Method and kit for enzymatic degradation of polythiourethanes

Abstract

We propose an enzymatic decomposition method for polythiourethane. [Solution] The present disclosure relates to an enzymatic degradation method for polythiourethanes containing thiourethane bonds of the formula -SC(=O)-N(H)- in all repeating units of the polymer chain, comprising contacting at least one enzyme of the present invention with a preferred form of polythiourethane for a sufficient amount of time and under non-toxic and environmentally friendly reaction conditions. This method may also be used to recover basic monomers and / or other compounds obtained by the enzymatic degradation process, which can then be used to obtain the polymer again, or to use them further as intermediates in the synthesis of other chemicals. The present disclosure also encompasses a kit for the enzymatic degradation of polythiourethanes.

Description

TECHNICAL FIELD

[0001] The present invention belongs to the field of polymer reuse, and more specifically relates to the field of polymer reuse by enzymatic degradation.

[0002] In particular, the present invention relates to a method for enzymatic degradation of polythiourethane (PTUR), which contains thiourethane bonds of formula -S-C(=O)-N(H)- in all repeating units of the polymer chain.

[0003] The present invention discloses methods and kits using enzymes capable of degrading PTUR under non-toxic and environmentally friendly conditions.

[0004] By degrading PTUR, the method of the present invention enables reduction of polymer waste and recovery of basic monomers and / or other chemicals, which can be further used to re-obtain the original PTUR, or can be further used as raw materials or intermediates for the synthesis of other chemicals. BACKGROUND ART

[0005] Plastics are ubiquitous synthetic polymers ever since their production started in the 1950s. Since that time, the production rate of plastics has been increasing to meet the global demand.

[0006] Environmental pollution caused by plastic waste has already been reported since the 1970s, and this material is resistant to natural biodegradation processes, so the accumulation of plastic waste in the environment is currently a major global problem.

[0007] In response to this problem, efforts to reduce plastic waste by disposing of plastics through separate collection and recycling are being strengthened. However, despite these efforts, only a small fraction of all plastics produced worldwide are currently being successfully addressed. The rest are released into the environment and are disposed of either in landfills, incinerated, or flow into the ocean, causing water pollution. All of these waste disposal strategies contribute to environmental pollution.

[0008] Even if plastics in the environment are broken down by photodepolymerization, biodepolymerization, and thermal oxidative depolymerization, and further by friction, such biodegradation can take a long time, ranging from 50 to over 100 years.

[0009] As a result, scientists began to consider ways to efficiently and quickly deal with the massive accumulation of plastics, compared to the aforementioned plastic waste disposal strategies.

[0010] Many microorganisms have been found to be capable of producing enzymes that can break down certain polymers (Tsushima et al., 2010). Enzymes known to break down plastic polymers belong to the "hydrolase" class. Enzymes in this class are involved in catalytic reactions that cause the breakdown of chemical bonds in their substrates in the presence of water. Hydrolases capable of breaking down certain plastics can hydrolyze one or more chemical bonds in many commonly used plastics. Enzymes identified to date include esterases, lipases, PETases, laccases, polyuretanases, and cutinases. Enzymatic degradation of plastic polymers yields simpler fragments, oligomers, or monomeric units. Microorganisms can further assimilate these smaller units into microbial cells, leading to further enzymatic degradation and release of metabolites such as CO2, H2O, CH4, and N2 (Amobonye et al., 2021). Many of these enzymes have been isolated from algae, actinomycetes, bacteria, and fungi (Urbanek et al., 2020).

[0011] The extraction of these enzymes and their modification to enhance their enzymatic activity is one of the key research areas in addressing the ever-increasing plastic pollution. Many types of plastics, such as polyethylene (PE), polyethylene terephthalate (PET), polylactic acid (PLA), polybutylene succinate (PBS), and polyurethane (PU), have been reported to degrade slowly in the presence of these enzymes (Mukai et al., 1993).

[0012] Ferris et al., 2010, disclose the enzymatic degradation of copolyurethanes. The enzymes disclosed therein are two proteolytic enzymes (papain and α-chymotrypsin) and two esterase enzymes (cholesterol esterase and pancreatic lipase). The copolyurethane disclosed in this publication is 1,4-di-S-benzyl- D,L-A linear sulfur-containing copolyurethane obtained using a sulfur atom-containing precursor, dithiothreitol (DTTSBn), which also contains sulfur atoms, is not a PTUR, i.e., it is not a polymer that contains a thiourethane bond of the formula -SC(=O)-N(H)- in each repeating unit of the polymer chain.

[0013] The use of such enzymes not only makes it possible to mitigate the problem of increasing plastic pollution, but also allows for the recovery and reuse of metabolic by-products of plastic degradation (some of which are certainly harmful) to obtain the original polymer again and / or synthesize other chemicals. This circular economy model makes it possible to reduce the environmental impact of plastics and thereby increase the sustainability of plastic production.

[0014] Polythiourethane (PTUR) is a highly versatile polymer due to its excellent physical, mechanical, and optical properties.

[0015] PTUR can be synthesized, for example, by reacting a polythiol compound with a polyisocyanate or polyisothiocyanate compound.

[0016] The versatility of PTUR stems from the possibility of altering its physical properties through the modification of the physical properties of polythiol compounds and / or polyisocyanates or polyisothiocyanate compounds.

