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\begin{document}

\title{Rational mutagenesis of Thermobifida fusca cutinase to modulate the
enzymatic degradation of polyethylene terephthalate}



\author[1]{Arpita Mrigwani}%
\author[1]{Madhav Pitaliya}%
\author[1]{Harman Kaur}%
\author[1]{Bhishem Thakur}%
\author[1]{Purnananda Guptasarma}%
\affil[1]{Indian Institute of Science Education and Research Mohali}%


\vspace{-1em}



  
  \date{May 27, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
\emph{Thermobifida fusca} cutinase ( \emph{Tf}C \emph{)} is a
carboxylesterase (CE) that degrades the environmental pollutant,
polyethylene terephthalate (PET). \emph{Tf}C also acts upon PET's
degradation intermediates (DIs), such as oligoethylene terephthalate
(OET), and bis-/mono-hydroxyethyl terephthalate (BHET/MHET), to convert
these into terephthalic acid (TPA), the terminal product of PET
degradation. We examined \emph{Tf}C's surface, compared it to that of
other CEs, and performed molecular docking and MD simulations with an
OET, 2HE-(MHET) \textsubscript{3}, to understand interactions between
\emph{Tf}C's surface and the OET, at \emph{Tf}C's active site as well as
vicinal regions. We mutated 17 residues on \emph{Tf}C's surface, mostly
individually, but sometimes using pairs of mutations, to see how these
modulate \emph{Tf}C's activity. Most mutants/variants showed a decrease
in activity against solid PET. Some killed activity completely. However,
three mutations (L90F, F209I and F249R), made using a background
mutation (G62A) already reported to improve activity by almost
\textasciitilde{}2.0-fold, yielded increases in activity that were
between \textasciitilde{}1.3- and \textasciitilde{}2.0-fold higher than
that of G62A \emph{Tf}C (which we found to display a
\textasciitilde{}1.7-fold increase in activity over \emph{Tf}C, in our
own experiments). \emph{Tf}C variants, G62A/F249R, and G62A/F209I,
exhibit the highest activities yet observed in any \emph{Tf}C
mutants/variants, against PET, and BHET, respectively.%
\end{abstract}%



\sloppy


\textbf{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 (\textasciitilde{}70
\selectlanguage{ngerman}°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)\emph{Humicola insolens} cutinase (HiC), and (iii)
\emph{Thermobifida fusca} cutinase (\emph{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 \emph{Tf} C through a detailed
examination of residues likely to contact PET. We compared \emph{Tf} C
with other PET hydrolases such as the thermostable cutinase, LCC
{[}Sulaiman et al., 2012{]}, and the meso-stable hydrolase, PETase, from
\emph{Ideonella sakaiensis}{[}Yoshida et al., 2016; Fecker et al.,
2018{]}. In terms of amino acid sequences, \emph{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 \emph{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 \emph{Tf} C mutant/variant displaying higher
turnover than \emph{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.

\textbf{2. Materials and methods:}

\emph{2.1 Bioinformatics studies} : Structural comparisons of \emph{Tf}
C with LCC and PETase were done using PYMOL (Schrödinger). Molecular
docking and Molecular Dynamics simulation studies, respectively, were
performed GLIDE (Schrödinger) and Desmond (Schrödinger), employing
parameters described previously {[}Mrigwani et al., 2022{]}. Docking was
performed using \emph{Tf} C (PDB ID: 4CG1) and
2HE-(MHET)\textsubscript{3}, containing three terephthalate moieties.
Structural alignments were done using the Tm align web server {[}Zhang
and Skolnick, 2005{]}.

