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.