Enzymes for biodegrading polyolefin-derived polymers and methods of using same

Abstract

An enzyme obtained from the saliva of waxworms (Galleria mellonella larvae) is disclosed 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 relates to enzymes and methods of use for the biodegradation of polyolefin-derived polymers, in particular within plastics, preferably polyolefin-derived plastics, more preferably polyethylene (PE). [Background technology]

[0002] Polyethylene (PE) accounts for 30% of synthetic plastic production and remains a major contributor to global plastic waste pollution. Along with polypropylene (PP), polystyrene (PS), and polyvinyl chloride (PVC), PE is one of the most resistant polymers, with extremely long C–C chains organized into a crystalline, dense structure. Given the accumulation of hundreds of millions of tons of plastic waste and the still-accelerating pace of plastic production, reusing plastic residues is a necessary path to mitigate the severity of the plastic pollution problem and simultaneously unlock the enormous potential carbon reservoir. Currently, only mechanical recycling is applied on a large scale. Several factors, such as the limited variety of plastics amenable to mechanical recycling and the low quality of secondary products, significantly limit potential solutions to the problem of plastic waste accumulation. Chemical recycling, as an alternative procedure, prioritizes plastic upcycling, such as decomposing polyolefin-derived plastics to utilize smaller intermediates. Although several techniques have been applied at the 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 a promising strategy for disposing of plastic residues. Biodegradation refers to environmental degradation by biological agents. The IUPAC defines biodegradation as "enzyme-catalyzed breakdown of substances in vitro or in vivo," but this definition 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 that 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 chain of events—oxidation of PE polymers—is typically driven by abiotic factors such as light or temperature. Once the long polymer molecules are degraded, this process is exposed to environmental factors in the wild for years, after which bacteria and fungi step in to continue the work. This is the current paradigm driving the field of biodegradation research. Within this paradigm, several bacterial and fungal strains capable of 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 uptake 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, pp. 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, AN et al. (2021). Environ.Sci.Technol 55, 12383-12392). Over the past decade, several microorganisms capable of acting on untreated PE17-23 have been reported, but they require significantly longer incubation times compared to experimental conditions using pre-oxidized PE.

[0004] Identifying microbial enzymes capable of degrading unmodified PE has proven challenging. In fact, no such enzymes have yet been identified, confirming the critical limiting role of oxidation in the entire biodegradation chain. Enzymes reported to be able to act on polyolefin-derived plastics require pretreatment of the plastic material (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, two reported laccases capable of chemically modifying PE require abiotic pretreatment or the addition of a redox mediator such as 1-hydroxybenzotriazole.

[0005] This scenario confirms that the synthetic nature of the compounds, along with their hydrophobic and inaccessible characteristics, make plastics less likely to be targeted by enzymatic activity from animals, fungi, and microorganisms. 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.

[0006] Zhang et al. (Sci. Total Environ., 704, 135931, 2020) reported that the fungus Aspergillus flavus, isolated from the viscera of the wax moth Galleria mellonella, biodegrades polyethylene microparticles through the action of two laccase-like multicopper oxidases (LMCOs). Ren et al. (Int. J. Environ. Res. Public Health, 16, 1941, 2019) reported that Enterobacter sp., isolated from the viscera of the wax moth Galleria mellonella, can degrade polyethylene. Memmel et al. (Insect Biochem. Molec. Biol., 22(4), pp. 333-342, 1992) disclose the nucleic acid sequence of the arylphorin gene from Galleria mellonella. Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, a continuing challenge in this field is to identify microbial enzymes capable of biodegrading untreated PE, particularly those capable of oxidizing untreated PE polymers, while avoiding the use of non-biological factors such as light and temperature, long incubation times, and aggressive pretreatment of PE. [Means for solving the problem]

[0008] In summary, we isolated an enzyme from the saliva of waxworms (Galleria mellonella larvae) with the unexpected ability to oxidize and degrade untreated polyolefin-derived polymers, such as polyethylene (PE), at room temperature (RT), neutral pH, and short incubation times. We achieved this by performing proteomic analysis and size-exclusion chromatography (SEC) of waxworm saliva and obtained a single enzyme, identified as belonging to the hexamerin / prophenoloxidase family and renamed Cibeles (g181563), with accession number XP_026756460.1 (NCBI), SEQ ID NO: 2 (the enzyme of the present invention). This enzyme's ability to oxidize and degrade polyolefin-derived polymers was tested on PE film (see the examples and figures in this patent application), demonstrating high degradation activity. This paves the way for potential solutions to the plastic waste pollution problem.

[0009] This effect on PE degradation is achieved after only a few hours of exposure at room temperature and physiological conditions (neutral pH). Therefore, long incubation times or aggressive pretreatment are not 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 oxidized aliphatic chains were identified, further confirming that the polymer had broken down into shorter molecules.

