Towards bio-upcycling of polyethylene terephthalate.

Over 359 million tons of plastics were produced worldwide in 2018, with significant growth expected in the near future, resulting in the global challenge of end-of-life management. The recent identification of enzymes that degrade plastics previously considered non-biodegradable opens up opportunities to steer the plastic recycling industry into the realm of biotechnology. Here, the sequential conversion of post-consumer polyethylene terephthalate (PET) into two types of bioplastics is presented: a medium chain-length polyhydroxyalkanoate (PHA) and a novel bio-based poly(amide urethane) (bio-PU). PET films are hydrolyzed by a thermostable polyester hydrolase yielding highly pure terephthalate and ethylene glycol. The obtained hydrolysate is used directly as a feedstock for a terephthalate-degrading Pseudomonas umsongensis GO16, also evolved to efficiently metabolize ethylene glycol, to produce PHA. The strain is further modified to secrete hydroxyalkanoyloxy-alkanoates (HAAs), which are used as monomers for the chemo-catalytic synthesis of bio-PU. In short, a novel value-chain for PET upcycling is shown that circumvents the costly purification of PET monomers, adding technological flexibility to the global challenge of end-of-life management of plastics.

Fast Turbidimetric Assay for Analyzing the Enzymatic Hydrolysis of Polyethylene Terephthalate Model Substrates.

Synthetic plastics such as polyethylene terephthalate (PET) can be cooperatively degraded by microbial polyester hydrolases and carboxylesterases, with the latter hydrolyzing the low-molecular-weight degradation intermediates. For the identification of PET-degrading enzymes, efficient and rapid screening assays are required. Here a novel turbidimetric method in a microplate format for the fast screening of enzyme activities against the PET model substrates with two ester bonds bis-(2-hydroxyethyl) terephthalate (BHET) and ethylene glycol bis-(p-methylbenzoate) (2PET) is reported. The carboxylesterase TfCa from Thermobifida fusca KW3 is used for validating the method. High correlation and regression coefficients between the experimental and fitted data confirm the accuracy and reproducibility of the method and its feasibility for analyzing the kinetics of the enzymatic hydrolysis of the PET model substrates. A comparison of the hydrolysis of BHET and 2PET by TfCa using a kinetic model for heterogeneous catalysis indicates that the enzyme preferentially hydrolyzes the less bulky molecule BHET. The high-throughput assay will facilitate the detection of novel enzymes for the biocatalytic modification or degradation of PET.

A dual enzyme system composed of a polyester hydrolase and a carboxylesterase enhances the biocatalytic degradation of polyethylene terephthalate films.

TfCut2 from Thermobifida fusca KW3 and the metagenome-derived LC-cutinase are bacterial polyester hydrolases capable of efficiently degrading polyethylene terephthalate (PET) films. Since the enzymatic PET hydrolysis is inhibited by the degradation intermediate mono-(2-hydroxyethyl) terephthalate (MHET), a dual enzyme system consisting of a polyester hydrolase and the immobilized carboxylesterase TfCa from Thermobifida fusca KW3 was employed for the hydrolysis of PET films at 60°C. HPLC analysis of the reaction products obtained after 24 h of hydrolysis showed an increased amount of soluble products with a lower proportion of MHET in the presence of the immobilized TfCa. The results indicated a continuous hydrolysis of the inhibitory MHET by the immobilized TfCa and demonstrated its advantage as a second biocatalyst in combination with a polyester hydrolase for an efficient degradation oft PET films. The dual enzyme system with LC-cutinase produced a 2.4-fold higher amount of degradation products compared to TfCut2 after a reaction time of 24 h confirming the superior activity of his polyester hydrolase against PET films.