polymers

Article
Degradation of PET Bottles by an Engineered Ideonella
sakaiensis PETase
Maria Eduarda Sevilla 1 , Mario D. Garcia 1, * , Yunierkis Perez-Castillo 2,3, * , Vinicio Armijos-Jaramillo 3,4 ,
Santiago Casado 1 , Karla Vizuete 5 , Alexis Debut 5,6 and Liliana Cerda-Mejía 1, *

1 Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología, Universidad Técnica de Ambato,

Ambato 180216, Ecuador
2 Área de Ciencias Aplicadas, Facultad de Ingeniería y Ciencias Aplicadas, Universidad de Las Américas,
Quito 170125, Ecuador
3 Grupo de Bio-Quimioinformática, Universidad de Las Américas, Quito 170125, Ecuador
4 Ingeniería en Biotecnología, Facultad de Ingeniería y Ciencias Aplicadas, Universidad de Las Américas,
Quito 170125, Ecuador
5 Centro de Nanociencia y Nanotecnología, Universidad de Las Fuerzas Armadas ESPE,
Sangolquí 171103, Ecuador
6 Departamento de Ciencias de la Vida y Agricultura, Universidad de Las Fuerzas Armadas ESPE,
Sangolquí 171103, Ecuador
* Correspondence: [Link]@[Link] (M.D.G.); [Link]@[Link] (Y.P.-C.);
[Link]@[Link] (L.C.-M.)

Abstract: Extensive plastic production has become a serious environmental and health problem due
to the lack of efficient treatment of plastic waste. Polyethylene terephthalate (PET) is one of the most
used polymers and is accumulating in landfills or elsewhere in nature at alarming rates. In recent
years, enzymatic degradation of PET by Ideonella sakaiensis PETase (IsPETase), a cutinase-like enzyme,
has emerged as a promising strategy to completely depolymerize this polymer into its building blocks.
Here, inspired by the architecture of cutinases and lipases homologous to IsPETase and using 3D
structure information of the enzyme, we rationally designed three mutations in IsPETase active site
for enhancing its PET-degrading activity. In particular, the S238Y mutant, located nearby the catalytic
Citation: Sevilla, M.E.; Garcia, M.D.;
triad, showed a degradation activity increased by 3.3-fold in comparison to the wild-type enzyme.
Perez-Castillo, Y.; Armijos-Jaramillo,
Importantly, this structural modification favoured the function of the enzyme in breaking down highly
V.; Casado, S.; Vizuete, K.; Debut, A.;
crystallized (~31%) PET, which is found in commercial soft drink bottles. In addition, microscopical
Cerda-Mejía, L. Degradation of PET
Bottles by an Engineered Ideonella
analysis of enzyme-treated PET samples showed that IsPETase acts better when the smooth surface
sakaiensis PETase. Polymers 2023, 15, of highly crystalline PET is altered by mechanical stress. These results represent important progress
1779. [Link] in the accomplishment of a sustainable and complete degradation of PET pollution.
polym15071779
Keywords: IsPETase; biodegradation; polyethylene terephthalate; PET; enzyme engineering
Academic Editors: Chi-Hui Tsou and
Manuel Reyes De Guzman

Received: 1 March 2023

Revised: 27 March 2023 1. Introduction
Accepted: 28 March 2023
Plastics have become a crucial component of modern society due to their exceptional
Published: 3 April 2023
mechanical and physical properties, including durability, flexibility, and impermeability [1].
The demand for plastics led to the production of 367 Mt in 2020 [2]. However, only 9%
of plastic waste generated undergoes conventional recycling processes, with the rest end-
Copyright: © 2023 by the authors.
ing up in landfills or elsewhere in the environment [3]. The continuing accumulation of
Licensee MDPI, Basel, Switzerland. plastic raises significant concerns about its impact on human and wildlife health. While
This article is an open access article approximately 100,000 marine animals are killed every year by entanglement or inges-
distributed under the terms and tion of macroplastics (>5 mm) [4], recent studies suggest that microplastic (1 µm–5 mm)
conditions of the Creative Commons contamination may reduce the survival of fish and cause oxidative stress and neurotoxic
Attribution (CC BY) license (https:// effects that lead to behavioural disorders [5,6]. In humans, plastic pollution and its by-
[Link]/licenses/by/ products induce negative sequels in different organs, including the lungs, skin, liver, brain
4.0/).