[0010] "The enzyme's ability to produce such modifications to PE films in a very short time at room temperature makes it a promising alternative to the abiotic oxidation of plastics, 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 an entirely new paradigm in the world of plastic degradation and even in the field of plastic waste management, paving the way for the design of new formulations / routes for the production of synthetic polymers." [Brief explanation of the drawings]

[0011] [Figure 1] Raman spectroscopy of Cibeles (g181563)-treated PE film and negative control. Timed analysis of treated PE film shows degradation of PE. Oxidation is observed between 1600-1800 cm-1 (carbonyl groups) and 3000-3500 cm-1 (hydroxyl groups). Control PE film shows typical PE peaks at 1061, 1128, 1294, 1440, 2846, and 2880 cm-1. [Figure 2] This figure shows the identification of degradation by-products of PE treated with Cibeles (g181563) by GC-MS. A. Chromatogram of the fragment ion m / z 58 from the methyl ketone of PE treated with the enzyme. The arrows indicate the peaks corresponding to 2-ketones with different carbon numbers. B. Changes in PE degradation by-products by GC-MS after various applications. The production of ketones as degradation products increased as the application of Cibeles to PE increased from four to eight times, and the production of 2-ketones increased by doubling the application of the enzyme to PE. [Figure 3-1] Sequence alignment and overall structure of four proteins present in G. mellonella saliva. (A) Amino acid sequence alignment color-coded by similarity. Key metal-coordinating residues are highlighted with boxes, glycosylation residues are marked with triangles, and disulfide bonds are indicated with asterisks. (B) Pairwise sequence identity percentages among the four factors. (C) Overall primary, tertiary, and quaternary structures of hemocyanin / phenoloxidase (Hc / PO) family members (Panulirus interruptus Hc, PDB code 1HCY, used as an example). The canonical copper-binding site is highlighted with a red sphere. [Figure 3-2]Sequence alignment and overall structure of four proteins present in G. mellonella saliva. (A) Amino acid sequence alignment color-coded by similarity. Key metal-coordinating residues are highlighted with boxes, glycosylation residues are marked with triangles, and disulfide bonds are indicated with asterisks. (B) Pairwise sequence identity percentages among the four factors. (C) Overall primary, tertiary, and quaternary structures of hemocyanin / phenoloxidase (Hc / PO) family members (Panulirus interruptus Hc, PDB code 1HCY, used as an example). The canonical copper-binding site is highlighted with a red sphere. DETAILED DESCRIPTION OF THE INVENTION

[0012] 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:2 for biodegrading, or oxidizing and / or depolymerizing, a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer.

[0013] In a further 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: 2 (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").

[0014] For example, preferably the enzyme is active in biodegrading, or oxidizing and / or depolymerizing, polyolefin-derived polymers or materials that include polyolefin-derived polymers.

[0015] 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:2, and compositions comprising said isolated enzyme.

[0016] In some embodiments, the enzyme of the invention is not the enzyme with database accession reference XP_026756460.1. In some embodiments, the enzyme of the present invention is an enzyme comprising a sequence that has 1% variation from the amino acid sequence of SEQ ID NO: 2. In some embodiments, the 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 enzyme comprises a sequence that has a 1 amino acid variation from the amino acid sequence of SEQ ID NO: 2. In some embodiments, the enzyme comprises a sequence that has a 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: 2.

[0017] Preferably, the enzyme has activity for biodegrading, or oxidizing and / or depolymerizing, polyolefin-derived polymers or materials comprising polyolefin-derived polymers. 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.

[0018] In some embodiments, the compositions of the present invention comprise one or more polyolefin-derived polymers or materials that comprise polyolefin-derived polymers. In some embodiments, the compositions of the present invention comprise one or more oxidized polyolefin-derived polymers.

[0019] 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.

[0020] 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. In some embodiments, the composition comprises a buffer, such as a HEPES buffer. More preferably, the composition comprises 150 mM NaCl, 20 mM Hepes, and 5% glycerol.

[0021] In view of the above, the present invention relates in one aspect to the use of 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: 2 or a functionally equivalent fragment thereof, or a nucleotide sequence encoding said enzyme, or a host cell comprising 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, for biodegrading or oxidizing and / or depolymerizing polyolefin-derived polymers or materials comprising polyolefin-derived polymers.

[0022] The enzyme of the present invention is preferably isolated from the larvae of Galleria mellonella, also known as waxworm (WW), which is an arylphorin subunit alpha, renamed Cibeles (g181563), and comprises an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 2 (accession reference number NCBI: XP_026756460.1).

