Enzymes and methods for biodegrading polyolefin-derived polymers - Patents.com

Abstract

The present invention relates to an enzyme from the saliva of waxworms (Galleria mellonella larvae) that has the unexpected ability to oxidize and depolymerize untreated polyolefin-derived polymers, such as polyethylene (PE), at room temperature (RT), neutral pH and short incubation times. The present invention also relates to a method for biodegrading polyolefin-derived polymers using this enzyme.

Description

[Technical field]

[0001] The present invention is in the field of biotechnology, in particular in the field of enzymatic biodegradation of plastics, preferably polyolefin-based plastics, more preferably polyethylene (PE). [Background technology]

[0002] Polyethylene (PE) accounts for 30% of synthetic plastic production and is a major contributor to plastic waste pollution on the planet to date. Together with polypropylene (PP), polystyrene (PS) and polyvinyl chloride (PVC), PE is one of the most resistant polymers, with very long C-C chains organized into crystalline dense structures. Given that hundreds of millions of tons of plastic waste have accumulated and the pace of plastic production is still accelerating, reuse of plastic residues is a necessary path to mitigate the severity of the plastic pollution problem and at the same time make huge potential carbon reservoirs available. Currently, only mechanical recycling is applied on a large scale. Several factors, such as the small number of plastic types amenable to mechanical recycling and the low quality of secondary products, significantly limit the potential solutions to the problem of plastic waste accumulation. Chemical recycling, as an alternative procedure, preferentially aims at upcycling plastics, such as decomposing polyolefin-derived plastics to utilize smaller intermediates. Although several techniques have been applied at laboratory scale, high energy costs may still hinder the scale-up of these technological tools.

[0003] In addition to mechanical and chemical recycling, biodegradation is widely considered as a promising strategy to dispose of plastic residues. Biodegradation refers to the environmental breakdown by biological agents. IUPAC defines biodegradation as "the breakdown of substances catalyzed by enzymes in vitro or in vivo", but it was later modified to "exclude abiotic enzymatic processes". In the case of PE, biodegradation requires the introduction of oxygen into the polymer chain; this leads to the formation of carbonyl groups, followed by the cleavage of long hydrocarbon chains with the generation of smaller molecules, which can then be metabolized by microorganisms (Albertsson, AC, Andersson, SO, and Karlsson, S. (1987). Polymer Degradation and Stability 18, pp. 73-87; Roy, PK, Hakkarainen, M., Varma, IK, and Albertsson, AC (2011). Environ Sci Technol 45, pp. 4217-4227). The crucial first step in this sequence of events, the oxidation of the PE polymer, is usually driven by abiotic factors such as light or temperature. Once the long polymer molecules are degraded, the process is exposed to environmental factors in the wild for years, after which bacteria and fungi step in to continue the job. This is the current paradigm driving the field of biodegradation research. Within this paradigm, several bacterial and fungal strains capable of performing some degree of PE degradation have been identified. However, in most cases, such degradation requires active pretreatment of PE (heating, UV light, etc.) to promote oxygen incorporation into the polymer, making abiotic oxidation the real bottleneck of the reaction (Wei, R., and Zimmermann, W. (2017). Microbial Biotechnology 10, pp. 1308-1322; Restrepo-Florez, J.-M., Bassi, A., and Thompson, M. R. (2014). International Biodeterioration & Biodegradation 88, 83-90; Amobonye, ​​A., Bhagwat, P., Singh, S., and Pillai, S. (2021).Sci Total Environ 759, 143536; Matjasic, T. et al., (2021). Science of the Total Environment 752; Walsh, A. N. et al., (2021). Environ.Sci.Technol 55, 12383-12392). In the last decade, several microorganisms have been reported that can act on untreated PE17-23, but they require significantly longer incubation times compared to the experimental conditions with pre-oxidized PE.

[0004] Identification of microbial enzymes capable of degrading untreated PE has proven to be a challenging task. In fact, such enzymes have not yet been identified, confirming the important limiting role of oxidation in the entire biodegradation chain. For enzymes reported to be able to act on polyolefin-derived plastics, pretreatment of the plastic material is required (Wei, R., and Zimmermann, W. (2017). Microbial Biotechnology 10, 1308-1322; Amobonye, ​​A., Bhagwat, P., Singh, S., and Pillai, S. (2021). Sci Total Environ 759, 143-536). For example, for the two reported laccases capable of chemically modifying PE, abiotic pretreatment or the addition of a redox mediator such as 1-hydroxybenzotriazole is required.

[0005] This scenario confirms that the synthetic nature of the compounds, together with their hydrophobic and inaccessible features, make plastics less likely to be targeted by enzymatic activity of animal, fungal, or microbial origin. Nevertheless, some lepidopteran and coleopteran insects have been found to have the unexpected ability to degrade untreated PE and PS, as described, for example, in Yang, Y., Wang, J., and Xia, M. (2020) Sci Total Environ 708, 135233. Summary of the Invention [Problem to be solved by the invention]

[0006] However, it is still necessary to identify microbial enzymes capable of biodegrading untreated PE, especially those capable of oxidizing untreated PE polymers, and avoid the use of abiotic factors such as light or temperature, long incubation times, or aggressive pretreatment of PE.

[0007] WO 2021 / 183867 discloses the use of enzymes such as laccase from Pseudomonas microorganisms and / or Trametes versicolor to reduce the molecular weight of polyethylene. However, WO 2021 / 183867 does not disclose enzymes from waxworms (Galleria mellonella larvae).

[0008] Bombelli, P., Howe, C.J., and Bertocchini, F.(2017). Current biology, 27(8), R292-R293, Kong, H.G., Kim, H.H., Chung, J.H., Jun, J., Lee, S., Kim, H.M., ···, and Ryu, C.M.(2019). Cell Reports, 26(9), 2451-2464 and Peydaei, A., Bagheri, H., Gurevich, L., de Jonge, N., and Nielsen, J.L.(2020). Comparative Biochemistry and Physiology Part D: Genomics and Proteomics, 34, 100678 disclose the degradation of polyethylene by larvae of the waxworm Galleria mellonella. However, these documents do not disclose the isolation of enzymes capable of biodegrading, oxidizing, and / or depolymerizing intact polyolefin-derived polymers.

[0009] German Patent No. 10041541 discloses proteins with some sequence identity to SEQ ID NO: 1 and XP026756396.1 is a database entry for an enzyme with the sequence of SEQ ID NO: 1. However, these disclosures provide no teaching towards proteins with activity to biodegrade, or oxidize and / or depolymerize, polyolefin-derived polymers or materials containing polyolefin-derived polymers. [Means for solving the problem]

[0010] The inventors isolated an enzyme from the saliva of waxworms (Galleria mellonella larvae) with the unexpected ability to oxidize and depolymerize raw polyolefin-derived polymers such as polyethylene (PE) at room temperature (RT), neutral pH and short incubation times. The inventors achieved this by performing proteomic analysis and size-exclusion chromatography (SEC) of waxworm saliva, obtaining one enzyme identified as arylphorin subunit α-like, renamed Demetra, with accession number XP_026756396.1 (NCBI), SEQ ID NO: 1 (the enzyme of the invention). The ability of this enzyme to oxidize / degrade polyolefin-derived polymers was tested on PE films (see examples and Figures 5 and 6 of this patent application) and showed high degradation activity. This paves the way for possibilities to solve the plastic waste pollution problem.

[0011] This effect on PE degradation is achieved after only a few hours of exposure at room temperature and physiological conditions (neutral pH). Therefore, no long incubation times or active pretreatments are required. The enzyme provided in the present invention can actually overcome the bottleneck step in PE biodegradation, namely the initial oxidation step. Using gas chromatography-mass spectrometry (GC-MS), degradation products such as small molecular oxidized aliphatic chains were identified, further confirming that the polymer was broken into shorter molecules.

[0012] The enzyme of the present invention belongs to the hexamerin / prophenoloxidase family and is not only able to oxidize PE after several hours of application at room temperature, but also physically degrade PE, releasing degradation by-products similar to those obtained from whole saliva.

[0013] The enzyme is able to produce such modifications on PE films in a very short time at room temperature, making it a promising alternative to the abiotic oxidation of plastics, which is the first and most difficult step in the degradation process. The identification of an invertebrate enzyme capable of oxidizing PE in a few hours presents a completely new paradigm in the world of plastic degradation and even in the field of plastic waste management, paving the way for designing new formulations / routes for synthetic polymer production. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] In one aspect, the present invention relates to an expression vector comprising a nucleotide sequence encoding an enzyme comprising an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:1 for biodegrading, or oxidizing and / or depolymerizing a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer.

