R E S E A R CH A R T I C L E Plastics degradation by hydrolytic enzymes: The plastics-active enzymes database—PAZy Patrick C. F. Buchholz1 | Golo Feuerriegel2 | Hongli Zhang2 | Pablo Perez-Garcia2 | Lena-Luisa Nover2 | Jennifer Chow2 | Wolfgang R. Streit2 | Jürgen Pleiss1 1Institute of Biochemistry and Technical Biochemistry, University of Stuttgart, Stuttgart, Germany 2Department of Microbiology and Biotechnology, University of Hamburg, Hamburg, Germany Correspondence Jürgen Pleiss, Institute of Biochemistry and Technical Biochemistry, University of Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany. Email: juergen.pleiss@itb.uni-stuttgart.de Wolfgang R. Streit, Department of Microbiology and Biotechnology, Universität Hamburg, Ohnhorststraße 18, D-22609 Hamburg, Germany. Email: wolfgang.streit@uni-hamburg.de Funding information Bundesministerium für Bildung und Forschung, Grant/Award Numbers: 031B867B, 031B0837B, 031B0571B, 031B0571A, 031B0562A Abstract Petroleum-based plastics are durable and accumulate in all ecological niches. Knowl- edge on enzymatic degradation is sparse. Today, less than 50 verified plastics-active enzymes are known. First examples of enzymes acting on the polymers polyethylene terephthalate (PET) and polyurethane (PUR) have been reported together with a detailed biochemical and structural description. Furthermore, very few polyamide (PA) oligomer active enzymes are known. In this article, the current known enzymes acting on the synthetic polymers PET and PUR are briefly summarized, their publi- shed activity data were collected and integrated into a comprehensive open access database. The Plastics-Active Enzymes Database (PAZy) represents an inventory of known and experimentally verified enzymes that act on synthetic fossil fuel-based polymers. Almost 3000 homologs of PET-active enzymes were identified by profile hidden Markov models. Over 2000 homologs of PUR-active enzymes were identified by BLAST. Based on multiple sequence alignments, conservation analysis identified the most conserved amino acids, and sequence motifs for PET- and PUR-active enzymes were derived. K E YWORD S hidden Markov model, hydrolases, metagenome, polyethylene terephthalate degradation, polyurethane degradation, sequence motif 1 | INTRODUCTION Today, we face the global challenge of plastics pollution in nearly all environments. The pollution has meanwhile reached levels that will ultimately have impact on our food chain and well-being within the next decades. A recent study implied that about 399 000 tons of plas- tics are present in the oceans alone, of which 69 000 tons are micro- plastics.1 Thus, urgent actions need to be implemented for removal of plastics from the environment and by reducing the steady input into the environment.2 Whereas it is perhaps more likely that large pieces can be removed mechanically from ocean surfaces or terrestrial sites, smaller particles (microplastics) will remain there unless microbial or chemical degradation (i.e., weathering) will occur.3–5 Plastic waste is a valuable raw material, therefore recycling is a promising alternative to incineration, either as a basis of synthesis of polymers or as a carbon source for fermentation.6 Petroleum-based plastics are in general extremely stable and durable; hence, it is widely accepted that plastics do not degrade well in nature,7 nor can be directly used in fermentation. The degradation processes described so far are slow, and it was shown that a PET Received: 30 November 2021 Revised: 1 February 2022 Accepted: 14 February 2022 DOI: 10.1002/prot.26325 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 https://orcid.org/0000-0001-5967-3777 https://orcid.org/0000-0003-2248-3544 https://orcid.org/0000-0002-7499-5325 https://orcid.org/0000-0001-7617-7396 https://orcid.org/0000-0003-1045-8202 mailto:juergen.pleiss@itb.uni-stuttgart.de mailto:wolfgang.streit@uni-hamburg.de http://creativecommons.org/licenses/by/4.0/ http://wileyonlinelibrary.com/journal/prot bottle remains up to 48 years in the ocean until it is decomposed by microbial degradation.8 Within this setting, it is reasonable to specu- late that prior to microbial and enzymatic degradation, mechanical treatment (waves, wind, friction) and photodegradation by UV light (especially for aromatic ring-containing polymers such as PET and PS) break down the debris into microplastics, thereby increasing the sur- face area, which mediates microbial degradation. For more details on microplastics-associated bacteria and fungi, we refer to excellent reviews of the field of plastics ecology.9–11 However, the colonization of microplastics does not necessarily indicate that the polymer is degraded, because additives are in general more bioavailable than the polymers. Therefore, measuring weight loss as an indicator for degra- dation might result in a false interpretation of the data,2 and in the conclusion that we already have many plastics-active enzymes from different microbial sources. A detailed search in the PubMed database revealed that today roughly 2500 publications address the topic of plastics degradation. However, less than 60 described the isolation and biochemical characterization of plastics-active enzymes (Tables 1 and S1–S3). Nevertheless, while this obvious challenge can be met by better analytical techniques, the by far greater risk for misinterpreta- tion of data comes from the unfiltered and noncritical use of the predicted plastic degrading microorganisms and consortia