[0023] SEQ ID NO: 2 (Civeles, g181563, enzyme of the invention)

[0024] [ka]

[0025] Alternatively, the enzymes of the present invention can be produced recombinantly according to techniques well known in the art. For example, using recognized techniques for 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, in which the enzyme is constructed by attachment to a resin such as Merrifield resin. In these synthetic procedures, the groups on the amino acids are generally protected using standard protecting groups such as t-butoxycarbonyl. If necessary, these protecting groups are cleaved once synthesis is complete. Chemical synthesis methods, such as solid-phase peptide synthesis, solution synthesis, and combinations of solid-phase synthesis with solution or enzymatic synthesis methods, are known to those skilled in the art. The enzymes of the present invention may also be produced by recombinant DNA procedures known in the art. Modifications may be introduced during or after the synthesis of the enzymes to include, for example, labels, purification tags, or other added tags for purification purposes.

[0026] In another aspect, the present invention provides an expression vector comprising a nucleic acid sequence encoding an enzyme of the present invention. 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, by standard sequence alignment algorithms known in the art, such as BLAST. 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.

[0027] Those skilled in the art understand that mutations in the nucleotide sequence of a gene that result 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 exhibits the same activity. These proteins are considered "functionally equivalent variants" of the sequence of SEQ ID NO: 2 and are included within the scope of the present invention. Therefore, as used herein, the term "functionally equivalent variant" refers to an enzyme derived from the native enzyme (SEQ ID NO: 2 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 exhibits 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 share at least 60% sequence identity with the amino acid sequence of SEQ ID NO: 2 and are capable of oxidizing untreated polyolefin-derived polymers are considered functionally equivalent variants in the context of the present invention. Examples of assays for checking whether a given protein is a functionally equivalent variant of the enzyme of SEQ ID NO: 2 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: 2.

[0028] 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: 2), 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: 2 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: 2.

[0029] In a preferred embodiment, the amino acid sequence of the enzyme of the invention comprises or consists of the sequence of SEQ ID NO: 2. As shown in Figure 3, the putative signal sequence of the enzyme of SEQ ID NO: 2 consists of amino acids 1 to 16, and the mature form of the enzyme consists of amino acids 17 to 702 (inclusive).

[0030] The nucleotide sequences of the present invention encoding the enzymes of the present invention may further comprise elements other 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 enabling more efficient enzyme purification.

[0031] The nucleotide sequence of the present 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 present invention.

[0032] As used herein, the term "genetic construct" or "vector" refers to a monocatenary or bicatenary nucleic acid molecule that has been isolated and modified to contain a nucleic acid segment in a manner not found 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 expression of a nucleotide sequence of the present invention. Thus, a 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, etc.

[0033] 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 that provide for its expression. The vector containing the nucleic acid sequence of the present invention can be introduced into a host cell so that the vector is maintained as a chromosomal integrant or as an autonomously replicating extrachromosomal vector.

[0034] The expression vector referred to in the present invention may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and is capable of producing expression of the nucleotide sequence of the present invention contained therein. The choice of vector will usually depend on the compatibility of the vector with the host cell into which it will be introduced. Expression vectors may be, for example, but are not limited to, plasmids, cosmids, phages, viruses or viral vectors, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), etc. Vectors within the context of the present invention may be linear or circular. Preferably, the expression vector of the present invention is a baculovirus expression vector, more preferably a P2 baculovirus vector.

[0035] As used herein, a "host cell" includes any cell type susceptible to transformation, transfection, transduction, etc. with the nucleotide sequences or expression vectors of the invention referenced above. The host cell may be prokaryotic or eukaryotic, preferably a 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.

[0036] Thus, a host cell of the invention comprises at least 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 a mature enzyme of the invention, or to a mature enzyme that must subsequently be processed to produce the mature enzyme.

[0037] Expression of the enzymes of the invention in the host cells of the invention may be induced by any procedure known in the art, such as transforming a suitable host cell with at least one nucleotide sequence of the invention or a vector of the invention to obtain a secreted functional enzyme, and culturing the transformed host cell under conditions that induce expression of the nucleotide sequence. Preferred conditions for inducing expression of the nucleotide sequence are incubation of the host cells at 27°C for 48 to 72 hours.

[0038] In some aspects, the invention relates to a method for expressing in a host cell 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:2, the method comprising culturing the host cell under conditions that induce expression of the expression vector to obtain the enzyme.

[0039] 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), electrophoresis (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction to obtain a substantially pure enzyme.

[0040] In another preferred embodiment, the enzyme of the present invention is recombinantly produced or isolated from the saliva of Galleria mellonella, particularly G. mellonella.

[0041] 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 and ion exchange chromatography) from waxworm saliva.

[0042] The compositions of the present invention comprise the enzymes of the present invention or functionally equivalent fragments thereof, or the host cells of the present 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.

[0043] Preferably, the compositions of the present invention further comprise one or more additional enzymes, such as one, two, or three additional enzymes, active for biodegrading, or oxidizing and / or depolymerizing, a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer.

[0044] Preferably, the compositions of the invention further comprise 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: 1, and more preferably, this additional protein comprises or consists of SEQ ID NO: 1. Preferably, the compositions of the invention further comprise 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 (amino acids 1 to 752 as shown in Figure 3).