[0017] The primary application of this class of polymers is the manufacture of optical materials due to their excellent optical and mechanical properties. Compared to polyurethane polymers (PU), the incorporation of sulfur atoms into the polymer chain increases flexibility, crystallinity, and other useful properties. Furthermore, the increased refractive index makes PTUR more suitable for optical applications.

[0018] Like most polymers, PTUR is primarily manufactured from non-renewable resources, which is problematic considering the large production volume of PTUR.

[0019] One way to enhance the sustainability of PTUR production is to shift from the current linear PTUR production model to a more circular model. A circular model would minimize the negative environmental impact of PTUR (i.e., reduce the carbon footprint of PTUR) and address the end-of-life issues of this polymer.

[0020] A circular economy approach can be realized through the development of biological depolymerization methods for PTUR. The idea behind biological depolymerization is to obtain raw materials (monomers, oligomers, polymer fragments, and / or other chemicals) through an energy-efficient and environmentally friendly polymer degradation process, which can then be used to obtain the original PTUR again and / or synthesize other polymers or chemicals. [Overview of the Initiative] [Problems that the invention aims to solve]

[0021] Despite the widespread use of PTUR and the resulting industrial interest, no enzymatic methods for the degradation of PTUR have been reported to date.

[0022] Such a method would be very interesting because it would enable the reuse of large quantities of PTUR generated in industry, and thus contribute to reducing the environmental impact of PTUR production and use. [Means for solving the problem]

[0023] As described in the experimental section below, the inventors discovered that a specific enzyme can decompose polythiourethane.

[0024] In light of this finding, the present invention aims to solve some or all of the above-mentioned problems in the prior art by proposing an enzymatic decomposition method for polythiourethane.

[0025] It should be noted that at the time of filing of the present application, no enzyme or method for enzymatic decomposition of polythiourethane has been disclosed.

[0026] As used herein, "comprise", or variants such as "comprises" or "comprising", as well as "including", "include", "includes" and "included" and other variants are non-limiting.

[0027] When used before a numerical value, the term "about" indicates that the value can vary within a reasonable range such as ±10%, ±5%, and ±1%. The expression "about x" includes the value "x".

[0028] As used herein, the term "polymer" refers to a compound comprising repeating units linked by covalent chemical bonds.

[0029] As used herein, the term "repeating unit" refers to a portion of a polymer, wherein a complete polymer chain (excluding terminal groups) is produced by repeating this portion through linking the repeating units to each other along the chain.

[0030] This repeating unit shall not be confused with the term "monomer", which refers to a small molecule from which a polymer is synthesized. In this sense, for example, a repeating unit can also be produced from two or more types of monomers.

[0031] As used herein, the term "oligomer" refers to the polymer of the present invention consisting of 2 to 50 repeating units, preferably 2 to 20 repeating units.

[0032] The polymer according to the present invention is a polythiourethane. As used herein, the term "polythiourethane" refers to a polymer in which all repeating units of the polymer chain contain a thiourethane bond of the formula -SC(=O)-N(H)-.

[0033] Polythiourethane is also known in the literature under the name polythiocarbamate.

[0034] The polythiourethanes used in the present invention can be prepared by any known method, for example, by reacting a polythiol compound with a polyisocyanate compound or a polyisothiocyanate compound.

[0035] Polythiol compounds can be typical examples used in the synthesis of polythiourethanes.

[0036] More specifically, polythiol compounds are given by formula (I): R 1 (SH) n1 (I) It is a compound in which n1 represents an integer in the range of 2 to 6, and R 1 This represents an aliphatic group, alicyclic group, heterocyclic group, or aromatic group.

[0037] For example, a polythiol compound may include one or more compounds selected from the group consisting of the following: pentaerythritol tetrakis(3-mercaptopropionate), pentaerythritol tetrakis(2-mercaptoacetate), tris(3-mercaptopropionate)trimethylolpropane, tris(mercaptoacetate)trimethylolpropane, 2,3-bis((2-mercaptoethyl)thio)-1-propanthol, ethylene glycol bis(3-mercaptopropionate), 2-mercaptoethyl 2-mercaptoacetate, 2-mercaptoethyl 3-mercaptopropionate, 5,7-dimercaptomethyl-1,11-dimercapto-3,6,9-trithiaundecane, 4,7-dimercaptomethyl-1,11-dimercapto-3,6,9-trithiaundecane, 4-mercaptomethyl-1,8-dimercapto-3,6-dithiaoctane, 1,2-bis(2-mercaptoethylthio)-3-mercaptopropane, 2,5-bis(mercaptomethyl)-1,4-dithiane, bis(mercaptoethyl)sulfide, 1,1,3,3-tetrakis(mercaptomethylthio)propane, 4,6-bis (mercaptomethylthio)-1,3-dithiane, 2-(2,2-bis(mercaptomethylthio)ethyl)-1,3-dithiethane, 1,1,2,2-tetrakis(mercaptomethylthio)ethane, 3-mercaptomethyl-1,5-dimercapto-2,4-dithiapentane, and tris(mercaptomethylthio)methane, dipentaerythritol hexakis(3-mercaptopropionate), tris[2-(3-mercaptopropionyloxy)ethyl]isocyanurate, butylene glycol dimercaptopropionate, and ethanedithiol.

[0038] Preferably, the polythiol compound may be at least one selected from the group consisting of 1,2-bis(2-mercaptoethylthio)-3-mercaptopropane and pentaerythritoltetrakis(3-mercaptopropionate).

