Enzymes for biodegradation of polyolefin-derived polymers and methods of use

Abstract

The present invention relates to enzymes for biodegradation of polyolefin-derived polymers and methods of use. The present invention relates to enzymes from the saliva of Cyclina melonella (Gallium melonella larva) having an unexpected ability to oxidize and depolymerize untreated polyolefin-derived polymers (e.g., polyethylene (PE)) at room temperature (RT), neutral pH, and short incubation time. The invention also relates to a method of biodegrading a polyolefin-derived polymer wherein the enzyme is used.

Description

Technical Field

[0001] The present invention relates to enzymes for biodegradation of polyolefin-derived polymers and methods of using the same, in particular enzymatic biodegradation of plastics, preferably enzymatic biodegradation of polyolefin-derived plastics, more preferably enzymatic biodegradation of polyethylene (PE). Background Art

[0002] Polyethylene (PE) accounts for 30% of synthetic plastic production and has significantly contributed to the plastic waste pollution on Earth to date. Together with polypropylene (PP), polystyrene (PS), and polyvinyl chloride (PVC), PE is one of the most resistant polymers, having very long C-C chains organized in a crystalline, dense structure. Given the accumulation of hundreds of millions of tons of plastic waste and the still accelerating plastic production rate, the reuse of plastic residues is a necessary way to mitigate the severity of the plastic pollution problem and at the same time provides a huge potential carbon reservoir that can be accessed. To date, only mechanical recycling is being applied on a large scale. Several factors, such as the small number of plastic types that are easily mechanically recycled and the low quality of secondary products, severely limit the potential of this solution to the plastic waste accumulation problem. As an alternative operation, chemical recycling is primarily aimed at plastic upcycling (e.g., decomposing polyolefin-derived plastics) to utilize smaller intermediates. Several techniques have been applied on a laboratory scale, but high energy costs may still hinder the scale-up of these technical tools.

[0003] In addition to mechanical and chemical recycling, biodegradation is widely regarded as a promising strategy for dealing with plastic waste. Biodegradation refers to the environmental degradation by biological agents. The IUPAC defines biodegradation as "the enzymatic decomposition of a substance, either in vitro or in vivo", which was later modified to "excluding 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 and subsequent cleavage of the long hydrocarbon chains to produce smaller molecules, which can then be metabolized by microorganisms (Albertsson, A.C., Andersson, S.O. and Karlsson, S. (1987). Polymer Degradation and Stability 18, 73-87; Roy, P.K., Hakkarainen, M., Varma, I.K., and Albertsson, A.C. (2011). Environ Sci Technol 45, 4217-4227). The key first step in this chain of events, namely the oxidation of the PE polymer, is usually carried out by abiotic factors (such as light or temperature). Once the long polymer molecules are broken down (a process that requires exposure to environmental factors in the wild for several years), bacteria or fungi interfere and continue the work. This is the current paradigm driving the field of biodegradation research. In this paradigm, several bacterial and fungal strains have been identified as being able to carry out a certain degree of PE degradation. However, in most cases, such degradation requires invasive (aggressive) pretreatment of the PE to accelerate the incorporation of oxygen into the polymer (heating, UV light, etc.), making abiotic oxidation the real bottleneck of the reaction (Wei, R., and Zimmermann, W. (2017). Microbial Biotechnology 10, 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; T., et al. (2021). Science of the Total Environment 752; Walsh, A.N., et al. (2021). Environ. Sci. Technol. 55, 12383 - 12392). In the past decade, several microorganisms have been described as being able to act on untreated PE17 - 23, but they require significantly longer incubation times compared to the experimental conditions in the case of pre - oxidized PE.

[0004] Identifying enzymes from microorganisms capable of degrading untreated PE has proven to be a difficult task. In fact, no such enzymes have been identified, which confirms the key limiting role of oxidation in the overall 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, 143536.). For example, two reported laccases capable of chemically modifying PE require abiotic pretreatment or the addition of redox mediators such as 1 - hydroxybenzotriazole.

[0005] This situation defines the synthetic nature of the compounds and the characteristics of hydrophobicity and inaccessibility, making plastics difficult targets for enzymatic activities from animals, fungi, or microorganisms. However, some Lepidoptera and Coleoptera insects show an unexpected ability to degrade untreated PE and PS, as described in Yang, Y., Wang, J., and Xia, M. (2020). Sci Total Environ 708, 135233.

[0006] Bombelli, P., Howe, C.J., & Bertocchini, F. (2017) Current Biology, 27(8), R292 - R293 and Sanluis - Verdes, A. et al., (2022) Nature Communications, 13(1) reported the degradation of polyethylene by the larvae of the waxworm Galleria mellonella. However, these documents did not disclose the isolation of enzymes capable of oxidizing and / or depolymerizing untreated polyolefin - derived polymers.

[0007] Therefore, there remains a problem in the art of identifying enzymes from microorganisms that are capable of biodegrading untreated PE, in particular enzymes that oxidize untreated PE polymers, while avoiding the use of abiotic factors such as light or temperature, long incubation times, or invasive pre-treatment of the PE. Summary of the Invention

[0008] Generally, the inventors have isolated such enzymes from the saliva of waxworms (Galleria mellonella larvae) that have the unexpected ability to oxidize and deteriorate untreated 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) on waxworm saliva, obtaining an enzyme identified as belonging to the hexamerin / prophenoloxidase family, renamed Cora (JHS), with accession number XP_026749149.1 (NCBI), SEQ ID NO: 1 (the enzyme of the present invention). The ability of this enzyme to oxidize / degrade polyolefin-derived polymers was tested on PE membranes (see the examples and figures of this patent application), showing high degradation activity. This opens up a high-speed route to the possibility of solving the problem of plastic waste pollution.

[0009] This effect on PE degradation was achieved after only a few hours of exposure at room temperature and physiological conditions (neutral pH). Therefore, long incubation times or invasive pre-treatment are no longer required. The enzyme provided in the present invention can indeed overcome the bottleneck step in PE biodegradation, namely the initial oxidation step. Degradation products such as small oxidized aliphatic chains were identified using Gas Chromatography-Mass Spectrometry (GC-MS), further confirming the fragmentation of the polymer into shorter molecules.