[0015] In one aspect, the present invention relates to: an enzyme comprising an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:1 (hereinafter referred to as "the enzyme or protein of the invention") or a functionally equivalent fragment thereof, or a nucleotide sequence encoding said enzyme, or a host cell (hereinafter referred to as "the host cell of the invention") comprising a vector comprising a nucleotide sequence encoding said enzyme, or a composition comprising said enzyme or a functionally equivalent fragment thereof, or said host cell (hereinafter referred to as "the composition of the invention").

[0016] For example, preferably the enzyme is active in biodegrading, or oxidizing and / or depolymerizing, polyolefin-derived polymers or materials that include polyolefin-derived polymers.

[0017] In some aspects, the present invention relates to an isolated enzyme comprising an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:1, and compositions comprising said isolated enzyme.

[0018] In some embodiments, the enzyme of the invention is not the enzyme of database accession reference NCBI:XP026756396.1.

[0019] In some embodiments, the enzyme of the invention is not the enzyme of database accession reference GSP:ABB77350.

[0020] In some embodiments, the enzyme of the present invention is an enzyme comprising a sequence with 1% variation from the amino acid sequence of SEQ ID NO: 1. In some embodiments, the enzyme comprises a sequence with 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40% variation from the sequence of SEQ ID NO: 1. In some embodiments, the enzyme comprises a sequence with 1 amino acid variation from the amino acid sequence of SEQ ID NO: 1. In some embodiments, the enzyme comprises a sequence with 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or 300 amino acid variation from the amino acid sequence of SEQ ID NO: 1.

[0021] Preferably, the enzyme has activity for biodegrading, or oxidizing and / or depolymerizing, polyolefin-derived polymers or materials comprising polyolefin-derived polymers.

[0022] In some embodiments, a composition of the invention comprises an enzyme of the invention or a functionally equivalent fragment thereof, or a host cell of the invention, and preferably at least one further component.

[0023] In some embodiments, the compositions of the present invention comprise one or more polyolefin-derived polymers or materials that comprise polyolefin-derived polymers.

[0024] In some embodiments, the compositions of the present invention comprise one or more oxidized polyolefin-derived polymers.

[0025] In some embodiments, the compositions of the present invention comprise one or more selected from the group of butane, 2,3-butanediol, trimethylsilyl (TMS) derivatives, sebacic acid, C10-C22 2-ketones, benzenepropanoic acid.

[0026] In some aspects of the invention, the enzyme or composition may be formulated into an enzyme granule. Preferably, the enzyme or composition of the invention may be formulated as an aqueous solution.

[0027] In some embodiments, the composition comprises a buffer, such as a HEPES buffer. More preferably, the composition comprises 150 mM NaCl, 20 mM HEPES, 5% glycerol.

[0028] In view of the above, the present invention relates in one aspect to the use of an enzyme or a functionally equivalent fragment thereof comprising an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID NO: 1, or a host cell comprising a nucleotide sequence encoding said enzyme, or a vector comprising a nucleotide sequence encoding said enzyme, or a composition comprising said enzyme or a functionally equivalent fragment thereof, or a host cell (hereinafter referred to as "use of the invention"), for biodegrading or oxidizing and / or depolymerizing polyolefin-derived polymers or materials comprising polyolefin-derived polymers. The enzyme of the invention is preferably isolated from the larvae of Galleria mellonella, also known as waxworm (ww). It is associated with the enzyme arylphorin subunit alpha-like, renamed Demetra, comprising an amino acid sequence having at least 60% sequence identity with the sequence of SEQ ID NO: 1 (accession reference number NCBI: XP_026756396.1).

[0029] SEQ ID NO:1 (Demetra, enzyme of the invention)

[0030] [ka]

[0031] Alternatively, the enzymes of the invention can be produced recombinantly according to techniques well known in the art. For example, using recognized techniques of chemical synthesis, the enzymes can be constructed from the N-terminus, or more commonly the C-terminus, using either a single amino acid or a peptide containing two or more amino acid residues. Specific techniques for synthesizing enzymes include classical methods, classical chemical synthesis amino acid by amino acid, and solid-phase peptide synthesis, where the enzyme is constructed by attachment to a resin, such as Merrifield resin. In these synthesis procedures, the groups on the amino acids are generally in protected form using standard protecting groups, such as t-butoxycarbonyl. If necessary, these protecting groups are cleaved once the synthesis is complete. Chemical synthesis methods, such as peptide synthesis in solid phase, solution synthesis, and combinations of solid-phase synthesis and solution or enzymatic synthesis methods, are known to those skilled in the art. The enzymes of the invention may be produced by recombinant DNA procedures known in the art. Modifications may be introduced during or after the synthesis of the enzymes, for example to include added labels, purification tags, for purification.

[0032] In another specific embodiment, the enzymes of the invention are encoded and expressed from a nucleotide sequence contained in an expression vector.

[0033] In the present invention, the term "identity" or "sequence identity" is understood to mean the degree of similarity between two nucleotide or amino acid sequences obtained by aligning the two sequences. Depending on the number of common residues between the aligned sequences, different degrees of identity, expressed as percentages, are obtained. The degree of identity between two amino acid sequences can be determined by conventional methods, for example, standard sequence alignment algorithms known in the art, such as BLAST [Altschul SF et al., Basic local alignment search tool. J Mol Biol. 1990 October 5;215(3):403-10]. BLAST programs, such as BLASTN, BLASTX, and TBLASTX, BLASTP, and TBLASTN, are in the public domain at the National Center for Biotechnology Information (NCBI) website.

[0034] Those skilled in the art understand that mutations in the nucleotide sequence of a gene resulting in conservative amino acid substitutions at positions not critical to the function of the protein are evolutionarily neutral mutations that do not affect its overall structure or its function, resulting in a protein that contains a different amino acid sequence but exerts the same activity. These proteins are considered "functionally equivalent variants" of the sequence of SEQ ID NO: 1 and are included within the scope of the present invention. Thus, the term "functionally equivalent variant" as used herein means an enzyme derived from the native enzyme (SEQ ID NO: 1 of the present invention) by one or more deletions, insertions, and / or substitutions of one or more amino acids at sites within its amino acid sequence and that exerts the same activity, i.e., retains the ability to oxidize untreated polyolefin-derived polymers, such as polyethylene (PE), at room temperature. Variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc. All proteins that have at least 60% sequence identity with the amino acid sequence of SEQ ID NO: 1 and are capable of oxidizing untreated polyolefin-derived polymers are considered functionally equivalent variants in the context of the present invention. Examples of assays to check whether a given protein is a functionally equivalent variant of the enzyme of SEQ ID NO: 1 are disclosed in the Examples of this patent application. Functionally equivalent variants of the invention have at least 20%, such as at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 100% of the oxidative activity of the native protein of SEQ ID NO: 1.

[0035] The present invention also encompasses functionally equivalent fragments of the enzymes of the present invention. The term "functionally equivalent fragment" refers to a polypeptide / protein having one or more (e.g., several) amino acids not present at the amino and / or carboxy terminus of the native protein (in the present invention, the sequence of SEQ ID NO: 1), and exhibits the same activity / function as the native protein (in the present invention, the ability to oxidize untreated polyolefin-derived polymers at room temperature). An example of an assay for determining whether a fragment of the protein of the present invention is a functionally equivalent fragment of SEQ ID NO: 1 is disclosed in the examples of this patent application. A functionally equivalent fragment of the present invention has at least 20%, for example at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the oxidation activity of the native protein SEQ ID NO: 1.

[0036] In a preferred embodiment, the amino acid sequence of the enzyme of the invention comprises or consists of the sequence SEQ ID NO:1.

[0037] The nucleotide sequences of the present invention encoding the enzymes of the present invention may further comprise other elements than the coding sequence, such as introns, non-coding sequences at the 3' and / or 5' ends, ribosome binding sites, etc. The nucleotide sequences of the present invention may also comprise sequences encoding additional amino acids useful for increasing the stability of the enzyme or for allowing more efficient enzyme purification.

[0038] The nucleotide sequence of the invention may be introduced into a vector or genetic construct, such as a cloning vector or an expression vector, to obtain a vector containing said nucleotide sequence. Preferably, said vector is a vector suitable for the expression and purification of the enzyme of the invention.

[0039] The term "genetic construct" or "vector" as used herein refers to a monocatenary or bicatenary nucleic acid molecule that has been isolated and modified to contain a nucleic acid segment in a manner that would not occur in nature. The term "nucleic acid construct" or "genetic construct" is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences required for the expression of the nucleotide sequence of the present invention. Thus, the genetic construct of the present invention may also contain one or more gene expression control or regulatory sequences, including, but not limited to, promoter sequences, leader sequences, terminator sequences, polyadenylation sequences, signal sequences, regulators, enhancers, and the like.