[0039] Polyisocyanates or polyisothiocyanate compounds may be typically used in the synthesis of polythiourethanes.

[0040] More specifically, polyisocyanates or polyisothiocyanates are given by formula (II): R 2 (NCX) n2 (II) It is a compound in which, in the formula, X represents O or S, n² represents an integer in the range of 2 to 6, and R 2 This represents an aliphatic group, alicyclic group, heterocyclic group, or aromatic group.

[0041] For example, a polyisocyanate compound or polyisothiocyanate compound may include one or more compounds selected from the group consisting of the following: toluene-2,4-diisocyanate, toluene-2,6-diisocyanate, diphenylmethane-4,4'-diisocyanate, diphenylmethane-2,4'-diisocyanate, paraphenylenediisocyanate, m-xylylenediisocyanate, biphenyl-diisocyanate, 3,3'-dimethyl-4,4'-diphenylenediisocyanate, tetramethylene-1,4-diisocyanate, hexamethylene-1,6-diisocyanate, 2,2,4-trimethylhexane-1,6-diisocyanate, lysine methyl ester diisocyanate, bis(isocyanatoethyl) fumarate, isophorone diisocyanate, etc. Diisocyanates, dodecane-1,12-diisocyanate, cyclobutane-1,3-diisocyanate, cyclohexane-1,3-diisocyanate, cyclohexane-1,4-diisocyanate, hexahydrotoluene-2,4-diisocyanate, tetramethylxylylene diisocyanate, perhydrodiphenylmethane-2,4'-diisocyanate, 4,4'-dicyclohexylmethane diisocyanate, bis(isocyanatomethyl)cyclohexane, dicyclohexylmethane diisocyanate, 2,5-bis(isocyanatomethyl)bicyclo-[2.2.1]-heptane, and 2,6-bis(isocyanatomethyl)bicyclo-[2.2.1]-heptane, naphthalene diisocyanate, pentamethylene diisocyanate, HDI trimers, and PDI trimers.

[0042] Preferably, the polyisocyanate compound or polyisothiocyanate compound may be at least one selected from the group consisting of m-xylylene diisocyanate and 2,5-bis(isocyanatomethyl)bicyclo[2.2.1]heptane.

[0043] In specific embodiments, the polythiourethane used in the present invention is either MR-7 (refractive index 1.67) or MR-8 (refractive index 1.60).

[0044] MR-7 is a polythiourethane that can be synthesized starting from the following: (i) m-xylylene diisocyanate (also known by the acronym "XDI"), and (ii) 1,2-bis(2-mercaptoethylthio)-3-mercaptopropane (also known by the alternative names 4-mercapto-methyl-3,6-dithia-1,8-octanedithiol and 2,3-bis((2-mercaptoethyl)thio)-1-propanthol).

[0045] MR-8 is a polythiourethane that can be synthesized starting from the following: (i) 2,5-bis(isocyanatomethyl)bicyclo[2.2.1]heptane (also known by the acronym "NDI", or by the alternative names norbornane-2,5-diylbis(methylene)diisocyanate, bicyclo[2.2.1]heptane, 2,5-bis(isocyanatomethyl)- and 2,5-bis(isocyanatomethyl)bicyclo-[2.2.1]-heptane), (ii) 1,2-bis(2-mercaptoethylthio)-3-mercaptopropane (also known by the alternative names 4-mercaptomethyl-3,6-dithia-1,8-octanedithiol and 2,3-bis((2-mercaptoethyl)thio)-1-propanethol), and (iii) Pentaerythritol tetrakis(3-mercaptopropionate).

[0046] Other polythiourethanes that can be enzymatically decomposed according to the method of the present invention are MR-6 and MR-10.

[0047] These optical materials and the monomers used in their preparation are described in particular in U.S. Patent Nos. 4,689,387, 4,775,733, 5,059,673, 5,087,758, and 5,191,055.

[0048] MR-6, MR-7, MR-8, and MR-10 are trademarks of Mitsui Chemicals, Inc.

[0049] The method of the present invention is applicable to any polythiourethane, regardless of the specific application considered. Advantageously, the polythiourethane decomposed according to the method of the present invention may originate from optical lenses.

[0050] Polythiourethane may contain at least one additive depending on the purpose, such as an internal release agent, reaction catalyst, heat stabilizer, UV absorber, blue cut agent (optionally associated with a color balancing agent), anti-reflective agent, mold release agent, colorant, antioxidant, color inhibitor, fluorescent whitening agent, and the like.

[0051] The polymers according to the present invention identify articles containing or derived from household, commercial, or industrial mixed or sorted waste flows or recovered plastic waste, and may feature any physical structure, for example, sheets, films, tubes, pipes, straws, rods, ropes, strings, lines, nets, mesh sheets, three-dimensional regular shapes, blocks, sheaths, fibers, membranes, woven and nonwoven fabrics, bags, containers, bottles, capsules, packaging, microspheres, (electrical)mechanical parts, clothing, coatings, and panels.

[0052] As used herein, the terms “enzymatic degradation,” “polymer degradation,” “depolymerization,” and “enzymatic hydrolysis” are used synonymously and refer to a chemical process in which a polymer material is degraded by one or more degrading enzymes that cleave one or more chemical bonds between the repeating units of the polymer. As a result of enzymatic degradation, degradation products are produced, which are the repeating units, monomers, oligomers, and / or fragments of the polymer.