by not veri- fied bioinformatic tools and pipelines. For instance, one recent study developed hidden Markov models (HMMs) for some plastic degrading enzymes and predicted a global distribution even though no such enzymes have been biochemically characterized.47 Others have developed phylogenetic trees and global distribution patterns by simply using automated literature searches without critical analyses of the data.48 These very recent studies in high-ranking journals are perhaps only the tip of the iceberg, but 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 gene sequences ultimately leads to incorrect conclusions on the avail- ability of plastic degrading enzymes and their role in nature. These studies do not only mislead research works, they furthermore suggest to environmentalists, policy and law makers, and even to the broader public audience that we would have solutions for the global plastics problem, which we however do not have. Within this framework, the proposed PAZy database will be a reliable and very useful tool giving an overview on truly functional enzymes. Notably today, only for polyethylene terephthalate (PET), poly- urethane ester-based (PUR), and polyamide (oligomers) (PA), a rather 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 plastics-degrading enzymes are hydrolases, often annotated as lipases, esterases, cutinases, amidases, or proteases (E.C. 3.1.x). However, we have still a limited understanding of the mechanism of enzymatic deg- radation. It is not clear to which extent bacteria have evolved specific enzymes that bind to the polymers and cleave the bonds similar to the processes that occur when cellulose or other biopolymers are degraded. It is supposed that plastics-degrading enzymes are exoenzymes, and it can be speculated that plastics-binding domains or proteins might contribute to degradation, similar to the role of cellu- lose binding domains or expansins in the degradation of cellulosic materials. To advance the research field, we have collected information of the currently known and verified plastics-active enzymes in the Plastics-Active Enzymes Database (PAZy). Because we mainly focus on synthetic fossil fuel-based plastics, enzymes degrading bioplastics such polylactide (PLAs) or polyhydroxyalkanoate-based polymers (PHAs) were excluded in this manuscript. For the latter and their global distribution, we refer to two excellent review articles on PHA hydrolases.49,50 Thus, we have included enzymes acting on low crystalline poly- mers PET and ester-based PUR. Since for none of the other fossil- based polymers (e.g., PE, PP, PVC, PS, ether-based PUR, larger PA polymers) truly functional enzymes are known, we have not yet included them. For nylon (PA), we have added the information on the few oligomer-active enzymes in the web-based version of the PAZy data base. Similarily, the web-based version contains information on enzymes acting on polylactic acid (PLA), polyhydroxyalkanoates (PHAs) and synthetic and natural rubber (NR, SR). Despite the obvious lack of enzymes acting on many of the fossil fuel-based polymers, already the current version of the PAZy data- base will serve as a comprehensive resource for the identification of further novel plastics-active enzymes, pathways, or microorganisms for plastics removal in industry and the environment. It will further help to advance improved circular use of the different plastic types. This data base will serve as a first platform and will be developed fur- ther on over the next few years. PAZy will in general be a valuable repository and tool in this emerging field of plastics research. 2 | METHODS 2.1 | Data selection Protein sequences with available UniProt identifiers and known activ- ity against PET or PUR were downloaded from the Plastics Microbial Biodegradation Database51 (PMBD, http://pmbd.genome-mining.cn/ home/) and NCBI GenBank. Based on available biochemical and/or structural data, in total 44 protein sequences were selected from the PMBD, with 34 and 10 UniProt identifiers for PET and PUR activity, respectively (as of November 2021). 2.2 | PETase homologs We are aware that there are controversial discussions about the term PETase, but we prefer to define all PET-active enzymes as PETases. Sixteen protein sequences for enzymes with known activity against PET were clustered using CD-HIT (version 4.6.8-1) at a threshold of 90% sequence identity and a word length of 5 to derive a reduced set of 12 centroid sequences.52,53 These protein sequences were aligned 1444 BUCHHOLZ ET AL. http://pmbd.genome-mining.cn/home/ http://pmbd.genome-mining.cn/home/ TABLE 1 Currently known and active PET hydrolases structural or molecular analyses Microbial host, enzyme, gene References Genbank, UniProt IDa PDB structure WT enzymes and variants tested Activity on PET foil, powder, bottleb Proteobacteria-affiliated PET active enzymes Ideonella sakaiensis 201-F6, IsPETase, ISF6_4831 12 A0A0K8P6T7 5XFY WT Oleispira antarctica RB-8, PET5, LipA 13 R4YKL9_OLEAN WT + Vibrio gazogenes, PET6, Gene: BSQ33_03270 13 A0A1Z2SIQ1_VIBGA WT + Polyangium brachysporum, PET12, Gene: AAW51_2473 13 A0A0G3BI90_9BURK WT + Pseudomonas pseudoalcaligenes DSM 50188,PpCutA 14 KU695574 n.d. P. pelagia DSM 25163 PpelaLip 14 KU695573 n.d. Pseudomonas aestusnigri VGXO14, B7O88_11480, PE-H 15 A0A1H6AD45 6SBN 6SCD WT, Y250S, S171A, D217A, H249A, G254S, S256N, I257S, Y258N, N259Q, ext.loop, Q294A, I219Y 6 mg MHET/L from PET film (WT and Y250S) in 24 h � 500 nM