Polymers 2023, 15, 1779. [Link] [Link]

Polymers 2023, 15, 1779 2 of 15

and gastrointestinal system, which triggers an immune response, cell toxicity, cancer and
neurological disorders, along with other health issues [7].
Polyethylene terephthalate (PET) (Figure 1), obtained by polycondensation of tereph-
thalic acid (TPA) and ethylene glycol (EG) or the transesterification of dimethyl terephtha-
late (DMT) and EG [8], is considered the most abundant thermoplastic material. Its low
price and remarkable durability and impermeability to liquids and gases make it ideal for
the fabrication of single-use soft drink bottles and packing [2]. Due to these favourable
properties, PET is highly resistant to biodegradation and accumulates in nature. New
materials with higher biodegradability, such as polylactic acid (PLA), polyhydroxyalka-
noate (PHA), and polybutylene succinate (PBS), have emerged as a measure to reduce
the environmental impact of PET [1]. However, certain applications in the food industry
require the use of packing materials exclusively made of PET [9] or using virgin PET [10] to
comply with safety regulations, which sustains PET consumption. This scenario highlights
the need for new strategies to recycle PET sustainably.

Figure 1. Chemical structure of IsPETase substrate and products. PET: polyethylene terephthalate,
BHET: bis(2-hydroxyethyl) terephthalic acid, MHET: mono(2-hydroxyethyl) terephthalic acid, TPA:
terephthalic acid, EG: ethylene glycol.

PET is mostly recycled through thermal treatments, but these processes cause degrada-
tion of the polymer structure and loss of some mechanical properties required for beverage
bottles [11]. Alternatively, PET is depolymerised by pyrolysis or gasification to yield fuels
used to produce heat and electricity. These processes are inexpensive and generate a high
profit margin, but they have significant environmental drawbacks, such as heavy pollution
and a contribution to the build-up of greenhouse gases [12]. Moreover, PET can be chem-
ically broken down into monomers using solvents, such as alcohol, and a catalyst. The
resulting monomers can be repolymerized into new PET with identical properties to virgin
plastic [13]. In spite of the benefits inherent to obtaining plastic with ideal properties, this
process is inconvenient due to its high cost, and therefore, thermal treatments remain the
most viable option. The discovery of the bacterium Ideonella sakaiensis 201-F6 and its ability
to use PET as the main carbon source for growth [14] has garnered significant attention in
recent years. I. sakaiensis employs two enzymes, IsPETase and IsMHETase, for breaking
down PET into its building blocks (TPA and EG) (Figure 1). To date, I. sakaiensis represents a
unique example where extracellular degradation of PET takes place in order to incorporate
PET degradation products into cellular catabolism. IsPETase is a cutinase-like enzyme
that catalyses the depolymerization of PET into mono(2-hydroxyethyl) terephthalic acid
Polymers 2023, 15, 1779 3 of 15

(MHET), with bis(2- hydroxyethyl) terephthalic acid (BHET) and TPA present in minor
amounts as by-products [14] (Figure 1). A similar PET degrading activity has also been
observed in cutinases from different organisms, including Thermobifida fusca (TfuCut) [15],
Thermobifida alba (TalCut) [16], and Thermofida cellulosilytica (ThcCut) [17], but these are not
as efficient as IsPETase. The second enzyme, IsMHETase, catalyses the conversion of MHET
into TPA and EG (Figure 1). The amounts of end-products generated in this pathway are
strongly influenced by the reaction performed by IsPETase. For that reason, multiple efforts
have focused on improving IsPETase activity and thermostability using rational approaches
that integrate critical architectural elements found in enzymes of the cutinase and lipase
families (Table 2). Nevertheless, the enzyme’s activity on highly crystalline PET, which is
found in most soft drink bottles, has not reached the threshold for industrial applicability
yet. Consequently, further investigation into novel modifications in the enzyme structure is
still required.

Table 1. Mutations of IsPETase structure for modifying its PET-degrading activity.

Mutation Effect on Enzymatic Activity Method Substrate Ref.

S238F/W159H 4.13% higher than wild type Absolute PET 14.8 ± 0.2%
[18]
W185A highly impaired performance relative to wild type crystallinity loss crystallinity
S160A
Disrupt the
D206A Not detected catalysis process
H237A
W159A Increased
W159H Increased
M161A Decreased Relative activity
W185A Decreased Influence the towards MHET PET drinking
substrate binding [19]
A209I No change observed and TPA bottle
Q119A Decreased production
S214H Increased
S238F Decreased
W97L Decreased
Change the
Q182L No change
hydrophobic
R123A Decreased
property
N241A Decreased
S160A Decreased
R132G Decreased
C203S Decreased
C239S Decreased
W185A Decreased
Expressed by
S214H Decreased
production levels BHET [20]
I208A Decreased
of MHET and TPA.
W159A Decreased
W159H Decreased
M161A Decreased
Y87A 80.73% MHET production; TPA production decreased
T88A Full activity in producing MHET; TPA production decreased
R61A Increased 1.6 times wild type activity
L88F Increased 2.0 times wild type activity Expressed by
I179F Increased 15.0 times wild type activity kinetic parameters PET film [21]
S178T Decreased to 29.7% of the activity of wild type (kcat /KM )
S209V Decreased to 38.2% of the activity of wild type
Polymers 2023, 15, 1779 4 of 15

Table 2. Mutations of IsPETase structure for modifying its PET-degrading activity.