[0053] The terms "complete decomposition" and "complete depolymerization" mean that the polymer according to the present invention is completely converted into the repeating units that constitute the polymer. Conversely, the terms "partial decomposition" and "partial depolymerization" mean that the polymer according to the present invention is only partially converted into the repeating units that constitute the polymer, that is, that a portion of the polymer is converted into oligomers and / or fragments.

[0054] As used herein, the term “degrading enzyme” refers to an enzyme capable of degrading polythiourethanes. This degrading enzyme may hydrolyze thiourethane bonds, ester bonds, or both.

[0055] According to the first embodiment, the present invention relates to a method for enzymatically digesting a polythiourethane having a thiourethane bond of the formula -SC(=O)-N(H)- in all repeating units of the polymer chain, wherein the molecule and the PTUR are degradable and the following: - This is enzyme 1, and its amino acid sequence is the following amino acid sequence: [ka] (Sequence ID 1 in the accompanying sequence listing) Enzyme 1, which shows at least 60% identity with, - This is enzyme 2, and its amino acid sequence is as follows: [ka] (Sequence ID 2 in the accompanying sequence listing) Enzyme 2, which shows at least 60% identity with, - This is enzyme 3, and its amino acid sequence is as follows: [ka] (Sequence ID 3 in the accompanying sequence listing) Enzyme 3, which shows at least 60% identity with, - Enzyme 4, which corresponds to lipase derived from Aspergillus oryzae. - Enzyme 5, which corresponds to the lipase derived from Thermomyces lanuginosus. - Enzyme 6, which corresponds to serine endopeptidase, - Enzyme 7, which corresponds to the protease derived from Bacillus licheniformis. - This is enzyme 8, and its amino acid sequence is as follows: [ka] (Sequence ID 4 in the accompanying sequence listing) Enzyme 8, which shows at least 60% identity with, - This is enzyme 9, and its amino acid sequence is as follows: [ka] (Sequence ID 5 in the accompanying sequence listing) Enzyme 9, which shows at least 60% identity with, - This is enzyme 10, and its amino acid sequence is as follows: [ka] (Sequence ID 6 in the accompanying sequence listing) Enzyme 10, which shows at least 60% identity with, - This is enzyme 11, and its amino acid sequence is as follows: [ka] (Sequence ID 7 in the accompanying sequence listing) Enzyme 11 exhibits at least 60% identity with [another enzyme]. The present invention relates to a method comprising contacting at least one enzyme selected from the group consisting of the following:

[0056] In the sense of the present invention, on the one hand, hydrolases or hydrolases capable of cleaving / hydrolyzing carbonate functional groups (e.g., lipase, protease, polymerase, cutinase, and esterase) are called degrading enzymes, and on the other hand, laccase is called a degrading enzyme.

[0057] The degrading enzymes selected from the group consisting of enzymes 1, 2, 3, 6, 8, 9, 10, and 11 may be of any suitable origin, for example, vegetable, animal, bacterial, fungal, and yeast origin. The origin may further be mesophilic or extremophilic (colophilic, cold-tolerant, thermophilic, barophilic, alkaliphilic, acidophilic, halophilic, etc.). In general, any microorganism capable of expressing the enzymes disclosed herein, whether natural or obtained by recombinant DNA technology and recombinant protein expression methods, may be suitable for carrying out the methods of the present invention. Preferably, the degrading enzymes originate from mesophilic organisms.

[0058] The enzymes used in this invention can, conveniently, be produced by recombinant technology using cell expression systems. Any suitable expression system can be used, such as expression systems involved in prokaryotic and eukaryotic cells. Suitable prokaryotic cells may be any Gram-positive or Gram-negative bacteria. Examples of Gram-positive bacteria include, but are not limited to, the following: Bacillus, Clostridium, Corynebacterium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, the following: Acinetobacter, Campylobacter, Escherichia coli (E. coli), Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma. Particularly preferred as heterologous hosts are strains of Escherichia coli (E. coli) that have been engineered to transport enzymes to the cell surface or extracellularly. Suitable eukaryotic cells may be selected from the group consisting of yeast, fungi, insect cells, mammalian cells, and plant cells.Suitable yeast cells may be selected from the following: Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), or Yarrowia. Suitable fungal cells may be selected from filamentous fungi, for example from the following genera: Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Muco, Miseliophtra. Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, and Trichoderma.

[0059] As used herein, the term “recombinant” refers to any type of substance produced by any technique of recombinant DNA technology, such as recombinant nucleic acid or polymerase chain reaction (PCR). Recombinant proteins are proteins produced by the expression of recombinant nucleic acid, and the enzymes disclosed in this application may be recombinant enzymes.

[0060] The enzymes used in this invention can also be similarly produced using other techniques known in the art, such as direct modification of isolated enzymes and direct protein synthesis.

[0061] Alternatively, the enzymes used in this invention may also be commercially available and used according to the manufacturer's instructions.

[0062] As used herein, the terms “sequence identity” or “identity” with respect to amino acid sequences define the number (or percentage, if expressed as a percentage) of identical amino acid residues between two polypeptide sequences. Sequence identity is determined by comparing sequences aligned to maximize overlap and identity while minimizing sequence gaps, and can be determined using any of the many alignment algorithms known to those skilled in the art. Specifically, sequence identity can be determined using a computer program (e.g., the BLASTP program, publicly available from NCBI and other sources) or direct sequence comparison.

