Proteins - 2022 - Buchholz - Plastics Degradation by Hydrolytic Enzymes The Plastics Active Enzymes Database PAZy

Received: 30 November 2021 Revised: 1 February 2022 Accepted: 14 February 2022
DOI: 10.1002/prot.26325
RESEARCH ARTICLE
Plastics degradation by hydrolytic enzymes: The plastics-active

enzymes database—PAZy
Patrick C. F. Buchholz1 | Golo Feuerriegel2 | Hongli Zhang2 |

2 2 2
Pablo Perez-Garcia | Lena-Luisa Nover | Jennifer Chow |
Wolfgang R. Streit2 | Jürgen Pleiss1
1
Institute of Biochemistry and Technical
Biochemistry, University of Stuttgart, Abstract
Stuttgart, Germany Petroleum-based plastics are durable and accumulate in all ecological niches. Knowl-
2
Department of Microbiology and
edge on enzymatic degradation is sparse. Today, less than 50 verified plastics-active
Biotechnology, University of Hamburg,
Hamburg, Germany enzymes are known. First examples of enzymes acting on the polymers polyethylene
terephthalate (PET) and polyurethane (PUR) have been reported together with a
Correspondence
Jürgen Pleiss, Institute of Biochemistry and detailed biochemical and structural description. Furthermore, very few polyamide
Technical Biochemistry, University of
(PA) oligomer active enzymes are known. In this article, the current known enzymes
Stuttgart, Allmandring 31, D-70569 Stuttgart,
Germany. acting on the synthetic polymers PET and PUR are briefly summarized, their publi-
Email: [email protected]
shed activity data were collected and integrated into a comprehensive open access
Wolfgang R. Streit, Department of
Microbiology and Biotechnology, Universität
database. The Plastics-Active Enzymes Database (PAZy) represents an inventory of
Hamburg, Ohnhorststraße 18, D-22609 known and experimentally verified enzymes that act on synthetic fossil fuel-based
Hamburg, Germany.
Email: [email protected]
polymers. Almost 3000 homologs of PET-active enzymes were identified by profile
hidden Markov models. Over 2000 homologs of PUR-active enzymes were identified
Funding information
Bundesministerium für Bildung und Forschung,
by BLAST. Based on multiple sequence alignments, conservation analysis identified
Grant/Award Numbers: 031B867B, the most conserved amino acids, and sequence motifs for PET- and PUR-active
031B0837B, 031B0571B, 031B0571A,
031B0562A
enzymes were derived.
KEYWORDS
hidden Markov model, hydrolases, metagenome, polyethylene terephthalate degradation,
polyurethane degradation, sequence motif
1 | I N T RO DU CT I O N can be removed mechanically from ocean surfaces or terrestrial sites,

smaller particles (microplastics) will remain there unless microbial or
Today, we face the global challenge of plastics pollution in nearly all chemical degradation (i.e., weathering) will occur.3–5 Plastic waste is a
environments. The pollution has meanwhile reached levels that will valuable raw material, therefore recycling is a promising alternative to
ultimately have impact on our food chain and well-being within the incineration, either as a basis of synthesis of polymers or as a carbon
next decades. A recent study implied that about 399 000 tons of plas- source for fermentation.6
tics are present in the oceans alone, of which 69 000 tons are micro- Petroleum-based plastics are in general extremely stable and
plastics.1 Thus, urgent actions need to be implemented for removal of durable; hence, it is widely accepted that plastics do not degrade well
plastics from the environment and by reducing the steady input into in nature,7 nor can be directly used in fermentation. The degradation
the environment.2 Whereas it is perhaps more likely that large pieces processes described so far are slow, and it was shown that a PET
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2022 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.
Proteins. 2022;90:1443–1456. wileyonlinelibrary.com/journal/prot 1443