[0040] An "expression vector" is a linear or circular DNA molecule that contains at least a nucleotide sequence of the present invention operably linked to additional nucleotides provided for its expression. This vector containing the nucleic acid sequence of the present invention can be introduced into a host cell such that the vector is maintained as a chromosomal component or as an autonomously replicating extrachromosomal vector.

[0041] The expression vector referred to in the present invention may be any vector (e.g., a plasmid or a virus) that can be conveniently subjected to recombinant DNA procedures and produce the expression of the nucleotide sequence of the present invention contained therein. The choice of vector usually depends on the compatibility of the vector with the host cell into which it is introduced. The expression vector may be, for example, but is not limited to, a plasmid, a cosmid, a phage, a virus or a viral vector, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), and the like. The vector within the context of the present invention may be linear or closed circular. Preferably, the expression vector of the present invention is a baculovirus expression vector, more preferably a P2 baculovirus vector.

[0042] As used herein, a "host cell" includes any cell type susceptible to transformation, transfection, transduction, etc. with the nucleotide sequence or expression vector of the present invention referenced above. The host cell may be prokaryotic or eukaryotic, preferably eukaryotic, such as a mammalian, insect, plant or fungal cell. In some embodiments, the host cell is a prokaryotic, preferably a bacterial cell, such as E. coli. In some embodiments, the host cell is a yeast cell. In a preferred embodiment, the host cell is an insect cell, more preferably an sf9 cell.

[0043] Thus, a host cell of the invention at least comprises a nucleic acid sequence of the invention, preferably by means of a vector of the invention in recombinant form. The nucleotide sequence of the invention or the vector of the invention is not naturally present in the cell, but has been deliberately introduced by genetic engineering procedures. The nucleotide sequence of the invention can encode a precursor protein consisting of a signal peptide linked to the mature enzyme of the invention, or to the mature enzyme which must subsequently be processed to produce the mature enzyme.

[0044] Expression of the enzyme of the invention in the host cell of the invention may be induced by any procedure known in the art, such as transformation of a suitable host cell with at least one nucleotide sequence of the invention or a vector of the invention, and culturing the transformed host cell under conditions inducing expression of the nucleotide sequence, to obtain a secreted functional enzyme. Preferred conditions for inducing expression of the nucleotide sequence are incubation of the host cells at 27° C. for 48 to 72 hours.

[0045] In some aspects, the invention relates to a method for expressing an enzyme having at least 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:1 in a host cell, the method comprising culturing the host cell under conditions inducing expression of an expression vector to obtain the enzyme.

[0046] The enzymes of the invention produced by the host cell hosts of the invention can be purified by a variety of procedures known in the art, including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic methods (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction to obtain a substantially pure enzyme. In another preferred embodiment, the enzymes of the invention are recombinantly produced or isolated from the saliva of Galleria mellonella, particularly G. mellonella.

[0047] Methods for isolating the enzyme of the present invention from Galleria mellonella saliva include, but are not limited to, chromatographic techniques (e.g., size exclusion chromatography, ion exchange chromatography, etc.) from the saliva of the waxworm.

[0048] The composition of the invention comprises the enzyme of the invention or a functionally equivalent fragment thereof, or the host cell of the invention, and optionally other elements necessary for their optimal activity or for their preservation. These additional elements can be, by way of example, buffers, e.g., HEPES, other enzymes, e.g., enzymes useful for the biodegradation of polyolefin-derived polymers, antibiotics, etc. Preferably, the composition of the invention further comprises a second enzyme comprising an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID NO:2, and more preferably, this additional protein comprises or consists of SEQ ID NO:2.

[0049] SEQ ID NO:2 (Ceres)

[0050] [ka]

[0051] Preferably, the second enzyme is active in biodegrading, or oxidizing and / or depolymerizing, a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer.

[0052] In some embodiments, the composition of the invention comprises an enzyme of the invention or a functionally equivalent fragment thereof, or a host cell of the invention, and preferably at least one further component, which in some embodiments is at least one of the additional components disclosed above.

[0053] Preferably, the composition of the invention further comprises an isolated second enzyme comprising an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:2.

[0054] In some embodiments, the second enzyme in the composition is not the enzyme with database accession reference number XP_026756459.1.

[0055] In some embodiments, the second enzyme in the composition is an enzyme that comprises a sequence that has 1% variation from the amino acid sequence of SEQ ID NO: 2. In some embodiments, the second enzyme comprises a sequence that has 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40% variation from the sequence of SEQ ID NO: 2. In some embodiments, the second enzyme comprises a sequence that varies by 1 amino acid from the amino acid sequence of SEQ ID NO: 2. In some embodiments, the second enzyme comprises a sequence that varies by 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or 300 amino acids from the amino acid sequence of SEQ ID NO: 2.

[0056] In one aspect, the present invention relates to a kit for biodegrading, or oxidizing and / or depolymerizing, a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer, said polyolefin-derived polymer comprising: Into the first container: an enzyme comprising an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:1 or a functionally equivalent fragment thereof; a host cell comprising a nucleotide sequence encoding said enzyme, or a vector comprising a nucleotide sequence encoding said enzyme, or a composition comprising said enzyme or a functionally equivalent fragment thereof, or said host cell; and instructions for using said enzyme, said host cell or said composition: a second enzyme comprising an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:2 or a functionally equivalent fragment thereof; a host cell comprising a nucleotide sequence encoding the second enzyme, or a vector comprising a nucleotide sequence encoding the second enzyme; or A composition comprising said second enzyme or a functionally equivalent fragment thereof, or said host cell. Includes.

[0057] In some embodiments, the kit comprises: In a further container: a second enzyme comprising an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:2 or a functionally equivalent fragment thereof; a host cell comprising a nucleotide sequence encoding the second enzyme, or a vector comprising a nucleotide sequence encoding the second enzyme; or A composition comprising said second enzyme or a functionally equivalent fragment thereof, or said host cell. Further includes:

[0058] In some embodiments of the invention, the second enzyme is an isolated enzyme.

[0059] In some embodiments, the enzyme, host cell or composition of SEQ ID NO:1 is used in a separate, sequential or simultaneous step with the enzyme, host cell or composition of SEQ ID NO:2 to biodegrade, or oxidize and / or depolymerize, a polyolefin-derived polymer or a material that includes a polyolefin-derived polymer.

[0060] In the most general aspect, "polyolefin-derived polymer" refers to any polyolefin polymer derived from olefin monomers.

[0061] In one aspect of the invention, the "polyolefin-derived polymer" is a polymer having the general formula (CH2CHR) n where R is an alkyl group. In some cases, R may be a hydrogen atom. They are usually derived from a small set of simple olefins (alkenes). The predominant commercial types are polyethylene and polypropylene. More specialized polyolefins include polyisobutylene and polymethylpentene. All of these are colorless or white oils or solids. The name of each polyolefin indicates the olefin from which it is prepared; for example, polyethylene is derived from ethylene and polymethylpentene is derived from 4-methyl-1-pentene.

[0062] In another preferred embodiment, the polyolefin-derived polymer referred to in the present invention is polyethylene (PE) or polypropylene (PP).

[0063] In another more preferred embodiment, the polyolefin derived polymer referred to in the present invention is polyethylene (PE).

[0064] "Polyethylene (PE)" or polyethylene (abbreviated PE; IUPAC name polyethylene or poly(methylene)) is the most common plastic in use today. It is a polymer, used primarily for packaging (plastic bags, plastic films, geomembranes, containers including bottles, etc.). Most have the chemical formula (C2H4) n Many types of polyethylene are known, with n = 1. PE is usually a mixture of similar polymers of ethylene, with various values ​​of n. It can be low density or high density: low density polyethylene is extruded using high pressure (1000-5000 atm) and high temperature (520 Kelvin), high density polyethylene is extruded using low pressure (6-7 atm) and low temperature (333-343 K). Polyethylene is usually thermoplastic, but can alternatively be modified to be thermoset, for example in crosslinked polyethylene. All types of PE are within the scope of the present invention.

[0065] In further preferred embodiments, the PE is selected from the list consisting of ultra-high molecular weight polyethylene (UHMWPE), ultra-low molecular weight polyethylene (ULMWPE or PE-WAX), high molecular weight polyethylene (HMWPE), high density polyethylene (HDPE), high density cross-linked polyethylene (HDXLPE), cross-linked polyethylene (PEX or XLPE), medium density polyethylene (MDPE), linear low density polyethylene (LLDPE), low density polyethylene (LDPE), very low density polyethylene (VLDPE), and chlorinated polyethylene (CPE). In a particular embodiment, the PE is LDPE, more preferably PE4000 or PE2000.