[0063] In specific embodiments, the amino acid sequence of the enzyme implemented in the present invention is at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, and even 100% identical to the amino acid sequence described in the sequence provided herein, and has the same enzymatic function.

[0064] In other words, such enzyme variants include different variants whose sequences retain the enzymatic activity of the enzyme described in the sequences provided herein, and which are determined by the addition, deletion, or substitution of one or more amino acid residues. These variants may have one or more conservation changes in which the substituted amino acid has similar structural or chemical properties (e.g., substitution of leucine with isoleucine). The modifications may occur at the amino-terminal or carboxy-terminal position of the reference polypeptide sequence, or anywhere between these terminal positions, and may be individually scattered among the amino acids in the reference sequence, or scattered within one or more consecutive groups in the reference sequence.

[0065] In certain embodiments, the amino acid sequence of enzyme 1 implemented in the present invention exhibits at least 60% identity with the amino acid sequence of SEQ ID NO: 1, and may exhibit at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, and even 100% identity.

[0066] Advantageously, the amino acid sequence of enzyme 1 used in the method according to the present invention includes or consists of SEQ ID NO: 1.

[0067] In more specific embodiments, enzyme 1 is lipase A, preferably lipase A derived from Candida antarctica, as is the case in the present invention.

[0068] In more specific embodiments, the enzyme 1 used in the present invention is the commercially available lipase Novor® AD-L. In other words, the enzyme 1 used in the present invention is commercially available under the name Novor® AD-L, which is manufactured and supplied, for example, by Strem Chemicals, Inc. or Sigma Aldrich (Merck).

[0069] As used herein, the terms “commercial” and “commercially available” refer to an enzyme having a given activity measured in units U, manufactured by a company in an essentially pure form and available for purchase from said company, where U is a unit of the catalytic activity of the enzyme, and 1 U is defined as the amount of enzyme that catalyzes the conversion of 1 micromolar of substrate per molecule under the conditions specified by the assay method.

[0070] Enzyme 1, as implemented in this invention, is capable of hydrolyzing thiourethane bonds, as demonstrated by the formation of m-xylylenediamine (mXDA), which is a signature of thiourethane bond hydrolysis, among the degradation products.

[0071] Consequently, the enzyme 1 implemented in the present invention is capable of hydrolyzing thiourethane bonds in polythiourethane, and can be used for this purpose to hydrolyze thiourethane bonds present in the molecule, which may be any molecule (monomer, polymer, etc.) containing at least one thiourethane bond.

[0072] In certain embodiments, the amino acid sequence of enzyme 2 implemented in the present invention exhibits at least 60% identity with the amino acid sequence of SEQ ID NO: 2, and may exhibit at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, and even 100% identity.

[0073] Advantageously, the amino acid sequence of enzyme 2 used in the method according to the present invention includes or consists of SEQ ID NO: 2.

[0074] In more specific embodiments, the enzyme 2 implemented in the present invention is a pancreatic lipase, preferably a porcine pancreatic lipase.

[0075] In more specific embodiments, the enzyme 2 used in this invention is a commercially available porcine pancreatic lipase. For example, the enzyme 2 used in this invention is a commercially available porcine pancreatic lipase under product number "L3126," which is manufactured and supplied by Sigma-Aldrich (Merck).

[0076] Enzyme 2, as implemented in this invention, is capable of hydrolyzing thiourethane bonds, as demonstrated by the formation of m-xylylenediamine (mXDA), which is a signature product of thiourethane bond hydrolysis, among the degradation products.

[0077] Consequently, the enzyme 2 implemented in the present invention is capable of hydrolyzing thiourethane bonds in polythiourethane, and can be used for this purpose to hydrolyze thiourethane bonds present in the molecule, which may be any molecule (monomer, polymer, etc.) containing at least one thiourethane bond.

[0078] In certain embodiments, the amino acid sequence of enzyme 3 implemented in the present invention exhibits at least 60% identity with the amino acid sequence of SEQ ID NO: 3, and may exhibit at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, and even 100% identity.

[0079] Advantageously, the amino acid sequence of enzyme 3 used in the method according to the present invention includes or consists of SEQ ID NO: 3.

[0080] In more specific embodiments, the enzyme 3 implemented in the present invention is an esterase, preferably an esterase derived from porcine liver, and more preferably an esterase derived from porcine liver having CAS number 9016-18-6.

[0081] In more specific embodiments, the enzyme 3 used in the present invention is a commercially available esterase derived from porcine liver. For example, the enzyme 3 used in the present invention is a commercially available porcine liver esterase under product number "E3019," which is manufactured and supplied by Sigma-Aldrich (Merck).

[0082] In certain embodiments, the enzyme 4 used in the present invention is Aspergillus oryzae lipase with CAS number 9001-62-1. In more specific embodiments, the enzyme 4 used in the present invention is commercially available Aspergillus oryzae lipase.

[0083] In more specific embodiments, the enzyme 4 used in the present invention is commercially available Aspergillus oryzae lipase, marketed under product number "L0777," which is manufactured and supplied by Sigma-Aldrich (Merck).

[0084] In yet another, more specific embodiment, the enzyme 4 implemented in the present invention is the commercially available lipase Novozym® 51032 derived from Aspergillus oryzae. In other words, the enzyme 4 implemented in the present invention is commercially available under the name Novozym® 51032, which is manufactured and supplied, for example, by Novozymes A / S or Strem Chemicals, Inc.