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1444 BUCHHOLZ ET AL.
bottle remains up to 48 years in the ocean until it is decomposed by exoenzymes, and it can be speculated that plastics-binding domains or
microbial degradation.8 Within this setting, it is reasonable to specu- proteins might contribute to degradation, similar to the role of cellu-
late that prior to microbial and enzymatic degradation, mechanical lose binding domains or expansins in the degradation of cellulosic
treatment (waves, wind, friction) and photodegradation by UV light materials.
(especially for aromatic ring-containing polymers such as PET and PS) To advance the research field, we have collected information of
break down the debris into microplastics, thereby increasing the sur- the currently known and verified plastics-active enzymes in the
face area, which mediates microbial degradation. For more details on Plastics-Active Enzymes Database (PAZy). Because we mainly focus
microplastics-associated bacteria and fungi, we refer to excellent on synthetic fossil fuel-based plastics, enzymes degrading bioplastics
reviews of the field of plastics ecology.9–11 However, the colonization such polylactide (PLAs) or polyhydroxyalkanoate-based polymers
of microplastics does not necessarily indicate that the polymer is (PHAs) were excluded in this manuscript. For the latter and their
degraded, because additives are in general more bioavailable than the global distribution, we refer to two excellent review articles on PHA
polymers. Therefore, measuring weight loss as an indicator for degra- hydrolases.49,50
2
dation might result in a false interpretation of the data, and in the Thus, we have included enzymes acting on low crystalline poly-
conclusion that we already have many plastics-active enzymes from mers PET and ester-based PUR. Since for none of the other fossil-
different microbial sources. A detailed search in the PubMed database based polymers (e.g., PE, PP, PVC, PS, ether-based PUR, larger PA
revealed that today roughly 2500 publications address the topic of polymers) truly functional enzymes are known, we have not yet
plastics degradation. However, less than 60 described the isolation included them. For nylon (PA), we have added the information on the
and biochemical characterization of plastics-active enzymes (Tables 1 few oligomer-active enzymes in the web-based version of the PAZy
and S1–S3). Nevertheless, while this obvious challenge can be met by data base. Similarily, the web-based version contains information on
better analytical techniques, the by far greater risk for misinterpreta- enzymes acting on polylactic acid (PLA), polyhydroxyalkanoates
tion of data comes from the unfiltered and noncritical use of the (PHAs) and synthetic and natural rubber (NR, SR).
predicted plastic degrading microorganisms and consortia by not veri- Despite the obvious lack of enzymes acting on many of the fossil
fied bioinformatic tools and pipelines. fuel-based polymers, already the current version of the PAZy data-
For instance, one recent study developed hidden Markov models base will serve as a comprehensive resource for the identification of
(HMMs) for some plastic degrading enzymes and predicted a global further novel plastics-active enzymes, pathways, or microorganisms
distribution even though no such enzymes have been biochemically for plastics removal in industry and the environment. It will further
47
characterized. Others have developed phylogenetic trees and global help to advance improved circular use of the different plastic types.
distribution patterns by simply using automated literature searches This data base will serve as a first platform and will be developed fur-
without critical analyses of the data.48 These very recent studies in ther on over the next few years. PAZy will in general be a valuable
high-ranking journals are perhaps only the tip of the iceberg, but repository and tool in this emerging field of plastics research.
clearly demonstrate that there is an urgent need for standardized and
verified enzyme databases in this rapidly developing field. The non-
critical and unfiltered use of many of the potential plastic degrading 2 | METHODS
gene sequences ultimately leads to incorrect conclusions on the avail-
ability of plastic degrading enzymes and their role in nature. These 2.1 | Data selection
studies do not only mislead research works, they furthermore suggest
to environmentalists, policy and law makers, and even to the broader Protein sequences with available UniProt identifiers and known activ-
public audience that we would have solutions for the global plastics ity against PET or PUR were downloaded from the Plastics Microbial
problem, which we however do not have. Within this framework, the Biodegradation Database51 (PMBD, http://pmbd.genome-mining.cn/
proposed PAZy database will be a reliable and very useful tool giving home/) and NCBI GenBank. Based on available biochemical and/or
an overview on truly functional enzymes. structural data, in total 44 protein sequences were selected from the
Notably today, only for polyethylene terephthalate (PET), poly- PMBD, with 34 and 10 UniProt identifiers for PET and PUR activity,
urethane ester-based (PUR), and polyamide (oligomers) (PA), a rather respectively (as of November 2021).
small number of degrading enzymes are known, but none for other
major fossil-fuel based polymers such as PVC, PE, PP, and PS, and
most of the ether-based PUR polymers. The known fossil-fuel based 2.2 | PETase homologs
plastics-degrading enzymes are hydrolases, often annotated as lipases,