[0010] The ability of this enzyme to act on PE membranes at room temperature and in a very short time to produce such modifications represents a promising alternative to abiotic oxidation of plastics (the first and most difficult step in the degradation process). Identifying invertebrate enzymes that can oxidize PE within a few hours represents a completely new paradigm in the field of plastic degradation and the broader field of plastic waste management, and opens up a high-speed route for designing new formulations / paths for synthetic polymer production.

[0011] In one aspect, the present invention relates to an expression vector comprising a nucleotide sequence encoding an enzyme, said 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:1, for biodegradation, or oxidation and / or depolymerization of polyolefin-derived polymers or materials containing polyolefin-derived polymers.

[0012] In another aspect, the present invention relates to: an enzyme (hereinafter referred to as "the enzyme or protein of the present invention") 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 functionally equivalent fragment thereof, or a host cell comprising a nucleotide sequence encoding said enzyme or a vector containing a nucleotide sequence encoding said enzyme, (hereinafter referred to as "the host cell of the present invention"), or a composition comprising said enzyme or a functionally equivalent fragment thereof or said host cell (hereinafter referred to as "the composition of the present invention"), which is used for biodegradation, or oxidation and / or depolymerization of polyolefin-derived polymers or materials containing polyolefin-derived polymers.

[0013] For example, preferably, said enzyme is active for biodegradation, or oxidation and / or depolymerization of polyolefin-derived polymers or materials containing polyolefin-derived polymers.

[0014] In some embodiments, 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 with SEQ ID NO:1, and a composition comprising said isolated enzyme.

[0015] In some embodiments, the enzyme of the present invention is not the enzyme with database accession number NCBI:XP026749149.1.

[0016] In some embodiments, the enzyme of the present invention is not the enzyme with database accession number A0A6J1WF64.

[0017] In some embodiments, the enzyme of the present invention is an enzyme comprising a sequence having a 1% variation from the amino acid sequence of SEQ ID NO:1. In some embodiments, the enzyme comprises a sequence having a 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 that differs from the amino acid sequence of SEQ ID NO:1 by 1 amino acid. In some embodiments, the enzyme comprises a sequence that differs from the amino acid sequence of SEQ ID NO:1 by 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or 300 amino acids.

[0018] Preferably, the enzyme is active in biodegradation, or oxidation and / or depolymerization of polyolefin-derived polymers or materials containing polyolefin-derived polymers.

[0019] In some embodiments, the composition of the present invention comprises the enzyme of the present invention or a functionally equivalent fragment thereof, or a host cell of the present invention, and preferably at least one additional component.

[0020] In some embodiments, the composition of the present invention comprises one or more polyolefin-derived polymers or materials containing polyolefin-derived polymers.

[0021] In some embodiments, the composition of the present invention comprises one or more oxidized polyolefin-derived polymers.

[0022] In some embodiments, the composition of the present invention comprises one or more selected from the group consisting of butane, 2,3-butanediol, trimethylslyl (TMS) derivatives, sebacic acid, C10 to C22 2-ketones, phenylpropionic acid.

[0023] In some embodiments of the present invention, the enzyme or composition can be formulated into enzyme particles. Preferably, the enzyme or composition of the present invention can be formulated as an aqueous solution.

[0024] In some embodiments, the composition comprises a buffer solution. For example, HEPES buffer. More preferably, the composition comprises 150 mM NaCl, 20 mM Hepes, 5% glycerol.

[0025] In view of the foregoing, 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 with SEQ ID NO:1, or a functionally equivalent fragment thereof, or A host cell comprising a nucleotide sequence encoding said enzyme or a vector containing a nucleotide sequence encoding said enzyme, or A composition comprising said enzyme or a functionally equivalent fragment thereof or said host cell, Use for biodegradation, or oxidation and / or depolymerization of a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer.

[0026] The enzyme of the present invention is preferably isolated from the saliva of Galleria mellonella larvae (also known as wax worm, ww). It is the juvenile hormone suppressing protein 1, renamed JHS (Cora), and comprises an amino acid sequence having at least 60% sequence identity with sequence SEQ ID NO:1 (accession number NCBI: XP_026749149.1).

[0027] SEQ ID NO:1 (JHS, Cora, the enzyme of the present invention)

[0028] Alternatively, the enzyme of the present invention can be recombinantly produced according to techniques well known in the art. For example, using well-recognized chemical synthesis techniques, the enzyme can be constructed from individual amino acids or peptides comprising two or more amino acid residues from the N-terminus or more commonly from the C-terminus. Specific techniques for synthesizing the enzyme include classical methods, classical chemical synthesis of amino acids one by one, and solid-phase peptide synthesis, where the enzyme is linked to a resin (such as Merrifield resin) for construction. In these synthetic operations, the groups on the amino acids will typically be in a protected form using standard protecting groups (such as tert-butoxycarbonyl). Once the synthesis is complete, these protecting groups are cleaved if necessary. Chemical synthesis methods, such as solid-phase peptide synthesis, solution synthesis, combinations of solid-phase synthesis with solution or enzymatic synthesis methods, are known to experts in the art. The enzyme of the present invention can also be produced by recombinant DNA procedures known in the art. Modifications can be introduced during or after enzyme synthesis, for example to include tags for purification, purification tags.

[0029] In another aspect, the present invention provides an expression vector comprising a nucleic acid sequence encoding the enzyme of the present invention.

[0030] 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. Different degrees of identity, expressed as a percentage, are obtained based on the number of residues shared between the aligned sequences. 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 prior art such as BLAST. The 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.