[0085] In certain embodiments, the enzyme 5 used in the present invention is a lipase derived from Thermomyces lanuginosus.

[0086] In more specific embodiments, the enzyme 5 used in the present invention is the commercially available lipase Lipozyme® TL 100L. In other words, the enzyme 5 used in the present invention is commercially available under the name Lipozyme® TL 100L, which is manufactured and supplied, for example, by Strem Chemicals, Inc. or Sigma Aldrich (Merck).

[0087] In certain embodiments, the enzyme 6 used in the present invention is a serine endopeptidase, preferably a commercially available serine endopeptidase.

[0088] In more specific embodiments, the enzyme 6 used in this invention is commercially available Savinase® 16L. In other words, the enzyme 6 used in this invention is commercially available under the name Savinase® 16L, which is manufactured and supplied, for example, by Novozymes A / S or Strem Chemicals, Inc.

[0089] In certain embodiments, the enzyme 7 used in the present invention is a protease derived from Bacillus licheniformis.

[0090] In more specific embodiments, the enzyme 7 used in this invention is a commercially available protease derived from Bacillus licheniformis. For example, the enzyme 7 used in this invention is a Bacillus licheniformis protease commercially available under product number "P4860," which is manufactured and supplied by Sigma-Aldrich (Merck).

[0091] In certain embodiments, the amino acid sequence of enzyme 8 implemented in the present invention exhibits at least 60% identity with the amino acid sequence of SEQ ID NO: 4, and may exhibit at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, and even 100% identity.

[0092] Advantageously, the amino acid sequence of enzyme 8 used in the method according to the present invention includes or consists of SEQ ID NO: 4.

[0093] In a more specific embodiment, enzyme 8 belongs to the carboxylesterase / lipase enzyme family.

[0094] In certain embodiments, the amino acid sequence of enzyme 9 implemented in the present invention exhibits at least 60% identity with the amino acid sequence of SEQ ID NO: 5, and may exhibit at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, and even 100% identity.

[0095] Advantageously, the amino acid sequence of the enzyme 9 used in the method according to the present invention includes or consists of SEQ ID NO: 5.

[0096] In more specific embodiments, enzyme 9 belongs to the putative lactonizing lipase family.

[0097] In a preferred embodiment, the enzyme 9, as defined according to any of the above embodiments, is used in the method of the present invention in the form of a purified protein or crude lysate.

[0098] In certain embodiments, the amino acid sequence of the enzyme 10 implemented in the present invention exhibits at least 60% identity with the amino acid sequence of SEQ ID NO: 6, and may exhibit at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, and even 100% identity.

[0099] Advantageously, the amino acid sequence of the enzyme 10 used in the method according to the present invention includes or consists of SEQ ID NO: 6.

[0100] In a more specific embodiment, enzyme 10 belongs to the triglyceride lipase enzyme family.

[0101] In certain embodiments, the amino acid sequence of the enzyme 11 implemented in the present invention exhibits at least 60% identity with the amino acid sequence of SEQ ID NO: 7, and may exhibit at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, and even 100% identity.

[0102] Advantageously, the amino acid sequence of the enzyme 11 used in the method according to the present invention includes or consists of SEQ ID NO: 7.

[0103] In a more specific embodiment, enzyme 11 belongs to the alpha / beta hydrolase superfamily.

[0104] In a preferred embodiment, the enzyme 11, as defined according to any of the above embodiments, is used in the method of the present invention in the form of a crude lysate.

[0105] According to the present invention, two or more enzymes as defined above (i.e., enzymes 1 to 11) can be used simultaneously for the enzymatic degradation of PTUR. The two or more enzymes may interact in the reaction mixture, and the result of these interactions may be additive (i.e., the degrading activity of the enzymes is the sum of the enzyme activities if they acted separately) or synergistic (i.e., the degrading activity of the enzymes is greater than the sum of the enzyme activities if they acted separately). If three or more enzymes as defined above (i.e., enzymes 1 to 11) are present, both additive and synergistic effects may act simultaneously. In addition to the enzymes disclosed in the present invention, other enzymes may also be present in the reaction mixture.

[0106] In this invention, the decomposed polythiourethane can be in the form of a mixture of two or more types of polythiourethane.

[0107] Prior to enzymatic degradation according to the method of the present invention, the polythiourethane may be subjected to a pretreatment step aimed at promoting or accelerating enzymatic degradation by altering the physical state of the polymer. This pretreatment step physically alters the structure of the polymer, for example, by increasing the surface area available for contact with the degrading enzyme.

[0108] Preferably, the pretreatment process is performed before or during contact with the enzyme carried out in the present invention, i.e., as a pretreatment or co-treatment step. Preferably, the pretreatment is performed as a pretreatment step.

[0109] Pretreatment may include, for example, mechanical treatment of the polymer by a process selected from the following: washing, centrifugation, cleaning, impact, sorting, grinding, homogenization, shredding, cutting, impacting, crushing, shearing, shredding, rotation on a rotary drum, fractionation, ultrasonic treatment, melting, extrusion, spinning, liquefaction, maceration, micronization, pelletizing, pressurization by screw, pressurization by piston, and granulation, or a combination of two or more of the above methods. Grinding is particularly preferred, optionally in conjunction with one or more of the other pretreatment steps listed above.