[0066] In another specific embodiment, the polyolefin-derived polymer or material comprising the polyolefin-derived polymer is not pre-treated with an abiotic agent (such as heat, UV light, etc.) prior to biodegradation by the enzymes of the invention, the host cells of the invention, or the compositions of the invention, i.e., the enzymes of the invention are capable of biodegrading untreated polyolefin-derived polymer or untreated material comprising polyolefin-derived polymer.

[0067] In another aspect, the present invention provides a method for biodegrading, oxidizing and / or depolymerizing a polyolefin-derived polymer, or a material comprising a polyolefin-derived polymer (hereinafter "method of the present invention"), comprising the steps of: The enzyme of the present invention, or A host cell of the invention, or Compositions of the Invention with a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer.

[0068] In a preferred embodiment of the method of the invention, said method is carried out at room temperature, preferably in an aqueous solution at neutral pH at room temperature.

[0069] "Room temperature" is 15°C to 30°C, preferably 22°C.

[0070] "Neutral pH" is between pH 7 and pH 8.

[0071] In a preferred embodiment, in the method of the invention, the enzyme is used in a volume of 2 to 10 μL, preferably 5 μL, at a concentration between 1 and 5 μg / μL.

[0072] In another preferred embodiment of the method of the present invention, the incubation time between the enzyme, host cell or composition of the present invention and the polyolefin-derived polymer is at least 60 to 120 minutes, preferably at least 90 minutes.

[0073] In a further preferred embodiment of the method of the invention, the enzyme is used in a volume of 5 μL or 10 μL, at a concentration between 1 and 5 μg / μL, preferably at a concentration of 1.2 μg / μL, and is applied to the polyolefin-derived polymer or to the material comprising the polyolefin-derived polymer at least 8 times, preferably 24 times, each time for 90 minutes.

[0074] In another preferred embodiment of the method of the invention, the enzyme of the invention comprises or consists of the sequence SEQ ID NO: 1. More preferably, the enzyme of the invention is isolated from G. mellonella, in particular from the saliva of G. mellonella.

[0075] In another preferred embodiment of the method of the present invention, the composition of the present invention further comprises a second enzyme comprising an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:2, more preferably comprises SEQ ID NO:2, even more preferably consists of SEQ ID NO:2.

[0076] In some embodiments, a second enzyme comprising an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:2 is contacted with the polyolefin-derived polymer or a material comprising a polyolefin-derived polymer in a separate, sequential, or simultaneous step with the enzyme comprising an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:1.

[0077] In another preferred embodiment of the method of the present invention, the polyolefin-derived polymer is polyethylene (PE) or polypropylene (PP).

[0078] In another more preferred embodiment of the method of the present invention, the polyolefin-derived polymer is polyethylene (PE).

[0079] In another preferred embodiment of the method of the present invention, the PE is selected from the list consisting of ultra-high molecular weight polyethylene (UHMWPE), ultra-low molecular weight polyethylene (ULMWPE or PE-WAX), high molecular weight polyethylene (HMWPE), high density polyethylene (HDPE), high density cross-linked polyethylene (HDXLPE), cross-linked polyethylene (PEX or XLPE), medium density polyethylene (MDPE), linear low density polyethylene (LLDPE), low density polyethylene (LDPE), very low density polyethylene (VLDPE), and chlorinated polyethylene (CPE). In a particular embodiment, the PE is LDPE, more preferably PE4000 or PE2000.

[0080] Another aspect of the invention is a method for pre-treating a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer, comprising the steps of: The enzyme of the present invention, or A host cell of the invention, or Compositions of the Invention with a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer to provide an oxidized polymer or material product.

[0081] In some embodiments, polyolefin-derived polymers or materials that contain polyolefin-derived polymers are pretreated using the method of the present invention before further decomposition steps.For example, further decomposition steps are carried out on oxidized polymers or material products.In some embodiments, polyolefin-derived polymers or materials that contain polyolefin-derived polymers are pretreated using the method of the present invention before applying one or more microbial decomposition steps.

[0082] Another aspect of the invention is a method for obtaining by-products derived from the biodegradation of polyolefin-derived polymers, comprising the steps of: (a) a polyolefin-derived polymer, The enzyme of the present invention, or A host cell of the invention, or Compositions of the Invention and (b) isolating by-products from the culture resulting from step (a). The present invention relates to a method comprising the steps of:

[0083] Another aspect of the invention is a method for preparing a plastic comprising the steps of: (a) a polyolefin-derived polymer, The enzyme of the present invention, or A host cell of the invention, or Compositions of the Invention and (b) isolating by-products from the culture resulting from step (a). (c) polymerizing the by-product isolated in step (b). The present invention relates to a method comprising the steps of:

[0084] "By-products" in the context of the present invention include, but are not limited to, butane, 2,3-butanediol, trimethylsilyl (TMS) derivatives, sebacic acid, 2-ketones having 10 to 22 carbon atoms, and small aromatic compounds recognized as benzenepropanoic acid. Preferably, the by-products obtained include C10 to C22 2-ketones.

[0085] The process conditions for the obtained by-products are those already described above for the processes for biodegrading, oxidizing and / or depolymerizing polyolefin-derived polymers or materials comprising polyolefin-derived polymers, for example, a second enzyme may be used in these processes.

[0086] Isolation of the by-products in step (b) of the process may be carried out by techniques well known in the art such as gas chromatography mass spectrometry (GC-MS).

[0087] Aspects of the present invention will now be described by way of example, and not by way of limitation, with reference to the accompanying drawings, in which: However, various further aspects and embodiments of the present invention will be apparent to those skilled in the art in light of the present disclosure.

[0088] As used herein, "and / or" is to be construed as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" is to be construed as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually set forth herein.

[0089] Unless the context indicates otherwise, the above feature descriptions and definitions are not limited to any particular aspect or embodiment of the invention, but apply equally to all aspects and embodiments described. [Brief description of the drawings]

[0090] [Figure 1] Figures showing collection and functional studies of Galleria mellonella saliva (GmSal). A. Diagram of collection and application of saliva. B, C, D, E. Raman analysis of PE films. B. PE film treated with GmSal: 3 applications of 30 μl each for 90 min. The peaks at 1500-2400 cm-1 show different collective stretching vibrations due to the presence of other organic compounds, a sign of PE degradation (red arrows). Oxidation is indicated between 1600-1800 cm-1 (carbonyl groups) and between 3000-3500 cm-1 (hydroxyl groups) (black arrows) (Larkin, 1998). C. Control PE film. Brackets indicate the picks characterizing PE (PE signature) corresponding to bands at 1061, 1128, 1294, 1440, 2846, and 2880 cm-1. D. Overlapping profiles (B and C). PE film treated with E. samia cynthia's saliva. [Diagram 2]Figure 1. HT-GPC analysis of PE treated with GmSal. Molecular weight distribution of PE film (A) and PE4000 (B). A. Control PE (black) and PE treated (red) are compared. Carbonyl index (CI) of control and experiment are shown. Mw control (red): 0, Mw experiment (black): 0.0882. B. Control PE (black), treated PE (15 applications) (blue), treated PE (30 applications) are compared. CI of each sample is shown. Mw control (black): 0.0106, Mw PE treated-15 applications (blue): 0.0500, Mw PE treated (30 applications) (red): 0.1039. Arrows indicate low molecular weight compounds in treated PE. [Diagram 3] Figure 1: Identification of PE degradation by-products by GC-MS. A, B, C. Chromatograms of PE treated with GmSal showing different compounds. A and B. Ketones of different lengths, indicated by the number of carbon atoms. C. 2,3-butanediol 2TMS derivative, benzenepropanoic acid TMS derivative, sebacic acid sTMS derivative. Compounds in C were identified by silylation (see Methods). IS: internal standard. [Figure 4] Figure 2. Functional characterization of GmSal contents. A. Electron microscopy negative staining of saliva sample, dilution 1:500. Protein complexes (10-15 nm size) are indicated by the boxes in the upper right. B. SDS-PAGE of GmSal sample, dilution 1:50. Molecular weight standards are listed on the left. Arrows indicate the major band at 75 kDa. CF. Raman of PE treated with IEX fractions (see Figure S4). C. Fraction 9, no activity (control PE film, see Figure 1 for details). DF. Profiles corresponding to IEX peaks 1 (fraction 29), 2 (fraction 34), and 3 (fraction 44) (see Figure 1 for details). The intense peak at 1085 cm-1 in E indicates amorphous PE. The reduction of peaks at 2845 and 2878 cm-1 indicates the presence of PE is less pronounced. G. Overlap of fractions C to F. [Diagram 5]Figure 1. Effect of Demetra on PE film. A, B. Control PE film. CE. Demetra on PE film. FI. Raman spectroscopy of control (F) and Demetra-treated PE film (G, H). F. Control PE film (see Figure 1 for details). G, H. Two different timed analyses in the crater displayed in E, showing PE degradation. See Figure 1 for details. Additionally, the bands at 845 and 916 cm-1 correspond to C-O-C and C-COO groups, respectively. In G, the presence of PE is negligible. [Figure 6] Figure 1. Formation of PE degradation by-products by Demetra. A. GC-MS chromatogram of PE treated with Demetra. Arrows indicate peaks corresponding to ketones with different carbon numbers. B. Application of Demetra to PE 5 to 10 times increases the formation of ketones as degradation products. EXAMPLES

[0091] I - Materials and Methods Waxworm saliva collection For saliva collection, 150–300 mg of Galleria mellonella larvae were used (GmSal). Briefly, a glass capillary tube connected to a mouth pipette was placed at the cheek opening and the liquid was collected. Saliva for PE application was used immediately. In some cases, frozen saliva alone could be used. For electron microscopy, saliva was diluted 1:1 in the following buffer: 10 mM Tris-Cl pH 8, 50 mM NaCl. For proteomic analysis, saliva was diluted 1:1 in 10 mM Tris-Cl, 50 mM NaCl, 2 mM DTT, 20% glycerol. As a control, final stage Samia cynthia larvae (kindly provided by InsectPark-Microfauna SL (Escorial, Madrid)) were used to collect saliva as previously described.