Mutation Effect on Enzymatic Activity Method Substrate Ref.

S160A Almost complete loss
D206A Almost complete loss
H237A Almost complete loss
Y87A 5% hydrolytic activity
M161A 52% hydrolytic activity
W185A 5% hydrolytic activity
I208A 46% hydrolytic activity Expressed
BHET [22]
W159A 8% hydrolytic activity hydrolytic activity
S238A Similar hydrolytic activity
N241A 18% hydrolytic activity
Similar hydrolytic activity, increased thermostability and
R280A PET degradation activity by 14-fold at 40 degrees Celsius;
when associated with E-121 and H-186.
W159H Dramatically decreased
S238F Dramatically decreased
C203A/C239A Dramatically decreased
S93M
Expressed 1-naphthyl
W159F Increases activity towards 1-naphthyl butyrate [23]
hydrolytic activity butyrate
N241F

In this study, a rational protein engineering based on an extensive analysis of the

structure of IsPETase and different homologous enzymes was carried out with the aim to
increase the enzyme activity. Molecular docking and molecular dynamic (MD) simulations
suggested that three different single point mutations improve PET binding compared to
the wild-type (WT) enzyme. Two of these mutations were introduced nearby the catalytic
residue H237, which favour the binding affinity of PET through the introduction of further
non-covalent interactions. A third mutation was introduced in the proximity of the active-
site cleft in order to alter the flexibility of the loops that contour the active site. Examination
of enzyme-treated PET films using scanning electron microscopy (SEM), atomic force
microscopy (AFM), and differential scanning calorimetry (DSC) showed that the IsPETase
mutant variants have an improved activity over crystalline PET films compared to the WT
enzyme. This work could provide an important contribution to solving the environmental
problem caused by plastic pollution through effective depolymerization of PET.

2. Materials and Methods

2.1. Molecular Docking
The PET substrate used for modelling purposes consisted of one subunit of BHET and
one subunit of MHET (PET dimer). One initial three-dimensional conformer of the substrate
was generated with Omega [24,25] and partial atomic charges of type am1-bcc were added
to this conformer with Molcharge [26]. The structure of the IsPETase enzyme was obtained
from the Protein Data Bank database (PDB code 6EQE). The PET dimer was docked into
the active site of the enzyme with Gold [27]. The ChemPLP scoring function was selected
for primary docking and scoring. A total of 30 docking solutions were generated and
rescored with the GoldScore, ChemScore, and Astex Statistical Potential (ASP) scoring
functions implemented in Gold. The best docking solution was selected from a consensus
scoring scheme employed in previous research [28]. The binding mode of the products was
generated by breaking the PET dimer and adopting the best docking solution into MHET
and BHET.

2.2. Molecular Dynamic Simulations and Estimation of the Free Energies of Binding
Molecular dynamic (MD) simulations were performed with Amber 2022 [29] following
a previously described protocol [30,31]. Briefly, the ff19SB and gaff2 force fields were used
for the parametrization of the protein and ligands, respectively. Complexes were enclosed
Polymers 2023, 15, 1779 5 of 15

in truncated octahedron boxes and solvated with Optimal Point Charge (OPC) water
molecules. Excess net charges were neutralized by adding sodium (Na+ ) and chloride (Cl− )
counterions at a concentration of 150 mM [32]. Two stages of energy minimization were
performed, with the solute restrained during the first of these and with no restrains during
the second one. The energy minimized systems were gradually heated from 0 to 300 K,
and equilibrated for 100 ps. The equilibrated systems were used as input to five short 4 ns
MD simulations performed in the isobaric-isothermal (NPT) ensemble. Each MD replica
was run with different random initial velocities for a better exploration of the complexes’
conformational space. The free energies of binding were estimated with the Molecular
Mechanics-Generalized Born and Surface Area (MM-GBSA) method, as implemented in
Amber 2022. For a complex, these calculations took place with 100 MD snapshots evenly
drawn from all five MD replicas and covering the 1 ns–4 ns time interval. For each variant
of the IsPETase enzyme, the free energies of binding were computed for the PET dimer and
the reaction products.

