Assessment of Four Engineered PET Degrading Enzymes Considering Large-Scale Industrial Applications

[Image: see text] In recent years, enzymatic recycling of the widely used polyester polyethylene terephthalate (PET) has become a complementary solution to current thermomechanical recycling for colored, opaque, and mixed PET. A large set of promising hydrolases that depolymerize PET have been found and enhanced by worldwide initiatives using various methods of protein engineering. Despite the achievements made in these works, it remains difficult to compare enzymes’ performance and their applicability to large-scale reactions due to a lack of homogeneity between the experimental protocols used. Here, we pave the way for a standardized enzymatic PET hydrolysis protocol using reaction conditions relevant for larger scale hydrolysis and apply these parameters to four recently reported PET hydrolases (LCC(ICCG), FAST-PETase, HotPETase, and PES-H1(L92F/Q94Y)). We show that FAST-PETase and HotPETase have intrinsic limitations that may not permit their application on larger reaction scales, mainly due to their relatively low depolymerization rates. With 80% PET depolymerization, PES-H1(L92F/Q94Y) may be a suitable candidate for industrial reaction scales upon further rounds of enzyme evolution. LCC(ICCG) outperforms the other enzymes, converting 98% of PET into the monomeric products terephthalic acid (TPA) and ethylene glycol (EG) in 24 h. In addition, we optimized the reaction conditions of LCC(ICCG) toward economic viability, reducing the required amount of enzyme by a factor of 3 and the temperature of the reaction from 72 to 68 °C. We anticipate our findings to advance enzymatic PET hydrolysis toward a coherent assessment of the enzymes and materialize feasibility at larger reaction scales.