[0045] SEQ ID NO: 1 (Cora, JHS)

[0046] [ka]

[0047] The compositions of the invention may further comprise an additional 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: 3, and more preferably, this additional protein comprises or consists of SEQ ID NO: 3. Preferably, the compositions of the invention further comprise 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: 3. As shown in Figure 3, the putative signal sequence for the enzyme of SEQ ID NO: 3 consists of amino acids 1 to 16, and the mature form of the enzyme consists of amino acids 17 to 700, inclusive. Without wishing to be bound by any particular theory, the inventors believe that the activity of the Cibeles enzyme is enhanced by forming a heterohexamer with the enzyme of SEQ ID NO: 3 (Demetra). The heterohexamer may be a 3:3 heterohexamer formed from a trimer of Cibeles-Demetra dimers.

[0048] SEQ ID NO: 3 (Demetra)

[0049] [ka]

[0050] The compositions of the invention may further comprise an additional 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: 4, and more preferably, this additional protein comprises or consists of SEQ ID NO: 4. Preferably, the compositions of the invention further comprise 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: 4. As shown in Figure 3, the putative signal sequence for the enzyme of SEQ ID NO: 4 consists of amino acids 1 to 18, and the mature form of the enzyme consists of amino acids 19 to 706, inclusive.

[0051] SEQ ID NO: 4 (Ceres)

[0052] [ka]

[0053] The compositions of the present invention can include one, two, or three additional enzymes or isolated enzymes that have the activity to biodegrade, or oxidize and / or depolymerize, polyolefin-derived polymers or materials that include polyolefin-derived polymers.

[0054] For example, the composition can include an additional 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.

[0055] In another example, the composition can include an additional 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 an additional 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:3.

[0056] For example, the composition can include an additional 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 an additional 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:4.

[0057] In another example, the composition can include an additional 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; an additional 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:3; and an additional 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:4.

[0058] In another example, the composition can include an additional 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:3.

[0059] In another example, the composition can include an additional 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:3, and an additional 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:4.

[0060] In another example, the composition can include an additional 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:4.

[0061] In some embodiments, the compositions of the invention comprise an enzyme of the invention or a functionally equivalent fragment thereof, or a host cell of the invention, and preferably at least one additional component, which in some embodiments is at least one of the additional components disclosed above.

[0062] In some embodiments, the additional enzyme in the composition is not the enzyme with database accession reference number XP026749149.1. In some embodiments, the additional 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 additional 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 additional enzyme comprises a sequence that differs by one amino acid from the amino acid sequence of SEQ ID NO: 2. In some embodiments, the additional 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.

[0063] In some embodiments, the additional enzyme in the composition is not the enzyme with database accession reference number XP_026756396. In some embodiments, the additional enzyme in the composition is an enzyme that comprises a sequence that has 1% variation from the amino acid sequence of SEQ ID NO: 3. In some embodiments, the additional 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: 3. In some embodiments, the additional enzyme comprises a sequence that varies by 1 amino acid from the amino acid sequence of SEQ ID NO: 3. In some embodiments, the additional 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: 3.

[0064] In some embodiments, the additional enzyme in the composition is not the enzyme with database accession reference XP093062524. In some embodiments, the additional enzyme in the composition is not the enzyme with database accession reference XP_026756459.1.

[0065] In some embodiments, the additional enzyme in the composition is an enzyme comprising a sequence having 1% variation from the amino acid sequence of SEQ ID NO: 4. In some embodiments, the additional enzyme comprises a sequence having 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40% variation from the sequence of SEQ ID NO: 4. In some embodiments, the additional enzyme comprises a sequence that varies by 1 amino acid from the amino acid sequence of SEQ ID NO: 4. In some embodiments, the additional 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: 4.

[0066] 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, 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 together with instructions for use of the enzyme, the host cell, or the composition: an additional 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 said additional enzyme, or a vector comprising a nucleotide sequence encoding said additional enzyme; or a composition comprising said additional enzyme or a functionally equivalent fragment thereof, or said host cell.

[0067] In some embodiments, the kit further comprises: In additional containers: an additional 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 said additional enzyme, or a vector comprising a nucleotide sequence encoding said additional enzyme, or A composition comprising said additional enzyme or a functionally equivalent fragment thereof, or said host cell.

[0068] In some embodiments, the kit further comprises: In additional containers: an additional 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: 3 or a functionally equivalent fragment thereof; a host cell comprising a nucleotide sequence encoding said additional enzyme, or a vector comprising a nucleotide sequence encoding said additional enzyme, or A composition comprising said additional enzyme or a functionally equivalent fragment thereof, or said host cell.