2.3. Construction of the IsPETase Mutants

The IsPETase mutant variants were designed based on a comparison of the IsPETase
substrate-binding site residues with those present in homologous enzymes, similar to
that reported previously [18]. First, the amino acid sequences of different α/β hydrolases
homologous to IsPETase were identified using PDBeFold and UniProt’s Basic Local Align-
ment Search Tool (BLAST) [33]. Then, the natural substitutions that occur in the residues
that contour the substrate-binding site in the homologous enzymes were identified by
multiple sequence alignment using ClustalOmega [34] and WebLogo [35] and assessed by
MD simulations.
All of the constructs for the mutants were obtained using the QuickChange® Site-
Directed Mutagenesis kit (Agilent, Foster City, CA, USA) as described previously [36]. In
brief, the wild-type IsPETase expression vector, which was previously codon optimized
for expression in Escherichia coli and cloned into pET21b(+) with a C-terminus His-tag
(pET21b(+)-IsPETase) [18], was obtained from Addgene. The pET21b(+)-IsPETase vec-
tor was amplified by polymerase chain reaction (PCR) using the PfuUltra polymerase
with the primers I208V-F: 50 -GAGAATGATAGCTGGGCACCGGTGAAC-30 , I208V-R: 50 -
GTTCACCGGTGCCCAGCTATCATTCTC-30 , N212A-F: 50 -ATTGCACCGGTGGCGAGCA
GCGCGCTG-30 , N212A-R: 50 -CAGCGCGCTGCTCGCCACCGGTGCAAT-30 , S238Y-F: 50 -
GGCGGTAGCCACTATTGTGCCAACTCT-30 , and S238Y-R: 50 -AGAGTTGGCACAATAGTG
GCTACCGCC-30 . The parental plasmid was then digested with 1 unit of Dpn I restriction
enzyme for 2 h at 37 ◦ C, and the resultant mutant IsPETase-containing constructs were
immediately transformed into E. coli DH5α chemically competent cells using the heat-shock
method [37]. The introduction of each single point mutation was confirmed by Sanger
sequencing.

2.4. Enzyme Expression and Purification

The wild type and mutant IsPETase variants were expressed and purified as reported
previously [18,20,38] with some modifications. All expression vectors were transformed
into E. coli BL21(DE3) Rosetta gami-B chemically competent cells. A single colony was trans-
ferred to 50 mL of Lysogeny Broth (LB) media supplemented with ampicillin (100 µg/mL)
and incubated overnight at 37 ◦ C with constant agitation (230 opm). This culture was
transferred to 1 L of Terrific Broth media and incubated at 37 ◦ C with constant agitation
(200 opm) and, when the optical density (OD600 ) reached 2.0, the expression of the protein
was induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 20 ◦ C and
200 opm during 18–24 h. The bacterial cells were harvested by centrifugation at 4 ◦ C,
3900× g for 20 min, and the cell pellet was collected and kept at −70 ◦ C. All subsequent
operations were performed at 4 ◦ C. For lysis, the pellet was resuspended in buffer A
(25 mM Tris, 150 mM sodium chloride, 20 mM imidazole, pH 7.5) supplemented with 0.5%
Triton X-100 and 10 µL/mL 0.5 M ethylenediaminetetraacetic acid (EDTA) and DNase. The
Polymers 2023, 15, 1779 6 of 15

cell suspension was incubated for 15 min and then sonicated in an ultrasonic cell crusher
(650 W, MRC, Harlow, UK for 10 × 10 s at a constant duty cycle, with rest intervals of 10 s.
The cell debris was removed by centrifugation at 21,100× g for 1 h. The supernatant was
loaded onto a HisTrap™HP column (Cytiva, Uppsala, Sweden), previously equilibrated
with buffer A, using a FPLC system (ÄKTA Start, Cytiva, Uppsala, Sweden). The unbound
proteins were removed with 10 column volumes (cv) of buffer A, and the enzyme was
eluted with a gradient of 20–500 mM imidazole. The flow-through was collected and
the fractions containing the enzyme were pooled and loaded onto a PD-10 desalting col-
umn (GE Health Care, Uppsala, Sweden) previously equilibrated with a buffer containing
25 mM Tris, 150 mM sodium chloride, and pH 7.5. The purified enzyme was stored in
50 µL aliquots at −70 ◦ C. Enzyme purity was assessed by denaturing polyacrylamide
gel electrophoresis (SDS-PAGE). All chemicals used were of analytical grade and were
purchased from Sigma-Aldrich (Merck, Darmstadt, Germany), unless otherwise stated.