esterases, cutinases, amidases, or proteases (E.C. 3.1.x). However, we We are aware that there are controversial discussions about the term
have still a limited understanding of the mechanism of enzymatic deg- PETase, but we prefer to define all PET-active enzymes as PETases.
radation. It is not clear to which extent bacteria have evolved specific Sixteen protein sequences for enzymes with known activity against
enzymes that bind to the polymers and cleave the bonds similar to the PET were clustered using CD-HIT (version 4.6.8-1) at a threshold of
processes that occur when cellulose or other biopolymers are 90% sequence identity and a word length of 5 to derive a reduced set
degraded. It is supposed that plastics-degrading enzymes are of 12 centroid sequences.52,53 These protein sequences were aligned
BUCHHOLZ ET AL. 1445
TABLE 1 Currently known and active PET hydrolases structural or molecular analyses
Microbial host, Genbank, PDB WT enzymes and variants Activity on PET foil, powder,
enzyme, gene References UniProt IDa structure tested bottleb
Proteobacteria-affiliated
PET active enzymes
Ideonella sakaiensis 201-F6, 12 A0A0K8P6T7 5XFY WT
IsPETase, ISF6_4831
Oleispira antarctica RB-8, 13 R4YKL9_OLEAN WT +
PET5, LipA
Vibrio gazogenes, 13 A0A1Z2SIQ1_VIBGA WT +
PET6,
Gene: BSQ33_03270
Polyangium brachysporum, 13 A0A0G3BI90_9BURK WT +
PET12,
Gene: AAW51_2473
Pseudomonas 14 KU695574 n.d.
pseudoalcaligenes DSM
50188,PpCutA
P. pelagia DSM 25163 14 KU695573 n.d.
PpelaLip
Pseudomonas aestusnigri 15 A0A1H6AD45 6SBN WT, Y250S, 6 mg MHET/L from PET film
VGXO14, 6SCD S171A, D217A, H249A, (WT and Y250S) in
B7O88_11480, PE-H G254S, S256N, I257S, 24 h 500 nM enzyme 1;
Y258N, N259Q, ext.loop, and 0.1 mg/ MHET from PET
Q294A, I219Y bottle for Y250S in
24 h 500 nM enzyme 1
Pseudomonas mendocina 16 N20M5AZM016 2FX5 solubilized 250-μm thick films
ATCC 53552, PmC in 96 h
Actinobacterial enzymes
LCC, leaf compost 17 G9BY57 12 mg TAeq. h 1 mg
metagenome AEV21261.1 enzyme 1 with WT enzyme
18 4EB0
19 N197Q, N266Q, N239G,
LCC-G
20 6THS S165A, ICCG-S165A 105.6 ± 3.9 mg TAeq. h 1
6THT F243I/D238C/ S283C/ mg enzyme 1, on
Y127G (ICCG), F243I/ commercial GF-PET with
D238C/ S283C/N246M best variant
(ICCM), F243W/D238C/
S283C/Y127G (WCCG) and
F243W/D238C/ S283C/
N246M (WCCM), F243I/
D238C/ S283C/T96M,
F243I/D238C/ S283C/
N246D, F243W/D238C/
S283C/T96M, F243W/
D238C/ S283C/N246D,
F243I/D238C/ S283C,
F243W/D238C/S283C,
D238C/S283C, T96M,
Y127G, F243I, F243W,
N246D, N246M
BhrPETase from HRB29 21 GBD22443 WT 0.17 mM BHET, 3.66 mM
bacterium MHET and 2.47 mM TA
from nanoparticles in 20 h
Thermobifida fusca 22–25 Q6A0I4_THEFU WT +
DSM43793, AJ810119
TfH, BTA-1
(Continues)
TABLE 1 (Continued)
T. fusca DSM43793, 22–25 AJ810119 +
TfH, BTA-2
T. fusca DSM 44342, 26 E9LVI0_THEFU 41 mmol TA/mmol enzyme in
TfH42_Cut1 ADV92528.1 120 h
T. fusca, (strain xy) 27–29 Q47RJ7_THEFY n.d.
WSH03-11, Tfu_0882
T. fusca, (strain xy) 27 Q47RJ6_THEFY n.d.
WSH03-11, Tfu_0883
T. fusca, 30 E5BBQ3_THEFU G62A/F209A 31 ± 0.1 nmol
TfCut-2 (Cut2-kw3) min 1 cm 2 on films; 12-fold
better than WT; lcPET film
(200 μm) 97% ± 1.8% in
30 h
31 4CG1
4CG2
4CG3
32 4CG1 G62A/I213S, G62A 42% weight loss after 50 h on
film G62A/I213S, G62A
2.7-fold better than WT
T. fusca NTU22, TfAXE 33 ADM47605.1 n.d.
T. fusca NRRL B-8184, Cut1 34 JN129499.1 n.d.
T. fusca NRRL B-8184, Cut2 34 JN129500.1 n.d.
Thermobifida cellulosilytica 26,35 ADV92526.1 5LUI 56 mmol TA/mmol enzyme in
DSM44535, Thc_Cut1 120 h
T. cellulosilytica DSM44535, 26,35 ADV92527.1 5LUJ R29N/A30V, 5 mmol TA/mmol enzyme in
Thc_Cut2 5LUL R19S/R29N/A30V 120 h
5LUK
Thermobifida alba AHL119, 36 F7IX06 6AID n.d.
Est119, est2 3WYN
3VIS
Thermomonospora curvata 37 D1A9G5 n.d.
DSM43183, ACY96861.1
Tcur_1278
T. curvata DSM43183, 37 ACY95991.1 n.d.
Tcur0390
Thermobifida halotolerans, 38 H6WX58 21.5 mmol TA mol
Thh_Est enzyme 1 in 2 h
T. alba, 39 BAI99230 n.d.
Est1 (Hydrolase 4)
Saccharomonospora 40–42 W0TJ64 4WFK S226P, S226P/R228S, 30% increase in Q138A/
(Thermoactinomyces) AB728484 4WFI S226P/R228S/ D250C-E296C/Q123H/
viridis AHK190, Cut190 4WFJ T262K, N202H variant, but no
7CTR Q138A/D250C-E296C/ kinetic data
7CTS Q123H/
N202H, disulfide bonds at
N250 and E296
Firmicutes
Bacillus subtilis 4P3-11, 43 ADH43200.1 n.d.
BsEstB
Bacteroidetes
Aequorivita sp. CIP111184, 44 WP_111881932 Active on foil and powder
PET27
Kaistella (Chryseobacteriu) 44 WP_039353427 7PZJ Active on foil and powder
jeonii, PET30
TABLE 1 (Continued)
Metagenome, probably
bacterial and
phylogenetically
unassigned
PET2, from metagenome, 13 C3RYL0 +
affiliated no obvious ACC95208.1
affiliation, lipIAF5-2
45 7ECB R47C/G89C/F105R/E110K/ 6.8-fold increase over WT
7EC8 S156P/G180A/T297P after 60 min in PET2 7 M
(PET2 7 M), F105R/E110K/ variant
S156P/G180A/T297P,
F105R/E110K/S156P/
T297P, R47C/G89C,
F105R/E110K, Y262C/
L298C, L265C/A295C,
D53P, F105R, E110K,
Q134Y, S155D, S156P,
W174H, G177A, G178A,
G179A, G180A, Q183R,
A192P, S202Q, T297P,
L298R
PET active enzymes from
Eukaryotes
Candida antarctica, 16 LIPB_PSEA2 WT Solubilized 250-μm thick films
lipase B, CalB in 96 h
46 WT +
Fusarium solani, 16 AAA33335.1 1OXM WT Solubilized 250-μm thick films
FsC in 96 h
Thermomyces (Humicola) 16 A0A075B5G4 4OYY, WT Solubilized 250-μm thick films
insolens, 4OYL in 96 h
HiC 46 WT +
Note: All WT enzymes listed were verified for their PET hydrolytic activities by either PET films, PET powder PET bottles, PET coupons, PET nanoparticles,
or various synthesized model polyester polymers. Only isolates were included that are available in public strain repositories. Recently published variants of
IsPETase and the LCC enzyme are summarized in Table S10.
