Journal of Hazardous Materials 460 (2023) 132449

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Journal of Hazardous Materials

journal homepage: www.elsevier.com/locate/jhazmat

Enzymatic polyethylene biorecycling: Confronting challenges and shaping

the future
Jin Jin a, Jane Arciszewski b, Karine Auclair b, Zongchao Jia a, *
a
Department of Biomedical and Molecular Sciences, Queen’s University, 18 Stuart Street, Kingston, ON KL7 3N6, Canada
b
Department of Chemistry, McGill University, 801 Sherbrooke St. West, Montréal QC H3A 0B8, Canada

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Biorecycling PE is challenging due to its

inert chemical structure.
• The discovery and development of PEa­
ses are vital for tackling plastic
pollution.
• More evidence is needed to support
enzymatic oxidation of untreated PE.
• Unconfirmed PEases from microorgan­
isms and insects need further study.
• PE biorecycling is likely to require a
multi-enzyme process with synergistic
effect.

A R T I C L E I N F O A B S T R A C T

Editor: Lingxin Chen Polyethylene (PE) is a widely used plastic known for its resistance to biodegradation, posing a significant
environmental challenge. Recent advances have shed light on microorganisms and insects capable of breaking
Keywords: down PE and identified potential PE-degrading enzymes (PEases), hinting at the possibility of PE biorecycling.
Polyethylene biodegradation Research on enzymatic PE degradation is still in its early stages, especially compared to the progress made with
PE-degrading enzymes
polyethylene terephthalate (PET). While PET hydrolases have been extensively studied and engineered for
Enzymatic oxidation
improved performance, even the products of PEases remain mostly undefined. This Perspective analyzes the
Synergistic effect
Evolutional engineering current state of enzymatic PE degradation research, highlighting obstacles in the search for bona fide PEases and
suggesting areas for future exploration. A critical challenge impeding progress in this field stems from the inert
nature of the C-C and C-H bonds of PE. Furthermore, breaking down PE into small molecules using only one
monofunctional enzyme is theoretically impossible. Overcoming these obstacles requires identifying enzymatic
pathways, which can be facilitated using emerging technologies like omics, structure-based design, and
computer-assisted engineering of enzymes. Understanding the mechanisms underlying PE enzymatic biodegra­
dation is crucial for research progress and for identifying potential solutions to the global plastic pollution crisis.

* Corresponding author.
E-mail address: jia@queensu.ca (Z. Jia).

https://doi.org/10.1016/j.jhazmat.2023.132449
Received 24 April 2023; Received in revised form 25 August 2023; Accepted 30 August 2023
Available online 1 September 2023
0304-3894/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-
nc/4.0/).
J. Jin et al.
1. Introduction
Among various types of plastics, polyethylene (PE) is a widely used,
low-cost, and versatile synthetic polymer, constituting 36% of the total
plastic market [36]. PE is a high molecular weight (MW, 30,000–6,000,
000 for commercial PE) hydrophobic compound, composed solely of
inert C-C and C-H bonds. PE can be broadly classified into two types:
low-density PE (LDPE) and high-density PE (HDPE), based on the
polymerization methods employed during synthesis [28]. LDPE is pro­
duced through high-pressure polymerization of gaseous ethylene,
resulting in a polymer with branched chains. It finds extensive appli­
cation in manufacturing packaging materials, plastic bags, and agri­
cultural mulching films. On the other hand, HDPE is produced by
subjecting petroleum to intense heat, converting ethylene gas into a
linear polymer. Due to its higher density, HDPE exhibits exceptional
durability and is therefore widely used in various industrial and
daily-life products, including detergent bottles, garbage containers, and
water pipes. LDPE and HDPE account for approximately 40% and 60%
of the PE market share, respectively.
The exceptional stability and low degradation rate of PE have led to a
significant accumulation of PE waste, causing severe environmental
impacts. The degradation rate of PE in nature varies depending on the
environment. For example, the estimated half-lives of HDPE range from
58 years (bottles) to 1200 years (pipes) in the marine environment and
from 250 years to 5000 years on land, respectively. The full degradation
of commonly used LDPE bags requires an estimated 3.4 years in the
marine environment and 4.6 years on land [11]. A total of 97 million
tons of PE waste were generated globally in 2015 [27], and the amount
of discarded PE continues to surge rapidly, aligning with the exponential
growth of plastic production. The accumulation of PE in the environ­
ment is a serious threat to both terrestrial and marine ecosystems, as
well as human health. As highlighted by Yao et al. [111], the presence of
floating PE particles of various sizes in marine environments can lead to
entanglement or ingestion by animals, resulting in organ damage, gen­
otoxicity, and even mortality. Likewise, the infiltration of PE micro­
particles into the soil can detrimentally affect soil quality and the
well-being of terrestrial organisms [88]. Furthermore, the presence of
PE microplastics or nanoplastics in the human body has been linked to
various health issues, including cancer, cardiovascular disease, and
chronic inflammation [3].
Traditional methods of managing PE waste encompass landfilling
and incineration, often leading to unintentional release into the envi­
ronment [74]. The breakdown of PE waste in landfills predominantly
happens through photodegradation, a time-consuming process known
for its low efficiency [112]. Incineration, liquefaction, and gasification
methods employed for PE waste treatment require elevated tempera­
tures at the cost of substantial energy consumption. These processes also
generate toxic gases due to incomplete combustion, including carbon
monoxide (CO), hydrogen sulfide (H2S), polycyclic aromatic hydrocar­
bons (PAHs), dioxins, and furans, thereby posing severe health risks and
contributing to global warming. Dioxins, for instance, can induce
harmful effects on various bodily systems, impacting the nervous sys­
tem, immune responses, and leading to carcinogenicity, hepatotoxicity,
and metabolic and enzyme toxicity [57]. Similarly, furans are widely
recognized as harmful chemicals with severe health implications,
including cancer development and compromised immunity [69].
Incineration, being a dominant method in plastic waste management,
results in remarkably high emissions and serves as the primary driver of
greenhouse gas emissions. Globally, plastic package incineration
contributed to approximately 16 million metric tons of CO2 emissions in
2015 [32]. Consequently, this has sparked interest in biodegradation
and biorecycling as more eco-friendly alternatives to landfilling and
incineration.
Numerous review articles have discussed PE biodegradation by mi­
croorganisms from soil, waste sludges, and marine sediments, including
algae [15], bacteria [108,123,28,48,86], and fungi [100,119,67]. In
2
Journal of Hazardous Materials 460 (2023) 132449
recent years, researchers have been investigating the PE chewing and
biodegrading abilities of insects such as worms and termites [49,82].
Additionally, they have explored the contribution of their gut microor­
ganisms in this process [110].
In this Perspective, we delve into the advancements of PE-degrading
enzymes (PEases) that facilitate the introduction of functional groups
into the PE polymer, thereby breaking down PE into smaller molecules.
These enzymes, found in diverse organisms, hold the potential to offer
an eco-friendly solution to plastic pollution. However, developing
practical and effective PEases poses formidable challenges due to PE’s
exceptional recalcitrance, requiring the coordinated action of multiple
enzymes for effective biodegradation. Our article strives to provide
comprehensive insights into these hurdles and guide future research in
this burgeoning field.
2. PE biodegradation pathways
The mechanism of PE biodegradation has been investigated, and
pathways have been proposed [112,114,123,25,86];), although they
remain largely incomplete and unconfirmed. For example, in the review
articles [114,120,121,123–125], the detailed explanations of the
mechanism mainly focused on the decomposition of short-chain alkanes
(C<24) in microorganisms or by enzymes. However, the original
modification and breakage of long-chain PE were only briefly attributed
to the synergistic actions of biotic and abiotic factors. In the research
papers [25,112], specific mechanisms of PE degradation were specu­
lated based on the metatranscriptomic analysis of PE-degrading micro­
organisms or the study of model substrate metabolism, lacking
experimental support. The most efficient PE biodegradation thus far
appears to be achieved by waxworms, likely due to the structural sim­
ilarity between PE and beeswax, a main food source of waxworms [9].
Under this circumstance, the mechanism of PE degradation in insects
and their intestinal microbiota has been explored, providing insights
into the enzymes involved in PE biodegradation [47]. The proposed PE
biodegradation pathway is divided into four stages based on the prod­
ucts of the enzymatic reactions: biodeterioration, depolymerization,
assimilation, and mineralization (Fig. 1). In the biodeterioration stage,
microbes adhere to the PE surface and use their extracellular enzymes to
introduce functional groups, such as hydroxyl, carbonyl, and ester
groups, through free radical and oxidation-reduction reactions. Hy­
droxylation is considered the starting point of PE biodegradation, with
Fig. 1. Proposed mechanism of PE enzymatic degradation.
J. Jin et al.
exogenous oxidation occurring in-chain and at the termini, similar to the
oxidation of shorter-chain alkanes by alkane hydroxylases. In-chain
hydroxylation is preferred due to its lower energy barrier, although
few oxidative enzymes have been discovered that cause in-chain
breakages in PE. After further oxidation reactions (e.g., to esters), the
modified PE polymer is depolymerized by extracellular hydrolases
and/or oxidases into small molecules, generating, for example, dicar­
boxylic acids, alcohols, fatty acids, ketones, aldehydes, and esters
(Fig. 2). The resulting products are expected to find their way into the
cell (assimilation), where several transformation steps generate useful
metabolites. Finally, in the mineralization stage, the metabolites are
broken down to form CO2 and H2O (Fig. 1).
PEases, as key enzymes engaged in PE biodegradation, facilitate the
modification of PE by introducing functional groups such as hydroxyl,
carbonyl, and ester groups, and subsequently catalyze the breakdown of
PE into smaller molecules. PEases encompass various types of enzymes,
including but not limited to hydroxylases, dehydrogenases, mono­
oxygenases, and hydrolases. The main challenge in the development of
enzymes for PE recycling lies in the intricate and multi-stage process of
PE biodegradation. Currently, the limited understanding of these path­
ways and of each individual step hampers the development of effective
enzymes for PE recycling. For instance, the PE degradability of Tenebrio
molitor has been assessed by using PE as its diet. PE degradation was
observed in the gut microbiota of mealworms, and alkene groups were
detected in the frass of PE-fed worms [10]. The formation of alkenes was
subsequently elucidated through the catalytic activities of CYP152
peroxygenases [80] and decarboxylase OleT [34], but the exact
Fig. 2. Enzymatic reactions involved in PE biodegradation. (AlkH: Alkane hydroxylase; AlcDH: Alcohol dehydrogenase; AldDH: Aldehyde dehydrogenase; BVMO: Baeyer-
Villiger monooxygenase.).
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Journal of Hazardous Materials 460 (2023) 132449
mechanism in PE degradation remains uncertain. In a study investi­
gating the degradability of PE by insects, the persistent weight of
Galleria mellonella served as evidence that the insect can utilize PE as its
exclusive energy source [47]. However, even though the host was
implicated in partial PE degradation, the evidence was inadequate to
definitively establish whether long-chain hydrocarbons (C>20) could be
metabolized by the larvae. To delve into comprehensive metabolic
pathways of aliphatic-compound degradation in bacteria, a blend of
isotope-tracking and proteomics analysis was employed. This approach
focused on Alcanivorax that was subjected to incubation with both PE
and alkanes of differing lengths [117]. Short and medium-chain alkanes
have been determined to undergo terminal hydroxylation catalyzed by
AlkB. Subsequently, it is believed that subterminal hydroxylated alkanes
are converted into esters through the action of the Baeyer-Villiger
monooxygenase (BVMO) AlmA. These esters are further hydrolyzed by
esterases. The hydroxylated alkanes resulting from this hydrolysis are
then oxidized into fatty acids, entering the β-oxidation pathway.
Regarding untreated PE, it is postulated that the primary chain scission
occurs through the generation of extracellular reactive oxygen species
(ROS) and nonspecific in-chain oxidation of high-molecular-weight PEs.
However, the mechanism of assimilating long-chain alkanes (such as
C50) remains elusive. Interestingly, a computer-based molecular dock­
ing study has meticulously analyzed the PE degradation pathway
involving individual enzymes and potential synergistic effects of mul­
tiple enzymes [92]. However, the lack of verification data for the ob­
jectives is notable. Moreover, the substrate utilized in this study,
dodecane, cannot faithfully represent the authentic degradation process
J. Jin et al.
of PE with a long carbon chain.
3. PE degradation by PEases: the status quo
Enzymes involved in the degradation of PE have been identified
through metagenomic studies, including transcriptomic and proteomic
analyses of microbes and insects cultured in the presence of PE (Fig. 3).
However, there have been very few reports of cell-free PEase activity,
especially from purified, recombinant enzymes (Table 1). In a recent
study [26], several PE- and PET-degrading enzymes, including lipases,
esterases, cutinases, and hydrolases, were proposed based on macro
transcriptomic profiling of a reconstituted bacterial community
composed of three strains isolated from marine sediments. Three of the
putative PEases were further produced recombinantly and shown to
cause morphological changes in PE films. Gao et al. [25] also applied
transcriptomic analysis to a marine fungus, Alternaria alternata FB1,
isolated from plastic debris, and detected PE degradation, leading to the
identification of enzyme sequences potentially associated with PE
degradation. The PE degradation activity of two of these enzymes, a
glutathione peroxidase and a laccase, was verified by scanning electron
microscopy (SEM) and gel permeation chromatography (GPC) analyses
of the reacted PE. Independently, Zhang et al. [120] monitored the ef­
fects of a laccase from Psychrobacter sp. NJ228 (PsLAC) on untreated PE
through various detection methods, revealing PE weight loss, reduction
in hydrophobicity and crystallinity, formation of oxygen-containing
polar functional groups, and morphological changes on the PE surface.
As the most efficient PE biodegraders currently known, waxworms
are an attractive source of potential PEases. Through proteomic analysis,
Sanluis-Verdes et al. [91] identified two enzymes from the saliva of the
waxworm G. mellonella larvae: Demetra (annotated as a phenoloxidase)
and Ceres (with high homology to hexamerins). Raman and Fourier
transform infrared spectroscopy (FTIR) analyses of PE after treatment
Fig. 3. Discovery and development of PEases.
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Journal of Hazardous Materials 460 (2023) 132449
with Demetra (no chemical or physical pre-treatment was applied)
revealed oxidation of PE to ketones at room temperature and neutral pH,
while treatment of PE with Ceres led to no visual deterioration. When
the PE-degrading effect of the waxworm G. mellonella was first reported
[9], some concerns were raised regarding the possibility of FTIR peaks
being due to protein contaminants and the absence of an inactive con­
trol; hence, a 13C-labelled experiment was recommended [107]. How­
ever, the recent report of PEase activity of the Demetra enzyme [91]
further supports the conclusions of Bombelli et al. [9]. More recently, a
laccase mediator system (LMS) consisting of two recombinant laccases
from Botrytis aclada (BaLac) and Bacillus subtilis (BsLac) along with three
mediators was applied to UV- and heat-treated LDPE. It was found that
this system enhanced the oxygen content of PE and decreased its average
MW by 40% [112]. The authors attributed the generation of carbonyl
groups on PE to the UV pre-treatment and found that PE underwent
greater oxidation with the formation of C-OH and C-O-C bonds in the
presence of the LMS compared to UV exposure alone. In a separate study,
the recombinant multi-copper oxidase abMCO from Rhyzopertha
dominica was reported to degrade PE films [123], based on the reduction
of the water contact angle (WCA) and the MW of PE.
Engineering bacterial strains to produce PEases is an important
strategy in the search for PE recycling methodologies. It has the
advantage of potentially combining multiple catalytic activities in one
agent. Cells engineered to heterologously express a specific alkane hy­
droxylase have been reported to modify PE, including Escherichia coli
BL21 harboring the alkane hydroxylase gene alkB from Pseudomonas sp.
E4, a strain that can degrade low MW PE (LMW-PE, i.e., MW of
1700–23,000, compared to 30,000–6,000,000 for commercial PE)
[115]. Further studies with an E. coli BL21 strain harboring the alkB gene
from P. aeruginosa E7 and the genes encoding rubredoxin and rubre­
doxin reductase revealed an enhanced LMW-PE degradability compared
to the E. coli cells that produced AlkB alone [37]. It is unclear whether
J. Jin et al. Journal of Hazardous Materials 460 (2023) 132449