[0092] Raman and FTIR analysis PE films were treated with 30 μl of GmSal for 90 min and applied three times for Raman. PE films were treated with 5 μl of GmSal for 90 min and applied nine times for FTIR. Recombinant proteins were applied as follows: 5 μl of protein (concentration between 1-5 μg / ml) was applied to the PE film eight times for 90 min each time. For the inactivated protein control, the recombinant protein was denatured at 100 degrees for 10 min. Size exclusion chromatography (SEC) and ion exchange chromatography (IEX) peak aliquots were applied six times for 30 min each and left overnight. Treated and control films were washed with water and ethanol. Raman analysis was performed on PE films (treated and control) using an Alpha300R-Alpha300A AFM Witec instrument with 5 mW power, 50x (NA0.8) objective, integration time 1, accumulation 30, and wavelength 532 nm. FTIR analysis was performed using a Jasco LE-4200 instrument with the following characteristics: interval 4000-400 cm -1 , resolution 4cm -1 , Scan 264 was performed.

[0093] High temperature gel permeation chromatography (HT-GPC) analysis.

[0094] HT-GPS was performed by Polymer Chart, Valencia, Spain. Briefly, the experimental conditions were as follows: Instrument: GPC-IR5_I Polymer Char; Solvent: TCB stabilized with 300 ppm BHT; Dissolution, detector and column temperatures: 160 degrees; Volume: 8 ml; Weight: 8 mg; Dissolution time: 60 min; Injection volume: 200 μl; Injection time: 55 min; Flow rate: 1 ml / min; Column: 3 μPL Gel Olexis Mix-Bed column (13 micron), 300x7.5 mm + guard column.

[0095] For the Carbonyl Index analysis, GPC_IR6 was used with the solvent o-DCB at a temperature of 150° C. PE film and PE4000 were treated with 100 μl of GmSal for 90 min. The treatment was repeated 15 times (film and PE4000) and 30 times (PE4000).

[0096] Gas chromatography-mass spectrometry (GC-MS) and tandem analysis.

[0097] A quantity of 20 mg of PE4,000 or 1.5 mg of PE2,000 was placed in a 1.5 mL Eppendorf tube. PE was exposed to 40 μL of G. mellonella saliva 9 times, 90 minutes each time, at room temperature, away from light. For long-term treatments (days 1, 2, 3, and 6), 3 applications of 100 μL of saliva were performed on each day, 90 minutes each time, at room temperature. Controls for each PE were performed using Milli-Q water instead of G. mellonella larval saliva, as well as using only G. mellonella larval saliva. PE was also exposed to 10 μL (1.2 mg / mL) of Demetra (SEQ ID NO: 1) 24 times for 90 minutes. Long-term treatments were also performed for Demetra (days 1 and 2), with 5 applications per day, 90 minutes each time, 10 μL (1.2 mg / mL). As a control, the same experiment was repeated using protein buffer. The samples were then centrifuged at 19,083 G for 30 s in an Eppendorf centrifuge 5810R, and the supernatant was transferred to a new 1.5 mL Eppendorf tube. Samples and controls were extracted using the QuEChERS (quick, easy, cheap, effective, and safe) method 1 based on 2 with some modifications. Briefly, 50 μL of diphenylphthalic acid (internal standard, IS) at a concentration of 1 mg / mL was added to each sample, which was then extracted with 300 μL of dichloromethane (DCM) and 5% (v / m) NaCl. The tubes were vortexed for 30 s, sonicated (50 / 60 Hz) in a water bath for 15 min at room temperature, and then centrifuged at 19,083 G for 10 min at 20 °C in an Eppendorf centrifuge 5810R. Finally, the DCM located as the supernatant was collected and placed in an insert before analysis. To determine the less volatile polar compounds that showed low detection sensitivity, a silylation reaction with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) was performed. A 50 μL aliquot of each sample was incubated with 50 μL of BSTFA at 60 °C for 20 min before analysis.

[0098] Dichloromethane (DCM; CAS number: 75-09-2) for gas chromatography-mass spectrometry (GC-MS) was of SupraSolv grade purity and was obtained from Sigma-Aldrich (Darmstadt, Germany). Sodium chloride (NaCl, ≥ 99.5%; CAS number: 7647-14-5) and ultrapure water from a Milli-Q system were supplied by Merck (Darmstadt, Germany).

[0099] Crystalline granular powdered polyethylene (PE4,000; CAS number: 9002-88-4) and analytical standard polyethylene (PE2,000; CAS number: 9002-88-4, details: Mw(Da) 1970; Mn(Da) 1700; Mp(Da) 1890; PD(Mw / Mn) 1.16) were supplied by Sigma-Aldrich (Saint Louis, USA).

[0100] Chromatographic analysis was performed using an Agilent Technologies gas chromatography-mass spectrometry system (GC-MS) 7980A-5975C. Separation of metabolites was performed on a polyimide-coated DB-5 column (30 m length, 0.25 mm inner diameter, 0.1 μm film thickness; Agilent Technologies, USA) for proper separation of substances, and helium (He) was used as carrier gas. Analysis was performed at 350 °C with an injection volume of 1 μL using a split injector. The ion source temperature was 230 °C and mass spectrometry was performed in scan mode, quadrupole temperature 150 °C, and fragmentation voltage 70 eV. The oven program was started at 60 °C for 3 min, then 20 °C / min to 350 °C in 1 min. The total run time was 18.5 min, and 19.5 min for the derivatized samples. The obtained chromatograms were processed using the software MSD ChemStation E.01.00.237 from Agilent Technologies and the NIST11 library was used for identification.

[0101] The assessment of long-term treatment was based on the relative abundance of each non-target compound, which was composed of the area under each compound's peak divided by the area under the IS peak.

[0102] Gas chromatography / tandem mass spectrometry on a BRUKER 456-GC SCION TQ was used to confirm non-targeted compounds. The experiments were performed at the Elemental and Molecular Analysis Facility of the University of Extremadura, Spain. Briefly, the injector port was set at 230 °C in split mode. Separation was achieved using HP 5MS, 30 m, 0.25 mm, and 0.25 μm columns. Helium (He) was utilized as the carrier gas. The column oven was programmed with the following conditions: 60 °C for 3 min, ramp to 325 °C at 20 °C / min for 1 min. Collision energy was 15 eV.

[0103] Electron microscopy analysis Larval saliva samples were diluted 1:50 in an appropriate buffer (see “Waxworm saliva collection”).

[0104] Samples were adsorbed onto glow-discharged carbon-coated grids, stained with 2% uranyl acetate, and then analyzed by electron microscopy (EM). Grids were viewed using a JEOL JEM-1230 EM operating at 100 kV and a nominal magnification of 40,000. EM images were taken under low-dose conditions using a CMOS Tvips TemCam-F416 camera at 2.84 Å / pixel.

[0105] Protein chromatography analysis For size-exclusion chromatography, waxworm (ww) saliva in the appropriate buffer (see "Waxworm Saliva Collection") was thawed, pooled and centrifuged. The supernatant was filtered (0.45 μm cutoff, Ultrafree Millipore) and loaded onto a size-exclusion chromatography column Superdex 200 5-150 (Cytiva) equilibrated with 10 mM Tris-Cl, 50 mM NaCl, 2 mM DTT.

[0106] For ion exchange chromatography, upon thawing, samples were diluted to 100 μL with 10 mM Tris-Cl pH 8, centrifuged, filtered and the supernatant was loaded onto a monoQ 5 / 50 GL ion exchange column (Cytiva). After a washing step, a 40 mL gradient with buffer A (10 mM Tris-Cl pH 8) and buffer B (same as A with the addition of 500 mM NaCl) was applied.