Abbreviations: , no activity observed; +, activity observed but not quantified; n.d., not determined.
a
Active link to NCBI Pubmed database.
b
Crystallinity of PET films, bottles, and samples and experimental conditions varied largely in the different studies and thus makes direct comparison
difficult.
in a structure-guided multiple sequence alignment by T-COFFEE (ver- PETase-profile HMM were selected from the HMMER results with a
sion 11.00.8cbe486-1).54 A profile HMM was derived from this multi- minimal score of 100, a minimal profile coverage of 95%, and a maxi-
ple sequence alignment by HMMER (version 3.1b2, http://hmmer. mum ratio of bias/score of 10%.
org). The profile HMM was trimmed by selecting alignment columns HMMER was also used to identify the C-terminal region for the
that corresponded to the region between amino acid positions 32 and Type IX secretion system sorting domain, using the profile HMM
274 in the PETase from Ideonella sakaiensis (IsPETase, UniProt identi- TIGR04183, which was derived from a multiple sequence alignment
fier A0A0K8P6T7) to avoid ambiguities at the N- and C-termini of 889 protein sequences in the TIGRFAM database (http://tigrfams.
(Figure S1; Table S4). The profile HMM and the underlying multiple jcvi.org/cgi-bin/index.cgi), with an E-value cut-off below 1.
sequence alignment can be downloaded from https://doi.org/10.
18419/darus-2055. This PETase-profile HMM was used to search
both the NCBI nonredundant (nr) protein database and the Protein 2.3 | PURase homologs
Data Bank (PDB) for an update of the Lipase Engineering Database
(LED, https://led.biocatnet.de), which was previously established as a Four protein sequences for enzymes with known activity against PUR
collection of protein sequences from α/β-hydrolases.55–57 Hits for the served as queries for BLAST (blastp, version 2.10.0+) against the
NCBI nonredundant (nr) protein database and the PDB.58 BLAST per- accessible at https://www.pazy.eu. Within the PAZy infrastructure,
formance was improved by multithreading with GNU/Parallel (version the LED (https://led.biocatnet.de) serves as the database for protein
20 170 622-1). 59
The BLAST results were filtered by an E-value sequences and structures from different superfamilies of α/-
threshold of 10 10
and a minimal coverage of 50% to further update β-hydrolases and their sequence annotations, since all currently
the LED. known enzyme activities toward PET or PUR were reported for
α/β-hydrolases.
For the update of the LED, 4887 entries were downloaded from
2.4 | Conservation analyses the NCBI nonredundant protein database, and 93 entries were down-
loaded from the PDB using the criteria mentioned in Section 2. The
The PETase-profile HMM was applied for a standard numbering updated LED contains 283 672 sequence entries and 1590 PDB
scheme, by aligning the 2930 sequences of PETase homologs from entries (an increase of 3034 and 33 entries compared to the previ-
the LED against the respective profile HMM and subsequently assig- ously published LED version from June 2019, respectively). For the
ning the position numbers from the IsPETase reference sequence as update of the LED, sequences that shared at least 50% similarity were
standard position numbers. For conservation analysis of PETase assigned to the same superfamily (Table S5). Sequences that shared at
homologs, the frequency of amino acid residues or gaps was counted least 60% similarity were assigned to a homologous family; otherwise,
at each standard position. they were assigned to a separate group containing all “singleton”
For the conservation analysis of PURase homologs from LED sequences. A new superfamily was introduced for PURase homologs
superfamilies 11 and 13, two multiple sequence alignments were gen- of PudA from D. acidovorans, as outlined in more detail below.
60
erated using Clustal Omega (version 1.2.4), and the frequency of
amino acid residues or gaps was counted at selected positions.
3.2 | PET active enzymes
2.5 | Protein sequence networks 3.2.1 | Biochemical properties
Sets of representative protein sequences were formed by clustering Our literature searches identified a total of over 35 wild-type enzymes
with CD-HIT to reduce the sample size and thus computational effort that have been shown to catalyze the partial degradation of PET to
for pairwise sequence alignments. Values of pairwise sequence iden- oligomers or even to monomers, originating from four different bacte-
tity or similarity were calculated by the Needleman–Wunsch algo- rial phyla and one eukaryotic lineage (Table 1). No archaeal PETases
rithm available in EMBOSS (version 6.6.0) with default gap opening have been functionally verified to date.
and gap extension penalties of 10 and 0.5, respectively, and the sub- Many of the currently known PETases are thermostable enzymes,
61,62
stitution matrix BLOSUM62. because the catalytic activity increases at temperatures close to the
Collections of protein sequences were represented as protein glass transition temperature (65 C) of PET due to the formation of
sequence networks that depicted sequences as nodes connected by flexible and thus enzyme-accessible amorphous domains.65 Notably,
edges (lines). The edges in a protein sequence network were weighted few enzymes are active at lower temperatures implying they may play
by values of pairwise sequence identity or similarity. A threshold of a role in cold-adapted PET degradation.66 However, all known native
the respective edge weights was chosen to select a subset of edges PETases have rather low catalytic activity toward PET.
for the network. Protein sequence networks were visualized in Cyto-
scape (version 3.8.2) with the prefuse-force directed layout algorithm,