Table 1
Cell-Free Enzymes with Verified PE Degradation Capability in Literature.
Putative PEase Original PE material Condition Verification results Reference

IR195_09930, hydrolase
(QPI65985.1)
IR195_04495, alpha/beta
hydrolase (QPI64981.1)
IR196_16650, alpha/beta
hydrolase (QPA27412.1)
Glutathione peroxidase (evm.
model.contig_3.359)
Laccase (evm.model.
contig_8.535)
rPsLAC, laccase (UVG67878.1)
Arylphorin subunit alpha
(Demetra), phenoloxidase
(XP_026756396.1)
Halomonas venusta PE film (Thickness: 0.25 mm), from pH7.5, 30 C,◦
• SEM: Degradation effects on PE films [26]
Good Fellow Company, UK 24 h
Halomonas venusta
Ochrobactrum
anthropi
Alternaria PE film (Thickness: 0.25 mm), from Sea water, • SEM: Cracks and signs [25]
alternata FB1 Good Fellow Company, UK 30◦ C, 48 h
• GPC: 18% and 7% reduction in Mn and Mw
• Two enzymes showed synergetic
degradation effect
Psychrobacter sp. PE particles (1000 μm)PE film pH3.0, 30◦ C, • Weight loss: 13.2% in PE particle [120]
NJ228 (30 mm×20 mm), from (Thermo 7d • WCA: Decreased from 97.2◦ to 74.6◦
Fisher Scientific) • FTIR: Carbonyl and carboxyl groups
• Crystallinity: 25% reduction
• SEM: Enormous damage to surface and
interior
Saliva of Galleria Commercial PE film, PE 4000, from RT, 5d • AFM-RAMAN: Demetra caused occasional [91]
mellonella Larvae Sigma-Aldrich visual effect and oxidation signature; Ceres
caused oxidation and deterioration of PE

Hexamerin acidic juvenile • GC-MS: 2-ketones from 10 to 22 carbons in

hormone-suppressible protein 1
(Ceres), hemocyanin
(XP_026756459.1)
BaLac, laccase (AFC76164.1)
BsLac, laccase (WP_003243170)
AbMCO, laccase,
(EHX0593423.1)
products
Botrytis aclada PE films (Thickness: 20 μm), from 5 days at 30◦ C • SEM: Changes in the microscopic [112]
PetroChina Company Limited UV, in a 150-rpm morphology of the PE films
70◦ C, 3d. With mediators: HBT, water bath
Bacillus subtilis ABTS, TEMPO • FTIR: Carbonyl and alcohol hydroxyl groups
• GPC: The average reduction in Mw by BaLac
is around 40% and, ranged from 31.3% to
52.6% for BsLac. The average reduction in Mn
by BaLac is around 21% and around 30% for
BsLac
Bacteria from LDPE film (a food fresh-keeping bag, 12 h at 42◦ C • SEM: Some damage [123]
Rhyzopertha Mw greater than 130 kD); LDPE
Dominica powder (Mw greater than 80,000) • WCA: Decreased from 78.2 to 68.1
◦ ◦

• GPC: Mn and Mw decreased by 52.20% and

1%
LfLAC, laccase (QDZ99871.1) Lysinibacillus LDPE film (4 × 4 cm) 8w at 28◦ C • Weight loss: 2.79% [121]
fusiformis • FTIR and XPS: -C=O, C=C-, and -C=N-,
group
• SEM: larger cracks and cavities

(AFM-RAMAN: Atomic Force Raman Spectroscopy; FTIR: Fourier transform infrared spectroscopy; GC-MS: Gas chromatography-mass spectrometry; GPC: Gel permeation
chromatography; Mn: Number-average molecular weight; Mw: Weight-average molecular weight; RT: room temperature; SEM: Scanning electron microscopy; WCA: Water
contact angle; XPS: X-ray photoelectron spectroscopy.)

AlkB catalyzes the hydroxylation reaction only or if it is involved in
other steps of PE degradation as well. Mineralization of PE to CO2, e.g.,
via β-oxidation, might be catalyzed by other enzymes of this engineered
E. coli strain. Besides alkane hydroxylases, cytochrome P450 enzymes
(CYPs) have also attracted attention due to their ability to hydroxylate
short degradation intermediates of PE (i.e., linear alkanes, alcohols, and
fatty acids of diverse chain lengths). It was recently proposed that bac­
teria engineered to produce specific CYPs might contribute to PE
degradation [114], although experimental data is needed to support this
hypothesis.
Secreted enzymes such as manganese peroxidases (MnP), lignin
peroxidases (LiP), and laccases have been suggested to play a role in the
oxidation of waste PE in the environment. The first report describing
extracellular enzyme activity on PE films attributed the degradability to
LiP [83]. Subsequently, partially purified MnP from the extracellular
matrix of the lignin-degrading fungus IZU-154 or from a Phanerochaete
chrysosporium culture was reported to contribute to the degradation of
PE, while the laccase and LiP purified from this extracellular matrix
were not [35]. Another study examined the degradation of chemically
oxidized PE using black liquor from the paper industry and identified a
synergistic effect between LiP and MnP activities [70]. MnP and laccase
secreted by Staphylococcus saprophyticus were also found to demonstrate
some PE degradation when exposed to LDPE [73]. While some of these
peroxidases have been characterized, such as the MnP from IZU-154
[63], more studies are needed with recombinantly expressed, purified
enzymes to further confirm and characterize their effects on the struc­
ture of PE.
Laccases are the most studied PEases, with some of the rare reports of
recombinant enzymes being used rather than enzymes isolated from
biological sources [112,120,25]. Treatment of PE with both a partially
purified laccase from Trametes versicolor IFO 6482 and hydroxybenzo­
triazole (HBT) caused a reduction in the elongation and tensile strength
of PE [23]. A crude laccase sample from the extracellular matrix of the
actinomycete Rhodococcus ruber was also reported to show oxidative and
degrading activity on PE [94]. In this study, UV irradiation was applied
first to increase the number of carbonyl groups before incubation with
the laccase from R. ruber. In contrast, a purified commercial laccase from
T. versicolor did not have this effect, suggesting that some isoforms are
more promising than others or that some mediators in the crude laccase
sample may play an important role in the reaction. Although hypo­
thetical PE biodegradation mechanisms have been proposed based on
the docking of dodecane to a MnP and a LiP, as well as a non-specific
peroxygenase [92], much remains unknown about the precise effect of
these enzymes on PE. Two laccase-like multicopper oxidases (LMCOs),