[0069] In some embodiments, the kit further comprises: In additional containers: an additional 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: 4 or a functionally equivalent fragment thereof; a host cell comprising a nucleotide sequence encoding said additional enzyme, or a vector comprising a nucleotide sequence encoding said additional enzyme, or A composition comprising said additional enzyme or a functionally equivalent fragment thereof, or said host cell.

[0070] In some embodiments, the kit comprises a first container as described above, and one, two, or three additional containers containing: i) an additional isolated enzyme comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO:2, or an additional host cell comprising an expression vector encoding said additional isolated enzyme, or an additional composition comprising said additional isolated enzyme or said additional host cell; ii) an additional isolated enzyme comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO:3, or an additional host cell comprising an expression vector encoding said additional enzyme, or an additional composition comprising said additional isolated enzyme or said additional host cell; and / or iii) an additional isolated enzyme comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO:4, or an additional host cell comprising an expression vector encoding said additional enzyme, or an additional composition comprising said additional isolated enzyme or said additional host cell. The kit can further comprise instructions for use of the additional isolated enzyme, host cell, or composition along with the additional isolated enzyme or enzymes, host cell, or composition or compositions.

[0071] In some embodiments of the present invention, the one or more additional enzymes are isolated enzymes. In some embodiments, the enzyme, host cell, or composition of SEQ ID NO: 1 is for use in a separate, sequential, or simultaneous step with the enzyme, host cell, or composition of SEQ ID NO: 2; and / or the enzyme, host cell, or composition of SEQ ID NO: 3; and / or the enzyme, host cell, or composition of SEQ ID NO: 4 to biodegrade, or oxidize and / or depolymerize, a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer.

[0072] In the most general aspect, "polyolefin-derived polymer" refers to any polyolefin polymer derived from olefin monomers. In one aspect of the present invention, the "polyolefin-derived polymer" is a polymer having the general formula (CH2CHR) nPolyolefins can be a type of polymer with the formula (where R is an alkyl group). In some cases, R can be a hydrogen atom. They are usually derived from a small set of simple olefins (alkenes). The most common commercially 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 made; for example, polyethylene is derived from ethylene and polymethylpentene from 4-methyl-1-pentene.

[0073] In another preferred embodiment, the polyolefin-derived polymer referred to in the present invention is polyethylene (PE). "Polyethylene (PE)" or polythene (abbreviated PE; IUPAC name polyethylene or poly(methylene)) is the most common plastic in use today. It is a type of polymer primarily used for packaging (plastic bags, plastic films, geomembranes, and containers, including bottles). Many types of polyethylene are known, most of which have the chemical formula (C2H4) n Polyethylene is usually a blend of similar polymers of ethylene, with varying values ​​of n. It can be of low or high density: low density polyethylene is extruded at high pressure (1000-5000 atmospheres) and high temperature (520 Kelvin), while high density polyethylene is extruded at low pressure (6-7 atmospheres) and low temperature (333-343K). Polyethylene is usually thermoplastic, but can alternatively be modified to be thermoset, e.g., cross-linked polyethylene. All types of PE are within the scope of the present invention.

[0074] 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 certain embodiments, the PE is LDPE, more preferably PE 4000 or PE 2000.

[0075] In another specific embodiment, the polyolefin-derived polymer or material comprising the polyolefin-derived polymer is not pretreated with a non-biological 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 polymers or untreated materials comprising polyolefin-derived polymers.

[0076] 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 "the method of the present invention"), said method comprising: an enzyme of the present invention, or a host cell of the invention, or The composition of the present invention contacting with a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer The present invention relates to a method, including:

[0077] In a preferred embodiment of the method of the present invention, said method is carried out at room temperature, preferably in an aqueous solution at neutral pH, at room temperature. "Room temperature" is 15°C to 30°C, preferably 22°C.

[0078] "Neutral pH" is pH 7 to pH 8. In a preferred embodiment, the enzyme is used in the method of the present invention in a volume of 2 to 10 μL, preferably 5 μL, and at a concentration between 1 and 5 μg / μL.

[0079] 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.

[0080] In a further preferred embodiment of the method of the present invention, the enzyme is used in a volume of 5 μL or 10 μL at a concentration of 1 to 5 μg / μL, preferably 1.2 μg / μL, and is applied to the polyolefin-derived polymer or material comprising the polyolefin-derived polymer at least 8 times, preferably 24 times, for 90 minutes each time.

[0081] In another preferred embodiment of the method of the invention, the enzyme of the invention comprises or consists of SEQ ID NO: 2. More preferably, the enzyme of the invention is isolated from G. mellonella, in particular from the saliva of G. mellonella.

[0082] In another preferred embodiment of the method of the invention, 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 to SEQ ID NO:1, more preferably comprising SEQ ID NO:1, and even more preferably consisting of SEQ ID NO:1.

[0083] In some embodiments, the additional 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, SEQ ID NO:3 or SEQ ID NO:4 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.

