Comparison of the enzymatic depolymerization of polyethylene terephthalate and AkestraTM using Humicola insolens cutinase

Abstract

The enzymatic depolymerization of synthetic polyesters has become of great interest in recycling plastics. Most of the research in this area focuses on the depolymerization of polyethylene terephthalate (PET) due to its widespread use in various applications. However, the enzymatic activity on other commercial polyesters is less frequently investigated. Therefore, AkestraTM attracted our attention, which is a copolymer derived from PET with a partially biobased spirocyclic acetal structure. In this study, the activity of Humicola insolens cutinase (HiCut) on PET and AkestraTM films and powder was investigated. HiCut showed higher depolymerization activity on amorphous PET films than on Akestra™ films. However, an outstanding performance was achieved on AkestraTM powder, reaching 38% depolymerization in 235h, while only 12% for PET powder. These results are consistent with the dependence of the enzymes on the crystallinity of the polymer since Akestra™ is amorphous while the PET powder has 14% crystallinity. On the other hand, HiCut docking studies and molecular dynamic simulations (MD) suggested that the PET-derived mono (hydroxyethyl)terephthalate dimer (MHET)2 is a hydrolyzable ligand, producing terephthalic acid (TPA), while the Akestra™-derived TPA-spiroglycol ester is not, which is consistent with the depolymerization products determined experimentally. MD studies also suggest ligand-induced local conformational changes in the active site.

Marine PET Hydrolase (PET2): Assessment of Terephthalate- and Indole-Based Polyester Depolymerization

“…However, PET2 showed greater activity than IsPETase on PBT and PHT. Regarding Akestra, IsPETase showed low activity, while no activity was detected for PET2 (Table 1), which significatively contrasts with the remarkable activity of HiC cutinase from Humicola insolens [38].…”

“…However, PET2 showed greater activity than IsPETase on PBT and PHT. Regarding Akestra, IsPETase showed low activity, while no activity was detected for PET2 (Table 1), which significatively contrasts with the remarkable activity of HiC cutinase from Humicola insolens [38]. To further investigate the activity of PET2 and Trx-IsPETase against PET, initial reaction rates were obtained with incubation of a high enzyme/substrate ratio (1.12 mg/mg) for 4 h. The thioredoxin fusion domain (Trx) allowed a significative higher yield production of Trx-IsPETase than IsPETase and a positive effect on PET depolymerization [35].…”

Acinetobacter guillouiaestrain isolated from sludge capable of partially degrade polyethylene terephthalate: genomic and biochemical insights

“…The extracellular enzymatic extract from Stenotrophomonas maltophilia also yields MEHT as the major PET powder degradation product (Din et al, 2023). The fungal cutinase HiC (HiCut) and Rhizomucor miehei lipase (RmL) produced TPA as the dominant PET degradation product (Aristizábal-Lanza et al, 2022; Brackmann et al, 2023), while Candida antarctica lipase (CaL) produced mainly BHET (Brackmann et al, 2023). However, the product’s ratio profile could vary depending on factors such as incubation time, temperature and enzyme/polymer ratio, among others (Wagner-Egea et al, 2021).…”

From Bulk to Binding: Decoding the Entry of PET into Hydrolase Binding Pockets

“…Furthermore, the computational methods in the previous studies comprise mainly docking 3,35,36,46,47 in combination with short molecular dynamics (MD) simulations. 14,40,[48][49][50][51] However, the dynamics of the binding process, which is composed of the flexibility of the binding site itself and the PET as well as the process of PET entry into the active site, cannot be adequately described by these methods, yet they have been suggested to be important factors for PET complexation and thus degradation. 4,48,49,[52][53][54] To fill this knowledge gap, we performed thorough computational simulations in combination with experimental studies on two metagenome-derived thermophilic PET hydrolases, LCC (catalytic triad: S165, D210, H242) and PES-H1 (catalytic triad: S130, D176, H208).…”

“…Furthermore, the computational methods in the previous studies comprise mainly docking 3,35,36,46,47 in combination with short molecular dynamics (MD) simulations. 14,40,48–51 However, the dynamics of the binding process, which is composed of the flexibility of the binding site itself and the PET as well as the process of PET entry into the active site, cannot be adequately described by these methods, yet they have been suggested to be important factors for PET complexation and thus degradation. 4,48,49,52–54…”