5
J. Jin et al.
named AFLA_006190 and AFLA_053930, from the Aspergillus flavus
fungus found in the G. mellonella wax worm’s gut were identified as
PEases based on a preliminary screening process [122]. However, their
ability to degrade PE requires further experimental verification.
PE enzymatic degradation is a rapidly growing area of research.
However, the understanding of these reported PEases is currently
confined to degradability assessment, mainly focusing on laccases.
Further in-depth characterization of PEases should encompass aspects
such as comprehensive validation, elucidating the catalytic mechanisms,
conducting kinetic analysis, performing structural investigations, and
undertaking endeavors like de novo enzyme design and engineering. In
this context, potential challenges might arise concerning the choice of
the expression system, improving expression yield, as well as the opti­
mization of solubility and stability of recombinant PEases.
4. Challenges in the search for PEases
4.1. Challenges in identifying authentic PEases through omics approaches
Currently, the main methods to identify PEases involve tran­
scriptomic and proteomic analyses of microbes that can grow in the
presence of PE. These approaches have been increasingly applied to
study PE degradation by bacteria, fungi, algae, as well as the gut
microbiota of insects and insect hosts (Fig. 2). Purohit et al. [84] con­
ducted a review on the application of metagenomic analysis of microbial
populations to expedite the discovery of PEases. Furthermore, a strategy
combining RNA-sequencing and lipidomic analyses has been developed
to identify R. ruber genes encoding potential PEases [31]. This led to the
discovery of a strong induction of pathways related to alkane degrada­
tion and β-oxidation of fatty acids in R. ruber. However, a bottleneck in
the ability of this bacterium to degrade high molecular weight PE
(HMW-PE) was observed, consistent with the extremely slow biodeg­
radation of PE in the environment. Transcriptomics studies of Rhodo­
coccus opacus R7 [118] identified genes potentially involved in PE
biodegradation through quantitative real-time PCR (RT-qPCR). Predic­
tive bioinformatics analyses revealed three up-regulated genes anno­
tated as laccase-like multicopper oxidases (LMCO1, 2, and 3), which
may be responsible for the first PE oxidation step. Other up-regulated
transcripts encoded oxygenases or oxidases that may participate in the
following oxidation steps. For example, the R7 alkane hydroxylase gene,
alkB, was up-regulated 100-fold when R. opacus was in the presence of
PE. Oberbeckmann et al. [71] employed an integrated
metagenomics-metaproteomics approach to assess the production of
PEases such as laccases and MnP during PE degradation. In another
study, Mishra et al. [66] utilized genomic sequencing to identify genes
associated with biofilm formation and PE biodegradation in the micro­
algae cyanobacteria Jacksonvillea sp. ISTCYN1. Their analysis predicted
the presence of several enzymes, including laccases, esterases, lipases,
thioesterases, and peroxidases, which play important roles in plastic
degradation. Transcriptomics studies of a PE-degrading bacterial com­
munity identified five putative PEases annotated as esterases, cutinases,
and hydrolases, of which the PE degradation effect of a putative esterase
and two hydrolases was claimed [26]. Subsequently, transcriptome
analysis revealed that, in Alternaria alternata FB1 cultured in the pres­
ence of PE, 153 potential PEases may be produced, including peroxi­
dases, laccases, hydroxylases, monooxygenases, oxygenases,
dehydrogenases, oxidoreductases, oxidases, reductases, esterases, li­
pases, and cutinases. In the presence of PE, the transcription levels of a
laccase-encoding gene, a peroxidase-encoding gene, and an
oxidoreductase-encoding gene were increased by 23, 44, and 102-fold,
respectively. The PE degradation activity of only two oxidative en­
zymes, a glutathione peroxidase and a laccase, was further investigated
[25]. Additional work is still required to confirm their PE-degrading
activities. Proteomics analysis of a marine bacterium Alcanivorax has
been performed following incubation with PE and its metabolic in­
termediates [117]. This study identified enzyme genes with altered
6
Journal of Hazardous Materials 460 (2023) 132449
expression and pinpointed specific nodes of their roles in the pathway.
Omics analysis has also been applied in studies of gut microbes and
saliva from PE-degrading insects. The functional analysis of insect gut
contents has shown promising results in identifying PEases, including
increased enolase activity during the metabolism of ingested PE [50].
Additionally, the protein composition of G. mellonella salivary glands
(after PE consumption) has been analyzed, revealing a total of 47 en­
zymes likely involved in PE metabolism, including oxidoreductases such
as (3 R)− 3-hydroxyacyl-CoA dehydrogenase, aldehyde dehydrogenase,
and catalase; transferases including acetyl-CoA C-acyltransferase and
glutathione transferase; and hydrolases such as multiple
inositol-polyphosphate phosphatase [78]. Transcriptome analysis of
T. molitor larvae revealed that mealworms act as downstream de­
composers of PE and that fatty acid degradation pathways may play an
important role in the breakdown of PE degradation intermediates [126].
In addition, as mentioned earlier, proteomics analysis was used to
explore enzymes responsible for PE degradation in the saliva of
G. mellonella, and two enzymes with PE oxidation activity were purified
by size exclusion and ion exchange chromatography [91].
The potential offered by omics analyses in PE biodegradation is
promising. However, the sheer volume of data acquired by omics anal­
ysis poses significant challenges. Moreover, the inaccuracy of annota­
tions can lead to a misunderstanding of the roles of these putative PEases
in the intricate process of PE biodegradation. Furthermore, the incom­
plete coverage of the entire genome and proteome may result in the
oversight of crucial enzymes. To address this problem, a specialized
database, PlasticDB, has been developed to implement analytical tools
that accept inputs, including genes, genomes, metagenomes, tran­
scriptomes, metatranscriptomes, and taxa data [24]. Despite recent
progress, the application of omics in PE biodegradation is still limited. In
the case of microbes, only 2% of environmental microbes can be
cultured in the laboratory [105], leaving a vast proportion of untapped
fungi, bacteria, and other extremophiles for future exploration. Con­
cerning insects, more precise omics analysis should extend across
various growth stages and tissue distributions to ascertain potential
PEases. Moreover, the integration of multiple omics holds the promise of
yielding more robust evidence. Additionally, the landscape becomes
further complex, as negative results with purified enzymes might not
have been published. This underscores the importance of transparently
sharing both positive and negative outcomes to foster a comprehensive
understanding of PE enzymatic degradation.
4.2. Debatable enzymatic degradation of untreated PE
The initial step of PE degradation, likely involving oxidation of inert
C-C and C-H bonds, is expected to be the limiting step based on the high
stability of these bonds. To date, most studies of PE biodegradation have
used PE plastic that had undergone abiotic pre-treatment consisting of
either thermo-irritation, UV photooxidation, or physicochemical treat­
ment. These processes are believed to initiate the oxidation of PE and
facilitate enzymatic degradation [28]. However, an opposing viewpoint
arises with the observation of new functional groups, such as carbonyls
produced from untreated PE by various microbes. This provides direct
evidence that enzymatic oxidation of PE is indeed possible [87]. Recent
studies have reported additional evidence of biodegradation of un­
treated PE by microbes and insects (Table S1). Further examples include
PE exposed to Stenotrophomonas sp. and Achromobacter sp. Dey et al.
[17], to Lysinibacillus sp. JJY0216 [38], to Agrocybe aegerita mycelium
[7], to a marine bacterial consortium composed of Oceanimonas, Vibrio,
and Paenibacillus [26], and to the marine fungus Alternaria alternata FB1
[25]. Additional PEases are expected to be discovered, particularly
through follow-up studies of the reports highlighted with an asterisk in
Table S1. Such studies would include omics analyses, enzymatic activity
classification, identification of microbes in the insect gut, and exami­
nation of PE metabolism in these promising organisms. Enzymatic
studies of PE degradation suggest that PE oxidation could possibly be
J. Jin et al.
initiated by enzymes. Enzymatic hydroxylation of C-H bonds may occur
at terminal (-CH3) and in-chain (-CH2-) positions in the main carbon
backbone, as well as the tertiary C-H bonds in the carbon chain branches
[114]. The PE degradability by bacteria containing the AlkB gene of
Pseudomonas sp., which encodes an alkane hydroxylase capable of hy­
droxylating alkanes, has offered compelling evidence for this hypothesis
on LMW-PE [37,115]. However, the precise position that AlkB attacks
remains unclear. Additionally, whether AlkB can hydroxylate long-chain
PE with higher MW remains to be determined. Furthermore, the use of
the recombinant PsLAC to degrade untreated PE [120], as well as the
detection of PE oxidation by two recombinant, purified enzymes iden­
tified from waxworm [91], provides more compelling evidence for the
enzymatic oxidation of untreated PE. Inert C-C bonds in PE have posed
further challenges for enzymatic degradation due to their high dissoci­
ation energy. Ligninolytic enzymes, such as laccases, which can cleave
C-C bonds present in lignin, have been documented as capable of
degrading PE [12,36]. PE degradability studies of PsLAC [120]
demonstrated that incubation with recombinant PsLAC for 7 days at
30 ◦ C resulted in a 13.2% weight loss of PE. AbMCO, a laccase from an
intestinal microbe of Rhyzopertha dominica, resulted in a 52.2% reduc­
tion in Mn and a 1% reduction in Mw of PE film at 42 ◦ C for 12 h [123].
Additional ligninolytic enzymes, such as LiP and MnP, have been sug­
gested to potentially contribute to PE degradation based on observations
of partially purified enzymes or attributed activities. It is noteworthy
that the strength of the C-C bonds in PE (330–370 kJ mol-1) exceeds that
of lignin (240–425 kJ mol-1) [12]. The involvement of mediators has
been reported to improve laccase-catalyzed PE degradation, overcoming
the hindrance derived from C-C bonds [112]. However, further enzy­
matic characterization and structural studies are required to explain the
mechanism.
The available experimental data confirming the direct degradation of
PE by annotated hydrolases or esterases is limited. Only one report,
based on three enzymes from a marine bacterial consortium, has stated
that a hydrolase can directly degrade untreated PE [26]. This assertion is
incredibly intriguing and demands deeper exploration, particularly
considering that PE lacks hydrolysable bonds. If these hydrolases are
unequivocally verified as bona fide PEases, they could serve as appealing
initial templates for protein engineering endeavors. Another study has
attributed the PE-degrading effect of three marine bacterial isolates [two
Marinobacter sp. (H-244 and H-246) and one Bacillus subtilis (H-248)] to
the production of three bacterial esterases. This conclusion based on the
significant increase in esterase activity observed during PE degradation
by all three strains [45]. Up-regulated expression of lipases, esterases,
and cutinases was detected during the PE degradation process, and es­
ters were detected in the products of PE degradation, indicating that
these hydrolases appear to be involved in PE degradation. The specific
step in which hydrolases or esterases are involved remains unclear. A
mechanism study of PE degradation conducted by Zadjelovic et al. [117]
revealed that esterases participate in hydrolysis after oxidized PE is
converted to an ester by BVMO. Interestingly, a docking study used
cutinase, a cutin hydrolase, as a negative control for PE degradation
[92]. The study found that cutinase exhibited the least negative
enzyme-PE score and the smallest enzyme cavity, indicating weaker
attraction for PE compared to the other tested enzymes. From these
findings, it is plausible that hydrolases are only involved in the PE
degradation steps that incorporate a hydrolysable group, as depicted in
Fig. 2. However, conducting further verification is imperative to ascer­
tain the specific role(s) of the esterases in the process of PE degradation.
4.3. Structural characterization of potential PEases
One obstacle in the design and optimization of PEases is the lack of
structural information about these enzymes, which could guide protein
engineering research. In the early years of this field, mutagenesis aiming
to improve the redox potential of laccase was performed but failed due
to limited structural knowledge [109]. The structure of a laccase from
7
Journal of Hazardous Materials 460 (2023) 132449
the fungus Trametes versicolor (Lv) has since been determined, revealing
four copper sites [81]. Additionally, the structure of glycosylated Lv in
complex with an arylamine, 2,5-xylidine, has been reported, providing a
model for engineering more efficient laccases or improving substrate