[0031] Those skilled in the art understand that mutations in the nucleotide sequence of a gene that result in conservative amino acid substitutions at positions that are not critical for protein functionality are evolutionarily neutral mutations that do not affect its overall structure or its functionality, thereby producing proteins that, although containing different amino acid sequences, exhibit the same activity. These proteins are considered to be "functionally equivalent variants" of SEQ ID NO:1 and fall within the scope of the present invention. Thus, the term "functionally equivalent variant" as used herein means an enzyme that is derived from the native enzyme (SEQ ID NO:1 in 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 exhibits the same activity, i.e., the enzyme retains the ability to oxidize untreated polyolefin-derived polymers such as polyethylene (PE) at room temperature. Any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc., can be used to prepare the variants. All proteins having at least 60% sequence identity with the amino acid sequence of SEQ ID NO:1 and capable of oxidizing untreated polyolefin-derived polymers are considered to be 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:1 are disclosed in the examples of this patent application. The functionally equivalent variants of the present invention have at least 20% of the oxidation activity of the native protein of SEQ ID NO:1, 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% oxidation activity.

[0032] The present invention also encompasses functionally equivalent fragments of the enzymes of the present invention. The term "functionally equivalent fragment" means a polypeptide / protein having one or more (e.g., several) amino acids deleted from the amino terminus and / or carboxy terminus of the native protein (in the present invention, the sequence SEQ ID NO:1) and showing the same activity / function as the native protein (in the present invention, the ability to oxidize untreated polyolefin-derived polymers at room temperature). Examples of assays for checking whether a protein fragment of the present invention is a functionally equivalent fragment of SEQ ID NO:1 are disclosed in the examples of this patent application. The functionally equivalent fragments of the present invention have at least 20% of the oxidation activity of the native protein SEQ ID NO:1, 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% oxidation activity.

[0033] In a preferred embodiment, the amino acid sequence of the enzyme of the present invention comprises or consists of the sequence SEQ ID NO:1.

[0034] The nucleotide sequence of the present invention encoding the enzyme of the present invention may also contain other elements in addition to the coding sequence, such as introns, non-coding sequences in the 3' and / or 5' termini, ribosome binding sites, etc. The nucleotide sequence of the present invention may also contain sequences encoding additional amino acids, which may be used to enhance the stability of the enzyme or allow for more efficient enzyme purification.

[0035] The nucleotide sequence of the present invention can be introduced into a vector or genetic construct, such as a cloning vector or an expression vector, to obtain a vector containing the nucleotide sequence. Preferably, the vector is a suitable vector for expressing and purifying the enzyme of the present invention.

[0036] The term "genetic construct" or "vector" as used herein refers to a single-stranded (monocatenary) or double-stranded (bicatenary) nucleic acid molecule that has been isolated and modified to contain nucleic acid segments in a manner not found in nature. When a nucleic acid construct contains the control sequences required for expressing the nucleotide sequence of the present invention, the term "nucleic acid construct" or "genetic construct" is synonymous with the term "expression cassette". Thus, the genetic construct of the present invention may also contain one or more control sequences or regulatory sequences for gene expression, such as, but not limited to, promoter sequences, leader sequences, terminator sequences, polyadenylation sequences, signal sequences, regulators, enhancers, etc.

[0037] "Expression vector" is a linear or circular DNA molecule containing at least the nucleotide sequence of the present invention, and the nucleotide sequence of the present invention is operably linked to additional nucleotides provided for its expression. Such a vector containing the nucleic acid sequence of the present invention can be introduced into a host cell in such a way that the vector is maintained as a chromosomal component or as a self-replicating extrachromosomal vector.

[0038] The expression vector referred to in the present invention can be any vector (such as a plasmid or a virus) that can be conveniently subjected to recombinant DNA procedures and can produce the expression of the nucleotide sequence of the present invention contained therein. The choice of the vector will generally depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The expression vector can be, for example but not limited to, a plasmid, a cosmid, a phage, a virus or a viral vector, an artificial bacterial chromosome (BAC), an artificial yeast chromosome (YAC), or the like. The vector in the context of the present invention can be linear or closed circular. Preferably, the expression vector of the present invention is a baculovirus expression vector, more preferably a P2 baculovirus vector.

[0039] "Host cell" as used herein includes any cell type that is easily transformed, transfected, transduced, etc. with the nucleotide sequence or expression vector of the present invention as described above. The host cell can be prokaryotic or eukaryotic, preferably eukaryotic, such as mammalian, insect, plant or fungal cells. In some embodiments, the host cell is prokaryotic, preferably a bacterial cell, such as Escherichia 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.

[0040] Therefore, the host cell of the present invention contains at least the nucleic acid sequence of the present invention, preferably contains the nucleic acid sequence of the present invention in a recombinant manner through the vector of the present invention. The nucleotide of the present invention or the vector of the present invention is not naturally present in the cell, but is intentionally introduced through a genetic modification procedure. The nucleotide sequence of the present invention can encode the mature enzyme of the present invention or a protein precursor composed of a signal peptide linked to the mature enzyme, and the protein precursor must then be processed to produce the mature enzyme.

[0041] The expression of the enzyme of the present invention in the host cell of the present invention can be induced by any procedure known in the art, for example, by transforming a suitable host cell with at least one nucleotide sequence of the present invention or with a vector of the present invention, and culturing the transformed host cell under conditions that induce the expression of the nucleotide sequence to obtain a secreted and functional enzyme. The preferred conditions for inducing the expression of the nucleotide sequence are to incubate the host cell at 27 °C for 48 to 72 hours.

[0042] In some aspects, the present invention relates to a method for expressing an enzyme in a host cell that has at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:1, the method comprising culturing the host cell under conditions that induce the expression of an expression vector to obtain the enzyme.

[0043] The enzyme of the present invention produced by the host cell of the present invention can be purified by a variety of procedures known in the art to obtain a substantially pure enzyme, the procedures including but not limited to chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocus, and size exclusion), electrophoresis procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction.

[0044] In another preferred embodiment, the enzyme of the present invention is recombinantly produced or it is isolated from the saliva of Galleria mellonella, particularly from the saliva of Galleria mellonella (G. mellonella).

[0045] The method for isolating the enzyme of the present invention from the saliva of Galleria mellonella is, but not limited to, chromatographic techniques from the saliva of waxworms (e.g., size exclusion and ion exchange chromatography).

[0046] The composition of the present invention comprises the enzyme of the present invention or a functionally equivalent fragment thereof, or the host cell of the present invention, and optionally other elements required for the optimal activity or storage of these enzymes. These additional elements can be, for example, buffers (e.g., HEPES), other enzymes (e.g., enzymes that can be used to biodegrade polyolefin-derived polymers), antibiotics, etc.

