You get what you screen for: a benchmark analysis of leaf branch compost cutinase variants for polyethylene terephthalate (PET) degradation

Enzymatic recycling of polyethylene terephthalate (PET) is an innovative and environmentally friendly way to promote a circular plastics economy and reduce the use of petroleum-based plastic monomers. Despite, or rather because of the large number of natural and engineered enzymes discovered in recent years, there are substantial difficulties in comparing PET degradation rates between studies. The present study therefore gathered knowledge about potential pitfalls and the question of how benchmarking for enzymatic PET degradation should be performed. Since there is no progress to establish a well-defined standard substrate as well as uniform reaction conditions for comparative studies of PET hydrolases, we propose to carry a well-characterized enzyme as a reference in all studies. In this way, variants of the leaf branch compost cutinase (LCC) were benchmarked in miniaturized stirred tank reactors. Different mutants with amino acid exchanges inspired by natural or engineered PET hydrolases were compared. The best enzyme resulted from a combination of mutations contained in the ICCG variant of the LCC and the so-called DuraPETase. The product release rate of the resulting variant ICCG(DAQI) was 27% higher than the reference enzyme ICCG. Although the relative performance of the different enzymes seemed to be independent of the PET material used (bottles, textile fibres), the absolute degradation rates were strongly influenced by the substrate properties. In particular, compared to bottle PET, post-industrial textile fibres were efficiently degraded releasing 40 mg terephthalic acid per hour and mg(enzyme), resulting in complete degradation within less than 24 hours.