promiscuity [8]. Furthermore, the structure of a thermostable laccase
from bacteria enabled the identification of an interdomain loop as a
main contributor to thermostability [21]. In parallel, a range of protein
modeling techniques has been used to identify diverse substrate binding
modes for T. versicolor laccase, confirming the important roles of
Asp-206 and His-458 in substrate recognition and lending promise to
future rational optimization [64]. Meanwhile, structural insights into
the oxygen reduction mechanism of laccase in the context of water
bioremediation have been documented [2].
Structural studies of laccases with PE degradability have been
limited. One of the few X-ray structures available is BaLac, which was
obtained using a Pichia pastoris expression system with subsequent
deglycosylation [75]. Additionally, the structure of a mutant variant of
BaLac, L499M, was resolved, revealing a structure remarkably similar to
the wildtype. Intriguingly, the mutant exhibited a 140 mV decrease in
redox potential. Another laccase, known as BsLac, had its structure
resolved [22]. In the subsequent study, the complex structure of BsLac
with the substrate ABTS, which may act as a mediator in PE enzymatic
degradation, was determined, providing insights into the binding sites
with ABTS [20]. Further structures containing a monovalent copper ion
(Cu+), dioxygen, or hydroxyl ion within BsLac were analyzed, revealing
the oxidation mechanism of the laccase [5,76]. Structural analyses of
mutants leading to alterations in redox potential and oxidation capacity
further contributed to explaining the oxidation mechanism of BsLac [14,
18], as well as revealing novel binding sites for ABTS [56]. Subsequent
to these structural studies, the PE degradability of BaLac and BsLac was
published [112]. However, the specific substrates targeted by different
laccases during PE transformation remain unclear. The question remains
whether these enzymes act on PE directly or on PE breakdown in­
termediates. Alternatively, laccases may enhance the redox potential of
mediators, potentially facilitating the degradation process.
The structure of the long-chain alkane monooxygenase LadA in
complex with its coenzyme has been published, suggesting a flavopro­
tein monooxygenase mechanism [54]. CYP153D17, an enzyme from
Sphingomonas sp., has been crystallized in complex with dodecane,
revealing several features of substrate binding by this enzyme [52].
Insights into how these enzymes catalyze the degradation of n-alkanes
and what determines their substrate specificity have also been obtained
[39]. A better understanding of alkane hydroxylases can shed light on
the mechanism of PEases and guide protein engineering efforts, yet
further structural studies are needed.
4.4. The complexity of PE degradation products limits the identification of
new PEases
The degradation of PE can lead to a highly complex mixture of
products, including both soluble compounds such as alcohols, alde­
hydes, ketones, ethers, and acids, as well as insoluble compounds such as
long-chain alkanes. The identification of these compounds requires a
combination of techniques, including high-performance liquid chro­
matography (HPLC), FTIR, X-ray photoelectron spectroscopy (XPS),
nuclear magnetic resonance (NMR), and gas chromatography-mass
spectrometry (GC-MS). Therefore, this severely complicates the mea­
surement of PEase activity. The products of untreated PE reacted with
the waxworm saliva enzyme Demetra were analyzed by GC-MS and GC-
MS/MS. This analysis revealed the presence of a mixture of 2-ketones
ranging from 10 to 22 carbons in length [91]. In another study, the
products of UV-pretreated PE reacted with BaLac and BsLac in the
presence of different mediators were monitored by GC-MS. The analysis
revealed oxygenated products such as ketones, alcohols, aldehydes,
acids, esters, and ethers. The carbon chain length of these molecules
ranged from C3 to C20 [112]. The type and distribution of oxidation
J. Jin et al.
products differed between different mediators, indicating that the
selectivity of the LMS may differ. The range of product carbon chain
lengths also varied between the two tested laccases, which harbored
different redox potentials.
Adding to this complexity, GC-MS analysis consistently detected the
presence of plastic additives, including stabilizers and plasticizers, when
commercial PE bags were used as substrates. This finding also furnishes
indirect evidence of PE degradation [91,112]. The effect of these addi­
tives on PE biodegradation remains unclear. Pro-oxidant additives
aimed at enhancing PE biodegradation have been reviewed [28].
Conversely, the impact of different additives, including
oxo-biodegradable and non-oxo additive, has been assessed. Yet, no
conclusive evidence has emerged to suggest that any of these additives
can effectively promote PE biodegradation, either in anaerobic or aer­
obic conditions [96]. Additive-free PE films have been utilized to
investigate the impact of additives on PE biodegradation and to isolate
specific effects of additives [26]. Notably, due to the complexity of the
product mixture, it has been difficult to develop high-throughput
screening methods to identify PEases, which also explains the lack of
PEase engineering studies. In summary, product analysis remains chal­
lenging, and much work is required to further clarify the specific role of
different PEases during PE breakdown.
4.5. Main research gaps in the discovery and catalytic mechanism of
PEases
In recent years, as our comprehension of the biodegradation mech­
anism of PE has steadily deepened and novel enzymes have come to
light, the investigation of PE biodegradation has increasingly focused on
the molecular level. Nonetheless, the number of PEases is still limited,
and their catalytic activity remains low, underscoring substantial
research gaps. Since the first publication claiming the degradation effect
of recombinant PEases [26], more than 10 PEases have been reported to
exhibit varying degrees of PE degradability (Table 1). However, this
number pales in comparison to the vast genetic changes analyzable
through omics methods, underlining the limitations of omics research.
Although omics approaches have provided insights into changes in
enzyme gene expression in the presence of PE, they fall short in
revealing the specific steps of degradation for which these enzymes are
responsible. Further research is required to explore these altered en­
zymes via recombinant expression, despite the potential pitfalls in
expression yield, solubility, and stability.
Furthermore, the evidence supporting initial hydroxylation as a po­
tential mechanism in PE degradation is steadily increasing (Table S1).
Research has primarily concentrated on the degradation of untreated PE
by various organisms. While evidence has emerged that PE can be
oxidized by a single enzyme [91], the precise molecular mechanism
remains unclear. Although the involvement of hydrolases in the PE
degradation process is gaining support [26], further confirmation of
their direct degradability on PE would require additional methods
beyond SEM.
Structural studies of PEases are still limited, despite years of ongoing
research on related enzymes like laccase, which has provided valuable
insights into redox mechanisms, thermal stability, and substrate recog­
nition. Structural analysis of other related enzymes such as the long-
chain alkane monooxygenase LadA and CYP153D17 has revealed their
binding mode with short-chain dodecane. Such structural insights into
PEases and their interactions with related substrates will be increasingly
important for a better understanding of the PE degradation process. The
pathway of PE enzymatic degradation has been proposed using
computer-based study of PEases from fungi [92] without experimental
validation. Further structural analysis of PEases would be required to
clarify the mechanism of PE enzymatic degradation.
Regarding the PETase discovery process, which stands at the fore­
front of plastic-degrading enzymes, research on screening platforms is
continually evolving. This encompasses quantitative assessment of total
8
Journal of Hazardous Materials 460 (2023) 132449
products using light absorption techniques [127], ranging from Fenton
fluorescence labeling of terephthalate acid (TPA) [79] to
dual-fluorescence methods [55]. These advanced detection approaches,
integrated with traditional HPLC detection, have significantly propelled
the development of PETases and provide insights for assessing the
degradation rates of other plastics. However, when evaluating the de­
gradability of PEases, the complexity and low throughput of the
degradation products present ongoing challenges in quantifying PE de­
gradability and hinder the discovery of PEases. To overcome these ob­
stacles, high-sensitivity, rapid, and high-throughput screening platforms
based on substrate design are required.
Additionally, several other crucial research gaps remain in the field
of PE biodegradation. For instance, the mechanisms underlying the
absorption of long-chain PE polymers by bacteria in insects warrant
exploration. Are there additional processes at play beyond the initial
oxidation step? Moreover, it is essential to investigate whether the
oxidative modification and degradation of long-chain PE polymers are
consistent with the oxidation patterns observed in short-chain hydro­
carbons. Addressing these questions will undoubtedly advance our un­
derstanding of PE degradation mechanisms. Determining the optimal
recycling point for PE biodegradation, considering the synthesis of
valuable chemicals, and assessing whether degrading to the final
monomer represents the most efficient recycling pathway are pivotal
inquiries in the pursuit of advanced recycling strategies. Moreover,
gaining a comprehensive understanding of the significance of parame­
ters like temperature, pH, salt, and metal ions in PE degradation requires
further investigation. Lastly, conducting comparative studies among
different enzymes involved in PE degradation could yield valuable in­
sights into their distinct functionalities and potential applications. As
well, investigating factors like enzyme adhesion to the PE surface and
the inhibition of intermediate products on PEases will be valuable for
enhancing degradation efficiency. Addressing these research gaps is
imperative in advancing our knowledge and fostering more effective and
sustainable strategies for PE biodegradation and recycling.
5. Opportunities and future perspectives
5.1. Emerging resources
PE-degrading microbes, such as algae, bacteria, fungi, and insects
like worms, termites, and beetles, provide abundant sources of potential
new PEases. Recent advancements in metadata analysis techniques, such
as transcriptomics and proteomics, have significantly contributed to the
identification of potential PEase candidates, and some promising leads
have been validated by selected methods [25,26,91]. However, despite
these initial discoveries, most candidates remain unexplored, and their
proposed PE degradability awaits verification and comprehensive
characterization. Moreover, numerous organisms exhibiting substantial
PE degradability have yet to undergo omics analysis, which holds great
promise for unveiling more PEases. Despite the omics approach, ho­
mologous comparisons between genomes of different species may offer
another promising avenue to identify additional PEases, as successfully
applied in the research of PETases. For instance, the renowned PET
hydrolase from Ideonella sakaiensis 201-F6 (IsPETase) was identified
through draft genome sequencing, followed by a homology comparison
with a known hydrolase from Thermobifida fusca (TfH). The PET hy­
drolysis activity of IsPETase was subsequently confirmed through re­
combinant protein experiments [116]. Additionally, investigating
enzymes that act on substrates resembling PE (e.g., polypropylene,
polystyrene, and polyvinyl chloride), as well as on shorter alkanes (e.g.,
paraffin, beeswax) [47] or long linear PE-derived alkenes, alcohols,
ketones, aldehydes, or ethers, holds the potential to reveal novel en­
zymes capable of functioning as PEases.
J. Jin et al.
5.2. Standardization of PE degradation experiments
As PE biocycling is a relatively new field, research groups employ
diverse experimental conditions and measurements, rendering it chal­
lenging, if not impossible, to directly compare data across various
publications [67]. Moreover, these studies use PE materials from
different suppliers, with varying density and crystallinity, in various
forms (powder, foil, sheet, nanoparticle, etc.), with diverse additives
(plasticizers, etc.), or subjected to different pre-treatments (UV, thermal,
chemical), all factors that can affect enzymatic breakdown. In most PE
biodegradation studies (as shown in Table S1), LDPE was the primary
substrate investigated. Only a limited number of studies explored the
degradation of both LDPE and HDPE, or solely focused on HDPE.
Notably, one publication demonstrated a higher weight loss from LDPE
(58%) compared to HDPE (46%) in the presence of a formulated mi­
crobial consortium composed of three strains of Brevibacillus sp. and
Aneurinibacillus sp. Skariyachan et al. [97]. Another study reported that
the two potential fungal strains Penicillium oxalicum NS4 and Penicillium
chrysogenum NS10 exhibit a higher activity on HDPE than on LDPE [72].
These findings indicate that microbes may employ distinct mechanisms