[0084] In another preferred embodiment of the method of the present invention, the polyolefin-derived polymer is polyethylene (PE) or polypropylene (PP). 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 specific embodiment, the PE is LDPE, more preferably PE4000 or PE2000.

[0085] 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: an 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.

[0086] In some aspects, the methods include contacting an enzyme of the invention and one, two, or three additional enzymes described herein with a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer to provide an oxidized polymer or material product.

[0087] In some embodiments, polyolefin-derived polymers or materials comprising polyolefin-derived polymers are pretreated using the methods of the present invention before further degradation steps. For example, further degradation steps are performed on oxidized polymers or material products. In some embodiments, polyolefin-derived polymers or materials comprising polyolefin-derived polymers are pretreated using the methods of the present invention before applying one or more microbial degradation steps.

[0088] Another aspect of the present invention is a method for obtaining by-products derived from the biodegradation of polyolefin-derived polymers, comprising: (a) a polyolefin-derived polymer an enzyme of the present invention, or a host cell of the invention, or contacting with a composition of the present invention; and (b) isolating the by-products from the culture obtained from step (a). The present invention relates to a method comprising:

[0089] In some aspects, step (a) of the method comprises contacting the polyolefin-derived polymer with an enzyme of the invention and one, two, or three additional enzymes as described herein.

[0090] Another aspect of the present invention is: (a) a polyolefin-derived polymer an enzyme of the present invention, or a host cell of the invention, or contacting with a composition of the present invention; and (b) isolating by-products from the culture obtained from step (a); and (c) polymerizing the by-product isolated in step (b). The present invention refers to a method for preparing a plastic, comprising:

[0091] In some aspects, step (a) of the method comprises contacting the polyolefin-derived polymer with an enzyme of the invention and one, two, or three additional enzymes as described herein.

[0092] "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.

[0093] The process conditions for the resulting 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.

[0094] Isolation of the by-products in step (b) of this process may be carried out by techniques well known in the art, such as gas chromatography mass spectrometry (GC-MS). Aspects of the present invention are now described by way of example and not by way of limitation. 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.

[0095] 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 individually set forth herein.

[0096] Unless the context dictates otherwise, the above feature descriptions and definitions are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described.

[0097] While the present invention has been described in conjunction with the above embodiments, many equivalent modifications and variations will be apparent to those skilled in the art given this disclosure. Accordingly, the described embodiments of the invention are considered to be illustrative and not limiting. Various changes to the described embodiments can be made without departing from the spirit and scope of the invention. All documents cited herein are expressly incorporated by reference in their entirety for all purposes. [Example]

[0098] PE decomposition experiment. I - Materials and Methods Generation of recombinant Cibeles (SEQ ID NO:2) Recombinant siveles enzyme (SEQ ID NO: 2) can be produced using known expression protocols.

[0099] Raman analysis The recombinant protein of SEQ ID NO: 2 was applied as follows: 5 μl of protein (concentration 1-5 μg / ml) was applied onto the PE film 8 times for 90 minutes each time.

[0100] Raman analysis was performed on PE films (treated and control) using an Alpha300R - Alpha300A AFM Witec instrument with a power of 5 mW, a 50x objective (NA 0.8), an integration time of 1, a total count of 30, and a wavelength of 532 nm. The results are shown in Figure 1.

[0101] Gas chromatograph mass spectrometer (GC-MS) The plants were exposed to PE with 10 μl (1.2 μg / mL) Cibeles (g181563) for 90 min 24 times. Long-term treatments were performed with 10 μl (1.2 μg / mL) four times per day for 90 min each (days 1 and 2). The same experiment was repeated using protein buffer as a control. The samples were then centrifuged at 19083 g for 30 s in an Eppendorf centrifuge 5810 R, 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 with some modifications. Briefly, 50 μl of 1 mg / ml diphenylphthalic acid (internal standard, IS) was added to each sample and 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 at room temperature for 15 min, and then centrifuged at 19083 g for 10 min in an Eppendorf centrifuge 5810R at 20 °C. Finally, the supernatant DCM was collected and placed in an insert before analysis. To determine less volatile polar compounds that exhibit 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.

[0102] 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). Crystalline granular powdered polyethylene (PE4000; CAS number: 9002-88-4) was supplied by Sigma-Aldrich (Saint Louis, USA).

[0103] Chromatographic analysis was performed using an Agilent Technologies gas chromatography-mass spectrometry system (GC-MS) 7980A-5975C. Metabolite separation was performed on a polyimide-coated DB-5 column (30 m long, 0.25 mm internal diameter, 0.1 μm film thickness; Agilent Technologies, USA) using helium (He) as the carrier gas for proper separation of the substances. Analysis was performed using a split injector at 350 °C with an injection volume of 1 μl. The ion source temperature was 230 °C, and mass spectrometry was performed in scan mode with a quadrupole temperature of 150 °C and a fragmentation voltage of 70 eV. The oven program started at 60 °C for 3 min, then ramped to 350 °C at 20 °C / min in 1 min. The total run time was 18.5 min, with 19.5 min for the derivatized sample. 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.