taking the edge weights into account63: edges of higher sequence 3.2.2 | Enzyme structures
identity or similarity were depicted preferably in closer vicinity to each
other. The Python NetworkX package (version 1.11) was used to store All known PETases are α/β-hydrolases and are either cutinases,
the metadata of protein sequence networks in GraphML format, avail- lipases, or esterases and grouped into EC 3.1.1.101; EC 3.1.1.1, EC
64
able for download at https://doi.org/10.18419/darus-2054. 3.1.1.2; EC 3.1.1.3; and EC 3.1.1.74. Recently, the PETase from
I. sakainens was placed in a distinct class EC 3.1.1.101. In solution,
PETases are supposed to be active as monomers.67 A total of 12 struc-
3 | RESULTS tures affiliated with different organisms and the wild-type enzymes
are available in the PDB. For the best characterized examples LCC
3.1 | Update of the LED and IsPETase, multiple entries of variants have been made. All PET-
ases consist of a single domain, the α/β-hydrolase fold, which is
We focus only on validated enzymes acting on the synthetic and fossil formed by a central twisted β-sheet, flanked by two layers of
fuel-based polymers PET and PUR. Detailed biochemical data of cata- α-helices,68 and thus belong to a class of small α/β-hydrolases that
lytically active enzymes (Tables 1 and S1–S3), analyzed sequences and consist only of the core domain without a mobile lid.57 For a few PET-
structures of homologous proteins are comprised in the PAZy and ase homologs from Bacteroidetes, an additional C-terminal sorting
domain for the Type IX secretion system has been annotated and was additional regions at the N- or C-termini (signal peptides or transport
verified in the single structure published (Figure S5; Table 1). The domains, respectively). In superfamily 1 of the LED, 31 560 sequences
69
Type IX secretion system comprises several protein components, were annotated as GX-type, but only 2930 sequences were identified
and the corresponding C-terminal domain was also found in other as PETase homologous by a profile HMM. At a threshold of 55%
polymer-active enzymes such as cellulases and endo-1,4-β sequence similarity, the bacterial PETase core domains formed a large
-glucanases.70–73 PETases share a conserved catalytic triad of serine, cluster, mainly originating from Actinobacteria or Proteobacteria
histidine, and aspartic acid, and a GX-type oxyanion hole, which stabi- (Figure 1). Most of the sequences from the PMBD were found in this
lizes the reaction intermediate.74 In the PETase homologs, the first cluster (Figure S3). In addition, a connected subgroup of PETase core
oxyanion hole residue X is mostly a conserved aromatic residue such domains from other bacterial phyla emerged, such as the PETase pro-
as tyrosine or phenylalanine. The second oxyanion-stabilizing residue teins from Bacteroidetes or Planctomycetes. Some homologs of PET-
is a conserved methionine following the serine of the catalytic triad. ase core domains occurred also in enzymes from extremophiles
For the PETase from I. sakaiensis, several residues were suggested for (Figure S4). The fungal PETase core domains such as the PETase
substrate binding, such as an aromatic clamp formed by the first resi- homologs from Fusarium were separated from the bacterial PETase
due of the oxyanion hole and a second aromatic residue.75 In addition core domains. At a higher threshold of 60% sequence similarity, the
to this subsite I, a second subsite II was proposed from the interaction sequences for PETase core domains from Bacteroidetes or
observed in a modeled complex with a PET monomer.76 Variants with Planctomycetes emerged as a separated cluster (Figure S5).
increased catalytic activity were designed and tested. The most active
enzymes are LCC variants, where the addition of disulfide bonds
increased thermostability.20 The two LCC quadruple variants F243 3.2.4 | Sequence motifs
[WI]-D238C-S283C-Y127G, which include the additional disulfide
bond, are among the most active PETases described to date. The PETase-profile HMM 13 was applied to analyze the conservation
of amino acid residues in the 2930 PETase core domains annotated in
the LED (Table S6) in comparison to the equivalent positions in the
3.2.3 | Sequence network PETase from I. sakaiensis (IsPETase, UniProt identifier A0A0K8P6T7)
and LCC (UniProt identifier G9BY57). The catalytic triad, the previ-
For the comparison of PETase sequences, the profile HMM for PET- ously suggested PET binding subsite I, which includes an aromatic
ases 13 was used to identify the PETase core domain, and the clamp for possible substrate interaction, and PET binding subsite II
sequences of all core domains were aligned without considering from Joo et al.76 were found to be highly conserved (Table 2). The
F I G U R E 1 Network representation for 869 protein sequences of the “PETase core domain” linked by 318 773 edges. The protein sequences
depicted here were selected by clustering at a threshold of 90% sequence identity. Edges (links) were selected at a threshold of 55% sequence
similarity. Nodes are colored according to their annotated source organisms, with Actinobacteria in red , Proteobacteria in blue , Fungi in cyan
, Bacteroidetes in orange , other bacteria from the FCB group in yellow , Planctomycetes in green , and unknown bacteria colored in white .
See Section 2 for more details on the network layout. See Figures S2 and S3 for supplementary figures
TABLE 2 A selection of amino acid positions in the 2930 PETase homologs from the LED
Position in IsPETase Amino acid or gap symbol Annotations or substitutions References