for the degradation of LDPE and HDPE. Therefore, conducting further
comparative studies is essential to delve deeper into the specificity of
microbial PE degradation.
Notably, the definition of "degradation" can vary among studies [33,
67]. Some define it as a reduction in MW, while others consider it as the
breakdown of the polymer into small molecules or surface modifica­
tions. Thus, meticulous evaluation of methods and results is crucial.
Given that the field of PE degradation is still in its early stages, norms
and standards in research have not yet been established and are ex­
pected to rapidly evolve. Standardizing the PE materials used, reaction
conditions, and detection methods is highly desirable at this early stage.
Moreover, conducting comparative studies between different PEases
within the same publication and independently reproducing published
results will further strengthen existing knowledge.
5.3. Synergistic effect of enzymes
PE is a highly recalcitrant polymer because it lacks reactive func­
tional groups. Therefore, its complete degradation requires the coordi­
nated action of multiple enzymes together with their cofactors. This is
illustrated by the fact that the degradation of PE by a microbial com­
munity is always more efficient than that of each individual species, and
microorganisms are more effective at breaking down PE than individual
enzymes. For example, a consortium of C. necator H16, P. putida LS46,
and P. putida IRN22 has been reported to cause significantly greater
LDPE weight loss than that induced by the individual bacteria [68]. In
another study, the breakdown of PE was more pronounced when a
marine bacterial community of three strains was used rather than any
single strain [26]. A separate investigation using a two-enzyme system
from a marine fungus confirmed that glutathione peroxidase and laccase
have a synergistic effect on PE film [25]. It is expected that microbial
proteins responsible for biofilm formation may facilitate PE degradation
by bringing the secreted enzymes and the substrate closer in proximity.
Fungi are known to secrete hydrophobins to help hyphae attach to the
surface of hydrophobic substrates like PE [90]. From this, it is envisaged
that future research seeking PE biorecycling systems should aim for a
tailored consortium of several different enzymes.
In addition, cofactors, metals, and mediators may also be essential
for optimal enzyme activity. Considering that PE is a partially crystal­
line, hydrophobic polymer, enzymes involved in its first oxidation step
would have to harbor a relatively exposed, hydrophobic active site. For
PET breakdown, a co-display yeast system of a hydrophobin and a
PETase significantly increased the degradation of PET films [13]. The
same strategy could be applied to PE degradation, as suggested by some
studies. For example, chemical surfactants such as Tween 20, Tween 80,
and CHAPSO help MnP degrade PE in the absence of H2O2 pre-treatment
9
Journal of Hazardous Materials 460 (2023) 132449
[19]. The biosurfactant known as surfactin was reported to enhance PE
degradation by Bacillus subtilis [104]. Besides surfactants, mediators
such as 1-hydroxybenzotriazole (HBT), 2,2’-azino-bis (3-ethyl­
benzothiazoline-6-sulfonic acid) (ABTS), and 2,2,6,6-tetramethylpiperi­
din-1-yloxy (TEMPO) have been used in LMS derived from two
recombinant laccases, BaLac from Botrytis aclada and BsLac from Bacillus
subtilis. The mediators were found to increase PE degradation, as sug­
gested by larger molecular weight reduction and the introduction of
more oxygen-containing groups [112]. It is also well-known that metal
ions can play an important role in enzyme-catalyzed reactions. For
example, laccases are multi-copper oxidoreductases, and the addition of
copper has been reported to increase their PE degradation activity [94].
PE degradation by MnP was found to be accelerated in the presence of
manganese ions [35]. Therefore, selecting the appropriate auxiliary
agents for PEase activity is a key consideration. Furthermore, techniques
combining living cells, such as whole-cell catalysts, yeast-display sys­
tems, and multiple gene engineering cells, are promising strategies to
take advantage of synergistic effects.
5.4. Structure study and enzyme design
Although no structural studies have been published for any of the
putative PEases in the presence of PE-related ligands, some potential
PEases, such as laccases, MnP, LiP, alkane hydroxylases, and CYPs, have
been structurally characterized in complex with small molecules or
other substrates not related to PE [52,54,65,81,99], providing confor­
mational information on the substrate binding pockets and
structure-stabilizing features. Further studies using X-ray crystallog­
raphy and cryo-EM are needed to explore the binding mode of PEases to
large polymeric substrates relevant to PE. In the meantime, structure
predictions, including those with computational servers such as Alpha­
Fold, Phyr2, and Rosetta [42,44,53], as well as programs predicting
enzyme-substrate interactions (e.g., docking and molecular dynamics),
can offer some guidance. The rise of artificial intelligence (AI) meth­
odologies, particularly machine learning, holds the potential to accel­
erate enzyme engineering and enhance the design of more efficient
PEases. AI techniques have already found application in the advance­
ment of PETase. A noteworthy instance is FAST-PETase, crafted through
machine learning, which represents a significant accomplishment by
showcasing remarkable enhancements in both thermostability and
degradation efficiency in comparison to the wildtype PETase [58].
5.5. Protein engineering and directed evolution of potential PEases
Directed evolution and site-specific saturation mutagenesis have
shown great potential for improving the efficiency of enzymatic PET
degradation, particularly with IsPETase [116,16,58,98], and with the
leaf-branch compost cutinase (LCC) [101]. Unfortunately, protein en­
gineering studies of PEases have yet to appear in the scientific literature.
For example, despite the considerable interest in laccases as potential
PEases, no protein engineering efforts have been documented that spe­
cifically aim to improve their PE-degrading activity. However, studies
have been conducted to improve the optimal pH range, thermostability,
and redox potential of laccases, using rational, semi-rational, and
directed evolution approaches [60,77]. Interestingly, a high redox po­
tential laccase (HRPL) from basidiomycete PM1 was engineered using
directed evolution and rational design, resulting in significantly
improved laccase activity [59]. Rational design has also been used to
enhance stability and confer affinity for aniline in a laccase [89,93].
Structure-based engineering and enzyme evolution have been combined
to improve the stability and activity of a chloride-tolerant HRPL from
the ascomycete B. aclada [95]. The thermostability of a fungal laccase
has been improved by swapping the second cupredoxin domain with
that of another fungal laccase, and by computationally designing re­
combinant chimeras to stabilize flexible surface loops [103]. Together,
computational design and directed evolution have enabled the
J. Jin et al.
generation of fungal HRPL variants with improved stability and redox
potential, resulting in increased activity toward high-redox-potential
mediators [62]. In another study, 27 stabilizing mutations were intro­
duced into an HRPL from basidiomycete PM1, yielding a variant with
remarkable thermostability [61]. However, whether these laccase var­
iants are more efficient in PE degradation remains to be explored. Future
laccase engineering studies focusing on improving their ability to
degrade PE are warranted.
5.6. Assessment of PE enzymatic degradation
As aforementioned, assessment of PE degradation varies significantly
from one lab to another, with techniques ranging from visual inspection
to weight loss measurement, GPC, FTIR, SEM, and GC-MS. Ideally, for
comprehensive product characterization of a given reaction and for
cross-validation purposes, these methods are best used in combination.
Although the products of PE degradation are complex, it is anticipated
that the intermediates and final products of PE enzymatic degradation
will be categorized and elucidated through the increasing application of
techniques such as FTIR, HPLC, and GC-MS. Isotope-labeled PE has
proven to be a powerful tool for tracking the metabolism of PE in or­
ganisms. For instance, the use of deuterated PE has been successfully
employed in conjunction with μFTIR hyperspectral imaging on cry­
osections of larvae fed with deuterated PE. This method not only facil­
itated the visualization of PE metabolization but also offered valuable
insights into the specific tissues where PE metabolites accumulate and
the potential synthesis of diverse biomolecules involving PE [85]. 13C
isotope-labeled PE tracer has been created to study the kinetics of
mineralization [30]. This strategy allows for the tracing of the 13C from
13
C-PE in the final product CO2, providing unequivocal proof of the
mineralization of PE-derived carbon by microbes. Furthermore, the
application of these techniques can provide solid evidence to distinguish
between authentic degradation of PE and products attributed to addi­
tives. Additionally, a library of PE metabolites may be created to help
establish the sequence of events and corresponding enzymes.
Model substrates that share some chemical features of PE or likely
intermediates of its degradation have been used to verify the specific
function of some putative PEases. For example, oleic acid and squalene
were used to determine whether double bonds are broken during
enzymatic PE breakdown [112]. These experiments indicated an easier
reaction after UV pre-treatment and confirmed the involvement of hy­
droxylation, ketone formation, epoxidation, and chain breakdown dur­
ing PE degradation by LMS. The discovery and utilization of new model
substrates, along with the corresponding product determination, can
provide a clearer understanding of the PE biodegradation pathway,
thereby identifying the specific activity of the enzymes involved.
Detection methods for total fatty acids and dicarboxylic acids in PE
degradation mixtures can be developed (if not assimilated by organisms,
the end products are expected to be mostly fatty acids and dicarboxylic
acids). Besides complete PE degradation, closed-loop recycling or
upcycling PE into value-added products, such as polyhydroxyalkanoate
(PHA) [41], contribute to environmental sustainability.
5.7. Future trends and perspectives
In the field of PE enzymatic degradation research, several impressive
developments are emerging. Harnessing the capabilities of structural
databases and cutting-edge tools like those in PDB, AlphaFold2, and
FoldSeek [102,42,6] for PEase mining holds the promise of revolution­
ary breakthroughs. These resources grant us the ability to scrutinize
existing structures, such as laccase, and systematically learn and accu­
mulate features in the laccases with PE degradability. Armed with this
wealth of knowledge, we can venture into designing novel enzymes,
whether they stem from biological sources or are engineered de novo. A
novel PEase from Lysinibacillus fusiformis has been identified using
computer-aided structure analysis combined with initial activity-based
10
Journal of Hazardous Materials 460 (2023) 132449
screening [121]. Further design strategies can be expected for
tailoring enzymes for specific properties. De novo enzyme design based
on deep learning would provide additional opportunities for highly
efficient PEases. In fact, there might already be existing examples of this
approach [51,113]. Additionally, directed evolution can further play
important roles in improving enzyme stability, degradation efficiency
and selectivity, and adhesion capability of PEases.
Fusing various proteins and domains with different functionalities
opens new avenues for novel PEases. For instance, combining PEases
with proteins with surface adhesion properties could increase their
attachment to the PE surface [121]. Another intriguing pursuit lies in the
development of chimeric dual-enzyme systems, where two enzymes
with distinct catalytic activities, such as oxidation-reduction and hy­
drolytic enzymes, are linked together. This strategy assists an enzyme
with relatively poor solubility and stability by coupling it with one that
has better properties. More importantly, it enables synergistic effects
due to their proximity. Similar strategies have been successfully applied
in PET degradation, such as the combination of a polyester hydrolase
and the immobilized carboxylesterase TfCa [4], as well as the chimeric
protein consisting of PETase and MHETase [46]. Important consider­
ations for this fusion strategy include the selection of PEases with
diverse activities, the choice of a PE-binding domain, optimization of
mutants, types of linkers, and fine-tuning their length. A recent publi­
cation reported the screening of candidate laccases and
carbohydrate-binding domains [29].
Designing multi-enzyme engineering bacteria and establishing
detection methods at various levels, ranging from the organismal level,
such as worms and bacteria, to molecular levels, like PEases, can pave
the way for more comprehensive insights in the field of PE degradation.
Importantly, establishing high-throughput detection methods becomes