2.5. PET Degradation Assay

Circular PET films (6 mm diameter, 0.213 mm thick) cut from transparent commercial
PET bottles were washed with sterile deionized water and dried at room temperature for
48 h before enzymatic treatment. The films were treated in 500 µL of a buffer containing
50 mM glycine-sodium hydroxide (NaOH), pH 9.4, with and without 500 nM enzyme. All
samples were incubated at 30 ◦ C for 72 h. The reaction was stopped by aqueous dilution
with 20 mM phosphate, 12% dimethyl sulfoxide (DMSO) pH 2.5, and the precipitated
enzyme was removed by centrifugation at 13,500× g for 10 min [14]. Then, the PET films
were washed with 1% sodium dodecyl sulphate (SDS), distilled water, and ethanol. Finally,
the films were air-dried for SEM, AFM, and DSC [18].

2.6. Scanning Electron Microscopy (SEM)

Dried samples were cleaned with nitrogen gas and then mounted on carbon tape and
secured with silver tape on a SEM stub and then metalized using a sputter coater (Q150R ES,
Quorum, Laughton, UK). Surface morphology of the control and IsPETase-treated samples
was evaluated using a field emission scanning electron microscope (MIRA 3, TESCAN,
Brno, Czech Republic) operating at 5 kV.

2.7. Atomic Force Microscopy (AFM)

Nanoscopic corrugation measurements were performed using an atomic force micro-
scope (XE7, Park Systems, Santa Clara, CA, USA). Samples were attached to the sample
holder with double-sided tape. Without any additional treatment, film surfaces were
scanned in the non-contact mode under ambient conditions using PPP-CONTSCR com-
mercial silicon cantilever tips (0.2 N/m, 25 kHz, <7 nm typical radius). All images were
acquired at 512 × 512 pixels. AFM image edition was restricted to only a single polynomial
levelling, performed using the Park System’s XEI software, version 5.1.6 build 1.

2.8. PET Crystallinity Assay

The thermal crystallinity of PET was assayed using a differential scanning calorimeter
(DSC3 STARe System, Mettler Toledo, Urdorf, Switzerland). In summary, 6 mg of PET
film was equilibrated at 25 ◦ C, heated to 300 ◦ C at 10 ◦ C/min, held at 300 ◦ C for 1 min,
and cooled down to 25 ◦ C at 10 ◦ C/min. The experiment was carried out in a nitrogen
(N2 ) atmosphere (50 mL/min). The percent crystallinity (%Xc ) of PET was determined as
described previously [39] using Equation (1).

∆Hm − ∆Hc
 
%X c = ∗ 100 (1)
∆Hom

where ∆Hm is the heat of melting, ∆Hc is the heat of cold crystallization, and ∆Hm ◦ is the
heat of melting of ideal PET crystals measured at the equilibrium melting point (140.1 J/g).
Polymers 2023, 15, 1779 7 of 15

3. Results and Discussion

3.1. IsPETase Engineering for Enhancing PET-Degrading Activity
To date, IsPETase constitutes the most efficient PET-degrading enzyme known. Com-
pared to other enzymes that are able to hydrolyse PET, such as TfuCut, leaf-branch com-
post cutinase (LCC) and Fusarium solani cutinase (FsCut), this enzyme exhibits between
9 and 19.3-fold increased activity when using low-crystallinity (1.9%) PET films as the
substrate [14]. However, IsPETase degrading activity is still insufficient for being used in
the PET recycling industry, and biodegradation of PET pollution remains a major challenge
due to the poor enzyme accessibility into the structure of highly crystallized PET used
to manufacture beverage bottles. To overcome these problems, based on the previously
reported crystal structure of IsPETase (PDB code 6EQE), molecular docking of PET and MD
simulations, we designed three different single point mutations inspired in the architecture
of 39 IsPETase homologous enzymes of the cutinase and lipase families (Figure 2A). MD
simulations showed that PET binds to a cleft contoured by 14 residues located at the protein
surface (Figure 2B). These include S160 and H237, which in consort with D206, conform the
catalytic triad present in other enzymes of the α/β hydrolase superfamily [18] (Figure S1).
We hypothesized that substitutions introduced nearby the catalytic residues could posi-
tively impact the binding affinity of the substrate, and consequently, the enzyme activity.
S160 is flanked by W159 and W185, a highly conserved residue critical for enzyme activity
(Table 2). On the other hand, H237 is flanked by I208 and S238, which also form part of
the binding pocket (Figure 2B,C). A multiple sequence alignment (Figure S1) showed that
most IsPETase homologous enzymes possess the W159H substitution, which confers an
increased PET-degrading activity alone [19] or in combination with S238F [18], compared
to the WT enzyme (Table 2). A greater variability was noted at positions I208 and S238,
with two and six potential substitutions, respectively. Overall, we noted that the mutations
I208V, S238F, and S238Y are more frequent in cutinases and lipases (Figure 2A). As stated
above, the mutations W159H and S238F have previously been tested (Table 2); therefore,
these were excluded in this study. A third position outside the binding pocket was also
considered with the aim to alter the flexibility of the loop (E204-S214) that connects the α5
helix and the β7 strand, where D206 is located. The residue N212 was chosen for protein
engineering due to its low level of conservation across all enzymes, and its position in the
protein structure, with the side chain atoms exposed to the solvent (Figure 2A,B).
MD calculations served to discriminate the best mutant candidates regarding the
values of free energy (∆G) of substrate and products binding (Table 3), compared with
those predicted when PET (−21.20 kcal/mol) or MHET (−17.94 kcal/mol) bind the WT.
Interestingly, engineering IsPETase to adopt a cutinase/lipase-like active site through the
introduction of I208V or S238Y mutations increased the binding affinity of PET by 20%
(−25.50 kcal/mol) without altering the affinity of the reaction products. A similar result
was observed when the asparagine at position 212 was mutated to alanine (∆G PET binding
= −28.36 kcal/mol) (Table 3). Thus, we generated the IsPETase I208V, S238Y, and N212A
variants using mutagenic PCR. The enzymes were then expressed in E. coli and isolated by
affinity chromatography.