[0107] Proteomic analysis Liquid chromatography-mass spectrometry (LC-MS) analysis. All peptide separations were performed on an Easy-nLC 1000 nano system (Thermo Fisher Scientific). For each analysis, samples were loaded onto a precolumn Acclaim PepMap 100 (Thermo Fisher Scientific) and eluted in an RSLC PepMap C18, 15 cm long, 50 μm inner diameter, and 2 μm particle size (Thermo Fisher Scientific). The mobile phase flow rate was 300 nl / min, with 0.1% formic acid in water (solvent A) and 0.1% formic acid and 100% acetonitrile (solvent B). The gradient profile was set as follows: 5%-35% solvent B for 45 min, 35%-100% solvent B for 5 min, 100% solvent B for 10 min. 4 μl of each sample was injected.

[0108] MS analysis was performed using a Q Exactive mass spectrometer (Thermo Fisher Scientific). For ionization, a junction voltage of 1900 V and a capillary temperature of 270 °C were used. A full scan method was employed with m / z 400–1500 mass selection, Orbitrap resolution of 70,000 (at m / z 200), a target automatic gain control (AGC) value of 3e6, and a maximum injection time of 100 ms. After a survey scan, the 15 most intense precursor ions were selected for MS / MS fragmentation. Fragmentation was performed with a normalized collision energy of 27 eV, and MS / MS scans were acquired with a starting mass of m / z 100, an AGC target of 2e5, a resolution of 17,500 (at m / z 200), an intensity threshold of 8e4, an isolation window of 2 m / z units, and a maximum IT of 100 ms. Charge state screening allowed the exclusion of unassigned, singly charged, and ≥7 protonated ions. A dynamic exclusion time of 20 s was used to differentiate between previously selected ions.

[0109] MS data analysis. Mass spectra *.raw files were searched against an in-house proprietary database for Galleria_Proteins (12715 protein entries) using the Sequest search engine by Proteome Discoverer (version 1.4.1.14) (Thermo Scientific). Search parameters included a maximum of two missed cleavages allowed, carbamidomethylation of cysteine ​​as fixed modification, and oxidation of methionine as variable modification. Precursor and fragment mass tolerances were set at 10 ppm and 0.02 Da, respectively. Identified peptides were validated using the Percolator algorithm with a q-value threshold of 0.01. Protein identification by nLC-MS / MS was performed at the Proteomics and Genomics Facility (CIB-CSIC), a member of the ProteoRed-ISCIII network 3.

[0110] Recombinant Protein Production and Use Arylphorin, arylphorin subunit alpha-like (Demetra, SEQ ID NO:1) and hexamerin (Ceres, SEQ ID NO:2) were produced by Genscript utilizing the baculovirus expression system in insect cells according to the manufacturer. Briefly, sf9 cells were infected with P2 baculovirus, flasks were incubated at 27 °C for 48-72 h and the medium was harvested. Cells were then removed and transfection medium was applied for purification. Produced proteins were resuspended in 150 mM NaCl, 20 mM Hepes, 5% glycerol and used for degradation assays. As a negative control, only the same buffer was used.

[0111] Galleria mellonella genome annotation To obtain the most useful information from mass spectrometry of proteins extracted from ww saliva, it is crucial to use a representative database of protein sequences. In addition to the official NCBI annotation of G. mellonella protein sequences, a new annotation was created that also leveraged information from G. mellonella salivary gland RNA-seq data (Bertocchini, unpublished). G. mellonella genome annotation was performed using genome sequences available at NCBI (accession number ASM258982v1). To obtain the most accurate annotation, a specific pipeline was developed to combine information from RNA-seq (Bertocchini, unpublished) with ab initio predictors. Briefly, RNA-seq data were mapped onto a reference genome using STAR (version 2.5 0c) in local mode and used to perform a reference-guided transcriptome assembly using Trinity (v2.11.0). The resulting transcript and mapping files were used as input for the Braker2 pipeline, combining the AUGUSTUS ab initio annotations and the transcriptome assembly to obtain annotations in GFF format along with the sequences of the transcripts and proteins. The proteins were used as input for the PANNZER2 pipeline to obtain description, gene ontology, and KEGG annotations. Approximately 32000 genes could be annotated for the Galleria genome. To analyze the corresponding proteins and evaluate the completeness, we performed a BLASTP alignment against the UniRef90 database and calculated the percentage of alignment. A similarity search against the UniRef90 (Nov2018) database showed that approximately 50% of the predicted proteins covered 100% (i.e., full length) of the corresponding hits, and approximately 80% of the predicted proteins covered at least 50% of the corresponding hits. As a further control, the proteins and genome were evaluated with the BUSCOv2 pipeline. The BUSCO database contains a set of single-copy highly conserved genes across a variety of taxa (i.e., eukaryotes or insects).By performing an analysis on the BUSCO database, it is possible to assess the completeness of the genome / proteome, the presence of duplications and / or fragmentation. This analysis was performed using the predicted proteome and an unannotated genome for comparison. By comparing the results of the unannotated and annotated genomes, a small portion of missing genes can be identified that are probably not present in the genome assembly and cannot be recovered from the current genome sequence. This explains some missing genes present in the later NCBI annotation.

[0112] II - Results Wax worm saliva oxidizes PE film Saliva, broadly defined here as the juice present in the anterior part of the digestive tract, was collected from the mouth of a w. maculatus and tested on a commercial PE film (Fig. 1A). After three consecutive applications of 30 μl of GmSal for 90 min each, confocal Raman microscopy / Raman spectroscopy (Raman) analysis showed that the polymer was highly oxidized, concomitant with an overall degradation of the film (Fig. 1B). This was evident from the overlap with the PE control (Fig. 1C,D), revealing the expected PE signature profile. As a further control, application of saliva from another lepidopteran insect larva, Samia cynthia, to the PE film did not result in oxidation (Fig. 1E). The changes produced by GmSal after a few hours of application are similar to those produced by environmental factors after exposure to weathering for months or years. Fourier transform infrared spectroscopy (FTIR) analysis confirmed the oxidation profile. Changes in the chemical composition of PE revealed by spectroscopic techniques suggest that molecules other than long PE polymer chains are formed upon contact with GmSal.

[0113] Waxworm saliva breaks down PE The molecular weight characteristics of PE before and after GmSal treatment were analyzed using high temperature gel permeation chromatography (HT-GPC). After several hours of application of GmSal to the PE film (15 applications, 90 min each), the molecular weight distribution became bimodal with a new peak in the low molecular weight region, indicating the cleavage of C-C bonds and the appearance of smaller compounds (Figure 2A). An increase in the carbonyl index (CI, Figure 2A) indicated the formation of C=O bonds as a result of chain scission. The weight average (M w ) only changed slightly, suggesting that the polymer was not homogeneously modified by GmSal, but that some parts were strongly depolymerized while others remained intact. The same results were obtained when PE4000 was used instead of the PE film, resulting in an increase in the carbonyl index of the GmSal-treated samples (Figure 2B, black and blue curves). These results indicate that exposure to GmSal resulted in the oxidation and depolymerization of PE to form low molecular weight oxidized molecules. To analyze the dynamics of the changes in PE after increased exposure to GmSal over time, the exposure time of PE4000 to GmSal was doubled (30 applications, 90 min each) (Figure 2B, red curve). The scission of large polymer chains accompanied by the formation of smaller molecules was significantly increased with longer exposure times, as shown by the comparison of the molecular weight distributions and the doubling of the CI (Figure 2B). These experiments confirmed that exposure to GmSal for several hours oxidized PE with concomitant depolymerization, an effect that increased with time, suggesting the presence of an unknown activity capable of degrading PE.

[0114] Identification and analysis of by-products of PE treated with waxworm saliva To gain insight into the degradation products resulting from contact of PE with saliva and released from the oxidized polymer, PE granules (crystalline polyethylene - PE4000) were exposed to GmSal and subsequently analyzed by gas chromatography-mass spectrometry (GC-MS) and identified in the NIST11 library for non-targeted compounds. After nine applications of 40 μL of GmSal for 90 min each at room temperature, new compounds were detected in the experimental samples (Figure 3). The detected compounds consisted of oxidized aliphatic chains such as 2-ketones with carbon numbers from 10 to 22. Ketones with carbon numbers from 10 to 18 were identified by comparing the fragmentograms of ion m / z 58 from methyl ketones corresponding to the McLafferty displacement on the carbonyl located at the second carbon of each ketone. Those not present in the library as 2-eicosanone and 2-docosanone showed the same fragmentogram m / z 58 and were defined by equidistant peaks along the retention time and their molecular weights. Furthermore, the presence of 2-ketones was confirmed by GC-MS / MS in MRM mode using the ions with the highest m / z as well as the exclusivity of each molecule, 212>58 for 2-tetradecanone, 240>59 for 2-hexadecanone, and 282>58 for 2-octadecanone. Also, butane, 2,3-butanediol, 2-trimethylsilyl (TMS) derivatives, and sebacic acid, 2TMS derivatives, were identified using sample silylation, indicating degradation of PE chains (Figure 3C). At the same time, benzenepropanoic acid, a TMS derivative, a small aromatic compound that can be recognized as a plastic antioxidant, was found. The derivative chemicals were also confirmed using GC-MS / MS at m / z 147>73, 331>73, and 104>75, respectively. The same results were obtained using monodisperse polyethylene PE2000, except for the ketones. The presence of this plastic antioxidant suggests that there are "openings" in the polymer structure that allow the release of small stabilizing compounds (plastic additives) normally present within the plastic.