crucial. Deuterated PE [85] and 13C isotope-labeled PE have been uti­
lized in the quantification of metabolites derived from PE breakdown.
The design of fluorescent and luminescent-based detection platforms
with rapid and high sensitivity will contribute to the kinetic analysis of
PEases and accurate assessment of degradability. Quantification of total
fatty acids and diacids will undoubtedly aid in accelerating the pace of
discoveries. Finally, implementing moist-solid methods has shown great
promise in significantly enhancing the efficiency of plastic degradation
enzymes [43].
6. Concluding remarks
Even though investigations of plastic waste biodegradation started 5
decades ago, research on PE in particular has been severely hampered
by: 1) the exceptional lack of reactivity of PE; 2) the numerous steps
required for PE biodegradation; 3) the challenges of characterizing the
multiple reaction products; 4) the limited number of enzymes known to
degrade or act on PE; 5) the difficulty in identifying pure enzymes with
true PE-degrading activity since multiple enzymes are likely needed; and
6) the lack of structural information about PEases. Currently, most
research focuses on the degradation of short-chain PE and alkanes, and
there are still many unknowns about how to transition from short-chain
PE to long-chain PE materials.
In recent years, the degradation of commercial PE by worms has
demonstrated the feasibility of PE biodegradation and has gradually
attracted researchers to explore its mechanism. While some promising
PEase hits have emerged, future research needs to focus on demon­
strating their ability to degrade PE to a specific product or family of
compounds, thereby providing bona fide targets for optimization
through enzyme engineering techniques. Such enzymes would likely
also be produced recombinantly and characterized structurally and
mechanistically, facilitating further enzyme design. High-throughput
screening methods are desperately needed to identify effective and
efficient PEases. Eventually, once enzymes are identified for each step of
PE breakdown, it might be possible to fuse them together to create one
multi-functional enzyme capable of breaking down PE cleanly to a few
J. Jin et al.
small molecules.
In the field of de novo protein design, AI techniques are emerging as a
critical force [1,106]. Particularly noteworthy is the establishment of a
machine-learning-driven framework for plastic enzymatic degradation,
which has demonstrated the capability to predict an enzyme’s
plastic-degrading potential for specific plastic types. This framework
unravels concealed patterns within protein sequences, introducing a
novel tool for the prediction and discovery of PEases [40]. Given the
urgent need to address the problem of plastic waste accumulation, it is
conceivable that AI-based design may soon be applied to the degrada­
tion of PE plastics. We anticipate that AI could be used to design
single-function PE-degrading enzymes with high activity and thermo­
stability, as well as multi-functional PEases. The ultimate goal would be
to develop an enzyme capable of catalyzing multiple steps of the PE
degradation process, thus enabling single-enzyme PE degradation.
Although there are still significant challenges facing PE biodegra­
dation research, there is much room for exciting discoveries. Projects
like Open Plastic (www.openplastic.com), which aims to identify and
engineer bacteria and enzymes capable of breaking down plastics into
recyclable components or valuable fine chemicals more effectively than
chemical conversion-based technologies, and MIX-UP (www.mix-up.
eu), which aims to convert plastics into value-added, sustainable bio­
materials using biotechnology, have brought together experts from
various disciplines and created multi-tech platforms to explore feasible
solutions for plastic recycling and upcycling. It is expected that
continued research and development in this field will lead to significant
advancements in the coming years, moving us closer to being able to
tackle PE waste accumulation through biorecycling.
CRediT authorship contribution statement
Jin Jin: Conceptualization, Writing – original draft, Writing – review
& editing. Jane Arciszewski: Writing – review & editing. Karine
Auclair: Writing – review & editing. Zongchao Jia: Conceptualization,
Writing – review & editing, Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was funded by the Government of Canada through
Genome Canada and Ontario Genomics (OGI-207). Cartoon figures were
created with BioRender.com.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.jhazmat.2023.132449.
References
[1] Anishchenko, I., Pellock, S.J., Chidyausiku, T.M., Ramelot, T.A., Ovchinnikov, S.,
Hao, J., Bafna, K., Norn, C., Kang, A., Bera, A.K., DiMaio, F., Carter, L., Chow, C.
M., Montelione, G.T., Baker, D., 2021. De novo protein design by deep network
hallucination. Nature 600 (7889), 547–552. https://doi.org/10.1038/s41586-
021-04184-w.
[2] Arregui, L., Ayala, M., Gomez-Gil, X., Gutierrez-Soto, G., Hernandez-Luna, C.E.,
Herrera de Los Santos, M., Levin, L., Rojo-Dominguez, A., Romero-Martinez, D.,
Saparrat, M.C.N., Trujillo-Roldan, M.A., Valdez-Cruz, N.A., 2019. Laccases:
structure, function, and potential application in water bioremediation. Micro Cell
Fact 18 (1), 200. https://doi.org/10.1186/s12934-019-1248-0.
[3] Azoulay, D., Villa, P., Arellano, Y., Gordon, M.F., Moon, D., Miller, K.A.,
Thompson, K., Kistler, A., 2019. Plastic & health: the hidden costs of a plastic
planet. CIEL Geneva, Switzerland. 〈https://www.ciel.org/reports/plastic-health-
the-hidden-costs-of-a-plastic-planet-february-2019/〉.
11
Journal of Hazardous Materials 460 (2023) 132449
[4] Barth, M., Honak, A., Oeser, T., Wei, R., Belisário-Ferrari, M.R., Then, J.,
Schmidt, J., Zimmermann, W., 2016. A dual enzyme system composed of a
polyester hydrolase and a carboxylesterase enhances the biocatalytic degradation
of polyethylene terephthalate films. Biotechnol J 11 (8), 1082–1087. https://doi.
org/10.1002/biot.201600008.
[5] Bento, I., Silva, C.S., Chen, Z., Martins, L.O., Lindley, P.F., Soares, C.M., 2010.
Mechanisms underlying dioxygen reduction in laccases. Structural and modelling
studies focusing on proton transfer. BMC Struct Biol 10 (1), 28. https://doi.org/
10.1186/1472-6807-10-28.
[6] Berman, H.M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T.N., Weissig, H.,
Shindyalov, I.N., Bourne, P.E., 2000. The protein data bank. Nucleic Acids Res 28
(1), 235–242. https://doi.org/10.1093/nar/28.1.235.
[7] Bertolacci, L., Goldoni, L., Zych, A., Athanassiou, A., 2022. Biocatalytic oxidation
of polyethylene by Agrocybe aegerita mycelium. Polym Degrad Stab 199,
109911. https://doi.org/10.1016/j.polymdegradstab.2022.109911.
[8] Bertrand, T., Jolivalt, C., Briozzo, P., Caminade, E., Joly, N., Madzak, C.,
Mougin, C., 2002. Crystal structure of a four-copper laccase complexed with an
arylamine: insights into substrate recognition and correlation with kinetics.
Biochemistry 41 (23), 7325–7333. https://doi.org/10.1021/bi0201318.
[9] Bombelli, P., Howe, C.J., Bertocchini, F., 2017. Polyethylene bio-degradation by
caterpillars of the wax moth Galleria mellonella. Curr Biol 27 (8), R292–R293.
https://doi.org/10.1016/j.cub.2017.02.060.
[10] Brandon, A.M., Gao, S.H., Tian, R., Ning, D., Yang, S.S., Zhou, J., Wu, W.M.,
Criddle, C.S., 2018. Biodegradation of polyethylene and plastic mixtures in
mealworms (larvae of tenebrio molitor) and effects on the gut microbiome.
Environ Sci Technol 52 (11), 6526–6533. https://doi.org/10.1021/acs.
est.8b02301.
[11] Chamas, A., Moon, H., Zheng, J., Qiu, Y., Tabassum, T., Jang, J.H., Abu-Omar, M.,
Scott, S.L., Suh, S., 2020. Degradation rates of plastics in the environment. ACS
Sustain Chem Eng 8 (9), 3494–3511. https://doi.org/10.1021/
acssuschemeng.9b06635.
[12] Chen, C.-C., Dai, L., Ma, L., Guo, R.-T., 2020. Enzymatic degradation of plant
biomass and synthetic polymers. Nat Rev Chem 4 (3), 114–126. https://doi.org/
10.1038/s41570-020-0163-6.
[13] Chen, Z., Duan, R., Xiao, Y., Wei, Y., Zhang, H., Sun, X., Wang, S., Cheng, Y.,
Wang, X., Tong, S., Yao, Y., Zhu, C., Yang, H., Wang, Y., Wang, Z., 2022.
Biodegradation of highly crystallized poly(ethylene terephthalate) through cell
surface codisplay of bacterial PETase and hydrophobin. Nat Commun 13 (1),
7138. https://doi.org/10.1038/s41467-022-34908-z.
[14] Chen, Z., Durão, P., Silva, C.S., Pereira, M.M., Todorovic, S., Hildebrandt, P.,
Bento, I., Lindley, P.F., Martins, L.O., 2010. The role of Glu498 in the dioxygen
reactivity of CotA-laccase from Bacillus subtilis. Dalton Trans 39 (11),
2875–2882. https://doi.org/10.1039/b922734b.
[15] Chia, W.Y., Ying Tang, D.Y., Khoo, K.S., Kay Lup, A.N., Chew, K.W., 2020.
Nature’s fight against plastic pollution: algae for plastic biodegradation and
bioplastics production. Environ Sci Ecotechnol 4, 100065. https://doi.org/
10.1016/j.ese.2020.100065.
[16] Cui, Y., Chen, Y., Liu, X., Dong, S., Tian, Ye, Qiao, Y., Mitra, R., Han, J., Li, C.,
Han, X., Liu, W., Chen, Q., Wei, W., Wang, X., Du, W., Tang, S., Xiang, H., Liu, H.,
Liang, Y., Houk, K.N., Wu, B., 2021. Computational REDesign of a PETase for
plastic biodegradation under ambient condition by the GRAPE Strategy. ACS
Catal 11 (3), 1340–1350. https://doi.org/10.1021/acscatal.0c05126.
[17] Dey, A.S., Bose, H., Mohapatra, B., Sar, P., 2020. Biodegradation of Unpretreated
Low-Density Polyethylene (LDPE) by Stenotrophomonas sp. and Achromobacter
sp., isolated from waste dumpsite and drilling fluid. Front Microbiol 11. https://
doi.org/10.3389/fmicb.2020.603210.
[18] Durão, P., Chen, Z., Silva, Catarina, S., Soares, Cláudio, M., Pereira, Manuela, M.,
Todorovic, S., Hildebrandt, P., Bento, I., Lindley, Peter, F., Martins, Lígia, O.,
2008. Proximal mutations at the type 1 copper site of CotA laccase: spectroscopic,
redox, kinetic and structural characterization of I494A and L386A mutants.
Biochem J 412 (2), 339–346. https://doi.org/10.1042/bj20080166.
[19] Ehara, K., Iiyoshi, Y., Tsutsumi, Y., Nishida, T., 2000. Polyethylene degradation
by manganese peroxidase in the absence of hydrogen peroxide. J Wood Sci 46 (2),
180–183. https://doi.org/10.1007/BF00777369.
[20] Enguita, F.J., Marçal, D., Martins, L.O., Grenha, R., Henriques, A.O., Lindley, P.F.,
Carrondo, M.A., 2004. Substrate and dioxygen binding to the endospore coat
laccase from bacillus subtilis. J Biol Chem 279 (22), 23472–23476. https://doi.
org/10.1074/jbc.M314000200.
[21] Enguita, F.J., Martins, L.O., Henriques, A.O., Carrondo, M.A., 2003. Crystal
structure of a bacterial endospore coat component: a laccase with enhanced
thermostability properties*. J Biol Chem 278 (21), 19416–19425. https://doi.
org/10.1074/jbc.M301251200.
[22] Enguita, F.J., Martins, L.O., Henriques, A.O., Carrondo, M.A., 2003. Crystal
structure of a bacterial endospore coat component. A laccase with enhanced
thermostability properties. J Biol Chem 278 (21), 19416–19425. https://doi.org/
10.1074/jbc.M301251200.
[23] Fujisawa, M., Hirai, H., Nishida, T., 2001. Degradation of polyethylene and nylon-
66 by the laccase-mediator system. J Polym Environ 9 (3), 103–108. https://doi.
org/10.1023/A:1020472426516.
[24] Gambarini, V., Pantos, O., Kingsbury, J.M., Weaver, L., Handley, K.M., Lear, G.,
2022. PlasticDB: a database of microorganisms and proteins linked to plastic
biodegradation. Database (Oxf) 2022. https://doi.org/10.1093/database/
baac008.
[25] Gao, R., Liu, R., Sun, C., 2022. A marine fungus Alternaria alternata FB1
efficiently degrades polyethylene. J Hazard Mater 431, 128617. https://doi.org/
10.1016/j.jhazmat.2022.128617.
J. Jin et al.
[26] Gao, R., Sun, C., 2021. A marine bacterial community capable of degrading poly
(ethylene terephthalate) and polyethylene. J Hazard Mater 416, 125928. https://
doi.org/10.1016/j.jhazmat.2021.125928.
[27] Geyer, R., Jambeck, J.R., Law, K.L., 2017. Production, use, and fate of all plastics
ever made. Sci Adv 3 (7), e1700782. https://doi.org/10.1126/sciadv.1700782.
[28] Ghatge, S., Yang, Y., Ahn, J.-H., Hur, H.-G., 2020. Biodegradation of
polyethylene: a brief review. Appl Biol Chem 63 (1), 27. https://doi.org/
10.1186/s13765-020-00511-3.
[29] Gollan, M., Black, G., Munoz-Munoz, J., 2023. A computational approach to
optimising laccase-mediated polyethylene oxidation through carbohydrate-
binding module fusion. BMC Biotechnol 23 (1), 18. https://doi.org/10.1186/
s12896-023-00787-5.
[30] Goudriaan, M., Morales, V.H., van der Meer, M.T.J., Mets, A., Ndhlovu, R.T., van
Heerwaarden, J., Simon, S., Heuer, V.B., Hinrichs, K.-U., Niemann, H., 2023.
A stable isotope assay with 13C-labeled polyethylene to investigate plastic
mineralization mediated by Rhodococcus ruber. Mar Pollut Bull 186, 114369.
https://doi.org/10.1016/j.marpolbul.2022.114369.
[31] Gravouil, K., Ferru-Clement, R., Colas, S., Helye, R., Kadri, L., Bourdeau, L.,
Moumen, B., Mercier, A., Ferreira, T., 2017. Transcriptomics and lipidomics of
the environmental strain rhodococcus ruber point out consumption pathways and
potential metabolic bottlenecks for polyethylene degradation. Environ Sci
Technol 51 (9), 5172–5181. https://doi.org/10.1021/acs.est.7b00846.
[32] Hamilton, L.A., Feit, S., 2019. Plastic & climate: the hidden costs of a plastic
planet. 〈https://www.ciel.org/plasticandclimate/〉.
[33] Harrison, J.P., Boardman, C., O’Callaghan, K., Delort, A.-M., Song, J., 2018.
Biodegradability standards for carrier bags and plastic films in aquatic
