Engineering and evaluation of thermostable IsPETase variants for PET degradation

Abstract

Polyethylene terephthalate (PET) is a mass‐produced petroleum‐based synthetic polymer. Enzymatic PET degradation using, for example, Ideonella sakaiensis PETase (IsPETase) can be a more environmentally friendly and energy‐saving alternative to the chemical recycling of PET. However, IsPETase is a mesophilic enzyme with an optimal reaction temperature lower than the glass transition temperature (Tg) of PET, where the amorphous polymers can be readily accessed for enzymatic breakdown. In this study, we used error‐prone PCR to generate a mutant library based on a thermostable triple mutant (TM) of IsPETase. The library was screened against the commercially available polyester‐polyurethane Impranil DLN W 50 for more thermostable IsPETase variants, yielding four variants with higher melting points. The most promising IsPETaseTMK95N/F201I variant had a 5.0°C higher melting point than IsPETaseTM. Although this variant showed a slightly lower activity on PET at lower incubation temperatures, its increased thermostability makes it a more active PET hydrolase at higher reaction temperatures up to 60°C. Several other variants were compared and combined with selected previously published IsPETase mutants in terms of thermostability and hydrolytic activity against PET nanoparticles and amorphous PET films. Our findings indicate that thermostability is one of the most important characteristics of an effective PET hydrolase.

Molecular and Biochemical Differences of the Tandem and Cold-Adapted PET Hydrolases Ple628 and Ple629, Isolated From a Marine Microbial Consortium

“…This is an indication that the main source of T release was the hydrolysis of the secondary degradation from other oligomers, including MHET itself. Is PETase degrades MHET but at a very slow rate, and this was observed for other Is PETase variants as well ( Brott et al, 2021 ).…”

Multiple Substrate Binding Mode-Guided Engineering of a Thermophilic PET Hydrolase

“…This dependence can be significantly mitigated by replacing the Ca 2+ binding residues by a disulfide bridge to generate variants that are thermostable in the absence of Ca 2+ . 5 , 19 , 11 , 17 , 20 − 22 Accordingly, we generated an R204C/S250C variant of PES-H1 and confirmed disulfide bond formation using Ellman’s reagent ( Figure S7 ). The T m of this variant was increased by 6.4 °C ( Figure 3 L, Table S5 ).…”

“… 13 , 16 − 18 Engineering of residues in the Ca 2+ binding sites has proven to be a useful approach for increasing the T m of several PET hydrolases by up to 26 °C. 5 , 11 , 17 , 19 − 22 Introduction of a disulfide bridge at this site (D238C/S283C) markedly increased the melting point ( T m ) of LCC from 84.7 to 94.5 °C. In a recent study, LCC ICCG was further engineered to obtain an A59K/V63I/N248P variant with a T m of 98.9 °C.…”

Biocatalysis as Key to Sustainable Industrial Chemistry

“…Therefore, the engineering of thermostable PETase variants is important for effective PET hydrolysis. [47]…”

“…This will improve the enzymatic degradation process and avoid any additional pre‐treatment. Therefore, the engineering of thermostable PETase variants is important for effective PET hydrolysis [47] …”