[0047] Preferably, the composition of the present invention further comprises one or more additional enzymes, for example, one, two or three additional enzymes, which are active for biodegradation, or oxidation and / or depolymerization of polyolefin-derived polymers or materials containing polyolefin-derived polymers.

[0048] Preferably, the composition of the present invention further comprises an additional enzyme, and the additional enzyme comprises 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. More preferably, such an additional protein comprises SEQ ID NO:2 or consists of SEQ ID NO:2. Preferably, the composition of the present invention further comprises an isolated enzyme, which comprises 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. As Figure 3 shown, the putative signal sequence of the enzyme of SEQ ID NO:2 consists of amino acids 1 to 16, and thus the mature form of this enzyme consists of amino acids 17 to 702 (including the endpoints).

[0049] SEQ ID NO:2 (Cibeles, g181563)

[0050] The composition of the present invention may further comprise an additional enzyme, and the additional enzyme comprises 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:3. More preferably, such an additional protein comprises SEQ ID NO:3 or consists of SEQ ID NO:3. Preferably, the composition of the present invention further comprises an isolated enzyme, and the isolated enzyme comprises 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:3. As Figure 3 shown, the putative signal sequence of the enzyme of SEQ ID NO:3 consists of amino acids 1 to 16, and thus the mature form of this enzyme consists of amino acids 17 to 700 (including the endpoints).

[0051] SEQ ID NO:3 (Demetra)

[0052] The composition of the present invention may further comprise an additional enzyme, 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 with SEQ ID NO:4. More preferably, such additional protein comprises SEQ ID NO:4 or consists of SEQ ID NO:4. Preferably, the composition of the present invention further comprises an isolated enzyme, the 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 with SEQ ID NO:4. As Figure 3 shown, the putative signal sequence of the enzyme of SEQ ID NO:4 consists of amino acids 1 to 18, and thus the mature form of the enzyme consists of amino acids 19 to 706 (including the endpoints).

[0053] SEQ ID NO:4 (Ceres)

[0054] The composition of the present invention may comprise one, two or three additional enzymes or isolated enzymes, which are active in biodegradation, or oxidation and / or depolymerization of polyolefin-derived polymers or materials containing polyolefin-derived polymers.

[0055] For example, the composition may comprise an additional enzyme, 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 with SEQ ID NO:2.

[0056] In another example, the composition may comprise an additional enzyme containing 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 an additional enzyme containing 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:3.

[0057] In another instance, the composition can comprise additional enzymes 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 additional enzymes 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 instance, the composition can comprise additional enzymes 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; additional enzymes 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 additional enzymes 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.

[0059] In another instance, the composition can comprise an additional enzyme, 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:3. In another instance, the composition can comprise additional enzymes 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 additional enzymes 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 instance, the composition may comprise an additional enzyme, 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 with SEQ ID NO:4.

[0061] In some embodiments, the composition of the present invention comprises the enzyme of the present invention or a functionally equivalent fragment thereof, or a host cell of the present invention, and preferably at least one additional component. In some embodiments, the component is at least one of the additional elements disclosed above.

[0062] In some embodiments, the additional enzyme in the composition is not the enzyme with database accession number XP026756460.1.

[0063] In some embodiments, the additional enzyme in the composition is an enzyme comprising a sequence having a 1% variation from the amino acid sequence of SEQ ID NO:2. In some embodiments, the additional enzyme comprises a sequence having a 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 from the amino acid sequence of SEQ ID NO:2 by 1 amino acid. In some embodiments, the additional enzyme comprises a sequence that differs from the amino acid sequence of SEQ ID NO:2 by 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or 300 amino acids.

[0064] In some embodiments, the additional enzyme in the composition is not the enzyme with database accession number XP026756396.

[0065] In some embodiments, the additional enzyme in the composition is an enzyme comprising a sequence having a 1% variation from the amino acid sequence of SEQ ID NO:3. In some embodiments, the additional enzyme comprises a sequence having a 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 differs from the amino acid sequence of SEQ ID NO:3 by 1 amino acid. In some embodiments, the additional enzyme comprises a sequence that differs from the amino acid sequence of SEQ ID NO:3 by 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or 300 amino acids.

[0066] In some embodiments, the additional enzyme in the composition is not the enzyme with database accession number XP093062524. In some embodiments, the additional enzyme in the composition is not the enzyme with database accession number XP_026756459.1.

[0067] In some embodiments, the additional enzyme in the composition is an enzyme comprising a sequence having a 1% variation from the amino acid sequence of SEQ ID NO:4. In some embodiments, the additional enzyme comprises a sequence having a 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 differs from the amino acid sequence of SEQ ID NO:4 by 1 amino acid. In some embodiments, the additional enzyme comprises a sequence that differs from the amino acid sequence of SEQ ID NO:4 by 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or 300 amino acids.

[0068] In one aspect, the present invention relates to a kit for biodegradation, or oxidation and / or depolymerization, of a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer, the kit comprising: in a 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 functional equivalent fragment thereof, or a host cell comprising a nucleotide sequence encoding the enzyme or a vector containing the nucleotide sequence encoding the enzyme, or a composition comprising the enzyme or a functional equivalent fragment thereof or the host cell; and instructions for using the enzyme, the host cell or the composition together with: 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 functional equivalent fragment thereof, or a host cell comprising a nucleotide sequence encoding the additional enzyme or a vector containing the nucleotide sequence encoding the additional enzyme, or a composition comprising the additional enzyme or a functional equivalent fragment thereof or the host cell.

[0069] In some embodiments, the kit further comprises: In a separate container: an additional enzyme having an amino acid sequence with 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 functional equivalent fragment thereof, or a host cell comprising a nucleotide sequence encoding said additional enzyme or a vector containing a nucleotide sequence encoding said additional enzyme, or a composition comprising said additional enzyme or a functional equivalent fragment thereof or said host cell.

[0070] In some embodiments, the kit further comprises: In a separate container: an additional enzyme having an amino acid sequence with 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 functional equivalent fragment thereof, or a host cell comprising a nucleotide sequence encoding said additional enzyme or a vector containing a nucleotide sequence encoding said additional enzyme, or a composition comprising said additional enzyme or a functional equivalent fragment thereof or said host cell.