\emph{2.2 Cloning, expression and protein purification of wild type Tf}
C\emph{and its mutants} : The gene encoding \emph{Tf} C was amplified
from the genomic DNA of \emph{Thermobifida fusca} (MTCC No.
1754\textsuperscript{T}) and cloned between Bam HI and Hind III
restriction sites in pQE-30 (Qiagen) for expression in fusion with an
N-terminal 6xHis tag. \emph{Tf} C variants/mutants were created through
site directed mutagenesis of this gene through splicing by overlap
extension PCR (SOE-PCR), prior to cloning into the same vector, between
the same sites. All clones were transformed into XL1-Blue for
constitutive expression. Transformed XL1-Blue cells (grown at 37 °C for
9 h at 220 rpm) were harvested and disrupted. Cell debris was separated
through centrifugation at 12,000 rpm for 1 h, and lysates subjected to
Ni-NTA (IMAC) chromatography. The second step of purification involved
size exclusion chromatography (SEC) on a GE Superdex-75 Increase 10/300
GL column, run on a GE AKTA Purifier-10 workstation, using 25 mM sodium
dihydrogen phosphate buffer of pH 8.0.

\emph{2.3 Circular dichroism (CD) studies} : An MOS-500 CD spectrometer
(BioLogic, France) was used to examine secondary structure in enzymes,
using concentrations of 0.2 mg/ml, and a quartz cuvette of 2 mm path
length. Mean Residue Ellipticity was calculated as MRE= (\selectlanguage{greek}θ\selectlanguage{ngerman}×mean residue
weight×100)/(1000×concentration in mg/ml×pathlength in cm); where, \selectlanguage{greek}θ \selectlanguage{english}is
the raw ellipticity. For chemical and thermal kinetics, enzyme was
incubated at various guanidium hydrochloride (Gdm.HCl) concentrations
and temperatures for 2 h, with monitoring of CD spectral intensity at
222 nm. Folded fractions were determined and used to determine rates of
unfolding and thermodynamic parameters of stability.

\emph{2.4 Differential Scanning Calorimetry (DSC) studies} : Using
VP-DSC (Microcal), thermal histories were created through repeated
heating/cooling (10-15 up/down scans) at 90 \selectlanguage{ngerman}°C/hr (upscan; 20-90°C) and
60 °C/hr (downscan; 90-20 °C). Then, buffer in the sample cell was
replaced by 0.5 mg/ml. One cycle of up/down scans was performed to
determine enzyme melting temperature, refolding ability and enthalpy
associated with unfolding, with raw data fitted using a non-2-state
cursor init model (Microcal).

\emph{2.5 PET-binding and activity against PET/BHET} : Prior to
binding/activity assays, circular discs of commercial PET film
(Goodfellow, Product code: GF25214475) were washed with 1\% SDS, water
and ethanol for 30 min, and air dried. For binding assays, 2 \selectlanguage{greek}µ\selectlanguage{english}M enzyme
was incubated with PET for 40 h at 60 \selectlanguage{ngerman}°C. Films were washed with buffer.
Enzyme fractions in solution, or bound to PET (extracted through boiling
with SDS-PAGE sample loading buffer) were determined through
electrophoresis, and densitometry using Image lab software (Bio-Rad).
For activity assays, films were incubated with 2 \selectlanguage{greek}µ\selectlanguage{english}M enzyme(s) at 60 \selectlanguage{ngerman}°C
for 50 h in phosphate buffer, pH 8.0. Ability to hydrolyse BHET (Sigma
Aldrich, Product Code 465151) was determined by incubating 250 \selectlanguage{greek}µ\selectlanguage{english}M BHET
with 1 \selectlanguage{greek}µ\selectlanguage{english}M enzyme at 60 \selectlanguage{ngerman}°C, for either 4 h, or 12 h. Reverse-phase HPLC
was used to quantify degraded species on a Shimadzu HPLC workstation
using previously described methods {[}Yoshida et al., 2016{]}.