environments: a critical review. R Soc Open Sci 5 (5), 171792. https://doi.org/
10.1098/rsos.171792.
[34] Hsieh, C.H., Huang, X., Amaya, J.A., Rutland, C.D., Keys, C.L., Groves, J.T.,
Austin, R.N., Makris, T.M., 2017. The enigmatic P450 decarboxylase OleT Is
capable of, but evolved to frustrate, oxygen rebound chemistry. Biochemistry 56
(26), 3347–3357. https://doi.org/10.1021/acs.biochem.7b00338.
[35] Iiyoshi, Y., Tsutsumi, Y., Nishida, T., 1998. Polyethylene degradation by lignin-
degrading fungi and manganese peroxidase. J Wood Sci 44 (3), 222–229. https://
doi.org/10.1007/BF00521967.
[36] Inderthal, H., Tai, S.L., Harrison, S.T.L., 2021. Non-hydrolyzable plastics - an
Interdisciplinary Look at Plastic Bio-Oxidation. Trends Biotechnol 39 (1), 12–23.
https://doi.org/10.1016/j.tibtech.2020.05.004.
[37] Jeon, H.J., Kim, M.N., 2015. Functional analysis of alkane hydroxylase system
derived from Pseudomonas aeruginosa E7 for low molecular weight polyethylene
biodegradation. Int Biodeterior Biodegrad 103, 141–146. https://doi.org/
10.1016/j.ibiod.2015.04.024.
[38] Jeon, J.-M., Park, S.-J., Choi, T.-R., Park, J.-H., Yang, Y.-H., Yoon, J.-J., 2021.
Biodegradation of polyethylene and polypropylene by Lysinibacillus species
JJY0216 isolated from soil grove. Polym Degrad Stab 191, 109662. https://doi.
org/10.1016/j.polymdegradstab.2021.109662.
[39] Ji, Y., Mao, G., Wang, Y., Bartlam, M., 2013. Structural insights into diversity and
n-alkane biodegradation mechanisms of alkane hydroxylases. Front Microbiol 4.
https://doi.org/10.3389/fmicb.2013.00058.
[40] Jiang, R., Shang, L., Wang, R., Wang, D., Wei, N., 2023. Machine learning based
prediction of enzymatic degradation of plastics using encoded protein sequence
and effective feature representation. Environ Sci Technol Lett 10 (7), 557–564.
https://doi.org/10.1021/acs.estlett.3c00293.
[41] Johnston, B., Adamus, G., Ekere, A.I., Kowalczuk, M., Tchuenbou-Magaia, F.,
Radecka, I., 2022. Bioconversion of plastic waste based on mass full carbon
backbone polymeric materials to value-added polyhydroxyalkanoates (PHAs).
Bioengineering 9 (9), 432. https://doi.org/10.3390/bioengineering9090432.
[42] Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O.,
Tunyasuvunakool, K., Bates, R., Zidek, A., Potapenko, A., Bridgland, A.,
Meyer, C., Kohl, S.A.A., Ballard, A.J., Cowie, A., Romera-Paredes, B., Nikolov, S.,
Jain, R., Adler, J., Back, T., Petersen, S., Reiman, D., Clancy, E., Zielinski, M.,
Steinegger, M., Pacholska, M., Berghammer, T., Bodenstein, S., Silver, D.,
Vinyals, O., Senior, A.W., Kavukcuoglu, K., Kohli, P., Hassabis, D., 2021. Highly
accurate protein structure prediction with AlphaFold. Nature 596 (7873),
583–589. https://doi.org/10.1038/s41586-021-03819-2.
[43] Kaabel, S., Therien, J.P.D., Deschenes, C.E., Duncan, D., Friscic, T., Auclair, K.,
2021. Enzymatic depolymerization of highly crystalline polyethylene
terephthalate enabled in moist-solid reaction mixtures. Proc Natl Acad Sci USA
118 (29). https://doi.org/10.1073/pnas.2026452118.
[44] Kelley, L.A., Mezulis, S., Yates, C.M., Wass, M.N., Sternberg, M.J., 2015. The
Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10
(6), 845–858. https://doi.org/10.1038/nprot.2015.053.
[45] Khandare, S.D., Agrawal, D., Mehru, N., Chaudhary, D.R., 2022. Marine bacterial
based enzymatic degradation of low-density polyethylene (LDPE) plastic.
J Environ Chem Eng 10 (3), 107437. https://doi.org/10.1016/j.
jece.2022.107437.
[46] Knott, B.C., Erickson, E., Allen, M.D., Gado, J.E., Graham, R., Kearns, F.L.,
Pardo, I., Topuzlu, E., Anderson, J.J., Austin, H.P., Dominick, G., Johnson, C.W.,
Rorrer, N.A., Szostkiewicz, C.J., Copié, V., Payne, C.M., Woodcock, H.L.,
Donohoe, B.S., Beckham, G.T., McGeehan, J.E., 2020. Characterization and
engineering of a two-enzyme system for plastics depolymerization. Proc Natl
Acad Sci USA 117 (41), 25476–25485. https://doi.org/10.1073/
pnas.2006753117.
[47] Kong, H.G., Kim, H.H., Chung, J.H., Jun, J., Lee, S., Kim, H.M., Jeon, S., Park, S.
G., Bhak, J., Ryu, C.M., 2019. The galleria mellonella hologenome supports
12
Journal of Hazardous Materials 460 (2023) 132449
microbiota-independent metabolism of long-chain hydrocarbon beeswax. e2455
Cell Rep 26 (9), 2451–2464. https://doi.org/10.1016/j.celrep.2019.02.018.
[48] Krueger, M.C., Harms, H., Schlosser, D., 2015. Prospects for microbiological
solutions to environmental pollution with plastics. Appl Microbiol Biotechnol 99
(21), 8857–8874. https://doi.org/10.1007/s00253-015-6879-4.
[49] Kumar, A., Kalleshwaraswamy, C.M., Sharma, R., Sharma, P., Poonia, A., 2022.
Biodegradation of plastic using termites and their gut microbiota: a mini review.
IOP Conf Ser: Earth Environ Sci 1057 (1), 012016. https://doi.org/10.1088/
1755-1315/1057/1/012016.
[50] Latour, S., Noël, G., Serteyn, L., Sare, A.R., Massart, S., Delvigne, F., Francis, F.,
2021. Multi-omics approach reveals new insights into the gut microbiome of
Galleria mellonella (Lepidoptera:Pyralidae) exposed to polyethylene diet.
bioRxiv, 2021.2006.2004.446152. 〈https://doi.org/10.1101/2021.06.04.44
6152〉.
[51] Laura, H.H., Niklas, G.M., Freja, T.T., Freja, R., Ioana-Malina, M., Evamaria, I.P.,
Peter, F., 2023. De novo Design of a Polycarbonate Hydrolase. bioRxiv,
2023.2003.2010.532063. 〈https://doi.org/10.1101/2023.03.10.532063〉.
[52] Lee, C.W., Yu, S.C., Lee, J.H., Park, S.H., Park, H., Oh, T.J., Lee, J.H., 2016.
Crystal structure of a putative cytochrome P450 alkane hydroxylase (cyp153d17)
from sphingomonas sp. PAMC 26605 and its conformational substrate binding.
Int J Mol Sci 17 (12). https://doi.org/10.3390/ijms17122067.
[53] Leman, J.K., Weitzner, B.D., Lewis, S.M., Adolf-Bryfogle, J., Alam, N., Alford, R.
F., Aprahamian, M., Baker, D., Barlow, K.A., Barth, P., Basanta, B., Bender, B.J.,
Blacklock, K., Bonet, J., Boyken, S.E., Bradley, P., Bystroff, C., Conway, P.,
Cooper, S., Correia, B.E., Coventry, B., Das, R., De Jong, R.M., DiMaio, F.,
Dsilva, L., Dunbrack, R., Ford, A.S., Frenz, B., Fu, D.Y., Geniesse, C.,
Goldschmidt, L., Gowthaman, R., Gray, J.J., Gront, D., Guffy, S., Horowitz, S.,
Huang, P.S., Huber, T., Jacobs, T.M., Jeliazkov, J.R., Johnson, D.K., Kappel, K.,
Karanicolas, J., Khakzad, H., Khar, K.R., Khare, S.D., Khatib, F., Khramushin, A.,
King, I.C., Kleffner, R., Koepnick, B., Kortemme, T., Kuenze, G., Kuhlman, B.,
Kuroda, D., Labonte, J.W., Lai, J.K., Lapidoth, G., Leaver-Fay, A., Lindert, S.,
Linsky, T., London, N., Lubin, J.H., Lyskov, S., Maguire, J., Malmstrom, L.,
Marcos, E., Marcu, O., Marze, N.A., Meiler, J., Moretti, R., Mulligan, V.K.,
Nerli, S., Norn, C., O’Conchuir, S., Ollikainen, N., Ovchinnikov, S., Pacella, M.S.,
Pan, X., Park, H., Pavlovicz, R.E., Pethe, M., Pierce, B.G., Pilla, K.B., Raveh, B.,
Renfrew, P.D., Burman, S.S.R., Rubenstein, A., Sauer, M.F., Scheck, A., Schief, W.,
Schueler-Furman, O., Sedan, Y., Sevy, A.M., Sgourakis, N.G., Shi, L., Siegel, J.B.,
Silva, D.A., Smith, S., Song, Y., Stein, A., Szegedy, M., Teets, F.D., Thyme, S.B.,
Wang, R.Y., Watkins, A., Zimmerman, L., Bonneau, R., 2020. Macromolecular
modeling and design in Rosetta: recent methods and frameworks. Nat Methods 17
(7), 665–680. https://doi.org/10.1038/s41592-020-0848-2.
[54] Li, L., Liu, X., Yang, W., Xu, F., Wang, W., Feng, L., Bartlam, M., Wang, L., Rao, Z.,
2008. Crystal structure of long-chain alkane monooxygenase (LadA) in complex
with coenzyme FMN: unveiling the long-chain alkane hydroxylase. J Mol Biol 376
(2), 453–465. https://doi.org/10.1016/j.jmb.2007.11.069.
[55] Liu, K., Xu, Z., Zhao, Z., Chen, Y., Chai, Y., Ma, L., Li, S., 2023. A dual
fluorescence assay enables high-throughput screening for poly(ethylene
terephthalate) hydrolases. ChemSusChem 16 (5), e202202019. https://doi.org/
10.1002/cssc.202202019.
[56] Liu, Z., Xie, T., Zhong, Q., Wang, G., 2016. Crystal structure of CotA laccase
complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) at a novel
binding site. Acta Crystallogr F Struct Biol Commun 72 (Pt 4), 328–335. https://
doi.org/10.1107/s2053230x1600426x.
[57] Loganathan, B.G., Masunaga, S., 2020. Chapter 18 - PCBs, dioxins, and furans:
human exposure and health effects. In: Gupta, R.C. (Ed.), Handbook of Toxicology
of Chemical Warfare Agents (Third Edition). Academic Press, Boston,
pp. 267–278. https://doi.org/10.1016/B978-0-12-819090-6.00018-0.
[58] Lu, H., Diaz, D.J., Czarnecki, N.J., Zhu, C., Kim, W., Shroff, R., Acosta, D.J.,
Alexander, B.R., Cole, H.O., Zhang, Y., Lynd, N.A., Ellington, A.D., Alper, H.S.,
2022. Machine learning-aided engineering of hydrolases for PET
depolymerization. Nature 604 (7907), 662–667. https://doi.org/10.1038/
s41586-022-04599-z.
[59] Mate, D., Garcia-Burgos, C., Garcia-Ruiz, E., Ballesteros, A.O., Camarero, S.,
Alcalde, M., 2010. Laboratory evolution of high-redox potential laccases. Chem
Biol 17 (9), 1030–1041. https://doi.org/10.1016/j.chembiol.2010.07.010.
[60] Mate, D.M., Alcalde, M., 2015. Laccase engineering: from rational design to
directed evolution. Biotechnol Adv 33 (1), 25–40. https://doi.org/10.1016/j.
biotechadv.2014.12.007.
[61] Mateljak, I., Alcalde, M., 2021. Engineering a highly thermostable high-redox
potential laccase. ACS Sustain Chem Eng 9 (29), 9632–9637. https://doi.org/
10.1021/acssuschemeng.1c00622.
[62] Mateljak, I., Rice, A., Yang, K., Tron, T., Alcalde, M., 2019. The generation of
thermostable fungal laccase chimeras by SCHEMA-RASPP Structure-guided
recombination in vivo. ACS Synth Biol 8 (4), 833–843. https://doi.org/10.1021/
acssynbio.8b00509.
[63] Matsubara, M., Suzuki, J., Deguchi, T., Miura, M., Kitaoka, Y., 1996.
Characterization of manganese peroxidases from the hyperlignolytic fungus IZU-
154. Appl Environ Microbiol 62 (11), 4066–4072. https://doi.org/10.1128/
aem.62.11.4066-4072.1996.
[64] Mehra, R., Muschiol, J., Meyer, A.S., Kepp, K.P., 2018. A structural-chemical
explanation of fungal laccase activity. Sci Rep 8 (1), 17285. https://doi.org/
10.1038/s41598-018-35633-8.
[65] Miki, Y., Calvino, F.R., Pogni, R., Giansanti, S., Ruiz-Duenas, F.J., Martinez, M.J.,
Basosi, R., Romero, A., Martinez, A.T., 2011. Crystallographic, kinetic, and
spectroscopic study of the first ligninolytic peroxidase presenting a catalytic
J. Jin et al.
tyrosine. J Biol Chem 286 (17), 15525–15534. https://doi.org/10.1074/jbc.
M111.220996.
[66] Mishra, A., Gupta, J., Kumari, T., Pal, R., Thakur, I.S., 2021. Unravelling the
attributes of novel cyanobacteria Jacksonvillea sp. ISTCYN1 by draft genome
sequencing. Bioresour Technol 337, 125473. https://doi.org/10.1016/j.
biortech.2021.125473.
[67] Montazer, Z., Habibi Najafi, M.B., Levin, D.B., 2020. Challenges with verifying
microbial degradation of polyethylene. Polym (Basel) 12 (1). https://doi.org/
10.3390/polym12010123.
[68] Montazer, Z., Habibi Najafi, M.B., Levin, D.B., 2021. In vitro degradation of low-
density polyethylene by new bacteria from larvae of the greater wax moth,
Galleria mellonella. Can J Microbiol 67 (3), 249–258. https://doi.org/10.1139/
cjm-2020-0208.
[69] Mukherjee, A., Debnath, B., Ghosh, S.K., 2016. A review on technologies of
removal of dioxins and furans from incinerator flue gas. Procedia Environ Sci 35,
528–540. https://doi.org/10.1016/j.proenv.2016.07.037.
[70] Mukherjee, S., Kundu, P.P., 2014. Alkaline fungal degradation of oxidized
polyethylene in black liquor: studies on the effect of lignin peroxidases and
manganese peroxidases. J Appl Polym Sci 131 (17). https://doi.org/10.1002/
app.40738.
[71] Oberbeckmann, S., Bartosik, D., Huang, S., Werner, J., Hirschfeld, C.,
Wibberg, D., Heiden, S.E., Bunk, B., Overmann, J., Becher, D., Kalinowski, J.,
Schweder, T., Labrenz, M., Markert, S., 2021. Genomic and proteomic profiles of
biofilms on microplastics are decoupled from artificial surface properties. Environ
Microbiol 23 (6), 3099–3115. https://doi.org/10.1111/1462-2920.15531.
[72] Ojha, N., Pradhan, N., Singh, S., Barla, A., Shrivastava, A., Khatua, P., Rai, V.,
Bose, S., 2017. Evaluation of HDPE and LDPE degradation by fungus,
implemented by statistical optimization. Sci Rep 7 (1), 39515. https://doi.org/
10.1038/srep39515.
[73] Oluwadamilola, E.O., Timinibefi, Z., Momoh, A., 2022. Production and
characterization of thermostable lignolytic enzymes produced from
Staphylococcus saprophyticus exposed to low-density polyethylene (LDPE.
J Biochem Int 9 (4), 18–27. https://doi.org/10.56557/jobi/2022/v9i47593.
[74] Orlando, M., Molla, G., Castellani, P., Pirillo, V., Torretta, V., Ferronato, N., 2023.
Microbial enzyme biotechnology to reach plastic waste circularity: current status,
problems and perspectives. Int J Mol Sci 24 (4), 3877. https://doi.org/10.3390/
ijms24043877.
[75] Osipov, E., Polyakov, K., Kittl, R., Shleev, S., Dorovatovsky, P., Tikhonova, T.,
Hann, S., Ludwig, R., Popov, V., 2014. Effect of the L499M mutation of the
ascomycetous Botrytis aclada laccase on redox potential and catalytic properties.
Acta Crystallogr D Biol Crystallogr 70 (Pt 11), 2913–2923. https://doi.org/
10.1107/s1399004714020380.
[76] Osipov, E.M., Polyakov, K.M., Tikhonova, T.V., Kittl, R., Dorovatovskii, P.V.,
Shleev, S.V., Popov, V.O., Ludwig, R., 2015. Incorporation of copper ions into
crystals of T2 copper-depleted laccase from Botrytis aclada. Acta Crystallogr F
Struct Biol Commun 71 (Pt 12), 1465–1469. https://doi.org/10.1107/
s2053230x1502052x.
[77] Pardo, I., Camarero, S., 2015. Laccase engineering by rational and evolutionary
design. Cell Mol Life Sci 72 (5), 897–910. https://doi.org/10.1007/s00018-014-
1824-8.
[78] Peydaei, A., Bagheri, H., Gurevich, L., de Jonge, N., Nielsen, J.L., 2020. Impact of