Table 3. Binding free energy calculations of substrate and products in complex with IsPETase.

∆G (kcal/mol)
IsPETase
PET Products
WT −21.20 −17.94
I208V −25.50 −18.58
N212A −28.36 −14.88
S238Y −25.50 −17.84
Polymers 2023, 15, 1779 8 of 15

Figure 2. Single point mutations introduced in IsPETase. (A) Logo representation subalignment of
IsPETase homologous enzymes. The * highlights the catalytic residues D206 and H237. (B) Connolly
surface and PET modelled in IsPETase active site. The polypeptide, the residues forming contacts
with the substrate and the mutations introduced in IsPETase are coloured light grey, cyan, and sky
blue, respectively. (C) Snapshot of the MD simulation of the PET binding mode in the IsPETase active
site. PET is represented in the ball and sticks model, while the protein amino acids are represented
in the stick models. The carbon atoms are coloured yellow (PET), cyan (catalytic residues, S160 and
H237), and sky blue (I208 and S238). Non-covalent interactions formed between the mutated residues,
and the substrate or the polypeptide are represented by dashed lines.

3.2. Modified IsPETase Exhibits Increased PET-Degrading Activity over Highly Crystallized PET
As mentioned above, in the original research report where IsPETase was isolated and
characterized, Yoshida et al. [14] used low crystallinity (1.9%) PET to examine the ability of
the enzyme to digest this polymer. In contrast, PET waste remains a challenge for recycling
because it usually shows a much higher degree of crystallinity, which reduces the enzyme’s
esterase activity. Here, we performed enzyme-mediated PET degradation assays using
coupons (6 mm diameter, 0.213 mm thick) mechanically cut from commercial soft-drink
PET bottles with 30.88% crystallinity (determined by DSC).
SEM microphotographs of the control sample and those treated with the WT and
mutant enzymes were acquired at different magnifications to evaluate their surface mor-
phology. SEM microphotographs showed the existence of surface defects in the form of
channels in all the samples (Figure 3, top left corner), possibly originated from the ma-
nipulation of the films. Considering this fact, the surface of the samples was evaluated
inside and outside the surface defects. It could be noticed that the control sample exhibits a
smoother surface (first row in Figure 3) compared to the enzyme-treated samples (second
to fifth rows in Figure 3). The enzymes induced a granular texture and pitting on the PET
film surface, a clear sign that these were degraded the polymer. These findings indicated
that the enzyme could break down PET that is highly crystalline, and that the process is
comparable to when the enzyme breaks down PET with lower crystallinity levels (1.9% [14]
or 14.8% crystallinity [18]). In addition, we observed that, in all enzyme-treated films, there
is a greater change in the morphology inside the surface defects. Therefore, we inferred that
the enzyme activity is initiated and accentuated in those parts that previously presented
Polymers 2023, 15, 1779 9 of 15

surface defects, most likely due to favoured polyester chain flexibility [40] induced by
mechanical stress. Amongst the three mutants, PET films treated with the I208V variant
showed fewer changes in the morphology of the PET surface compared to the WT and the
N212 and S238Y variants, suggesting a decreased PET-degrading activity. Interestingly, the
S238Y mutant induced a severe degradation of the PET film surface and the formation of
microscopic PET filaments in the order of several hundred of nanometers wide (typically
around 200 nm) inside and outside the surface defects (bottom row in Figure 3). This
finding constitutes the first report where an IsPETase mutant variant produced this type
of degradation of PET, since all previous data found in the literature has only shown a
prominent pitting [14,18].

