Engineering the thermal stability of a PET degrading enzyme

, Weigert Sebastian, Höcker Birte.

One of the most abundant plastics is polyethylene terephthalate (PET), a semi-crystalline polyester made from terephthalic acid and ethylene glycol. Its outstanding properties such as chemical resistance, lightweight and stability facilitate the use of the material in challenging applications like food and beverage packaging and for fibre production. But like for many other plastics these characteristics lead to the accumulation in the environment in the form of microplastic, which causes problems on many different levels. For PET-derived microplastic a new perspective arose with the discovery of PETase in Ideonella sakaiensis.(1) This enzyme is the key for the organism's ability to hydrolyse PET and use it as a nutrition source. It therefore features as the first known PET degrading enzyme with substantial activity at ambient temperatures. Although the enzymes moderate turnover rates fit its biological context, it limits its utilization for microplastic related applications like recycling and decontamination. One way to develop PETase into an enzyme with the desired properties is rational protein design. As a first step we focus on the thermal stability, which is often linked to kinetic stability of an enzyme. Here we used a fully automated algorithm for protein stabilization called PROSS (2) and yielded an increase in Tm of 8°C. We continued to combine this variant with an even more stable enzyme, designed by Cui et al. using their semi-automated clustering algorithm GRAPE, called DuraPETase.(3) Besides ranking the performance of the two algorithms, we are also looking for synergistic mutations from both algorithms to create a further improved PETase. These examples illustrate possible contributions of protein design to facilitate recycling and decontamination in the scope of microplastic. (1) Yoshida et al. (2016) DOI: 10.1126/science.aad6359 (2) Goldenzweig et al. (2016) DOI: 10.1016/j.molcel.2016.06.012 (3) Cui et al. (2019) bioRxiv DOI:

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