Efficient depolymerization of polyethylene terephthalate (PET) and polyethylene furanoate by engineered PET hydrolase Cut190

• Published in: AMB Express 2022-10, Vol.12 (1), p.134-134, Article 134
• Main Authors: Kawai, Fusako, Furushima, Yoshitomo, Mochizuki, Norihiro, Muraki, Naoki, Yamashita, Mitsuaki, Iida, Akira, Mamoto, Rie, Tosha, Takehiko, Iizuka, Ryo, Kitajima, Sakihito
• Format: Article
• Language: English
• Subjects: Binding; Biomedical and Life Sciences; Biotechnology; Bottles; Chains (polymeric); Chlorides; Chromatography; Cleavage; Cut190 variant; Cutinase; Degradability; Degradation; Degradation products; Depolymerization; Erosion mechanisms; High performance liquid chromatography; Hydrolase; Hydrolysis; Life Sciences; Liquid chromatography; Microbial Genetics and Genomics; Microbiology; Micronization; Milling; Original; Original Article; Packages; PET bottle; PET hydrolase; PET package; pH effects; Plastic debris; Polyethylene terephthalate; Polymers; Recycling; Surface area; Thermal stability
• ISSN: 2191-0855; 2191-0855
• DOI: 10.1186/s13568-022-01474-y

The enzymatic recycling of polyethylene terephthalate (PET) can be a promising approach to tackle the problem of plastic waste. The thermostability and activity of PET-hydrolyzing enzymes are still insufficient for practical application. Pretreatment of PET waste is needed for bio-recycling. Here, we analyzed the degradation of PET films, packages, and bottles using the newly engineered cutinase Cut190. Using gel permeation chromatography and high-performance liquid chromatography, the degradation of PET films by the Cut190 variant was shown to proceed via a repeating two-step hydrolysis process; initial endo-type scission of a surface polymer chain, followed by exo-type hydrolysis to produce mono/bis(2-hydroxyethyl) terephthalate and terephthalate from the ends of fragmented polymer molecules. Amorphous PET powders were degraded more than twofold higher than amorphous PET film with the same weight. Moreover, homogenization of post-consumer PET products, such as packages and bottles, increased their degradability, indicating the importance of surface area for the enzymatic hydrolysis of PET. In addition, it was required to maintain an alkaline pH to enable continuous enzymatic hydrolysis, by increasing the buffer concentration (HEPES, pH 9.0) depending on the level of the acidic products formed. The cationic surfactant dodecyltrimethylammonium chloride promoted PET degradation via adsorption on the PET surface and binding to the anionic surface of the Cut190 variant. The Cut190 variant also hydrolyzed polyethylene furanoate. Using the best performing Cut190 variant (L136F/Q138A/S226P/R228S/D250C-E296C/Q123H/N202H/K305del/L306del/N307del) and amorphous PET powders, more than 90 mM degradation products were obtained in 3 days and approximately 80 mM in 1 day. Graphical Abstract Key points The increased surface area of PET promotes its hydrolysis by Cut190, supporting the surface erosion mechanism by a two-step process (endo-type scission of a polymer chain and exo-type hydrolysis of depolymerized fragments). Dodecyltrimethylammonium chloride functioned as a binding module between Cut190 and PET surface, showing the higher hydrolysis rate at 65 °C than at 70 °C in the absence of the detergent. The best performing Cut190 variant produced more than 90 mM degradation products at 63 °C in 3 days and approximately 80 mM at 65 °C in one day, using amorphous PET powders. Additionally, polyethylene furanoate was highly hydrolyzed at 63 °C in 3 days by the Cut190 vari