enzyme�1; and 0.1 mg/ MHET from PET bottle for Y250S in 24 h � 500 nM enzyme�1 Pseudomonas mendocina ATCC 53552, PmC 16 N20M5AZM016 2FX5 solubilized 250-μm thick films in 96 h Actinobacterial enzymes LCC, leaf compost metagenome 17 G9BY57 AEV21261.1 12 mg TAeq. � h�1 � mg enzyme�1 with WT enzyme 18 4EB0 19 N197Q, N266Q, N239G, LCC-G 20 6THS 6THT S165A, ICCG-S165A F243I/D238C/ S283C/ Y127G (ICCG), F243I/ D238C/ S283C/N246M (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 105.6 ± 3.9 mg TAeq. � h�1 � mg enzyme�1, on commercial GF-PET with best variant BhrPETase from HRB29 bacterium 21 GBD22443 WT 0.17 mM BHET, 3.66 mM MHET and 2.47 mM TA from nanoparticles in 20 h Thermobifida fusca DSM43793, TfH, BTA-1 22–25 Q6A0I4_THEFU AJ810119 WT + (Continues) BUCHHOLZ ET AL. 1445 TABLE 1 (Continued) Microbial host, enzyme, gene References Genbank, UniProt IDa PDB structure WT enzymes and variants tested Activity on PET foil, powder, bottleb T. fusca DSM43793, TfH, BTA-2 22–25 AJ810119 + T. fusca DSM 44342, TfH42_Cut1 26 E9LVI0_THEFU ADV92528.1 41 mmol TA/mmol enzyme in 120 h T. fusca, (strain xy) WSH03-11, Tfu_0882 27–29 Q47RJ7_THEFY n.d. T. fusca, (strain xy) WSH03-11, Tfu_0883 27 Q47RJ6_THEFY n.d. T. fusca, TfCut-2 (Cut2-kw3) 30 E5BBQ3_THEFU G62A/F209A 31 ± 0.1 nmol 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 DSM44535, Thc_Cut1 26,35 ADV92526.1 5LUI 56 mmol TA/mmol enzyme in 120 h T. cellulosilytica DSM44535, Thc_Cut2 26,35 ADV92527.1 5LUJ 5LUL 5LUK R29N/A30V, R19S/R29N/A30V 5 mmol TA/mmol enzyme in 120 h Thermobifida alba AHL119, Est119, est2 36 F7IX06 6AID 3WYN 3VIS n.d. Thermomonospora curvata DSM43183, Tcur_1278 37 D1A9G5 ACY96861.1 n.d. T. curvata DSM43183, Tcur0390 37 ACY95991.1 n.d. Thermobifida halotolerans, Thh_Est 38 H6WX58 21.5 mmol TA � mol enzyme�1 in 2 h T. alba, Est1 (Hydrolase 4) 39 BAI99230 n.d. Saccharomonospora (Thermoactinomyces) viridis AHK190, Cut190 40–42 W0TJ64 AB728484 4WFK 4WFI 4WFJ 7CTR 7CTS S226P, S226P/R228S, S226P/R228S/ T262K, Q138A/D250C-E296C/ Q123H/ N202H, disulfide bonds at N250 and E296 30% increase in Q138A/ D250C-E296C/Q123H/ N202H variant, but no kinetic data Firmicutes Bacillus subtilis 4P3-11, BsEstB 43 ADH43200.1 n.d. Bacteroidetes Aequorivita sp. CIP111184, PET27 44 WP_111881932 Active on foil and powder Kaistella (Chryseobacteriu) jeonii, PET30 44 WP_039353427 7PZJ Active on foil and powder 1446 BUCHHOLZ ET AL. in a structure-guided multiple sequence alignment by T-COFFEE (ver- sion 11.00.8cbe486-1).54 A profile HMM was derived from this multi- ple sequence alignment by HMMER (version 3.1b2, http://hmmer. org). The profile HMM was trimmed by selecting alignment columns that corresponded to the region between amino acid positions 32 and 274 in the PETase from Ideonella sakaiensis (IsPETase, UniProt identi- fier A0A0K8P6T7) to avoid ambiguities at the N- and C-termini (Figure S1; Table S4). The profile HMM and the underlying multiple 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 Data Bank (PDB) for an update of the Lipase Engineering Database (LED, https://led.biocatnet.de), which was previously established as a collection of protein sequences from α/β-hydrolases.55–57 Hits for the PETase-profile HMM were selected from the HMMER results with a minimal score of 100, a minimal profile coverage of 95%, and a maxi- mum ratio of bias/score of 10%. HMMER was also used to identify the C-terminal region for the Type IX secretion system sorting domain, using the profile HMM TIGR04183, which was derived from a multiple sequence alignment of 889 protein sequences in the TIGRFAM database (http://tigrfams. jcvi.org/cgi-bin/index.cgi), with an E-value cut-off below 1. 2.3 | PURase homologs Four protein sequences for enzymes with known activity against PUR served as queries for BLAST (blastp, version 2.10.0+) against the TABLE 1 (Continued) Microbial host, enzyme, gene References Genbank, UniProt IDa PDB structure WT enzymes and variants tested Activity on PET foil, powder, bottleb Metagenome, probably bacterial and phylogenetically unassigned PET2, from metagenome, affiliated no obvious affiliation, lipIAF5-2 13 C3RYL0 ACC95208.1 + 45 7ECB 7EC8 R47C/G89C/F105R/E110K/ S156P/G180A/T297P (PET2 7 M), F105R/E110K/ 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 6.8-fold increase over WT after 60 min in PET2 7 M variant PET active enzymes from Eukaryotes Candida antarctica, lipase B, CalB 16 LIPB_PSEA2 WT Solubilized 250-μm thick films in 96 h 46 WT + Fusarium solani, FsC 16 AAA33335.1 1OXM WT Solubilized 250-μm thick films in 96 h Thermomyces (Humicola) insolens, HiC 16 A0A075B5G4 4OYY, 4OYL WT Solubilized 250-μm thick films in 96 h 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. aActive link to NCBI Pubmed database. bCrystallinity of PET films, bottles, and samples and experimental conditions varied largely in the different studies and thus makes direct comparison difficult. BUCHHOLZ ET AL. 1447 http://hmmer.org http://hmmer.org https://doi.org/10.18419/darus-2055 https://doi.org/10.18419/darus-2055 https://led.biocatnet.de http://tigrfams.jcvi.org/cgi-bin/index.cgi http://tigrfams.jcvi.org/cgi-bin/index.cgi NCBI nonredundant (nr) protein database and the PDB.58 BLAST per- formance was improved by multithreading with GNU/Parallel (version 20 170 622-1).59 The BLAST results were filtered by an E-value threshold of 10�10 and a minimal coverage of 50% to further update the LED. 2.4 | Conservation analyses The PETase-profile HMM was applied for a standard numbering scheme, by aligning the 2930 sequences of PETase homologs from the LED against the respective profile HMM and subsequently assig- ning the position numbers from the IsPETase reference sequence as standard position numbers. For conservation analysis of PETase homologs, the frequency of amino acid residues or gaps was counted at each standard position. For the conservation analysis of PURase homologs from LED superfamilies 11 and 13, two multiple sequence alignments were gen- erated using Clustal Omega (version 1.2.4),60 and the frequency of amino acid residues or gaps was counted at selected positions. 