[0071] In some embodiments, the kit further comprises: In a separate container: an additional enzyme having an amino acid sequence with 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 functional equivalent fragment thereof, or a host cell comprising a nucleotide sequence encoding said additional enzyme or a vector containing a nucleotide sequence encoding said additional enzyme, or a composition comprising said additional enzyme or a functional equivalent fragment thereof or said host cell.

[0072] In some embodiments, the kit comprises the first container as described above and one, two, or three additional containers, the additional containers comprising i) an additional isolated enzyme comprising an amino acid sequence having at least 60% sequence identity with SEQ ID NO:2, or an additional host cell comprising an expression vector encoding the additional isolated enzyme, or an additional composition comprising the additional isolated enzyme or the additional host cell; ii) an additional isolated enzyme comprising an amino acid sequence having at least 60% sequence identity with SEQ ID NO:3, or an additional host cell comprising an expression vector encoding the additional enzyme, or an additional composition comprising the additional isolated enzyme or the additional host cell; and / or iii) an additional isolated enzyme comprising an amino acid sequence having at least 60% sequence identity with SEQ ID NO:4, or an additional host cell comprising an expression vector encoding the additional enzyme, or an additional composition comprising the additional isolated enzyme or the additional host cell. The kit may further comprise instructions for use of the isolated enzyme, host cell, or composition and the additional isolated enzyme, host cell, or composition.

[0073] In some embodiments of the invention, one or more additional enzymes are isolated enzymes.

[0074] In some embodiments, the enzyme, host cell, or composition of SEQ ID NO:1 is used to biodegradably degrade, oxidize, and / or depolymerize a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer in a step separate from, sequential with, or simultaneous 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.

[0075] In its most general aspect, "polyolefin-derived polymer" refers to any polyolefin polymer derived from olefin monomers.

[0076] In one aspect of the invention, the "polyolefin-derived polymer" can be a polymer type having the general formula (CH2CHR) n wherein R is an alkyl group. In some cases, R can also be a hydrogen atom. They are generally derived from a small group of simple olefins (alkenes). Commercially dominant are polyethylene and polypropylene. More specialized polyolefins include polyisobutene and polymethylpentene. They are all 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.

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

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

[0079] "Polyethylene (PE)" or polythene (abbreviated as PE; IUPAC name is polyethylene or poly(methylene)) is the most commonly used plastic today. It is a polymer mainly used for packaging (such as plastic bags, plastic films, geomembranes, and containers including bottles, etc.). Many types of polyethylene are known, most of which have the chemical formula (C2H4) n . PE is usually a mixture of similar ethylene polymers with different n values. It can be of low density or high density: low-density polyethylene is extruded using high pressure (1000 to 5000 atm) and high temperature (520 kelvin), while high-density polyethylene is extruded using low pressure (6 to 7 atm) and low temperature (333 to 343 K). Polyethylene is usually thermoplastic, but it can be modified to be thermosetting, such as crosslinked polyethylene. All types of PE are covered within the scope of the present invention.

[0080] In an even more preferred embodiment, 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 PE 4000 or PE 2000.

[0081] In another specific embodiment, before biodegradation by the enzyme of the present invention, the host cell of the present invention, or the composition of the present invention, the polyolefin-derived polymer or the material containing the polyolefin-derived polymer is not pretreated with abiotic factors (such as heating, UV light, etc.), that is, the enzyme of the present invention can biodegrade the untreated polyolefin-derived polymer or the untreated material containing the polyolefin-derived polymer.

[0082] In another aspect, the present invention refers to a method for biodegradation, oxidation, and / or depolymerization of a polyolefin-derived polymer or a material containing a polyolefin-derived polymer, hereinafter referred to as "the method of the present invention", wherein the method comprises contacting the following with the polyolefin-derived polymer or the material containing the polyolefin-derived polymer: the enzyme of the present invention, or the host cell of the present invention, or the composition of the present invention.

[0083] In a preferred embodiment of the method of the present invention, the method is carried out at room temperature, preferably in an aqueous solution having a neutral pH at room temperature.

[0084] "Room temperature" is from 15°C to 30°C, preferably 22°C.

[0085] "Neutral pH" is from pH 7 to pH 8.

[0086] In a preferred embodiment, the amount of enzyme used in the method of the present invention is 2 to 10 μL, preferably 5 μL, and the concentration is 1 to 5 μg / μL.

[0087] 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.

[0088] In an even more preferred embodiment of the method of the present invention, the amount of enzyme used is 5 μL or 10 μL, the concentration is 1 to 5 μg / μl, preferably 1.2 μg / μL, and it is applied to the polyolefin-derived polymer or a material containing the polyolefin-derived polymer at least 8 times, preferably 24 times, for 90 minutes each time.

[0089] In another preferred embodiment of the method of the present invention, the enzyme of the present invention comprises the sequence SEQ ID NO:1 or consists of the sequence SEQ ID NO:1. More preferably, the enzyme of the present invention is isolated from Galleria mellonella, particularly from the saliva of Galleria mellonella.

[0090] In another preferred embodiment of the method of the present invention, the composition of the present invention further comprises a second enzyme, the 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, more preferably comprising SEQ ID NO:2, even more preferably consisting of SEQ ID NO:2.

[0091] In some embodiments, 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 with SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4 is contacted with the polyolefin-derived polymer or a material containing the polyolefin-derived polymer in a step separate from, sequential to or simultaneous 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 with SEQ ID NO:1.

[0092] In another preferred embodiment of the method of the present invention, the polyolefin-derived polymer is polyethylene (PE) or polypropylene (PP).

[0093] In another more preferred embodiment of the method of the present invention, the polyolefin-derived polymer is polyethylene (PE).

[0094] 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 PE 4000 or PE 2000.

[0095] Another aspect of the present invention relates to a method for pretreating a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer, the method comprising contacting the following with the polyolefin-derived polymer or the material comprising a polyolefin-derived polymer to provide an oxidized polymer or material product: the enzyme of the present invention, or the host cell of the present invention, or the composition of the present invention.