[0115] To verify whether increasing exposure time to GmSal causes increased degradation of PE, the PE4000 exposure experiment was repeated consecutively, applying 100 μL of GmSal four times a day for 90 min each at room temperature, for 1, 2, 3, and 6 consecutive days. Analysis of the supernatants revealed a gradual increase in the formation of 2-decanone, 2-dodecanone, 2-tetradecanone, and 2-hexadecanone. These data indicate that prolonged exposure to GmSal results in at least a two-fold increase in the relative abundance of the polymer and the extent of its degradation over time (1–6 days).

[0116] Study of saliva of wax worms (wax worn): Enzyme identification and functional studies To understand the nature of oral fluid, GmSal samples were analyzed by negative staining electron microscopy (EM), which revealed a high content of proteins or protein complexes (size: 10-15 nm) (Figure 4A). No other structures (like vesicles or bacteria) were detected. Electrophoretic analysis (SDS-PAGE) confirmed the presence of a protein with a prominent band at approximately 75 kDa (Figure 4B).

[0117] To assess whether GmSal contains the enzymes responsible for the detected PE modifications, a proteomic analysis of the GmSal contents was performed. More than 200 proteins were detected, including various enzymatic activities, transport and structural proteins, etc. To narrow the number of possible candidates, saliva samples were analyzed by size-exclusion chromatography (SEC). The elution profile showed a main single broad peak. An SDS-PAGE gel of the main fraction showed a strong band at approximately 75 kDa. The proteomic profile of this band revealed the presence of proteins known to be associated with transport or storage in arthropods.

[0118] To further identify whether the broad peak contained subspecies, ion exchange chromatography (IEX) was performed with a second saliva sample, which showed four distinct elution peaks. Analysis by SDS-PAGE showed that all contained proteins of similar molecular weight. To check which protein fractions retained PE degradation activity in both SEC and IEX, aliquots of the elution fractions were tested on PE film. Raman spectroscopy was used to analyze degradation activity from fractions of the four peaks in IEX (Figure 4C-G) and the main peak in SEC. Peaks marked 1, 2, and 3 showed a greater degree of PE oxidation than fraction 4, which apparently contained some residual activity (not shown). High degradation activity was also detected in the main peak in SEC.

[0119] Proteomics of IEX peaks 1, 2, and 3 revealed the presence of a few proteins belonging to the arthropod hexamerin / prophenoloxidase superfamily. This result, on the one hand, confirmed the results of the proteomics of the SEC fractions and, on the other hand, refined it by reducing the number of possible candidates present in each peak. This family contains oxidase activity and therefore represents a candidate for PE degradation capacity within GmSal. Using a recombinant expression system, these proteins, namely arylphorin subunit alpha, arylphorin subunit alpha-like, and hexamerin phosphate juvenile hormone inhibitory protein 1, were produced and tested for this capacity.

[0120] Identification of a waxworm enzyme as a PE oxidizer. To evaluate the activity of these proteins, 5 μL of each purified enzyme at concentrations of 1-5 μg / μL was applied separately on the PE film for eight consecutive times (90 min each). Whereas arylphorin subunit alpha had no effect on the PE film (not shown), arylphorin subunit alpha-like, renamed as Demetra (Accession number: XP_026756396.1 SEQ ID NO: 1), caused degradation of PE and occasionally showed noticeable visual effects on the film itself (Figure 5A-E). Raman confirmed the hallmarks of oxidized and damaged PE (Figure 5F-H). Hexamerin, renamed as Seres (Accession number: XP_026756459.1 SEQ ID NO: 2), caused oxidation / degradation of PE but no visual effects with Demetra. The same experiment was performed with inactivated enzyme and showed no modifications on the PE film as indicated by Raman analysis. These results support the original hypothesis based on the idea that oral secretions are the main source of plastic-degrading activity in ww.

[0121] To analyze the potential PE-oxidizing capacity of salivary proteins, GC-MS of PE granules (PE4000) exposed to Demetra (SEQ ID NO: 1) or Ceres (SEQ ID NO: 2) was performed. After 24 applications of Demetra (10 μL at 1.2 μg / μL, 90 min each), 2-ketones with carbon numbers from 10 to 22 were detected in the supernatant using GC-MS, fragment gram m / z 58, and retention time for identification (Figure 6A). With increasing treatments (10 vs. 5 applications, 90 min each), the relative abundance of 2-ketones with carbon numbers from 10 to 20 increased, with the appearance of 2-docosanone, which was not detected after 5 applications (Figure 6B). On the other hand, no by-products were detected in the supernatant after PE4000 treatment with Ceres, suggesting considerable differences between the two proteins, even though both share the ability to oxidize PE.

[0122] The present invention demonstrates that ww saliva can oxidize and depolymerize PE and that the ww enzymes contained therein are able to reproduce the effects observed in whole saliva. This is the first report of an enzymatic activity capable of attacking PE polymers without prior abiotic treatment. This ability is achieved by animal enzymes operating in aqueous solutions at neutral pH at room temperature. Under these conditions, the enzymatic action of ww saliva overcomes the recognized bottleneck step in PE degradation (i.e., oxidation) in a matter of hours.

[0123] The effect of the enzymes disclosed in this invention and present in the saliva of G. mellonella on PE is comparable to that of abiotic pretreatments.

[0124] The ability of these particular enzymes (SEQ ID NO:1 and SEQ ID NO:2) to rapidly and extensively oxidize PE, a macromolecular, compact, hydrophobic material, is unexpected. The presence of enzymes produced by insects, secreted from their mouths, and evolved to act on plastics at room temperature and neutral pH, offers a new paradigm for the biological degradation of PE. This new framework goes far beyond the current definition of biodegradation, which is based solely on the complete conversion of plastics to CO2 by microbial metabolic activity: on the one hand, the observed oxidation and degradation of PE does not depend on any microbial activity; on the other hand, the facile working conditions and the appearance of degradation products such as ketones and additives suggest the use of these enzymes for the degradation of plastic waste and the recycling or upcycling of plastic components. This possibility could be used as an alternative to metabolic conversion of plastics to CO2 or as a first oxidation step in combination with standard microbial degradation pathways.

[0125] Terms Clause 1. For biodegrading polyolefin-derived polymers or materials containing polyolefin-derived polymers, an enzyme comprising an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:1, or a host cell comprising a nucleotide sequence encoding said enzyme; or A composition comprising the enzyme or the host cell Use of.

[0126] Clause 2. The use of clause 1, wherein the amino acid sequence comprises or consists of the sequence of SEQ ID NO:1.

[0127] Clause 3. The use of clause 1 or 2, wherein the composition further comprises a second enzyme comprising an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:2.

[0128] Clause 4. The use according to any one of clauses 1 to 3, wherein the polyolefin derived polymer is polyethylene (PE).

[0129] Clause 5. The use according to clause 4, wherein the PE is selected from the list consisting of ultra high molecular weight polyethylene (UHMWPE), ultra low molecular weight polyethylene (ULMWPE or PE-WAX), high molecular weight polyethylene (HMWPE), high density polyethylene (HDPE), high density cross-linked polyethylene (HDXLPE), cross-linked polyethylene (PEX or XLPE), medium density polyethylene (MDPE), linear low density polyethylene (LLDPE), low density polyethylene (LDPE), very low density polyethylene (VLDPE), and chlorinated polyethylene (CPE).

[0130] Clause 6. The use according to any one of clauses 1 to 5, wherein the enzyme is isolated from Galleria mellonella, preferably from the saliva of G. mellonella.

[0131] Clause 7. A method for biodegrading a polyolefin-derived polymer, or a material comprising a polyolefin-derived polymer, comprising: an enzyme comprising an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:1, or a host cell comprising a nucleotide sequence encoding said enzyme; or A composition comprising the enzyme or the host cell with a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer.