polyethylene on salivary glands proteome in Galleria melonella. Comp Biochem
Physiol Part D Genom Proteom 34, 100678. https://doi.org/10.1016/j.
cbd.2020.100678.
[79] Pfaff, L., Breite, D., Badenhorst, C.P.S., Bornscheuer, U.T., Wei, R., 2021. Chapter
Twelve - Fluorimetric high-throughput screening method for polyester hydrolase
activity using polyethylene terephthalate nanoparticles. In: Weber, G.,
Bornscheuer, U.T., Wei, R. (Eds.), Methods in Enzymology. Academic Press,
pp. 253–270. https://doi.org/10.1016/bs.mie.2020.11.003.
[80] Pickl, M., Kurakin, S., Cantú Reinhard, F.G., Schmid, P., Pöcheim, A., Winkler, C.
K., Kroutil, W., de Visser, S.P., Faber, K., 2019. Mechanistic studies of fatty acid
activation by CYP152 peroxygenases reveal unexpected desaturase activity. ACS
Catal 9 (1), 565–577. https://doi.org/10.1021/acscatal.8b03733.
[81] Piontek, K., Antorini, M., Choinowski, T., 2002. Crystal structure of a laccase
from the fungus Trametes versicolor at 1.90-A resolution containing a full
complement of coppers. J Biol Chem 277 (40), 37663–37669. https://doi.org/
10.1074/jbc.M204571200.
[82] Pivato, A.F., Miranda, G.M., Prichula, J., Lima, J.E.A., Ligabue, R.A., Seixas, A.,
Trentin, D.S., 2022. Hydrocarbon-based plastics: progress and perspectives on
consumption and biodegradation by insect larvae. Chemosphere 293, 133600.
https://doi.org/10.1016/j.chemosphere.2022.133600.
[83] Pometto 3rd, A.L., Lee, B.T., Johnson, K.E., 1992. Production of an extracellular
polyethylene-degrading enzyme(s) by Streptomyces species. Appl Environ
Microbiol 58 (2), 731–733. https://doi.org/10.1128/aem.58.2.731-733.1992.
[84] Purohit, J., Chattopadhyay, A., Teli, B., 2020. Metagenomic exploration of plastic
degrading microbes for biotechnological application. Curr Genom 21 (4),
253–270. https://doi.org/10.2174/1389202921999200525155711.
[85] Réjasse, A., Waeytens, J., Deniset-Besseau, A., Crapart, N., Nielsen-Leroux, C.,
Sandt, C., 2022. Plastic biodegradation: do galleria mellonella larvae
bioassimilate polyethylene? a spectral histology approach using isotopic labeling
and infrared microspectroscopy. Environ Sci Technol 56 (1), 525–534. https://
doi.org/10.1021/acs.est.1c03417.
[86] Restrepo-Flórez, J.-M., Bassi, A., Thompson, M.R., 2014. Microbial degradation
and deterioration of polyethylene – a review. Int Biodeterior Biodegrad 88,
83–90. https://doi.org/10.1016/j.ibiod.2013.12.014.
13
Journal of Hazardous Materials 460 (2023) 132449
[87] Ru, J., Huo, Y., Yang, Y., 2020. Microbial degradation and valorization of plastic
wastes. Front Microbiol 11, 442. https://doi.org/10.3389/fmicb.2020.00442.
[88] Sa’adu, I., Farsang, A., 2023. Plastic contamination in agricultural soils: a review.
Environ Sci Eur 35 (1), 13. https://doi.org/10.1186/s12302-023-00720-9.
[89] Saez-Jimenez, V., Fernandez-Fueyo, E., Medrano, F.J., Romero, A., Martinez, A.
T., Ruiz-Duenas, F.J., 2015. Improving the pH-stability of versatile peroxidase by
comparative structural analysis with a naturally-stable manganese peroxidase.
PLoS One 10 (10), e0140984. https://doi.org/10.1371/journal.pone.0140984.
[90] Sánchez, C., 2020. Fungal potential for the degradation of petroleum-based
polymers: an overview of macro- and microplastics biodegradation. Biotechnol
Adv 40, 107501. https://doi.org/10.1016/j.biotechadv.2019.107501.
[91] Sanluis-Verdes, A., Colomer-Vidal, P., Rodriguez-Ventura, F., Bello-Villarino, M.,
Spinola-Amilibia, M., Ruiz-Lopez, E., Illanes-Vicioso, R., Castroviejo, P., Aiese
Cigliano, R., Montoya, M., Falabella, P., Pesquera, C., Gonzalez-Legarreta, L.,
Arias-Palomo, E., Sola, M., Torroba, T., Arias, C.F., Bertocchini, F., 2022. Wax
worm saliva and the enzymes therein are the key to polyethylene degradation by
Galleria mellonella. Nat Commun 13 (1), 5568. https://doi.org/10.1038/s41467-
022-33127-w.
[92] Santacruz-Juarez, E., Buendia-Corona, R.E., Ramirez, R.E., Sanchez, C., 2021.
Fungal enzymes for the degradation of polyethylene: molecular docking
simulation and biodegradation pathway proposal. J Hazard Mater 411, 125118.
https://doi.org/10.1016/j.jhazmat.2021.125118.
[93] Santiago, G., De Salas, F., Lucas, M.Ft, Monza, E., Acebes, S., Martinez, An.T.,
Camarero, S., Guallar, V., 2016. Computer-aided laccase engineering: toward
biological oxidation of arylamines. ACS Catal 6 (8), 5415–5423. https://doi.org/
10.1021/acscatal.6b01460.
[94] Santo, M., Weitsman, R., Sivan, A., 2013. The role of the copper-binding enzyme –
laccase – in the biodegradation of polyethylene by the actinomycete Rhodococcus
ruber. Int Biodeterior Biodegrad 84, 204–210. https://doi.org/10.1016/j.
ibiod.2012.03.001.
[95] Scheiblbrandner, S., Breslmayr, E., Csarman, F., Paukner, R., Führer, J.,
Herzog, P.L., Shleev, S.V., Osipov, E.M., Tikhonova, T.V., Popov, V.O.,
Haltrich, D., Ludwig, R., Kittl, R., 2017. Evolving stability and pH-dependent
activity of the high redox potential Botrytis aclada laccase for enzymatic fuel
cells. Sci Rep 7 (1), 13688. https://doi.org/10.1038/s41598-017-13734-0.
[96] Selke, S., Auras, R., Nguyen, T.A., Castro Aguirre, E., Cheruvathur, R., Liu, Y.,
2015. Evaluation of biodegradation-promoting additives for plastics. Environ Sci
Technol 49 (6), 3769–3777. https://doi.org/10.1021/es504258u.
[97] Skariyachan, S., Patil, A.A., Shankar, A., Manjunath, M., Bachappanavar, N.,
Kiran, S., 2018. Enhanced polymer degradation of polyethylene and
polypropylene by novel thermophilic consortia of Brevibacillus sps. and
Aneurinibacillus sp. screened from waste management landfills and sewage
treatment plants. Polym Degrad Stab 149, 52–68. https://doi.org/10.1016/j.
polymdegradstab.2018.01.018.
[98] Son, H.F., Cho, I.J., Joo, S., Seo, H., Sagong, H.-Y., Choi, S.Y., Lee, S.Y., Kim, K.-J.,
2019. Rational protein engineering of thermo-stable PETase from ideonella
sakaiensis for highly efficient PET degradation. ACS Catal 9 (4), 3519–3526.
https://doi.org/10.1021/acscatal.9b00568.
[99] Sundaramoorthy, M., Gold, M.H., Poulos, T.L., 2010. Ultrahigh (0.93A) resolution
structure of manganese peroxidase from Phanerochaete chrysosporium:
implications for the catalytic mechanism. J Inorg Biochem 104 (6), 683–690.
https://doi.org/10.1016/j.jinorgbio.2010.02.011.
[100] Temporiti, M.E.E., Nicola, L., Nielsen, E., Tosi, S., 2022. Fungal enzymes involved
in plastics biodegradation. Microorganisms 10 (6). https://doi.org/10.3390/
microorganisms10061180.
[101] Tournier, V., Topham, C.M., Gilles, A., David, B., Folgoas, C., Moya-Leclair, E.,
Kamionka, E., Desrousseaux, M.L., Texier, H., Gavalda, S., Cot, M., Guemard, E.,
Dalibey, M., Nomme, J., Cioci, G., Barbe, S., Chateau, M., Andre, I., Duquesne, S.,
Marty, A., 2020. An engineered PET depolymerase to break down and recycle
plastic bottles. Nature 580 (7802), 216–219. https://doi.org/10.1038/s41586-
020-2149-4.
[102] van Kempen, M., Kim, S.S., Tumescheit, C., Mirdita, M., Lee, J., Gilchrist, C.L.M.,
Söding, J., Steinegger, M., 2023. Fast and accurate protein structure search with
Foldseek. Nat Biotechnol. https://doi.org/10.1038/s41587-023-01773-0.
[103] Vicente, A.I., Viña-Gonzalez, J., Mateljak, I., Monza, E., Lucas, F., Guallar, V.,
Alcalde, M., 2020. Enhancing thermostability by modifying flexible surface loops
in an evolved high-redox potential laccase. AIChE J 66 (3), e16747. https://doi.
org/10.1002/aic.16747.
[104] Vimala, P.P., Mathew, L., 2016. Biodegradation of polyethylene using bacillus
subtilis. Procedia Technol 24, 232–239. https://doi.org/10.1016/j.
protcy.2016.05.031.
[105] Wade, W., 2002. Unculturable bacteria–the uncharacterized organisms that cause
oral infections. J R Soc Med 95 (2), 81–83. https://doi.org/10.1177/
014107680209500207.
[106] Wang, J., Lisanza, S., Juergens, D., Tischer, D., Watson, J.L., Castro, K.M.,
Ragotte, R., Saragovi, A., Milles, L.F., Baek, M., Anishchenko, I., Yang, W.,
Hicks, D.R., Exposit, M., Schlichthaerle, T., Chun, J.H., Dauparas, J., Bennett, N.,
Wicky, B.I.M., Muenks, A., DiMaio, F., Correia, B., Ovchinnikov, S., Baker, D.,
2022. Scaffolding protein functional sites using deep learning. Science 377
(6604), 387–394. https://doi.org/10.1126/science.abn2100.
[107] Weber, C., Pusch, S., Opatz, T., 2017. Polyethylene bio-degradation by
caterpillars. Curr Biol 27 (15), R744–R745. https://doi.org/10.1016/j.
cub.2017.07.004.
[108] Wei, R., Zimmermann, W., 2017. Microbial enzymes for the recycling of
recalcitrant petroleum-based plastics: how far are we? Micro Biotechnol 10 (6),
1308–1322. https://doi.org/10.1111/1751-7915.12710.
J. Jin et al.
[109] Xu, F., Berka, R.M., Wahleithner, J.A., Nelson, B.A., Shuster, J.R., Brown, S.H.,
Palmer, A.E., Solomon, E.I., 1998. Site-directed mutations in fungal laccase: effect
on redox potential, activity and pH profile. Biochem J 334 (Pt 1), 63–70. https://
doi.org/10.1042/bj3340063.
[110] Yang, X.G., Wen, P.P., Yang, Y.F., Jia, P.P., Li, W.G., Pei, D.S., 2022. Plastic
biodegradation by in vitro environmental microorganisms and in vivo gut
microorganisms of insects. Front Microbiol 13, 1001750. https://doi.org/
10.3389/fmicb.2022.1001750.
[111] Yao, C., Xia, W., Dou, M., Du, Y., Wu, J., 2022. Oxidative degradation of UV-
irradiated polyethylene by laccase-mediator system. J Hazard Mater 440, 129709.
https://doi.org/10.1016/j.jhazmat.2022.129709.
[112] Yao, Z., Seong, H.J., Jang, Y.-S., 2022. Environmental toxicity and decomposition
of polyethylene. Ecotoxicol Environ Saf 242, 113933. https://doi.org/10.1016/j.
ecoenv.2022.113933.
[113] Yeh, A.H.-W., Norn, C., Kipnis, Y., Tischer, D., Pellock, S.J., Evans, D., Ma, P.,
Lee, G.R., Zhang, J.Z., Anishchenko, I., Coventry, B., Cao, L., Dauparas, J.,
Halabiya, S., DeWitt, M., Carter, L., Houk, K.N., Baker, D., 2023. De novo design
of luciferases using deep learning. Nature 614 (7949), 774–780. https://doi.org/
10.1038/s41586-023-05696-3.
[114] Yeom, S.J., Le, T.K., Yun, C.H., 2022. P450-driven plastic-degrading synthetic
bacteria. Trends Biotechnol 40 (2), 166–179. https://doi.org/10.1016/j.
tibtech.2021.06.003.
[115] Yoon MG, J.H., Kim, M.N., 2012. Biodegradation of Polyethylene by a Soil
Bacterium and AlkB Cloned Recombinant Cell. J Bioremediation Biodegrad 3,
145. https://doi.org/10.4172/2155-6199.1000145.
[116] Yoshida, S., Hiraga, K., Takehana, T., Taniguchi, I., Yamaji, H., Maeda, Y.,
Toyohara, K., Miyamoto, K., Kimura, Y., Oda, K., 2016. A bacterium that degrades
and assimilates poly(ethylene terephthalate. Science 351 (6278), 1196–1199.
https://doi.org/10.1126/science.aad6359.
[117] Zadjelovic, V., Erni-Cassola, G., Obrador-Viel, T., Lester, D., Eley, Y., Gibson, M.I.,
Dorador, C., Golyshin, P.N., Black, S., Wellington, E.M.H., Christie-Oleza, J.A.,
2022. A mechanistic understanding of polyethylene biodegradation by the marine
bacterium Alcanivorax. J Hazard Mater 436, 129278. https://doi.org/10.1016/j.
jhazmat.2022.129278.
[118] Zampolli, J., Orro, A., Manconi, A., Ami, D., Natalello, A., Di Gennaro, P., 2021.
Transcriptomic analysis of Rhodococcus opacus R7 grown on polyethylene by
RNA-seq. Sci Rep 11 (1), 21311. https://doi.org/10.1038/s41598-021-00525-x.
14
Journal of Hazardous Materials 460 (2023) 132449
[119] Zeghal, E., Vaksmaa, A., Vielfaure, H., Boekhout, T., Niemann, H., 2021. The
potential role of marine fungi in plastic degradation – a review. Front Mar Sci 8.
https://doi.org/10.3389/fmars.2021.738877.
[120] Zhang, A., Hou, Y., Wang, Q., Wang, Y., 2022. Characteristics and polyethylene
biodegradation function of a novel cold-adapted bacterial laccase from Antarctic
sea ice psychrophile Psychrobacter sp. NJ228. J Hazard Mater 439, 129656.
https://doi.org/10.1016/j.jhazmat.2022.129656.
[121] Zhang, A., Hou, Y., Wang, Y., Wang, Q., Shan, X., Liu, J., 2023. Highly efficient
low-temperature biodegradation of polyethylene microplastics by using cold-
active laccase cell-surface display system. Bioresour Technol 382, 129164.
https://doi.org/10.1016/j.biortech.2023.129164.
[122] Zhang, J., Gao, D., Li, Q., Zhao, Y., Li, L., Lin, H., Bi, Q., Zhao, Y., 2020.
Biodegradation of polyethylene microplastic particles by the fungus Aspergillus
flavus from the guts of wax moth Galleria mellonella. Sci Total Environ 704,
135931. https://doi.org/10.1016/j.scitotenv.2019.135931.
[123] Zhang, Y., Lin, Y., Gou, H., Feng, X., Zhang, X., Yang, L., 2022. Screening of
polyethylene-degrading bacteria from rhyzopertha dominica and evaluation of its
key enzymes degrading polyethylene. Polymers 14 (23). https://doi.org/
10.3390/polym14235127.
[124] Zhang, Y., Pedersen, J.N., Eser, B.E., Guo, Z., 2022. Biodegradation of
polyethylene and polystyrene: from microbial deterioration to enzyme discovery.
Biotechnol Adv 60, 107991. https://doi.org/10.1016/j.biotechadv.2022.107991.
[125] Zhang, Y., Plesner, T.J., Ouyang, Y., Zheng, Y.-C., Bouhier, E., Berentzen, E.I.,
Zhang, M., Zhou, P., Zimmermann, W., Andersen, G.R., Eser, B.E., Guo, Z., 2023.
Computer-aided discovery of a novel thermophilic laccase for low-density
polyethylene degradation. J Hazard Mater 458, 131986. https://doi.org/
10.1016/j.jhazmat.2023.131986.
[126] Zhong, Z., Nong, W., Xie, Y., Hui, J.H.L., Chu, L.M., 2022. Long-term effect of
plastic feeding on growth and transcriptomic response of mealworms (Tenebrio
molitor L.). Chemosphere 287 (Pt 1), 132063. https://doi.org/10.1016/j.
chemosphere.2021.132063.
[127] Zhong-Johnson, E.Z.L., Voigt, C.A., Sinskey, A.J., 2021. An absorbance method
for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate)
films. Sci Rep 11 (1), 928. https://doi.org/10.1038/s41598-020-79031-5.