Insights into the effect of substrate crystallinity on the enzymatic degradation of Poly(ethylene terephthalate)

Plastic pollution has become a global environmental threat, affecting both aquatic and terrestrial ecosystems. Despite these concerns, the demand for plastics continues to rise, leading to a substantial production of 390 Mt in 2021. A key contributor to plastic pollution is Poly(ethylene terephthalate) (PET), a semi-crystalline plastic polyester material with a global production volume of 83 Mt/year. Currently, less than 10% of all PET is recycled. This recycling, by conventional methods, does furthermore results in lower quality products. Hence, the development of novel recycling technologies is essential for establishing a truly circular economy. Recently, it was demonstrated that certain enzymes may catalyze the degradation of PET. As enzymatic recycling of PET enables the synthesis of new high-quality PET, it has the potential for the establishment of a circular economy of PET. The enzymatic degradation rate is, however, strongly influenced by the properties of PET, notably the degree of crystallinity (X_C). As most post-consumer PET products (Plastic bottles and textiles etc.) have an X_C > 20% a pretreatment step is required for efficient biorecycling of PET. This pretreatment is however very energy-demanding, thus lowering the sustainable potential of biorecycling.

This Ph.D. thesis aimed to study how substrate-related properties affected the enzymatic degradation of PET. For this purpose, we developed a standardized methodology, for controlling the X_C. This method was based on thermal-induced crystallization of commercially available amorphous PET, via annealing at 115 ºC. This substrate was subsequently used to study how several benchmark PET hydrolases were affected by increasing X_C. The initial degradation of PET, denoted as the lag phase, did not result in any formation of soluble products. Once the lag phase was surpassed, the concentration of soluble products, resulting from the enzymatic treatment, was released at a constant rate, denoted as the steady-state rate. The steady state reaction rate was also heavily affected by the X_C. This negative effect becomes particularly profound once X_C exceeds a certain threshold, ~20%. This threshold was, however, affected by several factors, including reaction temperature, extent of reaction, and the enzyme catalyzing the reaction. We found that LCC, LCC_ICCG, and DuraPETase were more prone to increasing levels of X_C compared to HiC, TfC, and PHL7. This increased tolerance was caused by a presumable broader substrate specificity, leading to higher rates and maximal degradation yields at higher X_C. By studying the lag phase during enzymatic treatment we found that the duration of this phenomenon was heavily prolonged at increasing X_C (up to 5 days at X_C > 20%), and lower steady-state rates. We ascribed the lag phase to an initial endo-type degradation pattern, which would not yield any soluble products. This mechanism was consolidated by studying the proton release during this initial stage, which confirmed hydrolysis by the enzyme during the lag phase.

We furthermore studied the effect of the glass transition temperature (T_g) on the enzyme hydrolysis rate. This was done by lowering the T_g of a PET material from 75 ºC to 60ºC by soaking it in water. By assaying these disks at 69ºC (between the T_g of the substrates) we found that the reaction rate was unaffected by the lower T_g.