Assessment of IsPETase-Assisted Depolymerization of Terephthalate Aromatic Polyesters and the Effect of the Thioredoxin Fusion Domain

Abstract

Terephthalate polyesters such as poly(ethylene terephthalate) (PET) have been massively produced over the last few decades due to their attractive properties in multiple applications. However, due to their limited biodegradability, they have accumulated in landfills and oceans, posing an environmental threat. Enzymatic recycling technologies are predicted to generate long-term socioeconomic benefits. In the present work, we compared the IsPETase (from Ideonella sakaiensis 201-F6) activity on a series of polyesters, including poly(butylene) terephthalate (PBT), poly(hexamethylene) terephthalate (PHT) and Akestra™, with PET. The IsPETase showed remarkable activity toward PET (39% degradation of the original polyester) that was higher than that toward Akestra™ (0.13%), PBT (0.25%) and PHT (0.13%) after 72 h. Thus, based on experimental data and computational analysis, we report insights into IsPETase activity on a series of terephthalate-based polyesters. Aside from that, the fusion domain (Trx) effect in the production and activity of a recombinant Trx-IsPETase is reported.

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

“…Amorphous PET film was purchased from Goodfellow (product number ES30-FM-000145), RamaPET N180 from Indorama, and Akestra TM courtesy of Perstorp AB. The called powder polymers were prepared through a dissolutionprecipitation pretreatment as previously described (Wagner-Egea et al, 2021). The physical properties of the polymers used in this study are summarized in Table 1.…”

“…Protein unfolding was monitored by measuring the intrinsic fluorescence at emission wavelengths of 330 and 350 nm. (Ronkvist et al, 2009;Wagner-Egea et al, 2021). Triplicates, including negative control (reaction mixture without the enzyme), were incubated at 70 °C and 200 rpm for 235 h. Samples at various times were retrieved by diluting 200 µL of sample in 500 µL of DMSO, filtered with a 0.2 µm PES sterile membrane, and transferred to glass vials for further HPLC analyses.…”

“…Reaction products of the enzymatic degradation were analyzed by High-Performance Liquid Chromatography (HPLC) and Liquid Chromatography-Mass Spectrometry (LC-MS) with methods described previously (Wagner-Egea et al, 2021). The HPLC used is an Ultimate 3000 RS (Dionex) equipped with a UV/Vis detector (SPD-20A) and the LC-MS is an Ultimate 3000 RS HPLC-system (ThermoFisher Scientific) coupled with an LTQ Velos Pro Ion trap mass spectrom (ThermoFisher Scientific) using a heated electrospray ionization source (HESI-II).…”

“…The amorphous samples were heated from room temperature to 200 °C at the rate of 10 °C/min and PET powder was heated from room temperature to 300 °C at the rate of 10 °C/min under nitrogen with a purge rate of 50 ml min −1 . The crystallinity of PET powder was calculated according to previously reported method (Wagner-Egea et al, 2021).…”

“…Among the newly developed biobased polyesters, a partially biobased polymer Akestra TM (Perstorp AB) presents a high T g (90-110 °C) and transparency, which shows high potential in various applications, such as hot-filling food packaging, bottles, teapots, and others. Previously, enzymatic treatments of Akestra TM using Ideonella sakaiensis PETase (IsPETase) and a marine PET hydrolase (PET2) showed only 0.13% of depolymerization (Wagner-Egea et al, 2021) and no detectable depolymerization products (Wagner-Egea et al, 2022), respectively. Both enzymatic processes were performed at 37 °C (IsPETase) and 55 °C (PET2), temperatures significantly lower than the glass transition temperature of Akestra TM .…”