Figure 3. SEM micrographs of PET films degraded by IsPETase WT and mutant variants. All films
were incubated in buffer 50 mM glycine-NaOH pH 9.4, with or without enzyme, at 30 ◦ C for 72 h.

A similar type of PET surface morphology modifications was observed in AFM char-
acterizations. AFM confirmed SEM surface observations by permitting topographic profile
measurements on samples without any coating. We analyzed the PET film surface using
Polymers 2023, 15, 1779 10 of 15

2D and 3D views and the AFM profile. The 2D AFM topographic images represent zenithal
views of the height data recorded at each sample using a colour code to describe the ele-
vation values. The 3D AFM images display the same surface using a three-dimensional
perspective. Profile diagrams depict linear altitudes measured along the line highlighted
on the 2D corresponding picture. A comparison at the nanoscale between enzyme-treated
samples and the buffer-treated PET films revealed important topographic modifications
of the PET surfaces in agreement with a previous report [41]. These can be observed at
the profile corrugation contrast between control and sample scans presented in Figure 4.
Furthermore, the results show that the WT and N212A mutant apparently attack the PET
sample heterogeneously, degrading some regions more intensively than others and pro-
ducing a hollowed surface (see WT and N212A images in Figure 4). This suggests that, at
the nanoscale, PET bottles may show some degree of heterogeneity, and IsPETase could
attack first spots with more flexible polymer chains exposed on the original PET surface.
On the contrary, the I208V mutant may damage the PET more homogeneously, creating
a flatter but granular structure, as displayed in picture I208I of Figure 4. In the particular
case of S238Y, filamentous degradation protrusions beyond 17 times above the highest
corrugation measured on the other samples were detected, in agreement with SEM images
shown previously. In addition, PET surfaces treated with the S238Y variant exhibit hollows
15-fold deeper compared to those observed for the WT-treated PET film. In a previous
report [41], AFM observations suggested that the enzymatic degradation of PET induces
the corrugation of the film surface in a time-dependent manner and attributed this change
to the accumulation of the BHET product on the polymer surface. Here, our SEM and AFM
Polymers 2023, 15, x
measurements suggest a similar type of corrugation for all enzyme variants, except11for the
of 15
S238Y mutant that induced a hollowed surface. This could indicate that the activity of the
S238Y mutant does not produce significant amounts of BHET and principally yields MHET
or TPA.

Figure 4. Cont.
Polymers 2023, 15, 1779 11 of 15

Figure 4. AFM surface topographic 3D and 2D characterizations of PET films degraded by IsPETase
WT and
Figure mutant
4. AFM variants.
surface The profiles
topographic 3D andextracted from the depicted
2D characterizations of PETsolid
films lines reveal
degraded bythe height
IsPETase
WT and mutant variants. The profiles extracted from the depicted solid lines reveal the height dif-
differences.
ferences.
In order to further characterize the PET-degrading activity of the mutant variants,
changes on the structure of the polymer were analysed using DSC. Similar to that reported
previously [18], IsPETase induced a reduction in the crystallinity of PET. All three mutant
variants performed differently than the WT enzyme under this observation, with the S238Y
and the N212A mutants showing 3.3- and 1.4-fold increased activity, respectively. In
contrast, the I208V mutant barely altered the crystalline structure of PET (Figure 5).
These results provided some clues about the mechanism of action of the IsPETase
mutant variants created in this study. First, our MD calculations predicted an increased
binding affinity of PET to the I208V mutant compared to the WT enzyme (Table 3). How-
ever, in vitro assays showed a decreased degrading activity. A possible explanation to
these results is that the substitution of isoleucine for valine disrupts several non-covalent
interactions formed between the Cδ of the isoleucine and the side chain atoms of H237, a
key residue part of the catalytic triad (Figures 2 and 6). These interactions orient the imida-
zole ring of H237 towards S160, allowing its deprotonation [18,20]. Thus, the substitution
I208V may have contributed to a greater flexibility of H237, reducing the enzyme turnover.
On the other hand, the S238Y mutation allows the aromatic side chain of the introduced
tyrosine residue to position favourably for a π-π stacking interaction with the PET substrate
(Figure 6), similar to that observed when this residue is mutated to phenylalanine [18,42].
This interaction could lead to a better affinity of the mutated enzyme for PET relative to
the WT serine residue. Finally, despite N212 not being directly involved in the recognition
of the substrate, its mutation to alanine could have a favourable structural impact on the
Polymers 2023, 15, 1779 12 of 15

enzyme’s mechanism. In the WT enzyme, N212 is located at the beginning of a α-helix and
points to the solvent. The N212A mutation could make this portion of the helix move closer
to the rest of the protein, affecting the loop 204–210 that we speculate moves to narrow the
PET binding channel. In addition, the displacement of the 204–210 loop could position the
catalytic residue D206 in a more favourable location for catalysis (Figure 6).