\textbf{3. Results and discussion:}

\emph{3.1 Bioinformatics-based determination of Tf} C \emph{residues
crucial for PET binding}

Figure 1A shows a \emph{Tf} C-2HE-(MHET)\textsubscript{3} complex.
Hydrophobic, electrostatic, hydrogen bonding and van der Waals
interactions between \emph{Tf} C and 2HE-(MHET)\textsubscript{3} in this
docked structure are illustrated in Figure 1B. Residues observed to
participate in interactions with 2HE-(MHET)\textsubscript{3} were
confirmed using Molecular Dynamics (MD) simulations. The MD simulation
interaction diagram (SID) is shown in Figure 1C, plotting
fraction/extent of interactions during 75 ns simulations. With PETase
(PDB ID: 6ANE) {[}Fecker et al., 2018{]}, and LCC (PDB ID: 4EB0)
{[}Sulaiman et al., 2012{]}, respectively, \emph{Tf} C (PDB ID: 4CG1)
{[}Roth et al., 2014{]} shows RMSD alignment values of
\textasciitilde{}1.26 Å, and \textasciitilde{}1.04 Å. We
compared\emph{Tf} C's structure with those of PETase, and LCC, in
respect of analogous residues (in and around the active site) that make
contact with 2HE-(MHET)\textsubscript{3} in docking and MD studies, to
decide upon mutations of \emph{Tf} C residues into residues present at
structurally-analogous locations in PETase, or LCC, with a view to
examining the effect of such mutations upon \emph{Tf} C's activity.
Notably, this approach is analogous to recent work in which residues in
PETase were replaced by residues in \emph{Tf} C {[}Fecker et al.,
2018{]}\emph{.} Our objectives were (i) to obtain mutants/variants
superior to \emph{Tf} C, and (ii) to analyse reductions or improvements
in activity, in light of mechanistic and catalytic insights.

The mutants made are listed below. \textbf{G62A} : G62 is located in the
PET-binding groove (Figure 1A). Mutation G62A reduces inhibition
of\emph{Tf} C by MHET and increases activity by
\textasciitilde{}2.0-fold {[}Wei et al., 2016{]}, ostensibly through
disruption of steric clashes preventing interactions (required for
release of product after hydrolysis) of residue G59's backbone nitrogen
atom with a carbonyl oxygen in PET. We used G62A as a control, and also
as a background (base) mutation for other mutations. \textbf{L90A, L90F}
(F125 in LCC, otherwise conserved): L90 is distal to \emph{Tf} C's PET
binding groove (Figure 1A and Supporting Information Figure S1). L90A
shows a 5-fold increase in activity upon cutin, over \emph{Tf} C {[}Dong
et al., 2020{]}. Mutations L90A and L90F were made to examine effects on
activity. \textbf{H129W} (W132 in PETase {[}Fecker et al., 2018{]}):
PETase is more catalytically active than \emph{Tf} C between 30 and 40
°C. To mimic PETase's catalytic environment around S130 in \emph{Tf} C,
mutation H129W was performed (Supporting Information Figure S2A), in
place of H129A, already reported to reduce PET hydrolysis by 80\%
{[}Fecker et al., 2018{]}. \textbf{W155F} (W conserved in all PET
hydrolases), \textbf{H184A, and H184S} (S187 in PETase): These mutations
were made with a view to create more space in \emph{Tf} C's
OET/PET-binding cleft towards more efficient PET binding (Supporting
Information Figure S2A). H184 was made to allow a nearby tryptophan to
wobble between open and closed conformers, as in PETase {[}Han et al.,
2017{]}. \textbf{A173C/A210C} (C174/C210 in PETase)
and\textbf{A173C/A206C} : These mutations were jointly made to create a
disulphide bond below \emph{Tf} C's catalytic site, mimicking such a
bond in PETase {[}Han et al., 2017{]}, to study its role in catalysis
(Supporting Information Figure S2B). \selectlanguage{greek}\textbf{ΔV164}\selectlanguage{english} : V164 exists in a
loop following a beta strand leading to D176 (a member of \emph{Tf} C's
catalytic triad). This was deleted to bring D176 closer to the other two
residues in the triad, to promote better catalysis by minimizing
distance between D176 and H208 (Supporting Information Figure
S3).\textbf{F209I} (S211 in PETase), \textbf{F249R} , and \textbf{F249A}
(not conserved): These residues lie either near or somewhat away
from\emph{Tf} C's active site. Mutations were made to reduce surface
hydrophobicity without compromising catalysis (Supporting Information
Figure S1). \textbf{General} : L90, F209 and F249 (Figure 1A), were
mutated to either increase hydrophobicity (e.g., L90F) or reduce it
(e.g., F209I, F249R). Other residues shown in red (Figures 1B and 1C)
were mutated to gain mechanistic insights.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1:} Molecular docking and molecular dynamics simulations
of \emph{Tf} C and 2HE-(MHET)\textsubscript{3}. (A) Surface
representation of \emph{Tf} C docked with 2HE-(MHET)\textsubscript{3},
showing catalytic triad residues in orange and potential regions
selected for altering PET binding affinity shown in pink. Residues
in\emph{Tf} C participating in interaction with
2HE-(MHET)\textsubscript{3}obtained from (B) Molecular docking and (C)
Molecular dynamics simulations performed for 75 ns.