2.5 | Protein sequence networks Sets of representative protein sequences were formed by clustering with CD-HIT to reduce the sample size and thus computational effort for pairwise sequence alignments. Values of pairwise sequence iden- tity or similarity were calculated by the Needleman–Wunsch algo- rithm available in EMBOSS (version 6.6.0) with default gap opening and gap extension penalties of 10 and 0.5, respectively, and the sub- stitution matrix BLOSUM62.61,62 Collections of protein sequences were represented as protein sequence networks that depicted sequences as nodes connected by edges (lines). The edges in a protein sequence network were weighted by values of pairwise sequence identity or similarity. A threshold of the respective edge weights was chosen to select a subset of edges 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 identity or similarity were depicted preferably in closer vicinity to each other. The Python NetworkX package (version 1.11) was used to store the metadata of protein sequence networks in GraphML format, avail- able for download at https://doi.org/10.18419/darus-2054.64 3 | RESULTS 3.1 | Update of the LED We focus only on validated enzymes acting on the synthetic and fossil fuel-based polymers PET and PUR. Detailed biochemical data of cata- lytically active enzymes (Tables 1 and S1–S3), analyzed sequences and structures of homologous proteins are comprised in the PAZy and accessible at https://www.pazy.eu. Within the PAZy infrastructure, the LED (https://led.biocatnet.de) serves as the database for protein sequences and structures from different superfamilies of α/- β-hydrolases and their sequence annotations, since all currently known enzyme activities toward PET or PUR were reported for α/β-hydrolases. For the update of the LED, 4887 entries were downloaded from the NCBI nonredundant protein database, and 93 entries were down- loaded from the PDB using the criteria mentioned in Section 2. The updated LED contains 283 672 sequence entries and 1590 PDB entries (an increase of 3034 and 33 entries compared to the previ- ously published LED version from June 2019, respectively). For the update of the LED, sequences that shared at least 50% similarity were assigned to the same superfamily (Table S5). Sequences that shared at least 60% similarity were assigned to a homologous family; otherwise, they were assigned to a separate group containing all “singleton” sequences. A new superfamily was introduced for PURase homologs of PudA from D. acidovorans, as outlined in more detail below. 3.2 | PET active enzymes 3.2.1 | Biochemical properties Our literature searches identified a total of over 35 wild-type enzymes that have been shown to catalyze the partial degradation of PET to oligomers or even to monomers, originating from four different bacte- rial phyla and one eukaryotic lineage (Table 1). No archaeal PETases have been functionally verified to date. Many of the currently known PETases are thermostable enzymes, because the catalytic activity increases at temperatures close to the glass transition temperature (65�C) of PET due to the formation of flexible and thus enzyme-accessible amorphous domains.65 Notably, few enzymes are active at lower temperatures implying they may play a role in cold-adapted PET degradation.66 However, all known native PETases have rather low catalytic activity toward PET. 3.2.2 | Enzyme structures All known PETases are α/β-hydrolases and are either cutinases, lipases, or esterases and grouped into EC 3.1.1.101; EC 3.1.1.1, EC 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- tures affiliated with different organisms and the wild-type enzymes are available in the PDB. For the best characterized examples LCC and IsPETase, multiple entries of variants have been made. All PET- ases consist of a single domain, the α/β-hydrolase fold, which is formed by a central twisted β-sheet, flanked by two layers of α-helices,68 and thus belong to a class of small α/β-hydrolases that consist only of the core domain without a mobile lid.57 For a few PET- ase homologs from Bacteroidetes, an additional C-terminal sorting 1448 BUCHHOLZ ET AL. https://doi.org/10.18419/darus-2054 https://www.pazy.eu https://led.biocatnet.de domain for the Type IX secretion system has been annotated and was verified in the single structure published (Figure S5; Table 1). The Type IX secretion system comprises several protein components,69 and the corresponding C-terminal domain was also found in other polymer-active enzymes such as cellulases and endo-1,4-β -glucanases.70–73 PETases share a conserved catalytic triad of serine, histidine, and aspartic acid, and a GX-type oxyanion hole, which stabi- lizes the reaction intermediate.74 In the PETase homologs, the first oxyanion hole residue X is mostly a conserved aromatic residue such as tyrosine or phenylalanine. The second oxyanion-stabilizing residue