase and MHETase. We see that a dual-enzyme system consisting of PETase and MHETase can digest PET in nature. Therefore we hypothesize that this dual-enzyme system will also be capable of digesting PET plastics in our project.
The ultimate goal of this project is to use a protein engineering approach to develop a dual-enzyme system in which two enzymes act synergistically to completely degrade PET into its constituting monomers. These monomers can be further synthesized back into PET and other useful products, allowing the plastic industry to develop more sustainably. We propose the usage of two enzymes, PETase and MHETase, to depolymerize PET. Using PETase, PET is first broken down into three monomers: bis(2-hydroxyethyl) terephthalic acid (BHET), mono(2-hydroxyethyl) terephthalic acid (MHET), and terephthalic acid (TPA). MHETase further catalyzes the breakdown of MHET into TPA and ethylene glycol (EG) (Figure 1).
Figure 1. The breakdown process of PETing usPETase and MHETase. Left: PETase depolymerizes PET into BHET, MHET and TPA. Right: MHETase depolymerizes MHET into TPA and EG.
In 2019, our team created two successful PETase mutants that can increase the enzymatic activity of PETase. The single-mutant S245I, and the double-mutant W159H/S245I proved to have a higher depolymerization activity as compared with the wild type. This year, we did some follow-up experiments to confirm if our PETase mutant S245I can successfully digest PET. We used a Scanning Electron Microscope (SEM) to observe the pitting of the digested PET film surface. The HPLC result shows the levels of the intermediate product of digestion, MHET, and the monomer, TPA. We showed that when only wild type PETase and the S245I PETase is present in the digestion process, MHET was detected, which suggests that PET is not completely depolymerized by PETase (Table 1). Therefore, we hypothesize the presence of MHETase in our enzyme system can increase the degradation rate of PET into its constituting monomers.
Figure 2. HPLC data of the products obtained after 96 hours of PET digestion at 30°C. The retention time of TPA and MHET HPLC standards were at 4.64 minutes and 5.17 minutes respectively. Left: PETase digestion using wild type PETase as the only enzyme. Right: PETase digestion using S245I PETase as the only enzyme.
We hypothesize that adding MHETase with PETase will synergize the PET depolymerization process. We propose to develop a dual-enzyme of PETase and MHETase system in forms of chimeric proteins and enzyme cocktails. For the chimeric proteins of PETase (including PETase mutants) and MHETase, we link the C-terminus of PETase to the N-terminus of MHETase using a 12 amino acid serine-glycine linker. We expect the efficiency of the degradation of PET into its final monomers, TPA and EG, will be increased. For the protein cocktails of PETase (including PETase mutants) and MHETase, we mix PETase with MHETase in a single reaction. We would also like to compare the depolymerization activities of the chimeric protein and the protein cocktail.
In the start of our project, we designed our new constructs, cut using the restriction enzyme and did PCR screening. Following that, we did protein induction to express the protein, and protein extraction and purification. We did Bradford protein assay to test the concentration of protein, and SDS-PAGE to confirm if our protein is expressed. Finally, we did PET film digestion and used HPLC and SEM equipment borrowed from HKU to analyse the results of the experiment (See Figure 3).
References [1]: “EU plastics production and demand first estimates for 2020” [2]: ? [3]: The New Plastics Economy, Rethinking The Future of Plastics (Rep.). (2016). Geneva, Switzerland: The World Economic Forum. [4]: [5]: [6]: Austin, H. P., Allen, M. D. et al. (2018). Characterization and engineering of a plastic-degrading aromatic polyesterase. Proceedings of the National Academy of Sciences, 115(19). doi:10.1073/pnas.1718804115 [7]: Yoshida, S., Hiraga, K. et al. (2016). A bacterium that degrades and assimilates poly(ethylene terephthalate). Science,351(6278), 1196-1199. doi:10.1126/science.aad6359 [8]: Knott, B. C., Erickson, E. et al. (2020). Characterization and engineering of a two-enzyme system for plastics depolymerization, pnas.org. doi:10.1073/pnas.2006753117 [9]: Han, X., Liu, W. et al. (2017). Structural insight into catalytic mechanism of PET hydrolase, nature communications. DOI:10.1038/s41467-017-02255-z [10]: Joo, S., Cho, I. J. et al. (2018). Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation. Nature Communications, 9(1). doi:10.1038/s41467-018-02881-