[0132] Clause 8. The method of clause 7, carried out at room temperature, preferably in an aqueous solution at neutral pH, at room temperature.

[0133] Clause 9. The method of clause 7 or 8, wherein said amino acid sequence comprises or consists of the sequence of SEQ ID NO:1.

[0134] Clause 10. The method of any one of clauses 7 to 9, wherein said composition further comprises a second enzyme comprising an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:2.

[0135] Clause 11. The method of any one of clauses 7 to 10, wherein the polyolefin derived polymer is polyethylene (PE).

[0136] Clause 12. The method of clause 11, wherein the PE is selected from the list consisting of ultra high molecular weight polyethylene (UHMWPE), ultra low molecular weight polyethylene (ULMWPE or PE-WAX), high molecular weight polyethylene (HMWPE), high density polyethylene (HDPE), high density cross-linked polyethylene (HDXLPE), cross-linked polyethylene (PEX or XLPE), medium density polyethylene (MDPE), linear low density polyethylene (LLDPE), low density polyethylene (LDPE), very low density polyethylene (VLDPE), and chlorinated polyethylene (CPE).

[0137] Clause 13. The method of any one of clauses 7 to 12, wherein the enzyme is isolated from G. mellonella, preferably from the saliva of G. mellonella.

[0138] Clause 14. A method for obtaining by-products resulting from the biodegradation of polyolefin-derived polymers, comprising the steps of: (a) a polyolefin-derived polymer, - an enzyme comprising an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 1, or a host cell comprising a nucleotide sequence encoding said enzyme; or A composition comprising the enzyme or the host cell and (b) isolating by-products from the culture resulting from step (a). A method comprising:

[0139] Clause 15. The method of clause 14, wherein the by-products are selected from the list consisting of butane, 2,3-butanediol, trimethylsilyl (TMS) derivatives, sebacic acid, 2-ketones having 10 to 22 carbon atoms, and small aromatic compounds recognizable as benzenepropanoic acid.

Claims

1. 1. A method for biodegrading a polyolefin-derived polymer, or a material comprising a polyolefin-derived polymer, comprising: An enzyme comprising an amino acid sequence having at least 60% sequence identity with SEQ ID NO:1, or a host cell comprising a nucleotide sequence encoding said enzyme; or A composition comprising the enzyme or the host cell with a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer.

2. 2. The method of claim 1, which is carried out at room temperature, preferably in an aqueous solution at neutral pH at room temperature.

3. 3. The method of claim 1 or 2, wherein the enzyme comprises an amino acid sequence having at least 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:

1.

4. The method according to any one of claims 1 to 3, wherein the amino acid sequence comprises or consists of the sequence of SEQ ID NO:

1.

5. The method of any one of claims 1 to 4, wherein the composition further comprises a second enzyme comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO:

2.

6. 6. The method of claim 5, wherein the second enzyme comprises an amino acid sequence having at least 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:

2.

7. 7. The method of claim 5 or 6, wherein the second enzyme is contacted with the polyolefin-derived polymer or a material comprising a polyolefin-derived polymer in a separate, consecutive, or simultaneous step with the step of contacting with the enzyme of claim 1.

8. The method according to any one of claims 1 to 7, wherein the enzyme is isolated from G. mellonella, preferably from the saliva of G. mellonella.

9. An expression vector comprising a nucleotide sequence encoding an enzyme for biodegrading or oxidizing and / or depolymerizing a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer, said enzyme comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO:

1.

10. A host cell comprising the expression vector of claim 9.

11. 11. A method for expressing an enzyme in a host cell according to claim 10, comprising culturing the host cell according to claim 10 under conditions inducing expression of the expression vector to obtain the enzyme.

12. 1. An isolated enzyme for biodegrading, or oxidizing and / or depolymerizing, a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer, comprising an amino acid sequence having at least 60% sequence identity with SEQ ID NO:1, The enzyme is an isolated enzyme that is not the enzyme of database accession reference NCBI:XP026756396.1 or accession reference GSP:ABB77350.

13. 13. The isolated enzyme of claim 12, wherein the enzyme is obtained from G. mellonella, preferably from the saliva of G. mellonella.

14. 14. The isolated enzyme of claim 12 or 13, comprising an amino acid sequence having at least 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:

1.

15. A composition comprising the host cell of claim 10 or an isolated enzyme comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO:1 and at least one further component for biodegrading or oxidizing and / or depolymerizing a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer.

16. 16. The composition of claim 15, wherein the amino acid sequence has at least 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:

1.

17. 17. The composition of claim 15 or 16, comprising a second isolated enzyme comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO:

2.

18. 18. The composition of claim 17, wherein the second isolated enzyme comprises an amino acid sequence having at least 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:

2.

19. The composition of any one of claims 15 to 18, comprising one or more polyolefin-derived polymers or materials comprising polyolefin-derived polymers.

20. 20. The composition of any one of claims 15 to 19, comprising one or more of butane, 2,3-butanediol, trimethylsilyl (TMS) derivatives, sebacic acid, C10-C22 2-ketones, benzenepropanoic acid.

21. The composition of any one of claims 15 to 20, comprising an oxidized polyolefin-derived polymer.

22. 1. A kit for biodegrading a polyolefin-derived polymer, or a material comprising a polyolefin-derived polymer, comprising: Into a first container: An isolated enzyme according to any one of claims 12 to 14, or A host cell according to claim 10, or A composition according to any one of claims 15 to 21; In a further container: A second isolated enzyme comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO:2; or a second host cell comprising an expression vector encoding the second enzyme; or a second composition comprising the second isolated enzyme or the second host cell; and Instructions for using said isolated enzyme, host cell or composition with a second isolated enzyme, host cell or composition. Including the kit.

23. The method, vector, enzyme, cell, composition or kit according to any one of claims 1 to 22, wherein the polyolefin-derived polymer is polyethylene (PE) or polypropylene (PP).

24. 24. The method, vector, enzyme, cell, composition or kit of claim 23, wherein the polyolefin derived polymer is polyethylene (PE) selected from the list of ultra high molecular weight polyethylene (UHMWPE), ultra low molecular weight polyethylene (ULMWPE or PE-WAX), high molecular weight polyethylene (HMWPE), high density polyethylene (HDPE), high density cross-linked polyethylene (HDXLPE), cross-linked polyethylene (PEX or XLPE), medium density polyethylene (MDPE), linear low density polyethylene (LLDPE), low density polyethylene (LDPE), very low density polyethylene (VLDPE), and chlorinated polyethylene (CPE).

25. 1. A method for obtaining by-products resulting from the biodegradation of polyolefin-derived polymers, comprising: (a) a polyolefin-derived polymer, An enzyme comprising an amino acid sequence having at least 60% sequence identity with SEQ ID NO:1, or a host cell comprising a nucleotide sequence encoding said enzyme; or A composition comprising the enzyme or the host cell and (b) isolating by-products obtained from the culture resulting from step (a). A method comprising:

26. 28. The method of claim 27, wherein the by-products are selected from the group of butane, 2,3-butanediol, trimethylsilyl (TMS) derivatives, sebacic acid, C10-C22 2-ketones, benzenepropanoic acid.

27. 1. A method for pretreating a polyolefin-derived polymer, comprising: An enzyme comprising an amino acid sequence having at least 60% sequence identity with SEQ ID NO:1, or a host cell comprising a nucleotide sequence encoding said enzyme; or A composition comprising the enzyme or the host cell with a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer to oxidize the polymer.

28. For biodegrading polyolefin-derived polymers or materials containing polyolefin-derived polymers, An enzyme comprising an amino acid sequence having at least 60% sequence identity with SEQ ID NO:1, or a host cell comprising a nucleotide sequence encoding said enzyme; or A composition comprising the enzyme or the host cell Use of.

29. 29. The use according to claim 28, wherein the amino acid sequence comprises or consists of the sequence of SEQ ID NO:

1.

30. 30. The use of claim 28 or 29, wherein the composition further comprises a second enzyme comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO:

2.

31. The use according to any one of claims 28 to 30, wherein the polyolefin-derived polymer is polyethylene (PE) or polypropylene (PP).

32. 32. The use according to claim 31 , wherein the PE is selected from the list consisting of ultra high molecular weight polyethylene (UHMWPE), ultra low molecular weight polyethylene (ULMWPE or PE-WAX), high molecular weight polyethylene (HMWPE), high density polyethylene (HDPE), high density cross-linked polyethylene (HDXLPE), cross-linked polyethylene (PEX or XLPE), medium density polyethylene (MDPE), linear low density polyethylene (LLDPE), low density polyethylene (LDPE), very low density polyethylene (VLDPE), and chlorinated polyethylene (CPE).

33. Use according to any one of claims 28 to 32, wherein the enzyme is isolated from Galleria mellonella, preferably from the saliva of G. mellonella.

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