is a conserved methionine following the serine of the catalytic triad. For the PETase from I. sakaiensis, several residues were suggested for substrate binding, such as an aromatic clamp formed by the first resi- due of the oxyanion hole and a second aromatic residue.75 In addition to this subsite I, a second subsite II was proposed from the interaction observed in a modeled complex with a PET monomer.76 Variants with 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 [WI]-D238C-S283C-Y127G, which include the additional disulfide bond, are among the most active PETases described to date. 3.2.3 | Sequence network For the comparison of PETase sequences, the profile HMM for PET- ases 13 was used to identify the PETase core domain, and the sequences of all core domains were aligned without considering additional regions at the N- or C-termini (signal peptides or transport domains, respectively). In superfamily 1 of the LED, 31 560 sequences were annotated as GX-type, but only 2930 sequences were identified as PETase homologous by a profile HMM. At a threshold of 55% sequence similarity, the bacterial PETase core domains formed a large cluster, mainly originating from Actinobacteria or Proteobacteria (Figure 1). Most of the sequences from the PMBD were found in this cluster (Figure S3). In addition, a connected subgroup of PETase core domains from other bacterial phyla emerged, such as the PETase pro- teins from Bacteroidetes or Planctomycetes. Some homologs of PET- ase core domains occurred also in enzymes from extremophiles (Figure S4). The fungal PETase core domains such as the PETase homologs from Fusarium were separated from the bacterial PETase core domains. At a higher threshold of 60% sequence similarity, the sequences for PETase core domains from Bacteroidetes or Planctomycetes emerged as a separated cluster (Figure S5). 3.2.4 | Sequence motifs 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 PETase from I. sakaiensis (IsPETase, UniProt identifier A0A0K8P6T7) and LCC (UniProt identifier G9BY57). The catalytic triad, the previ- ously suggested PET binding subsite I, which includes an aromatic clamp for possible substrate interaction, and PET binding subsite II from Joo et al.76 were found to be highly conserved (Table 2). The F IGURE 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 BUCHHOLZ ET AL. 1449 extension of the second α-helix and the extended loop region, which were described previously as functionally relevant in IsPETase, were also found in several PETase homologs in the LED. Using the position numbers from IsPETase, we suggest a typical PETase sequence motif written as follows (X stands for an arbitrary aa): [YF]87, Q119, X3 139–141, S160, M161, W185, D206, H237, X6 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 (NCBI: WP_116302080.1) were found to comprise the PETase sequence motif and W238, which was affiliated with improved activ- ity and substrate binding. Further additional sequences (from Caldimonas manganoxidans, NCBI: WP_019560450.1, from Caldimonas taiwanensis, NCBI:WP_062195544.1, from Rhizobacter gummiphilus, NCBI:WP_085749610.1, and from Aquabacterium sp., NCBI: MBI3384080.1) were found to comprise the PETase sequence motif and M241, which was mentioned as an aa substitution for improved thermostability. These six different and novel protein sequences, each selected by a sequence motif of 17 amino acid posi- tions in total, are proposed for upcoming studies on PETase activity. 3.3 | Polyurethanes (PUR) active enzymes 3.3.1 | Biochemical properties Polyurethanes comprise numerous possible polymers of diverse com- position, such as combinations of different isocyanates with different 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 75 140 Gap 80% Extension in α-helix 2 75 141 Gap 90% 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 76 246 �89% Extended loop 76 247 �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. 1450 BUCHHOLZ ET AL. polyethers, polyesters, or polycarbonates.77 The best studied PURases are α/β-hydrolases, as reviewed in more detail in Magnin et al.77 Only 10 characterized PUR-degrading enzymes (PURases) have been reported, yet. Four recently identified enzymes (LCC, TfCut-2, Tcur_1278T, and Tcur0390) are cutinases from Actinomyces, which are also active on PET and have a broad substrate profile.78 Further bacterial lipases from Betaproteobacteria have been identified and characterized, such as PueA and PueB from Pseudomonas chlororaphis. Whereas all the above-mentioned studies identified lipases or ester- ases, earlier studies reported that commercially available peptidases and proteases might also degrade thin PUR films.79 Notably, none of the above-mentioned enzymes is able to act on ether bonds. 