Figure 5. Engineered IsPETase provoke changes in the crystalline structure of PET. The crystallinity
of enzyme-treated PET was analysed by DSC.

Figure 6. Stereoview of the superposition of IsPETase WT (tan) and the mutant variants, I208V
(magenta), N212A (green), and S238Y (yellow) with PET bound. PET is represented as balls and
sticks, while the mutated residues are depicted as sticks models. The catalytic residue S160 is also
shown.

4. Conclusions
In the present study, IsPETase mutants were engineered for enhancing PET-degrading
activity using a computational rational design approach of the enzyme’s active site. This
was achieved by comparing the 3D structure of IsPETase with that of homologous esterase-
like enzymes (cutinases and lipases). In particular, the S238Y substitution provoked a
substantially increased PET-degrading activity, expressed in the form of modifications of
the PET surface and crystallinity loss, when using highly crystalline PET from commercial
PET bottles as substrate. To date, PET recycling is mostly conducted via thermal treatments
that produce dangerous gas emissions, linked to severe health and environmental issues.
The results presented here prove the concept that taking advantage of IsPETase’s recent
Polymers 2023, 15, 1779 13 of 15

evolution to adopt a PET-degrading activity could facilitate PET pollution management in

a sustainable manner and that the introduction of single-point mutations in the enzyme
architecture can significantly impact its applicability in the recycling industry. Moreover, the
methodology presented in this study provides a first step towards rapidly identifying new
potential mutations of the enzyme for enhancing its activity. This investigation adds new
information about the important role of I208 in catalysis and contributes to understanding
the mechanism of PET degradation by IsPETase.
It has been shown that treatment of the PET surface with physical or chemical agents
(detergents, organic solvents, heat, or alkali) enhances IsPETase activity, most likely by
promoting an amorphous conformation of the polymer chains and improving the enzyme’s
accessibility to the substrate. Here, we identified that the performance of IsPETase is
improved when PET suffers mechanical stress over its smooth surface. This finding could
be highly important in the recycling context since it provides new information for designing
pre-treatments for the enzymatic treatment of PET pollution that accumulates in landfills
and the environment.

Supplementary Materials: The following supporting information can be downloaded at: https:
//[Link]/article/10.3390/polym15071779/s1, Figure S1: Multiple sequence alignment of
IsPETase homologous enzymes.
Author Contributions: Conceptualization, M.D.G. and L.C.-M.; methodology, M.D.G., Y.P.-C., S.C.,
A.D. and L.C.-M.; formal analysis, M.E.S., M.D.G., Y.P.-C., V.A.-J., S.C., K.V., A.D. and L.C.-M.;
investigation, M.E.S., M.D.G., Y.P.-C., V.A.-J., S.C., K.V., A.D. and L.C.-M.; writing—original draft
preparation, M.E.S., M.D.G. and L.C.-M.; writing—review and editing, M.E.S., M.D.G., Y.P.-C., V.A.-
J., S.C., K.V., A.D. and L.C.-M.; supervision, M.D.G. and L.C.-M.; project administration, L.C.-M.;
funding acquisition, M.D.G., Y.P.-C. and L.C.-M. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was funded by Corporación Ecuatoriana para el Desarrollo de la Investi-
gación y Academia-CEDIA, grant “Degradación de polímeros orgánicos mediante el uso de enzimas
recombinantes”, and Universidad de las Américas-UDLA, grant number [Link].20.03.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors would like to thank to Corporación Ecuatoriana para el Desarrollo
de la Investigación y Academia-CEDIA for the financial support given to the present research,
development, and innovation work through its CEPRA program, especially for the “Degradación de
polímeros orgánicos mediante el uso de enzimas recombinantes” fund, supported also by Universidad
Técnica de Ambato (UTA) (UTA-CONIN-2022-0156-R). In addition, this work has been possible thanks
to the equipment provided by the Ecuador–Spain Debt Swap Program, UTA’s Department of Research
and Development (DIDE), ESPE’s Centre for Nanoscience and Nanotechnology and UDLA.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.

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