[0096] In some embodiments, the method comprises contacting the enzyme of the present invention and one, two, or three additional enzymes as described herein with the polyolefin-derived polymer or the material comprising a polyolefin-derived polymer to provide an oxidized polymer or material product.

[0097] In some embodiments, the method of the present invention is used to pretreat a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer, followed by a further degradation step. For example, a further degradation step is performed on the oxidized polymer or material product. In some embodiments, the method of the present invention is used to pretreat a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer, followed by the application of one or more microbial degradation steps.

[0098] Another aspect of the present invention relates to a method for obtaining biodegradable by-products derived from polyolefin-derived polymers, which comprises: (a) contacting the polyolefin-derived polymer with the following: the enzyme of the present invention, or the host cell of the present invention, or the composition of the present invention, and (b) Separate the by-products obtained from the culture resulting from step (a).

[0099] In some embodiments, step (a) of the method comprises contacting the polyolefin-derived polymer with the enzyme of the present invention and one, two or three additional enzymes as described herein.

[0100] Another aspect of the present invention relates to a method for preparing plastics, which comprises: (a) Contacting the polyolefin-derived polymer with: the enzyme of the present invention, or the host cell of the present invention, or the composition of the present invention, and (b) Separating the by-products obtained from the culture resulting from step (a), and (c) Polymerizing the by-products separated in step (b).

[0101] In some embodiments, step (a) of the method comprises contacting the polyolefin-derived polymer with the enzyme of the present invention and one, two or three additional enzymes as described herein.

[0102] In the context of the present invention, "by-products" are, but not limited to, butane, 2,3-butanediol, trimethylsilyl (TMS) derivatives, sebacic acid, 2-ketones having 10 to 22 carbons, and small aromatic compounds recognizable as phenylpropionic acids. Preferably, the by-products obtained comprise C10-C22 2-ketones.

[0103] The conditions for the method related to the by-products obtained are those of the methods for biodegradation, oxidation and / or depolymerization of polyolefin-derived polymers or materials containing polyolefin-derived polymers as explained above. For example, a second enzyme can be used in these methods.

[0104] The separation of the by-products in step (b) of the method can be carried out by techniques well known in the art such as Gas Chromatography-Mass Spectroscopy (GC-MS).

[0105] Embodiments of the present invention will now be described by way of example and not limitation with reference to the accompanying drawings. However, in view of the present disclosure, various additional aspects and embodiments of the present invention will be apparent to those skilled in the art.

[0106] As used herein, "and / or" is considered to specifically disclose each of the two indicated features or components with or without the other. For example, "A and / or B" is considered to specifically disclose (i) A, (ii) B, and (iii) each of A and B, as if each were listed separately herein.

[0107] Unless otherwise indicated in the context, the descriptions and definitions of the features listed above are not limited to any particular aspect or embodiment of the present invention and equally apply to all aspects and embodiments described. Description of the Drawings

[0108] Figure 1 . Raman analysis of PE films treated with purified recombinant Cora. A, B. PE films treated with Cora. Peaks from 1500 to 2400 cm -1 indicate PE degradation. Oxidation is indicated at 1600 to 1800 cm -1 (carbonyl) and 3000 to 3500 cm -1 (hydroxyl) (apparent in B). C. Control PE film. The PE characteristics are characterized by peaks at 1061, 1128, 1294, 1440, 2846, and 2880 cm -1 . D. Overlaid normalized spectra (A, B, and C).

[0109] Figure 2 . Identification of degradation by-products of PE treated with Cora by GC-MS. A. Chromatogram of the fragmentgram of the ion m / z 58 of methyl ketones from enzyme-treated PE. Arrows indicate the peaks corresponding to 2-ketones with different carbon numbers. B. Changes in PE degradation by-products by GC-MS after different applications. During 6 to 12 applications of Cora to PE, the formation of ketones as degradation products increased, and the formation of 2-ketones increased by doubling the application of the enzyme to PE.

[0110] Figure 3 . Sequence alignment and overall structure of four proteins present in the saliva of Galleria mellonella. (A) Amino acid sequence alignment colored by similarity. The major metal-coordinating residues are highlighted with squares, the glycosylated residues are marked with triangles, and the disulfide bridges are indicated by asterisks. (B) Percentage of pairwise sequence identity between the four factors. (C) Overall primary, tertiary, and quaternary structures of members of the hemocyanin / phenoloxidase (Hc / PO) family (the Hc of Panulirus interruptus, PDB code 1HCY, used as an example). The typical copper-binding sites are highlighted with red spheres. Example. PE degradation experiment. I - Materials and methods Generation of recombinant Cora (SEQ ID NO:1)

[0111] Recombinant Cora enzyme (SEQ ID NO:1) can be generated using known expression protocols. Raman analysis

[0112] The recombinant protein of SEQ ID NO:1 was applied as follows: 5 μl of the protein (at a concentration of 1 to 5 μg / ml) was applied eight times on a PE membrane, each time for 90 minutes.

[0113] Raman analysis was performed on (treated and control) PE membranes using an Alpha300R - Alpha300AAFM Witec device at a power of 5 mW, with a 50× (NA 0.8) objective, integration time 1, accumulation 30, and wavelength 532 nm. The results are shown in Figure 1 as follows. Gas chromatography - mass spectrometry (GC - MS)

[0114] PE was exposed to 10 μl (1.2 μg / mL) of Cora (JHS) 24 times for 90 minutes. Long - term treatment (on day 1 and day 2) was carried out with 4 applications of 10 μl (1.2 μg / mL) each time for 90 minutes per day. As a control, the same experiment was repeated using protein buffer. Then, the samples were centrifuged at 19083 g for 30 seconds using an Eppendorf centrifuge 5810R, and the supernatant was transferred to a new 1.5 ml Eppendorf tube. The samples and controls were extracted using a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method. Briefly, 50 μl of diphenyl phthalate (internal standard; IS) at a concentration of 1 mg / ml was added to each sample, and the samples were extracted with 300 μl of dichloromethane (DCM) and 5% (v / m) NaCl. The tubes were vortexed for 30 seconds at room temperature and sonicated (50 / 60 Hz) in a bath for 15 minutes, followed by centrifugation at 20 °C and 19083 g for 10 minutes using an Eppendorf centrifuge 5810R. Finally, the DCM in the supernatant was collected and placed in an insert for subsequent analysis. Silylation reaction was carried out with N,O - bis(trimethylsilyl)trifluoroacetamide (BSTFA) to determine low - volatile polar compounds that showed low detection sensitivity. 50 μL aliquots of each sample were incubated with 50 μL of BSTFA at 60 °C for 20 minutes and then analyzed.