[0110] Alternatively, or in addition, PTUR may be physically treated by a process selected from the following: irradiation, e.g., drying, UV irradiation, amorphous formation, aggregation, heating (e.g., microwave heating), cooling, freezing, dehydration, or a combination of two or more of the aforementioned methods.

[0111] In the method according to the present invention, PTUR may be brought into contact with at least one enzyme as defined above in any suitable manner, that is, in a manner that allows the enzyme to interact with PTUR so that it can exert degradation activity. Non-limiting examples of bringing an enzyme into contact with a polymer are as follows: - PTUR may be dispersed in an enzyme-containing solution. In this case, the polymer may be in any form, for example, added as a film, sheet, or granular material. Other polymer forms (e.g., molded articles) may be added as a whole or in a pulverized form. Coated or bonded materials, or materials with a polymer coating deposited on them (e.g., paper or cardboard, and coated paper or coated cardboard) may be added to the enzyme-containing solution as a whole or in a pulverized form; - Enzyme solutions can be sprayed onto PTUR: an enzyme-containing aqueous solution can be sprayed or deposited onto the polymer to be decomposed. In this case, the enzyme solution, regardless of its form, should completely cover the polymer. - PTUR can be brought into direct contact with enzyme-expressing organisms. Unpurified (cell lysate) or semi-purified culture supernatant containing the enzyme can also be brought into contact with the polymer.

[0112] Therefore, PTUR may be present in bulk or as a solution, preferably as an aqueous solution or water. The aqueous solution required to carry out the method according to the present invention may be buffered. Since the activity of each enzyme is optimal at different pH and temperature values, the pH and temperature are determined by the specific enzyme used. The pH is generally 2 to 12, preferably 5 to 9, and most preferably 6 to 8. The temperature at which enzymatic decomposition is carried out is generally 5°C to 60°C; preferably 20°C to 50°C, and most preferably 30°C to 50°C. Non-aqueous solvent / water mixtures may also be used as solvents. The non-aqueous solvent may be an organic solvent, a deep eutectic solvent, or a natural deep eutectic solvent.

[0113] The following are examples of buffers that may be used in accordance with the present invention: citrate, acetate, phosphate, formate, carbonate, tris-hydroxymethylaminomethate, triethanolamine, imidazole, oxalate, tartrate, fumarate, maleate, phthalate, succinate, and ethylenediamine, and a selection thereof. Preferably, acetates and phosphates are used as buffers.

[0114] PTUR can be contacted with the enzyme defined above at a certain concentration and temperature for a period of time sufficient to achieve the desired amount of degradation. Suitable time ranges from several hours to several days and can be selected to achieve the desired amount of conversion. Examples of reaction times include 1 day, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days. In some embodiments, the reaction time may be one week or several weeks. Those skilled in the art can determine an appropriate reaction time by referring to the physical state of the polymer, the selected enzyme, and the desired degree of degradation.

[0115] This method may include stirring the reaction mixture to improve contact between the enzyme and the polymer, thereby promoting the adsorption of the enzyme onto the PTUR. This step may include, for example, continuous stirring at a speed of 100 revolutions per minute (rpm) to 5000 rpm.

[0116] Chemical elements and / or compounds that may be necessary for or may have a positive effect on the enzymatic reaction may also be added to the reaction mixture. Preferably, these chemical elements and / or compounds are selected and added to the reaction mixture to have a positive effect on the enzyme activity. Examples of chemical elements and / or compounds that have a positive effect on enzyme activity include, but are not limited to, metal ions (e.g., sodium or calcium ions) and salts (e.g., added to adjust the ionic strength of the solution).

[0117] The entire process can be carried out in any suitable vessel (for example, a large reaction vessel equipped with a mixer and surrounded by a heating mantle to regulate the temperature).

[0118] The enzyme concentration should be sufficient to quantitatively degrade the polymer. This degradation may be partial or complete. The amount of enzyme required to carry out the method of the present invention can be readily determined by those skilled in the art, in particular, by referring to the relative amount of the polymer and / or the number of any additional enzymes present in the reaction mixture.

[0119] For example, in a preferred embodiment, the enzyme used in the present invention is used in an amount of up to 5% by weight of PTUR, more preferably in an amount of up to 1% by weight of the polymer, even more preferably in an amount of up to 0.1%, and even more preferably in an amount of up to 0.05%. For example, the amount of the enzyme of the present invention may be in the range of 0.001% to 5% by weight of the polymer, preferably in the range of 0.002% to 1% by weight of the polymer, more preferably in the range of 0.003% to 0.1%, and even more preferably in the range of 0.005% to 0.05%.

[0120] According to another embodiment of the method according to the present invention, at least one of the previously defined degrading enzymes is used in the form of a pure protein, a crude lysate, or an immobilized form on a carrier.

[0121] In this specification, “pure form” is used to mean “substantially pure form” (i.e., purity that enables effective use of the protein in the methods described herein). For the protein to be most useful in the methods of the present invention or any method utilizing the enzyme described herein, in most cases the protein is substantially free of impurities, other proteins, and / or chemicals that may interfere with or would interfere with its use in the present method (e.g., which may interfere with enzyme activity). This form may also be known as a “cell-free lysate” obtained after centrifugation and filtration of the lysate obtained during classical protein overexpression.

[0122] As used herein, the expression “substantially pure form” means that the enzyme should be at least 90% pure, preferably 95% pure, and more preferably 98% pure.