87 Y 51%, F 46% Subsite I, aromatic clamp, oxyanion hole 75,76
88 T 76% (V 7%, L 5%, M 1%) Subsite II 76
Thermostability: T96M in LCC 20
89 A 70% (G 12%, S 10%) Subsite II 76
119 Q 69% (F8%, Y4%, W2%) Subsite I 76
Thermostability: Y127G in LCC 20
139 Gap 91% Extension in α-helix 2 75
159 H 87% (W9%) Subsite II 75,76
160 S 100% Catalytic triad 75
161 M 94% Subsite I, oxyanion hole 76
185 W 77% (Y12%) Subsite I, aromatic clamp, “wobbly tryptophane” 75,76
206 D 100% Catalytic triad 75
208 V 54% (I 36%) Substrate interaction 75
237 H 100% Catalytic triad 75
238 F 64% (L 11%, S 8%, Y 3%) Subsite II 75,76
Binding & activity: F243I in LCC 20
Binding & activity: F243W in LCC 20
241 N 56% (Q 12%) Subsite II 76
Thermostability: N246D in LCC 20
Thermostability: N246M in LCC 20
242 (S 36%, T 32%, G 5%, I 4%) Extended loop 75,76
243 (S 40, G 11%, T 8%) Extended loop 76
244 N 74% (G 3%, Y 3%) Extended loop 76
245 87% Extended loop 76
Note: Amino acid residues that occurred in less than 50% of the sequences are listed in brackets for comparison with their previous mentions in literature.
Amino acid substitutions “in LCC” refer to the position numbers used in Tournier et al.20 Some positions only occurred in a small subset of all PETase
homologs and are thus indicated as gap symbols ( ). Percentages indicate values rounded to integers. Sixteen positions and their corresponding amino acid
symbols marked in red indicate the suggested PETase sequence motif, whereas the ones marked in green indicate additional positions that were used to
select the sequences mentioned in the main text.
extension of the second α-helix and the extended loop region, which gummiphilus, NCBI:WP_085749610.1, and from Aquabacterium sp.,
were described previously as functionally relevant in IsPETase, were NCBI: MBI3384080.1) were found to comprise the PETase sequence
also found in several PETase homologs in the LED. motif and M241, which was mentioned as an aa substitution for
Using the position numbers from IsPETase, we suggest a typical improved thermostability. These six different and novel protein
PETase sequence motif written as follows (X stands for an arbitrary sequences, each selected by a sequence motif of 17 amino acid posi-
aa): [YF]87, Q119, X3 139–141, S160, M161, W185, D206, H237, X6 tions in total, are proposed for upcoming studies on PETase activity.
242–247, followed by one of the previously published aa substitu-
tions from Tournier et al.20 Interestingly, two sequences from an
uncultured bacterium (NCBI: ACC95208.1) and Alkalilimnicola ehrlichii 3.3 | Polyurethanes (PUR) active enzymes
(NCBI: WP_116302080.1) were found to comprise the PETase
sequence motif and W238, which was affiliated with improved activ- 3.3.1 | Biochemical properties
ity and substrate binding. Further additional sequences (from
Caldimonas manganoxidans, NCBI: WP_019560450.1, from Polyurethanes comprise numerous possible polymers of diverse com-
Caldimonas taiwanensis, NCBI:WP_062195544.1, from Rhizobacter position, such as combinations of different isocyanates with different
polyethers, polyesters, or polycarbonates.77 The best studied PURases complex, which is an additional challenge for the identification of the
are α/β-hydrolases, as reviewed in more detail in Magnin et al.77 Only substrate binding site.81 Interestingly, most of the substrate binding
10 characterized PUR-degrading enzymes (PURases) have been residues predicted for PueA80 are conserved (Table S7).
reported, yet. Four recently identified enzymes (LCC, TfCut-2, The PURase from D. acidovorans (UniProt identifier Q9WX47)
Tcur_1278T, and Tcur0390) are cutinases from Actinomyces, which does not belong to the GX type hydrolases, but has a sequence simi-
are also active on PET and have a broad substrate profile.78 Further larity to carboxylesterases of superfamily 13 and to the family
bacterial lipases from Betaproteobacteria have been identified and PF00135 in Pfam, and thus belongs to the GGGX-type hydrolases.74
characterized, such as PueA and PueB from Pseudomonas chlororaphis. Other carboxylesterases in the LED are members of superfamily
Whereas all the above-mentioned studies identified lipases or ester- 4, which have a mobile lid between β-strand 4 and 3 of the core
ases, earlier studies reported that commercially available peptidases domain. Because the PURase from D. acidovorans shared less than
and proteases might also degrade thin PUR films.79 Notably, none of 50% sequence similarity to the sequences in the LED and due to the
the above-mentioned enzymes is able to act on ether bonds. lack of experimental structure information, it was assigned to a new
superfamily.
3.3.2 | Enzyme structure