\emph{3.2 Activity assessments of various Tf} C \emph{mutants upon PET
and BHET}

Figure 2A compares activities of mutants and \emph{Tf} C against
commercially-sourced PET (film). G62A was \textasciitilde{}1.7-fold more
active than \emph{Tf} C, corroborating its reported
\textasciitilde{}2.0-fold higher activity {[}Wei et al., 2016{]}. D12L,
and E47F, designed to enhance surface hydrophobicity at regions distal
to the substrate-binding site, showed activity levels similar to that
of\emph{Tf} C. W155F, H184S, H184A, and F209S, designed to create more
space at or near PET binding sites, and H129W, designed to increase
hydrophobicity (in imitation of PETase), showed drastic reductions in
activity against PET, indicating that mutations in the active site
impact catalysis negatively, through changes in binding site geometry
that are not tolerated, despite conservation of overall enzyme fold.
Importantly, a previous study showed higher (rather than lower) activity
in H129W {[}Furukawa et al., 2019{]}. Since \emph{Tf} C contains S136 as
a vicinal residue of H129W, and S136 happened to be T136 in our\emph{Tf}
C clone, we mutated T136 to S136 to create H129W/T136S which, however,
also showed lower (rather than higher) activity, in contradiction of the
earlier report. We propose that H129 influences catalysis in a
previously-unsuspected manner.

W155F and H184S were non-active against commercial-sourced PET films of
high crystallinity (\textasciitilde{}250 microns thickness). Both
mutants, however, showed activity against thin PET films of low
crystallinity (\textasciitilde{}10 microns thickness), made through
dissolution of PET granules (each of \textasciitilde{}30 mg) and
evaporation of the solvent (HFIP), on the walls of a micro-centrifuge
tube. Interestingly, using such thin films, A173C showed
\textasciitilde{}40\% improvement over \emph{Tf} C. However, using
commercially-sourced PET films, A173C was 30\% less active than\emph{Tf}
C, suggesting differences in how these enzymes see PET backbones in
films of different thickness and crystallinity. A173 is situated just
below the `catalytic triad' residues D176 and H208 (Supporting
Information Figure S2B). The A173C mutation could be anticipated to
decrease space in the binding cleft, facilitating better catalysis upon
thin PET films containing accessible PET chains (Supporting Information
Figure S4) but giving rise to poorer catalysis with crystalline or
semi-crystalline (thicker) commercial PET films (Figure 2A) at the
reaction temperature of 60 @\selectlanguage{english}C. A173C/A210C, and A173C/A206C, created to
introduce a disulphide bond below the catalytic cleft (mimicking such a
bond in PETase) led to loss of activity, suggesting distortion of
\emph{Tf} C's binding site. Deletion of V164 in a loop connecting
\selectlanguage{greek}β-\selectlanguage{english}strands below the active site also led to loss of activity.

