Introduction
Since plastics emerged in the 1950s, over 8.3 billion metric tons of plastic have accumulated in the planet’s ecosystems [Geyer et al., 2017], with PET being mainly responsible for this pollution [Ellis et al., 2021]. Clearly, an urgent need exists to develop both linear (valorisation-based) and circular (recycling-based) economies around PET degradation, to reduce carbon footprints created through inefficient PET degradation and production of PET from petrochemicals [Ru et al., 2020; Sarah and Gloria, 2021]. Degradation of PET by enzymatic means provides a ‘green’ route to breaking down solid plastic PET into pure terephthalic acid (TPA) using environmentally-friendly, low cost reactions [Kawai et al., 2019]. Thermostable cutinases (EC 3.1.1.74) and their structural homologs are enzymes that degrade aliphatic esters such as cutin (found upon leaf surfaces). Cutinases are now known to also hydrolyse ester bonds in PET chains, to release terephthalic acid (TPA), and various degradation intermediates (DIs) of PET such as oligoethylene terephthalate (OET) chains of varying lengths, bis-2-hydroxyethyl terephthalate (BHET), and mono-2-hydroxyethyl terephthalate (MHET) [Kawai et al., 2019]. Cutinases have also emerged as promising enzymes for hydrolysis of ester bonds in non-PET plastics such as polybutylene succinate, polyethylene furanoate, and polycaprolactone [Maurya et al., 2020].
The best enzymatic degradation reactions involving PET are carried out at (or near) PET’s glass transition temperature (~70 °C), which is the temperature at which chains become mobile and accessible to the active site(s) of thermophilic hydrolases [Kawai et al., 2020]. Of various thermophilic cutinases identified thus far, (i) metagenomically-derived leaf branch compost cutinase (LCC), (ii)Humicola insolens cutinase (HiC), and (iii) Thermobifida fusca cutinase (Tf C), are all capable of hydrolysing PET in the range of 60-70 °C [Kawai et al., 2020]. In the present study, we engineered Tf C through a detailed examination of residues likely to contact PET. We compared Tf C with other PET hydrolases such as the thermostable cutinase, LCC [Sulaiman et al., 2012], and the meso-stable hydrolase, PETase, from Ideonella sakaiensis[Yoshida et al., 2016; Fecker et al., 2018]. In terms of amino acid sequences, Tf C displays 57.4 %, and 51 %, similarity, respectively, with LCC and PETase. All of these enzymes host a conserved GXSXG motif, a conserved catalytic triad of Ser-His-Asp residues, and an alpha-beta hydrolase fold [Buchholz et al., 2022].
Degradation of PET is coarsely described as repeating cycles of five successive kinetic steps: (i) enzyme binding to solid PET, through general hydrophobic interactions; (ii) enzyme binding to PET chain backbones, through involvement of active site(s); (iii) rearrangements in the covalent bonds of PET; (iv) release of product [which could be OET, BHET, MHET, or TPA, as well as ethylene glycol (EG)]; and (v) enzyme dissociation from solid PET [Wei et al., 2022]. Enzymes such as LCC or Tf C are required to bind to PET through attractive forces facilitating formation of enzyme-substrate complexes. According to Sabatier’s principle, intermediate enzyme binding strength leads to most efficient degradation, since too low an affinity makes for weak enzyme-substrate complexes, as well as low rates of degradation, whereas too high an affinity makes for strong enzyme-substrate complexes, poor product/enzyme release, and poor turnover [Jensen et al., 2022]. Therefore, an intermediate binding affinity which is sufficient for enzyme-substrate complex formation is thought to be the best for high turnover. In search of a Tf C mutant/variant displaying higher turnover than Tf C, we describe below the obtaining of such mutants/variants through rational mutagenesis involving either (i) creation or reduction of space at the active site, or (ii) increase or decrease of hydrophobicity, at the active site, or vicinal to the active site.