3.3.3 | Sequence network
The 10 known PURases belong to two groups (Table S1): four belong
to the cutinases (LCC, TfCut-2, Tcur_1278T, and Tcur0390) and are A threshold of 60% similarity was used to construct protein sequence
similar to PETases. No crystal structures are available for the PURases networks for LED superfamilies 11 and 13 whose members originated
PueA and PueB from P. chlororaphis, but structures of homologs indi- from Proteobacteria only (Figure 2). Two disconnected sequence
cate that they belong to superfamily 11 of the LED, which in addition networks emerged: a network of 127 representative sequences of
to the core domain has a mobile N-terminal lid, which might mediate superfamily 11, which contains the GX-type PURases PueA and
binding to the substrate interface and substrate access, and an addi- PueB from P. chlororaphis, and a network of 15 representative
tional C-terminal β-sandwich domain. The four PETases and PueA and sequences of superfamily 13, which contains the GGGX-type, the
PueB from P. chlororaphis are GX types.74 Recently, a modeling study PURase PudA from D. acidovorans. In both superfamilies 11 and
on the PURases from P. chlororaphis predicted putative substrate 13, the sequences originated mainly from Gammaproteobacteria,
80
binding sites for PUR-like substrates. However, a rearrangement of with the genus Pseudomonas being most frequently annotated in
the substrate was observed upon the molecular simulation of the superfamily 11.
F I G U R E 2 Network representation for 142 complete protein sequences similar to PURases linked by 6419 edges. The protein sequences
depicted here were selected by clustering at a threshold of 90% sequence identity. Edges (links) were selected at a threshold of 60% global
sequence similarity, without defining a core domain region. Nodes are colored according to their annotated source organisms, with
Proteobacteria in blue and unknown bacteria in white. The network on the left represents sequences with an N-terminal lid and a C-terminal
β-sandwich domain and contains 127 nodes connected by 6314 edges. Diamonds represent sequences originating from the genus Pseudomonas
(from the class Gammaproteobacteria). The network on the right represents sequences similar to carboxylesterases and contains 15 nodes
connected by 105 edges. Squares represent sequences originating from the class of Betaproteobacteria. See Section 2 for more details on the
network layout
3.3.4 | Sequence motifs several positions in the putative PUR binding region were found to be
conserved, including mostly hydrophobic amino acids (Table S8).
82
Two sequence motifs were reported previously in Howard et al. for
the PURase from P. chlororaphis, PueA (Table 3). The occurrence of
the serine hydrolase motif (GXSXG) and the secretion signal sequence 4 | DI SCU SSION
motif (GGXGXDXXX) were confirmed for the vast majority of all 2054
sequence entries in a multiple sequence alignment for superfamily 4.1 | Sequence annotations of plastics-active
11 of the LED. Most PURase homologs from superfamily 11 have a enzymes
GHSLG motif flanking the catalytic serine and secretion motif
GGKGNDYLE. For sequences from superfamily 11 in the LED, the Enzymes of different EC classes have been proposed to contribute to
catalytic triad is formed by serine, aspartate, and histidine. In addition, degradation of PET or PUR. α/β-hydrolases annotated as cutinases,
most sequences in superfamily 11 (2039 out of 2054) matched the esterases, or lipases were described to catalyze the hydrolysis of ester
profile HMM for an RTX calcium-binding nonapeptide repeat (PFAM bonds and were collected in the PMBD,51 and peroxidases and
PF00353), which supports the previous suggestion of a Type I secre- laccases have been reported to enhance degradation of PUR.85 For
83
tion system for protein translocation. most types of plastics, however, knowledge on enzymatic degradation
Prominent amino acid positions in superfamily 13, which com- is missing, although materials such as polypropylene (PP) are produced
prises homologs of the PURase PudA from D. acidovorans, include the
GXSXG serine hydrolase motif, a catalytic triad of serine, aspartate,
and histidine, and a putative PUR binding region at PudA positions T A B L E 4 Amino acid positions mentioned in Nomura et al.84 and
347–395.84 Many of these positions were found to be conserved adjacent amino acid positions that occur in at least 50% of the 44
within the sequences of superfamily 13 (Table 4). Most PURase PURase homologs from the LED (superfamily 13)
homologs from superfamily 13 have the motif GESAG flanking the Position
catalytic serine, the motif VPX3G[ST]X2DE at the catalytic glutamate, in PudA Amino acid Annotation
and the motif AXHX3[LI]XY flanking the catalytic histidine. In addition, 223 G 100% Serine hydrolase motif GXSXG
224 E 90% Serine hydrolase motif GXSXG
225 S 100% Serine hydrolase motif GXSXG,
T A B L E 3 Amino acid positions mentioned in Howard et al.82 and catalytic triad
their occurrence in 2054 PURase homologs from the LED 226 A 97% Serine hydrolase motif GXSXG
(superfamily 11)
227 G 100% Serine hydrolase motif GXSXG
Position 340 V 100%
in PueA Amino acid Annotation
341 P 97%
204 G 100% Serine hydrolase motif GXSXG 342 V 64%
205 H 99% Serine hydrolase motif GXSXG 343 (I 33%, V 30%, M
206 S 100% Serine hydrolase motif 26%)
GXSXG, catalytic triad
345 G 100%
207 L 99% Serine hydrolase motif GXSXG 346 S 50% (T 42%)
208 G 100% Serine hydrolase motif GXSXG 347 N 71%
254 D 100% Catalytic triad
349 D 92%
312 H 100% Catalytic triad 350 E 100% Catalytic triad
381 G 99% Secretion motif GGXGXDXXX 457 A 95%
382 G 98% Secretion motif GGXGXDXXX 458 A 66%
383 K 39%, R 18%, A Secretion motif GGXGXDXXX 459 H 100% Catalytic triad
16%, S 13%
462 E 78%
384 G 98% Secretion motif GGXGXDXXX
463 L 69% (I 30%)
385 N 78%, A 16% Secretion motif GGXGXDXXX
464 Q 83%
386 D 99% Secretion motif GGXGXDXXX
465 Y 92%
387 Y 57%, F 42% Secretion motif GGXGXDXXX
466 L 83%
388 L 72%, I 27% Secretion motif GGXGXDXXX
Note: Position numbers refer to PudA from Delftia acidovorans, that is,
389 E 91% Secretion motif GGXGXDXXX
UniProt identifier Q9WX47. Similar amino acid residues occurring in less
Note: Position numbers refer to PueA from Pseudomonas chlororaphis, that than 50% of the sequences are indicated in brackets for comparison.
is, UniProt identifier A1Z374. Percentages indicate values rounded to Positions of a putative substrate binding region are listed in Table S5 for
integers. Position in the motif is marked in bold. comparison. Position in the motif is marked in bold.
in large scales and contribute extensively to global plastics pollution. based polymers. Thus one of the major challenges will be the identifi-
In this article, we focus on PET- and PUR-degrading α/β-hydrolases cation of novel enzymes for polymers for which none are currently
due to the availability of sequence information and detailed experi- known.2,87 Thus, we urgently need enzymes acting on PE, PP, PVC,