3.3.2 | Enzyme structure The 10 known PURases belong to two groups (Table S1): four belong to the cutinases (LCC, TfCut-2, Tcur_1278T, and Tcur0390) and are similar to PETases. No crystal structures are available for the PURases PueA and PueB from P. chlororaphis, but structures of homologs indi- cate that they belong to superfamily 11 of the LED, which in addition to the core domain has a mobile N-terminal lid, which might mediate binding to the substrate interface and substrate access, and an addi- tional C-terminal β-sandwich domain. The four PETases and PueA and PueB from P. chlororaphis are GX types.74 Recently, a modeling study on the PURases from P. chlororaphis predicted putative substrate binding sites for PUR-like substrates.80 However, a rearrangement of the substrate was observed upon the molecular simulation of the complex, which is an additional challenge for the identification of the substrate binding site.81 Interestingly, most of the substrate binding residues predicted for PueA80 are conserved (Table S7). The PURase from D. acidovorans (UniProt identifier Q9WX47) does not belong to the GX type hydrolases, but has a sequence simi- larity to carboxylesterases of superfamily 13 and to the family PF00135 in Pfam, and thus belongs to the GGGX-type hydrolases.74 Other carboxylesterases in the LED are members of superfamily 4, which have a mobile lid between β-strand �4 and �3 of the core domain. Because the PURase from D. acidovorans shared less than 50% sequence similarity to the sequences in the LED and due to the lack of experimental structure information, it was assigned to a new superfamily. 3.3.3 | Sequence network A threshold of 60% similarity was used to construct protein sequence networks for LED superfamilies 11 and 13 whose members originated from Proteobacteria only (Figure 2). Two disconnected sequence networks emerged: a network of 127 representative sequences of superfamily 11, which contains the GX-type PURases PueA and PueB from P. chlororaphis, and a network of 15 representative sequences of superfamily 13, which contains the GGGX-type, the PURase PudA from D. acidovorans. In both superfamilies 11 and 13, the sequences originated mainly from Gammaproteobacteria, with the genus Pseudomonas being most frequently annotated in superfamily 11. F IGURE 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 BUCHHOLZ ET AL. 1451 3.3.4 | Sequence motifs Two sequence motifs were reported previously in Howard et al.82 for the PURase from P. chlororaphis, PueA (Table 3). The occurrence of the serine hydrolase motif (GXSXG) and the secretion signal sequence motif (GGXGXDXXX) were confirmed for the vast majority of all 2054 sequence entries in a multiple sequence alignment for superfamily 11 of the LED. Most PURase homologs from superfamily 11 have a GHSLG motif flanking the catalytic serine and secretion motif GGKGNDYLE. For sequences from superfamily 11 in the LED, the catalytic triad is formed by serine, aspartate, and histidine. In addition, most sequences in superfamily 11 (2039 out of 2054) matched the profile HMM for an RTX calcium-binding nonapeptide repeat (PFAM PF00353), which supports the previous suggestion of a Type I secre- tion system for protein translocation.83 Prominent amino acid positions in superfamily 13, which com- 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 347–395.84 Many of these positions were found to be conserved within the sequences of superfamily 13 (Table 4). Most PURase homologs from superfamily 13 have the motif GESAG flanking the catalytic serine, the motif VPX3G[ST]X2DE at the catalytic glutamate, and the motif AXHX3[LI]XY flanking the catalytic histidine. In addition, several positions in the putative PUR binding region were found to be conserved, including mostly hydrophobic amino acids (Table S8). 4 | DISCUSSION 4.1 | Sequence annotations of plastics-active enzymes Enzymes of different EC classes have been proposed to contribute to degradation of PET or PUR. α/β-hydrolases annotated as cutinases, esterases, or lipases were described to catalyze the hydrolysis of ester bonds and were collected in the PMBD,51 and peroxidases and laccases have been reported to enhance degradation of PUR.85 For most types of plastics, however, knowledge on enzymatic degradation is missing, although materials such as polypropylene (PP) are produced TABLE 3 Amino acid positions mentioned in Howard et al.82 and their occurrence in 2054 PURase homologs from the LED (superfamily 11) Position in PueA Amino acid Annotation 204 G 100% Serine hydrolase motif GXSXG 205 H 99% Serine hydrolase motif GXSXG 206 S 100% Serine hydrolase motif GXSXG, catalytic triad 207 L 99% Serine hydrolase motif GXSXG 208 G 100% Serine hydrolase motif GXSXG 254 D 100% Catalytic triad 312 H 100% Catalytic triad 381 G 99% Secretion motif GGXGXDXXX 382 G 98% Secretion motif GGXGXDXXX 383 K 39%, R 18%, A 16%, S 13% Secretion motif GGXGXDXXX 384 G 98% Secretion motif GGXGXDXXX 385 N 78%, A 16% Secretion motif GGXGXDXXX 386 D 99% Secretion motif GGXGXDXXX 387 Y 57%, F 42% Secretion motif GGXGXDXXX 388 L 72%, I 27% Secretion motif GGXGXDXXX 389 E 91% Secretion motif GGXGXDXXX Note: Position numbers refer to PueA from Pseudomonas chlororaphis, that is, UniProt identifier A1Z374. Percentages indicate values rounded to integers. Position in the motif is marked in bold. TABLE 4 Amino acid positions mentioned in Nomura et al.84 and adjacent amino acid positions that occur in at least 50% of the 44 PURase homologs from the LED (superfamily 13) Position in PudA Amino acid Annotation 223 G 100% Serine hydrolase motif GXSXG 224 E 90% Serine hydrolase motif GXSXG 225 S 100% Serine hydrolase motif GXSXG, catalytic triad 226 A 97% Serine hydrolase motif GXSXG 227 G 100% Serine hydrolase motif GXSXG 340 V 100% 341 P 97% 342 V 64% 343 (I 33%, V 30%, M 26%) 345 G 100% 346 S 50% (T 42%) 347 N 71% 349 D 92% 350 E 100% Catalytic triad 457 A 95% 458 A 66% 459 H 100% Catalytic triad 462 E 78% 463 L 69% (I 30%) 464 Q 83% 465 Y 92% 466 L 83% Note: Position numbers refer to PudA from Delftia acidovorans, that is, UniProt identifier Q9WX47. Similar amino acid residues occurring in less than 50% of the sequences are indicated in brackets for comparison. Positions of a putative