[0115] Dichloromethane (DCM; CAS-No: 75-09-2) for gas chromatography-mass spectrometry (GC-MS) was of SupraSolv grade purity and obtained from Sigma-Aldrich (Darmstadt, Germany). Sodium chloride (NaCl; ≥99.5%; CAS-No: 7647-14-5) and ultrapure water from a Milli-Q system were provided by Merck (Darmstadt, Germany). Crystalline granular powder polyethylene (PE4000; CAS-No: 9002-88-4) was provided by Sigma-Aldrich (Saint Louis, USA).

[0116] Chromatographic analysis was performed using a gas chromatography-mass spectrometry system (GC-MS) 7980A-5975C from Agilent Technologies. The separation of metabolites was carried out on a DB-5 polyimide-coated column (30 m in length, 0.25 mm inner diameter, and 0.1 μm film thickness; Agilent Technologies, USA), and helium (He) was used as the carrier gas. Analysis was performed using a split injector at 350 °C, and the injection volume was 1 μL. The ion source temperature was 230 °C, the mass spectrometry was carried out in scan mode, the quadrupole temperature was 150 °C, and the fragmentation voltage was 70 eV. The oven program was started at 60 °C for 3 minutes and then ramped to 350 °C at 20 °C / min for 1 minute. The total run time for the samples used for derivatization was 18.5 minutes and 19.5 minutes. The resulting chromatograms were processed using software MSD ChemStation E.01.00.237 from Agilent Technologies, Inc., and identification was performed using the NIST11 library. th The evaluation of the long-term treatment was based on the relative abundance of each non-target compound, which consisted of the quotient of the peak area under the curve of each compound divided by the peak area under the curve of the IS. The results are shown in

[0117] the Figure 2 shown Protein buffer

[0118] The resulting protein was resuspended in 50 mM Tris-Cl, 500 mM NaCl, 10% glycerol, 0.5% sodium lauroyl sarcosinate, 2 mM TCEP, 2 mM glutathione (pH 7.5) and used for the degradation assay. A separate identical buffer was used as a negative control. II - Results Cora oxidized PE film

[0119] The ability of purified recombinant Cora (SEQ ID NO:1) to oxidize PE was studied. After eight consecutive applications of 5 μl of protein for 90 minutes each, confocal Raman microscopy / Raman spectroscopy (Raman) analysis showed highly oxidized polymer, accompanied by general deterioration of the membrane ( Figure 1 A and 1B). This was evident in the overlap with the PE control ( Figure 1 C and 1D), which revealed the expected characteristic spectrum of PE. As a negative control, the protein buffer alone was applied to the PE membrane and no oxidation occurred ( Figure 1 C). The changes produced by Cora during a multi-hour application were similar to those produced by environmental factors after months or years of exposure to weathering. The changes in the chemical composition of PE revealed by spectroscopic techniques indicated that molecules other than long PE polymer chains were formed after contact with Cora. Identification of Cora as an oxidizer of PE

[0120] To analyze the potential of Cora in oxidizing PE, GC-MS was performed on PE particles (PE4000) exposed to Cora (SEQ ID NO:1). After 24 applications of Cora (10 μL at 1.2 μg / μL for 90 minutes each), 2-ketones with 10 to 22 carbons were detected in the supernatant using GC-MS, and the fragment graph m / z 58m and retention time were used for identification ( Figure 2 A).

[0121] Increased treatment (twelve applications compared to six applications, 90 minutes each) showed an increase in the relative abundance of 2-ketones with 14 to 18 carbons and the appearance of 2-dodecanone, which was not detected after six applications ( Figure 2 B). III - Summary

[0122] The present invention demonstrates that Cora (SEQ ID NO:1) oxidizes and depolymerizes PE. This is the first report of this enzyme attacking PE polymers without any prior abiotic treatment. This is achieved by the action of the enzyme at room temperature and in an aqueous solution with neutral pH. Under these conditions, the enzymatic action of Cora overcomes the recognized bottleneck step (i.e., oxidation) in PE degradation within hours.

[0123] The action of the Cora enzyme, which is disclosed in the present invention and present in the saliva of the greater wax moth, on PE is equivalent to that of abiotic treatment.

[0124] The ability of the enzyme of SEQ ID NO:1 to rapidly and extensively oxidize the polymeric dense hydrophobic substance PE was unexpected. The existence of enzymes produced by insects, secreted from the mouth and evolved to act on plastics at room temperature and neutral pH provides a new paradigm for the biodegradation of PE. This new framework goes far beyond the current definition of biodegradation, which is only based on the complete conversion of plastics to CO2 through the metabolic activity of microorganisms: on the one hand, the observed oxidation and deterioration of PE do not depend on any microbial activity; on the other hand, the easy 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, the present inventors believe that the Cora enzyme will be active against other types of polyolefins. This potential can be used as an alternative to the metabolic conversion of plastics to CO2, or as an initial oxidation step in combination with the standard microbial degradation pathway. Clause Clause 1. Use of an enzyme or a host cell or a composition for the biodegradation of a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer, said 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:1, said host cell comprising a nucleotide sequence encoding said enzyme, said composition comprising said enzyme or said host cell. Clause 2. The use according to clause 1, wherein the amino acid sequence comprises or consists of the sequence SEQ ID NO:1. Clause 3. The use according to clause 1 or 2, wherein the composition further comprises a second enzyme, said 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. Clause 4. The use according to any one of clauses 1 to 3, wherein the polyolefin-derived polymer is polyethylene (PE). 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 crosslinked polyethylene (HDXLPE), crosslinked 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). Clause 6. The use according to any one of clauses 1 to 5, wherein the enzyme is isolated from Galleria mellonella, preferably isolated from the saliva of Galleria mellonella. Clause 7. A method for biodegradation of a polyolefin-derived polymer, or a material comprising a polyolefin-derived polymer, comprising contacting the following with the polyolefin-derived polymer or the material comprising the 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 the enzyme, or a composition comprising the enzyme or the host cell. Clause 8. The method according to clause 7, wherein the method is carried out at room temperature, preferably in an aqueous solution having a neutral pH at room temperature. Clause 9. The method according to clause 7 or 8, wherein the amino acid sequence comprises or consists of sequence SEQ ID NO:1. Clause 10. The method according to any one of clauses 7 to 9, wherein the composition further comprises a second enzyme, the 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. Clause 11. The method according to any one of clauses 7 to 10, wherein the polyolefin-derived polymer is polyethylene (PE). Clause 12. The method according to 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). Clause 13. The method according to any one of Clauses 7 to 12, wherein the enzyme is isolated from Galleria mellonella, preferably from the saliva of Galleria mellonella.

