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The 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 developing solutions to solve the global problem of plastic pollution and arousing public awareness of this issue.
We believe that PET plastic is the main contributing factor of global plastic pollution. PET contributes 93% by weight in a plastic bottle [4]. It also contributes to 20% of global plastic production in 2020 [5][6].
In 2016, a species of PET-digesting bacterium, Ideonella sakaiensis, was discovered in a Japanese recycling plant located in Sakai. This bacterium was found to secrete PETase and MHETase, in which PETase is capable of hydrolysing PET into reaction intermediates, and MHETase capable of digesting the reaction intermediates into the constituting monomers of PET [7]. We see that nature is evolving to depolymerize PET plastics with a dual-enzyme system. Therefore we hypothesized that this dual-enzyme system is also capable of degrading PET plastics in our project.
Two enzymes, PETase and MHETase, are used 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 PET using PETase 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 WT PETase. This year, we perform PET film digestion and High Performance Liquid Chromatography (HPLC) of PET digestion eluent to study the effect of PETase and S245I on PET degradation. This is to confirm the performance of our engineered mutant. Both the Scanning Electron Microscope (SEM) images and quantification of reaction products by HPLC indicate that our engineered mutant, S245I outperforms the wild type PETase, as seen by the increased surface erosion of PET film and the increased release of monomeric products. HPLC profiles of products released from PET film digestions reveal considerable amounts of intermediate product, MHET, suggesting that PET cannot be completely depolymerized by PETase alone (Figure 2). Thus, we hypothesized that the presence of a second enzyme, MHETase, in the PET depolymerization system synergizes 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: PET film digestion using wild type PETase as the only enzyme. Right: PET film digestion using S245I PETase as the only enzyme.
In our project, we synergize the PET depolymerization process by adding MHETase with PETase. A dual-enzyme system of PETase and MHETase is developed in the form of either as chimeras or enzyme cocktails. For the chimeric proteins of PETase (including PETase mutant, S245I) and MHETase, we link the C-terminus of PETase to the N-terminus of MHETase using a 12 amino acid serine-glycine linker. For the enzyme cocktails of PETase (including a PETase mutant, S245I) and MHETase, PETase is mixed with MHETase in a single reaction. We compare the depolymerization activities of the chimeric protein and the enzyme cocktail.
The ultimate goal of our 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, reducing the reliance on fossil fuels and allowing the plastic industry to develop more sustainably. We also hope that by combining PETase with a second enzyme, MHETase, the PET degradation process will be fast enough to handle tonnes of PET plastics wastes drowning the planet.
[1]: “EU plastics production and demand first estimates for 2020”, https://www.plasticseurope.org/en/newsroom/news/eu-plastics-production-and-demand-first-estimates-2020 [2]: National Geographic Society. (2019, February 22). Ocean Trash: 5.25 Trillion Pieces and Counting, but Big Questions Remain. https://www.nationalgeographic.org/article/ocean-trash-525-trillion-pieces-and-counting-big-questions-remain/ [3]: The New Plastics Economy, Rethinking The Future of Plastics (Rep.). (2016). Geneva, Switzerland: The World Economic Forum. [4]: PET & HDPE. (2020, November 23). New Life Plastics. https://www.nlplastics.com.hk/pet-hdpe/ [5]: Statista. (2021, September 10). Global plastic production 1950–2020. https://www.statista.com/statistics/282732/global-production-of-plastics-since-1950/ [6]: Statista. (2021a, January 27). Global polyethylene terephthalate production 2014–2020. https://www.statista.com/statistics/650191/global-polyethylene-terephthalate-production-outlook/ [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