substrate binding region are listed in Table S5 for comparison. Position in the motif is marked in bold. 1452 BUCHHOLZ ET AL. in large scales and contribute extensively to global plastics pollution. In this article, we focus on PET- and PUR-degrading α/β-hydrolases due to the availability of sequence information and detailed experi- mental data from literature and public databases.51 Many PETase homologs were found in the NCBI nonredundant protein database, due to the sequencing efforts of the cutinase- expressing bacterial phyla such as Actinobacteria and Proteobacteria. In contrast, sequences from fungal origin are unrepresented. The usage of metagenomics is expected to further broaden the scope of currently known PETases.13 The PETase sequence motif suggested herein (Table 2) is based on current knowledge from literature, such as the occurrence of an additional flexible loop region and amino acids that seem relevant for interaction with PET. The seven suggested PETase candidates can be used as starting points for wet-lab experi- ments, such as protein design experiments for improved PETase activ- ity or thermostability. Although PETases and PURases share the α/β-hydrolase fold as catalytic domain, their structure and their oxyanion hole types differ. All the 2930 PETase homologs belong to a large family (36 936 sequences) of GX-types,74 which consist of the core domain only (superfamily 1 in the Lipase Engineering Database57), whereas PURases belong to either of three superfamilies: superfamily 1, super- family 11, which consists of GX-types with two additional domains, an N-terminal mobile lid and a C-terminal β-sandwich domain (2054 sequences), or to superfamily 13, which are carboxylesterases of the GGGX-type (44 sequences). Ample structural information is available for PET-degrading α/β-hydrolases, especially for LCC and IsPETase, whereas the structures of PUR-degrading α/β-hydrolases have not been resolved, yet. The former has also inspired the design of improved PETase variants, as recently demonstrated for variants based on LCC.20 Previously, conserved subsite I, subsite II, and an 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 for PETases and the derived standard numbering scheme is available at the PAZy. It will help in the identification and comparison of amino acid positions reported in literature and will facilitate the design of new PETase variants. The design and construction of variants has already led to a number of highly active enzymes. The most active var- iants and their functional properties are summarized in Table S9. Notably, the engineered DuraPETase had an almost 300-fold increased activity as compared to the wild-type enzyme. Likewise for PURases, homology models predict substrate binding regions.80,81 Recently, a putative PURases was identified in the Prote- obacterium Serratia liquefaciens.86 Similarly, most putative PURases in the PAZy are from Proteobacteria (mainly from P. chlororaphis and D. acidovorans). 4.2 | Major challenges in searches for plastics- active enzymes Today, identifying truly plastic-active genes and enzymes is a very challenging task. This is especially true for enzymes acting on fossil- based polymers. Thus one of the major challenges will be the identifi- cation of novel enzymes for polymers for which none are currently known.2,87 Thus, we urgently need enzymes acting on PE, PP, PVC, but also on polymeric PA and PU ether bonds. Within this framework, the identification of plastic-specific binding domains, expansins, or loosenins will be of high relevance. Once these are identified they can be used to further improve enzymes acting PET and PUR. Thereby, a combination of modern synthetic biology approaches together with enzyme engineering approaches will be the appropriate tools to gen- erate the best performing enzymes. Further we realized the lack of a common PET or PUR model sub- strate, which would allow the direct comparison of the kinetic param- eters of different plastics-active enzymes. In contrast, kinetic analysis is generally performed for typical esterase substrates such as pNP- caproate. This data, however, does not allow a reliable prediction of the actual plastics activity. The few enzymes that have been charac- terized using polymers were tested on different polymer types, and pretreatment was used to enable better degradation (see Table 1 for references). In addition, all kinetic data were recorded using single point measurements, thus the hydrolysis of the polymer could not be separated from the attachment of the enzyme to the polymer surface or from the hydrolysis of the resulting oligomers. Within this frame- work, a characterization of surface area and a control of surface prop- erties such as crystallinity would be favorable to obtain better and more reliable kinetic data on the actual polymer. The accumulation of verified plastics-active enzymes in databases with a reliable structure–function analysis will allow more predictive searches to rapidly and reliably identify novel and more active enzymes. Thereby, it will allow to foster the search and development of novel pathways to create designer bugs using to solve the plastics problem. 