L90, situated distal to the binding site, was mutated to A90 (less
hydrophobic), and also to F90 (more hydrophobic), analogous to F125 in
LCC. L90A showed reduced activity upon PET (data not shown). In
contrast, G62A/L90F showed a \textasciitilde{}2.3-fold increase in
activity, compared to \emph{Tf} C. G62A/F209I (corresponding to F243I
variant of LCC {[}Tournier et al., 2020{]}), showed a
\textasciitilde{}3.17-fold improvement. The variant G62A/F249R showed
the highest hydrolytic potential against commercial-sourced PET films,
with a \textasciitilde{}3.5-fold improvement over \emph{Tf} C.
G62A/F209I and G62A/F249R are anticipated to show reduced PET binding,
due to the replacement of phenylalanine by a less hydrophobic residue,
or a charged residue. This may allow a higher fraction of the \emph{Tf}
C population to remain in solution and, therefore, to act upon
degradation intermediates accumulating in solution, so as to function
synergistically with the bound enzyme fraction of \emph{Tf} C (invading
and degrading solid PET), to generate TPA. This could explain the
enhancement of activity in these two variants, through a reduction in
non-specific PET binding, or faster dissociation from PET, without any
alterations in the active site.

Most mutants showing loss of activity against commercial PET film also
showed reduced catalytic degradation of BHET. In Figure 2B, H129W shows
the least activity with BHET, with H129W/T136S showing improved
degradation of BHET into TPA. L90A and A173C/A206C showed BHET
hydrolysis comparable to \emph{Tf} C. This suggests that the inability
of these mutants to degrade PET owes to compromised PET-binding, and not
to compromise hydrolysis of ester bonds. The G62A variant along with
four other binding-altered variants made using G62A as background (i.e.,
G62A/L90F, G62A/F209I, G62A/F249A and G62A/F249R) showed
\textasciitilde{}1.6 to \textasciitilde{}2.0-fold improvements in
conversion of BHET into TPA. The mutants showed similar relative trends
in their ability to hydrolyse BHET over 12 h (Figure 2B) or 4 h
(Supporting Information Figure S5). F209I showed maximum enhancement of
BHET conversion, \textasciitilde{}2 fold higher than \emph{Tf} C after
12 h of incubation.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2:} Comparison of activity of various \emph{Tf} C mutants
with respect to \emph{Tf} C, against (A) commericial PET films, (B)
BHET. The error bars show standard deviation based on tripicate
measurements.

\emph{3.3 Spectroscopic, chromatographic and PET-binding characteristics
of mutants.}

\emph{Tf} C is an \selectlanguage{greek}α/β \selectlanguage{english}hydrolase which shows a CD spectrum typical of
proteins with mixed \selectlanguage{greek}α/β \selectlanguage{english}structures, with a spectral shape dominated by
negative bands at 208 and 222 nm, and spectral intensity characterized
by substantially lower MRE than is associated with \selectlanguage{greek}α-\selectlanguage{english}helices. CD spectra
of all mutants/variants overlapped substantially with that of\emph{Tf}
C\emph{,} demonstrating that the enzyme tolerated the mutations, and
remained folded in a native-like structural format (Figure 3A). Size
exclusion chromatographic behaviour of all mutants/variants was
identical to that of \emph{Tf} C, with elution occurring at the same
volume (\textasciitilde{}13.5 ml) from the column used (data not shown
for all mutants in Figure 3B). Only L90F eluted at a later volume
(\textasciitilde{}14.1 ml), due to hydrophobic interactions with the
column matrix, owing to the enhanced surface hydrophobicity (Figure 3B).