[0104] The evaluation of long-term treatment was based on the relative abundance of each non-target compound, which was calculated by dividing the area under each compound's peak by the area under the IS peak. The results are shown in Figure 2. Protein Buffer The purified protein was resuspended in 150 mM NaCl, 20 mM HEPES, 5% glycerol and used in the degradation assay. The same buffer alone served as a negative control.

[0105] II - Results Cibeles oxidizes PE film The ability of purified recombinant Cibeles (SEQ ID NO: 2) to oxidize PE was examined. After eight consecutive 90-min applications of 5 μl of protein, confocal Raman microscopy / Raman spectroscopy (Raman) analysis demonstrated that the polymer was highly oxidized, accompanied by overall film degradation (Figures 1A and 1B). This was also evident from the overlay with a PE control, revealing the expected PE signature profile (Figures 1C and 1D). As a negative control, protein buffer alone was applied to the PE film, resulting in no oxidation (Figure 1C). The changes induced by Cibeles after a few hours of application resemble those caused by environmental factors after months or years of exposure to weathering. The changes in the chemical composition of PE revealed by spectroscopic techniques suggest that molecules other than long PE polymer chains were formed upon contact with Cibeles.

[0106] Identification of Cibeles as a PE oxidizer To analyze the potential PE-oxidizing ability of Cibeles, we performed GC-MS on PE granules (PE4000) exposed to Cibeles (SEQ ID NO: 2). After 24 applications of Cibeles (10 μL at 1.2 μg / μL, 90 min each), 2-ketones with carbon numbers 10 to 22 were detected in the supernatant using GC-MS, fragmentogram m / z 58m, and retention time for identification (Figure 2A).

[0107] Increasing the treatment (four applications vs. eight applications, 90 min each) showed an increase in the relative abundance of 2-ketones with 12–18 carbons, with the appearance of 2-decanone and icosan-2-one, which were not detected after four applications ( Fig. 2B ).

[0108] III - Summary The present invention demonstrates that Cibeles (SEQ ID NO: 2) oxidizes and depolymerizes PE. This is the first report of this enzyme attacking PE polymers without prior non-biological treatment. This is achieved by the enzyme operating at room temperature and in aqueous solution at neutral pH. Under these conditions, the enzymatic action of Cibeles overcomes the step recognized as the bottleneck in PE degradation (i.e., oxidation) within a few hours.

[0109] The effect of the Cibeles enzyme disclosed in this invention and present in the saliva of G. mellonella on PE is comparable to that of non-biological treatments. The ability of the enzyme SEQ ID NO:2 to rapidly and extensively oxidize PE, a polymeric, compact, hydrophobic material, was unexpected. The existence of an enzyme produced by insects, secreted from their mouths, and evolved to act on plastics at room temperature and neutral pH provides 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 through 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 operating 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. Based on these results, we believe that the Cibeles enzyme will also be active against other types of polyolefins. This potential could be used as an alternative to metabolic conversion of plastics to CO2 or as an initial oxidation step in combination with standard microbial degradation pathways.

[0110] Terms 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:2; or a host cell comprising a nucleotide sequence encoding said enzyme; or A composition comprising the enzyme or the host cell Use of.

[0111] 2. The use according to claim 1, wherein the amino acid sequence comprises or consists of the sequence of SEQ ID NO:2. 3. The use according to claim 1 or 2, wherein the polyolefin-derived polymer is polyethylene (PE).

[0112] 4. The use of claim 3, 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).

[0113] 5. Use according to any one of claims 1 to 4, wherein the enzyme is isolated from Galleria mellonella, preferably from the saliva of G. mellonella. 6. 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:2; 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.

[0114] 7. The method of claim 6, which is carried out at room temperature, preferably in an aqueous solution at neutral pH and at room temperature. 8. The method of claim 6 or 7, wherein the amino acid sequence comprises or consists of the sequence of SEQ ID NO:2.

[0115] 9. The method according to any one of claims 6 to 8, wherein the polyolefin-derived polymer is polyethylene (PE). 10. The method of claim 9, 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).

[0116] 11. The method according to any one of claims 6 to 10, wherein the enzyme is isolated from G. mellonella, preferably from the saliva of G. mellonella.

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: 2, 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. 10. 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:

2.

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

2.

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

1.

6. 6. The method of claim 5, wherein the additional 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.

7. 10. The method of any one of the preceding claims, wherein the composition further comprises an additional enzyme comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO:

3.

8. 8. The method of claim 7, wherein the additional 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:

3.

9. 10. The method of any one of the preceding claims, wherein the composition further comprises an additional enzyme comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO:

4.