4.3 | How can databases contribute to a solution of the plastics waste problem? For an efficient enzymatic degradation of plastics, we see four chal- lenges. First, enzyme families other than α/β-hydrolases should be considered. For instance, laccases or peroxidases can also act on PUR.77 First reports have been published but fell short in the identifi- cation of proteins and genes. Enzymatic or nonenzymatic degradation of other plastics components such as dyes or additives from commer- cial sources might need further investigations, too. Second, there is an increasing need for comparable data on plastics degradation. The comparability and reproducibility of data on enzymatic plastics degra- dation is impeded by the variety of possible substrates, for example, 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, number of additives, or crystallinity. Finally, incorrect annotation of genome and metagenome datasets has resulted in the accumulation of many false positive plas- tic degrading enzymes in various publications but also in PMBD. Removing these from the databases is a major task. By manual BUCHHOLZ ET AL. 1453 curation, the PAZy lists only enzymes that degrade synthetic polymers as verified by biochemical or genetical complementation analyses. Thus, all genes and enzymes listed in PAZy are truly functional. In con- trast, PMDB also contains putative enzymes, microbes, or microbial consortia, which are active on short oligomers and additives. Within this setting, the PAZy database provides information on several well studied enzymes supplemented by the protein sequences and structures of homologous sequences, which enables the search for novel candidates and the design of enzyme variants. Most protein sequences and functional data are available for PETases, for which several positions were already assessed experimentally for substrate binding or thermostability in earlier studies from literature (Table 2). The standard numbering scheme of PETase homologs facilitates the comparison of different amino acid positions from literature and the identification of sequence motifs for PETases, as shown for the com- parison between IsPETase, LCC, and other PETase homologs. In upcoming studies, the PAZy database will be updated to cover sequences from other protein family databases than the LED, depending on the availability of structures and biochemical data. Fur- ther, the underlying web platform of the PAZy database makes it eas- ier to share knowledge on various plastics degrading enzymes in a more comparable way. Thus, it can be expected that the suggested sequence motifs for PETases or PURases will be refurbished, as more experimental data on residues for substrate binding or thermostability are made available in the future. ACKNOWLEDGMENTS This work was supported in part by the Bundesministerium für Bildung und Forschung within the programs MarBiotech (031B0562A), MetagenLig (FKZ 031B0571A and 031B0571B), LipoBiocat (031B0837B), and PlastiSea (031B867B). GF was in part funded through the University of Hamburg and the Excellence strategy of the BMBF. Open access funding enabled and organized by Projekt DEAL. DATA AVAILABILITY STATEMENT The data that support the findings of this study are openly available in PAZy—The Plastics-Active Enzymes Database at https://pazy.eu. ORCID Patrick C. F. Buchholz https://orcid.org/0000-0001-5967-3777 Pablo Perez-Garcia https://orcid.org/0000-0003-2248-3544 Jennifer Chow https://orcid.org/0000-0002-7499-5325 Wolfgang R. Streit https://orcid.org/0000-0001-7617-7396 Jürgen Pleiss https://orcid.org/0000-0003-1045-8202 REFERENCES 1. Hohn S, Acevedo-Trejos E, Abrams JF, Fulgencio de Moura J, Spranz R, Merico A. The long-term legacy of plastic mass production. 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Proteins. 2022; 90(7):1443-1456. doi:10.1002/prot.26325 1456 BUCHHOLZ ET AL. info:doi/10.1111/febs.15071 info:doi/10.1016/S0022-2836(05)80360-2 info:doi/10.1038/msb.2011.75 info:doi/10.1016/j.cocis.2008.07.002 info:doi/10.1101/gr.1239303 info:doi/10.1016/S0032-3861(02)00236-7 info:doi/10.3389/FMICB.2021.803896 info:doi/10.1016/j.polymdegradstab.2012.02.003 info:doi/10.1016/j.polymdegradstab.2012.02.003 info:doi/10.1093/protein/5.3.197 info:doi/10.3389/fcimb.2017.00215 info:doi/10.1038/s41598-019-52675-8 info:doi/10.1371/journal.pone.0018814 info:doi/10.1111/1462-2920.13770 info:doi/10.1007/s12010-010-9070-5 info:doi/10.1016/S1381-1177(00)00092-8 info:doi/10.1016/j.bpj.2018.02.005 info:doi/10.1038/s41467-018-02881-1 info:doi/10.1016/j.biotechadv.2019.107457 info:doi/10.3390/POLYM9020065 info:doi/10.1002/jbm.820210207 info:doi/10.1016/j.jmgm.2019.03.001 info:doi/10.1007/s00894-021-04671-x info:doi/10.1016/S0964-8305(01)00042-7 info:doi/10.1016/j.molcel.2016.03.018 info:doi/10.1016/j.molcel.2016.03.018 info:doi/10.1016/S0922-338X(99)89001-1 info:doi/10.1016/j.indcrop.2011.03.010 info:doi/10.1016/j.indcrop.2011.03.010 info:doi/10.1016/j.foodchem.2020.127954 info:doi/10.1111/1751-7915.12710 info:doi/10.1002/prot.26325 Plastics degradation by hydrolytic enzymes: The plastics-active enzymes database-PAZy 1 INTRODUCTION 2 METHODS 2.1 Data selection 2.2 PETase homologs 2.3 PURase homologs 2.4 Conservation analyses 2.5 Protein sequence networks 3 RESULTS 3.1 Update of the LED 3.2 PET active enzymes 3.2.1 Biochemical properties 3.2.2 Enzyme structures 3.2.3 Sequence network 3.2.4 Sequence motifs 3.3 Polyurethanes (PUR) active enzymes 3.3.1 Biochemical properties 3.3.2 Enzyme structure 3.3.3 Sequence network 3.3.4 Sequence motifs 4 DISCUSSION 4.1 Sequence annotations of plastics-active enzymes 4.2 Major challenges in searches for plastics-active enzymes 4.3 How can databases contribute to a solution of the plastics waste problem? 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