Highly Crystalline Post‐Consumer PET Waste Hydrolysis by Surface Displayed PETase Using a Bacterial Whole‐Cell Biocatalyst

Abstract

The enzymatic degradation of polyethylene terephthalate (PET) is a promising new approach for the environmentally friendly recycling of PET waste, but so far, low degradation rates paired with releatively high costs have limited the economic feasibility of this method. Here we present the construction of a new bacterial whole‐cell biocatalyst utilizing a new inverse autotranspoter based surface display of PETase in E. coli. The resulting catalyst allows an extremely easy production of large amounts of enzyme and a five times more effective degradation of PET at a lower temperature of 25 °C compared to free PETase at 30 °C. Additionally, we demonstrate how rhamnolipids, which are environmentally benign and can be cost‐effectively produced using strains of E. coli, can be used to amplify the hydrolysis rates of PET even further, presumably by acting as mediators between PETase and PET. Cells displaying PETase in combination with externally supplied rhamnolipids outperformed free PETase by a factor of 16, allowing the degradation of highly crystalline post‐consumer PET waste to an extent of 8 % within 3 days at room temperature.

Enzymatic Degradation of Polyethylene Terephthalate Plastics by Bacterial Curli Display PETase

“…Notably, a degradation efficiency reached 9.1% for highly crystalline PET (crystallinity > 20%) under the mild conditions at 30 °C used here, while a chemical method using more harsh conditions (100 vol % EtOH with 10 wt % NaOH at 50 °C) had PET degradation efficiency of 11.9% . There is still room for improvement of BIND-PETase compared with another study on enzymatic PET depolymerization (which reported 8% depolymerization at 25 °C in 3 days) . Collectively, our results demonstrated the applicability of the BIND-PETase biocatalyst in depolymerizing highly crystalline PET waste, which is promising for plastic recycling applications.…”

Enzyme cascade converting cyclohexanol into ε‐caprolactone coupled with NADPH recycling using surface displayed alcohol dehydrogenase and cyclohexanone monooxygenase on E. coli

“…This pH optimum is higher than the pH optima that have been determined for purified CHMOs, which were reported to be best active between pH 7 and 9 (Trudgill, 1990; Sheng et al ., 2001). Alterations in substrate specificities (Jose and von Schwichow, 2004) or turnover number (Schumacher et al ., 2012) by the surface display of enzymes have been reported before, as well as that surface displayed enzymes showed an altered pH optimum (Bussamara et al ., 2012; Gercke et al ., 2021). In addition, as shown by the example of cellulases, the same enzyme exhibited different pH optima when displayed on different host cells (Obeng et al ., 2018).…”

“…4A). The use of the YeeJ autotransporter translocation domain for the surface display of enzymes has been reported just recently (Gercke et al ., 2021). In consequence, a plasmid was constructed (pYeeJ‐ADH) encoding a YeeJ‐ADH autotransporter fusion protein.…”

“…The β‐barrel of the translocation unit serves as an anchor for the catalytically active enzyme. More recently, it has been shown that recombinant enzymes can be displayed by a so‐called inverse autotransporter domain as well on the surface of a Gram‐negative bacterium (Gercke et al ., 2021). Inverse autotransporters belong to secretion type Ve and have a different domain organization compared to classical autotransporters (Oberhettinger et al ., 2015).…”

“…This may be of consequence for the enzyme activity of the passenger, depending on the location of the active site or whether flexibility is required for e.g. substrate uptake at the C‐terminus or the N‐terminus (Gercke et al ., 2021). The whole biocatalyst obtained by autodisplay, independent of using a classical or an inverse autotransporter, can easily be separated from any kind of solution by centrifugation.…”