51
mental data from literature and public databases. but also on polymeric PA and PU ether bonds. Within this framework,
Many PETase homologs were found in the NCBI nonredundant the identification of plastic-specific binding domains, expansins, or
protein database, due to the sequencing efforts of the cutinase- loosenins will be of high relevance. Once these are identified they can
expressing bacterial phyla such as Actinobacteria and Proteobacteria. be used to further improve enzymes acting PET and PUR. Thereby, a
In contrast, sequences from fungal origin are unrepresented. The combination of modern synthetic biology approaches together with
usage of metagenomics is expected to further broaden the scope of enzyme engineering approaches will be the appropriate tools to gen-
currently known PETases.13 The PETase sequence motif suggested erate the best performing enzymes.
herein (Table 2) is based on current knowledge from literature, such Further we realized the lack of a common PET or PUR model sub-
as the occurrence of an additional flexible loop region and amino acids strate, which would allow the direct comparison of the kinetic param-
that seem relevant for interaction with PET. The seven suggested eters of different plastics-active enzymes. In contrast, kinetic analysis
PETase candidates can be used as starting points for wet-lab experi- is generally performed for typical esterase substrates such as pNP-
ments, such as protein design experiments for improved PETase activ- caproate. This data, however, does not allow a reliable prediction of
ity or thermostability. the actual plastics activity. The few enzymes that have been charac-
Although PETases and PURases share the α/β-hydrolase fold as terized using polymers were tested on different polymer types, and
catalytic domain, their structure and their oxyanion hole types differ. pretreatment was used to enable better degradation (see Table 1 for
All the 2930 PETase homologs belong to a large family (36 936 references). In addition, all kinetic data were recorded using single
sequences) of GX-types,74 which consist of the core domain only point measurements, thus the hydrolysis of the polymer could not be
(superfamily 1 in the Lipase Engineering Database57), whereas separated from the attachment of the enzyme to the polymer surface
PURases belong to either of three superfamilies: superfamily 1, super- or from the hydrolysis of the resulting oligomers. Within this frame-
family 11, which consists of GX-types with two additional domains, an work, a characterization of surface area and a control of surface prop-
N-terminal mobile lid and a C-terminal β-sandwich domain (2054 erties such as crystallinity would be favorable to obtain better and
sequences), or to superfamily 13, which are carboxylesterases of the more reliable kinetic data on the actual polymer.
GGGX-type (44 sequences). Ample structural information is available The accumulation of verified plastics-active enzymes in databases
for PET-degrading α/β-hydrolases, especially for LCC and IsPETase, with a reliable structure–function analysis will allow more predictive
whereas the structures of PUR-degrading α/β-hydrolases have not searches to rapidly and reliably identify novel and more active
been resolved, yet. The former has also inspired the design of enzymes. Thereby, it will allow to foster the search and development
improved PETase variants, as recently demonstrated for variants of novel pathways to create designer bugs using to solve the plastics
20
based on LCC. Previously, conserved subsite I, subsite II, and an problem.
extended loop region were identified in IsPETase and used for a sys-
tematic comparison with its homologs,76 which was confirmed by our
conservation analysis of 2930 protein sequences. The profile HMM 4.3 | How can databases contribute to a solution
for PETases and the derived standard numbering scheme is available of the plastics waste problem?
at the PAZy. It will help in the identification and comparison of amino
acid positions reported in literature and will facilitate the design of For an efficient enzymatic degradation of plastics, we see four chal-
new PETase variants. The design and construction of variants has lenges. First, enzyme families other than α/β-hydrolases should be
already led to a number of highly active enzymes. The most active var- considered. For instance, laccases or peroxidases can also act on
iants and their functional properties are summarized in Table S9. PUR.77 First reports have been published but fell short in the identifi-
Notably, the engineered DuraPETase had an almost 300-fold cation of proteins and genes. Enzymatic or nonenzymatic degradation
increased activity as compared to the wild-type enzyme. of other plastics components such as dyes or additives from commer-
Likewise for PURases, homology models predict substrate binding cial sources might need further investigations, too. Second, there is an
regions.80,81 Recently, a putative PURases was identified in the Prote- increasing need for comparable data on plastics degradation. The
obacterium Serratia liquefaciens.86 Similarly, most putative PURases in comparability and reproducibility of data on enzymatic plastics degra-
the PAZy are from Proteobacteria (mainly from P. chlororaphis and dation is impeded by the variety of possible substrates, for example,
D. acidovorans). in case of plastics built from several types of monomers. Furthermore,
physical properties of plastic materials can differ remarkably among
different commercial suppliers, for example, thickness of plastic foils,
4.2 | Major challenges in searches for plastics- number of additives, or crystallinity.
active enzymes Finally, incorrect annotation of genome and metagenome
datasets has resulted in the accumulation of many false positive plas-
Today, identifying truly plastic-active genes and enzymes is a very tic degrading enzymes in various publications but also in PMBD.
challenging task. This is especially true for enzymes acting on fossil- Removing these from the databases is a major task. By manual
curation, the PAZy lists only enzymes that degrade synthetic polymers 4. Mohammadian M, Allen NS, Edge M, Jones K. Environmental degra-
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Received: 30 November 2021 Revised: 1 February 2022 Accepted: 14 February 2022

Plastics degradation by hydrolytic enzymes: The plastics-active

Patrick C. F. Buchholz1 | Golo Feuerriegel2 | Hongli Zhang2 |

1 | I N T RO DU CT I O N can be removed mechanically from ocean surfaces or terrestrial sites,

Proteins. 2022;90:1443–1456. wileyonlinelibrary.com/journal/prot 1443

2.5 | Protein sequence networks 3.2.1 | Biochemical properties

Position in IsPETase Amino acid or gap symbol Annotations or substitutions References

3.3.2 | Enzyme structure

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