10. 10. The method of claim 9, wherein the additional 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:

4.

11. 11. The method of any one of claims 5 to 10, wherein the additional enzyme or enzymes are contacted with the polyolefin-derived polymer or material comprising a polyolefin-derived polymer in a separate, consecutive or simultaneous step with the enzyme of any one of claims 1 to 4.

12. 12. The method according to any one of claims 1 to 11, wherein the enzyme is isolated from G. mellonella, preferably from the saliva of G. mellonella.

13. 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, wherein the enzyme comprises an amino acid sequence having at least 60% sequence identity with SEQ ID NO:

2.

14. A host cell comprising the expression vector of claim 13.

15. 15. A method for expressing an enzyme in a host cell according to claim 14, comprising culturing the host cell according to claim 14 under conditions that induce expression of the expression vector to obtain the enzyme.

16. 1. An isolated enzyme for biodegrading, or oxidizing and / or depolymerizing, a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer, the enzyme comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO:2; wherein the enzyme of the invention is not the enzyme with database accession reference number NCBI:XP026756460.

1.

17. 17. The isolated enzyme of claim 16, wherein the enzyme is obtained from G. mellonella, preferably from the saliva of G. mellonella.

18. 18. The isolated enzyme of claim 16 or 17, 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:

2.

19. 15. A composition comprising the host cell of claim 14 or an isolated enzyme comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 2 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.

20. 20. The composition of claim 19, 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:

2.

21. 21. The composition of claim 19 or 20, comprising an additional isolated enzyme comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO:

1.

22. 22. The composition of claim 21 , wherein the additional 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:

1.

23. 23. The composition of any one of claims 19 to 22, comprising an additional isolated enzyme comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO:

3.

24. 24. The composition of claim 23, wherein the additional 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:

3.

25. 25. The composition of any one of claims 19 to 24, comprising an additional isolated enzyme comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO:

4.

26. 26. The composition of claim 25, wherein the additional 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:

4.

27. The composition of any one of claims 19 to 26, comprising one or more polyolefin-derived polymers or materials comprising polyolefin-derived polymers.

28. 28. The composition of any one of claims 19 to 27, comprising one or more of butane, 2,3-butanediol, trimethylsilyl (TMS) derivatives, sebacic acid, C10 to C22 2-ketones, benzenepropanoic acid.

29. The composition of any one of claims 19 to 28, comprising an oxidized polyolefin-derived polymer.

30. 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 13 to 15, or The host cell of claim 11 or A composition according to any one of claims 16 to 23; and In a further container: an additional isolated enzyme comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 1; or an additional host cell comprising an expression vector encoding said additional isolated enzyme; or an additional composition comprising said additional isolated enzyme or said additional host cell; and / or In a further container: an additional isolated enzyme comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO:3; or an additional host cell comprising an expression vector encoding said additional enzyme; or an additional composition comprising said additional isolated enzyme or said additional host cell; and / or In a further container: an additional isolated enzyme comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO:4; or an additional host cell comprising an expression vector encoding said additional enzyme; or an additional composition comprising the additional isolated enzyme or the additional host cell; and A kit comprising instructions for using the isolated enzyme, host cell or composition with an additional isolated enzyme or enzymes, host cell or host cells or composition or compositions.

31. 10. The method, vector, enzyme, cell, composition or kit of any one of the preceding claims, wherein the polyolefin-derived polymer is polyethylene (PE) or polypropylene (PP).

32. 32. The method, vector, enzyme, cell, composition or kit of 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. 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: 2, or a host cell comprising a nucleotide sequence encoding said enzyme; or A composition comprising the enzyme or the host cell contacting the (b) isolating the by-products obtained from the culture resulting from step (a). A method comprising:

34. 34. The method of claim 33, wherein the by-products are selected from the group consisting of butane, 2,3-butanediol, trimethylsilyl (TMS) derivatives, sebacic acid, C10-C22 2-ketones, and benzenepropanoic acid.

35. 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: 2, 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.

36. 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: 2, or a host cell comprising a nucleotide sequence encoding said enzyme; or A composition comprising the enzyme or the host cell Use of.

37. 37. The use according to claim 36, wherein the amino acid sequence comprises or consists of SEQ ID NO:

2.

38. 38. The use of claim 36 or 37, wherein the composition further comprises an additional enzyme comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO:

1.

39. The use according to any one of claims 36 to 38, wherein the composition further comprises an additional enzyme comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO:

3.

40. 40. The use according to any one of claims 36 to 39, wherein the composition further comprises an additional enzyme comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO:

4.

41. The use according to any one of claims 36 to 40, wherein the polyolefin-derived polymer is polyethylene (PE) or polypropylene (PP).

42. 42. The use of claim 41, 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).

43. Use according to any one of claims 36 to 42, wherein the enzyme is isolated from Galleria mellonella, preferably from the saliva of G. mellonella.

Images