We performed PET-binding assays, based on SDS-PAGE analyses of enzyme
populations seen in lanes corresponding to reaction supernatants, or
extracts of PET-bound populations (Figure 3C), followed by densitometric
analyses (Figure 3D). These assays were performed for mutants/variants,
G62A/L90F, G62A/F209I and G62A/F249R. Binding of L90F to PET was noted
to be comparable to that of \emph{Tf} C, in that the partitioning of
enzyme populations between PET (lane 1 \emph{versus} lane 3) and
solution (lane 2 \emph{versus} lane 4) were similar. In contrast, with
the G62A/F209I variant, significantly less enzyme was seen to have
remained bound to PET (lane 6 and Figure 3D), with proportionate
increase in the enzyme left in solution (lane 7). Figure 3D shows that
PET binding by G62A/F249R is intermediate to that of G62A/L90F and
G62A/F209I. The differences would appear to owe to differences in
overall surface hydrophobicity, wrought through the protein engineering
carried out.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3: (A)} Secondary structure assessment for \emph{Tf} C
and different mutants. (B) Size exclusion chromatography of binding
altered variants of \emph{Tf} C. (C) SDS-PAGE analysis for partitioning
of \emph{Tf} C after 40 h incubation with PET film. Lane 1: G62A bound
to PET, Lane 2: G62A in solution, Lane 3: G62A/L90F bound to PET, Lane
4: G62A/L90F in solution, Lane 5: Protein marker, Lane 6: G62A/F209I
bound to PET, Lane 7: G62A/F209I in solution, Lane 8: G62A/F249R bound
to PET, Lane 9: G62A/F249R in solution. (D) Densitometric quantification
of proteins in lanes 1, 3, 6, 8.

\emph{3.4 Thermal and chemical (kinetic and thermodynamic) stability of
Tf} C

Thermal unfolding of \emph{Tf} C was examined using CD and DSC. Changes
in \emph{Tf} C's secondary structure were measured during heating (20 \selectlanguage{ngerman}°C
to 90 °C), at 1 °C/min, and 0.2 °C/min, respectively (Figure 4A). At the
faster unfolding rate of 1 °C/min, \textasciitilde{}70\% denaturation
was observed at 90°C, whereas at the slower unfolding rate of 0.2
°C/min, \textasciitilde{}100 \% unfolding was observed, with a
T\textsubscript{m} of \textasciitilde{}85 °C. This indicates
that\emph{Tf} C is kinetically (thermally) stabilized, displaying a
resistance to unfolding that leads to unfolding equilibrium being
achieved only through slower heating. To further \emph{Tf} C's unfolding
kinetics, changes in CD were monitored over 120 min incubations, at
temperatures flanking the T\textsubscript{m} (Figure 4B). The Arrhenius
plot obtained from rates of unfolding in the range of 68-87.5 °C
(Supporting Information Figure S6A) yielded an activation energy,
E\textsubscript{a}, of 217.557 kJ/mol (i.e. 52 kcal/mol). Thermodynamic
parameters determined through construction of an Eyring plot (Supporting
Information Figure S6B) were: (1) \selectlanguage{greek}Δ\selectlanguage{english}H = 214.643 kJ/mol; (2) \selectlanguage{greek}Δ\selectlanguage{english}S = 314.4346
J/mol*K; and (3) \selectlanguage{greek}Δ\selectlanguage{english}G = 120.96 kJ/mol (i.e. 28.9 kcal/mol). DSC
measurements (Figure 4C) corroborated the T\textsubscript{m} determined
by CD, while establishing two proximal unfolding transitions at
\textasciitilde{}78.5 \selectlanguage{ngerman}°C and \textasciitilde{}86.7 °C, supporting the
high thermal, kinetic, and thermodynamic stability of \emph{Tf} C, and
causing \emph{Tf} C to be suited to PET degradation at temperatures
close to PET's glass transition temperature over hours, or tens of
hours.\emph{Tf} C's chemical stability was explored through overnight
incubation in urea (0 to 8 M), or guanidium hydrochloride, Gdm.HCl (0 to
6M), based on measurements of changes in the CD signal at 222
nm.\emph{Tf} C is resistant to urea, but not to Gdm.HCl (Figure 4D),
which is a more potent denaturant (disrupting electrostatic interactions
and hydrogen bonding, unlike urea which primarily disrupts only hydrogen
bonding). Gdm.HCl causes unfolding with an apparent C\textsubscript{m}of
3.9 M. Kinetics of denaturation by Gdm.HCl were determined through 2 h
incubations of \emph{Tf} C at 5M, 5.5M, 6M, 6.5M and 7M Gdm.HCl (Figure
4E). The rate of unfolding in absence of denaturant,
K\textsubscript{u,w}, was determined from a half-chevron plot
(Supporting Information Figure S7), to be
3.77*10\textsuperscript{-11}s\textsuperscript{-1}, indicative of slow
unfolding. This resistance to thermal and chemical denaturation bodes
well for \emph{Tf} C's utility under industrial conditions.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4:} Thermal and chemical denaturation studies. (A) MRE at
222 nm at temperatures from 20 \selectlanguage{ngerman}°C to 90°C at rates of 0.2 °C/min and 1
°C/min , measured by CD. (B) Thermal kinetics demonstrating fraction
folded during 2 hour incubations at different temperatures (68 °C to
87.5 °C), measured by CD. (C) DSC showing two-transition unfolding
of\emph{Tf} C. (D) MRE at 222 nm at varying urea (0 M to 8 M) and GdmCl
(0 M to 6 M) concentrations, measured by CD. (E) Chemical kinetics
demonstrating fraction folded during 2 h incubations at different
concentrations of GdmCl (5 M to 7 M), measured by CD.