Claims

1. A method for biodegradation of a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer, the method comprising contacting the following with the polyolefin-derived polymer or the material comprising the 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 the enzyme, or a composition comprising the enzyme or the host cell.

2. The method according to claim 1, wherein the method is carried out at room temperature, preferably in an aqueous solution having a neutral pH at room temperature.

3. The method according to 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 with 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 SEQ ID NO:

1.

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

2.

6. The method according to 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 with SEQ ID NO:

2.

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

3.

8. The method according to 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 with SEQ ID NO:

3.

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

4.

10. The method according to 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 with SEQ ID NO:

4.

11. The method according to any one of claims 5 to 10, wherein the additional enzyme is contacted with the polyolefin-derived polymer or the material comprising the polyolefin-derived polymer in a step separate from, sequential to, or simultaneous with the enzyme according to claim 1.

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

13. An expression vector comprising a nucleotide sequence encoding an enzyme for biodegradation, or oxidation and / or depolymerization, of 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 with SEQ ID NO:

1.

14. A host cell comprising the expression vector according to claim 13.

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

16. An isolated enzyme comprising an amino acid sequence having at least 60% sequence identity with SEQ ID NO:1, for biodegradation, or oxidation and / or depolymerization, of a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer; wherein the enzyme of the present invention is not the enzyme with database accession number NCBI: XP026749149.1 or accession number A0A6J1WF64.

17. The isolated enzyme according to claim 16, wherein the enzyme is obtainable from Galleria mellonella, preferably from the saliva of Galleria mellonella.

18. The isolated enzyme according to 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 with SEQ ID NO:

1.

19. A composition comprising the host cell according to claim 14 or an isolated enzyme containing an amino acid sequence having at least 60% sequence identity with SEQ ID NO:1, for biodegradation, or oxidation and / or depolymerization, of a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer, and at least one additional component.

20. The composition according to 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 with SEQ ID NO:

1.

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

2.

22. The composition according to 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 with SEQ ID NO:

2.

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

3.

24. The composition according to 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 with SEQ ID NO:

3.

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

4.

26. The composition according to 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 with SEQ ID NO:

4.

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

28. The composition according to any one of claims 19 to 27, which comprises one or more of the following: butane, 2,3-butanediol, trimethylsilyl (TMS) derivatives, sebacic acid, C10 to C22 2-ketones, phenylpropionic acid.

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

30. A kit for biodegradation of a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer, which comprises: In a first container: The isolated enzyme according to any one of claims 13 to 15, or, The host cell according to claim 11, or, The composition according to any one of claims 16 to 23; And In a further container: An additional isolated enzyme, which comprises an amino acid sequence having at least 60% sequence identity with SEQ ID NO:2, or An additional host cell, which comprises an expression vector encoding the additional isolated enzyme, or An additional composition, which comprises the additional isolated enzyme or the additional host cell; And / or In a further container: An additional isolated enzyme, which comprises an amino acid sequence having at least 60% sequence identity with SEQ ID NO:3, or An additional host cell, which comprises an expression vector encoding the additional enzyme, or Additional compositions, which comprise the additional isolated enzyme or the additional host cell; and / or In a further container: An additional isolated enzyme, which comprises an amino acid sequence having at least 60% sequence identity with SEQ ID NO:4, or An additional host cell, which comprises an expression vector encoding the additional enzyme, or An additional composition, which comprises the additional isolated enzyme or the additional host cell; and Instructions for use of the isolated enzyme, host cell or composition and the additional isolated enzyme, host cell or composition.

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

32. The method, vector, enzyme, cell, composition or kit 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 crosslinked polyethylene (HDXLPE), crosslinked 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. A method for obtaining by-products derived from the biodegradation of a polyolefin-derived polymer, which comprises: (a) contacting the polyolefin-derived polymer with: 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 the enzyme, or a composition comprising the enzyme or the host cell, and (b) isolating the by-products obtained from the culture resulting from step (a).

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

35. A method for pretreating a polyolefin-derived polymer, which comprises contacting the following with the polyolefin-derived polymer or a material comprising the polyolefin-derived polymer to oxidize the 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 the enzyme, or a composition comprising the enzyme or the host cell.

36. Use of an enzyme or host cell or composition for the biodegradation of a polyolefin-derived polymer or a material comprising a polyolefin-derived polymer: the enzyme comprises an amino acid sequence having at least 60% sequence identity with SEQ ID NO:1, the host cell comprises a nucleotide sequence encoding the enzyme, the composition comprises the enzyme or the host cell.

37. The use according to claim 36, wherein the amino acid sequence comprises SEQ ID NO:1 or consists of SEQ ID NO:

1.

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

2.

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

3.

40. The use according to any one of claims 36 to 39, wherein the composition further comprises an additional enzyme, and the additional enzyme comprises an amino acid sequence having at least 60% sequence identity with 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. The use according to 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 crosslinked polyethylene (HDXLPE), crosslinked 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. The use according to any one of claims 36 to 42, wherein the enzyme is isolated from Galleria mellonella, preferably from the saliva of Galleria mellonella.

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