\textbf{4. Conclusions:}

We created 17 mutants of \emph{Tf} C, with a view to (a) creating or
removing space in the active site, to modulate catalysis, or (b)
increasing or decreasing hydrophobicity in-and-around the binding site,
or at distal locations, to modulate \emph{Tf} C's binding of PET. In
every instance attempting to alter catalytic efficiency through changes
made to residues at the active site, we only observed a reduction or
complete abolition of activity upon solid PET, sometimes without a
reduction in activity against BHET, indicating the sensitivity
of\emph{Tf} C's active site to binding to crystalline PET chains. In
stark contrast, we achieved significant success with mutations designed
to reduce non-specific (hydrophobic) interactions of \emph{Tf} C with
PET, at locations distal to the active site, where our intention was to
facilitate either (i) improved dissociation of enzyme from PET,
following catalysis, to free up enzyme for further rounds of binding and
catalysis, or (ii) the improved presence of enzyme in solution, to
concomitantly convert OETs, BHET or MHET into TPA. Mutants G62A/F209I,
G62A/F249R, and G62A/L90F displayed between \textasciitilde{}2.3-fold
and \textasciitilde{}3.0-fold enhancement in PET degradation
over\emph{Tf} C, and between \textasciitilde{}1.3-fold and
\textasciitilde{}2.0-fold enhancement over G62A \emph{Tf} C, which is
known to be the currently best-performing mutant/variant of \emph{Tf} C.

\textbf{Acknowledgements:} AM thanks DBT, India, and HK and BT thank
CSIR, India, for doctoral fellowships. MP was a Masters' thesis student.
PG thanks DBT, India, for grant BT/PR/31706/PBD/26/705/2019. The work
was mainly conceived and executed by AM under the supervision of PG, and
the first draft was written up by AM and then edited by AM and PG. MP
and HK worked under the immediate supervision of AM and the overall
supervision of PG. MP produced and studied four of the seventeen
mutants. HK worked with AM in determining the activities of mutants
against BHET and PET. BT participated in discussions about the work,
suggested the making of certain mutants, and helped with
trouble-shooting in the production of genes/proteins in respect of
several mutants.

\textbf{Declaration of conflicting interests:} The authors have no
conflicting interests.

\textbf{Supplementary Data:} The seven figures in the supplementary data
for this article can be viewed online.

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