[0123] As used herein, the terms “crude lysate” and “cell lysate” are synonymous and can be used interchangeably. Both terms refer to specific products obtained during the overexpression of classical proteins.

[0124] Specifically, the crude lysate is obtained when host cells (e.g., Escherichia coli cells) are overexpressed with the target protein in culture medium, and then lysed using one of the methods known in the art to release the soluble overexpressed protein present in the cells into the supernatant.

[0125] Depending on the type of host cell and the amount of host cell to be lysed, various methods may be used. Examples of cell lysis methods include liquid shear pressure techniques (e.g., French press and homogenizer), sonication, freezing and thawing (e.g., in combination with enzymatic lysis using lysozyme), glass beads, osmotic shock, and chemical lysis (e.g., by using detergent).

[0126] Despite containing proteins in an impure form, cell lysates can, in certain cases, be effectively used as a substitute for their pure enzyme forms, as all cellular debris and chemicals remain present. This can occur, for example, when the enzyme concentration is particularly high. The main advantage of using cell lysates is that it avoids further steps of protein purification, which require additional time and increase the cost of the degradation process.

[0127] According to the present invention, enzymes may be present in an immobilized form in a composition. Immobilization is often performed to improve the stability of the enzyme. In this case, the enzyme is immobilized on a suitable carrier by any immobilization method. For example, the enzyme can be immobilized on a solid carrier, matrix, or particles (e.g., glass beads, and minerals, polymers, or metal particles, or a matrix selected from these).

[0128] The immobilization method is based on either physical techniques such as adsorption, capture, and encapsulation, or chemical bonding processes such as covalent bonding and crosslinking.

[0129] The reaction products of enzymatic degradation carried out according to the method of the present invention include one or more compounds selected from at least a compound exhibiting a thiol functional group, at least a compound exhibiting an amine functional group, at least one compound exhibiting a carboxyl functional group, at least an alcohol functional group, and CO2. One or more of these compounds can be identified by suitable analytical techniques to evaluate the course of the reaction. Therefore, by identifying one or more of such compounds, it becomes advantageously possible to determine whether or not enzymatic degradation has occurred.

[0130] Compounds exhibiting at least a thiol functional group can be conveniently measured, for example, by high-performance liquid chromatography (HPLC) after or after derivatization with benzoyl chloride (BzCl), or by using an Elmann assay based on the use of Elmann's reagent (5,5'-dithiobis-(2-nitrobenzoic acid) or DTNB). Other techniques known to those skilled in the art can also be used.

[0131] Compounds exhibiting at least an amine functional group can be easily detected, for example, by high-performance liquid chromatography (HPLC) after or after derivatization with benzoyl chloride (BzCl). Other techniques known to those skilled in the art can also be used in a similar manner.

[0132] Compounds exhibiting at least one carboxyl functional group can be easily detected, for example, by a bromothymol blue assay (described in the experimental section below). Other techniques known to those skilled in the art can also be used.

[0133] Compounds exhibiting at least an alcohol functional group can be easily detected, for example, by gas chromatography-mass spectrometry (GC-MS) or HPLC-MS. Other techniques known to those skilled in the art can also be used.

[0134] CO2 can be easily detected, for example, by a bromothymol blue assay. Other techniques known to those skilled in the art can also be used.

[0135] It will be understood that the enzymatic degradation by enzymes according to the method of the present invention does not require the complete depolymerization of the polymer, and that the nature and extent of the degradation can be determined in particular by the selected enzyme, reaction conditions, and the chemical properties and / or physical form of the polymer.

[0136] More specifically, factors that control the rate at which polymer materials decompose include enzyme concentration, the enzymatic reaction rate for polymer degradation, the polymer matrix, the polymer shape (for example, changes in this shape can affect the rate of substrate transport into and out of the material), the environment in which the reaction occurs (for example, this environment can be influenced by temperature, pH, ionic strength, and other factors that alter enzyme activity), and the fluid dynamics of the external environment. Other factors that can alter the rate of polymer degradation are readily available to those skilled in the art.

[0137] Therefore, it is clear from the above that enzymatic degradation mediated by the enzyme according to the present invention can generate fragments, monomers, and / or oligomers of PTUR.

[0138] Consequently, the method according to the present invention may further include the step of fractionating, isolating, or purifying the obtained degradation products. Exemplary methods for the purification / isolation / separation of degradation products include: filtration, distillation, solvent extraction (e.g., liquid / liquid extraction), precipitation, crystallization, evaporative concentration, evaporative crystallization, affinity chromatography, ion exchange chromatography, solvent extraction, centrifugation, electrophoresis, electrodialysis, hydrophobic interaction chromatography, gel filtration chromatography, reversed-phase chromatography, differential solubilization, high-performance liquid chromatography (HPLC), and / or reversed-phase HPLC, as well as combinations of two or more of the above methods.

[0139] The reaction product can also be recovered and reused to obtain other compounds of interest.

[0140] Therefore, the present invention relates to the use of already defined methods for generating fragments, oligomers, monomers, or repeating units derived from polythiourethane that can be recovered and reused to obtain the original polythiourethane.

[0141] According to another embodiment, the present invention also relates to a kit for the enzymatic degradation of polythiourethanes having thiourethane bonds of the formula -SC(=O)-N(H)- in all repeating units of the polymer chain, comprising at least one already defined enzyme.

[0142] According to another embodiment, the kit further comprises at least one reagent for the decomposition reaction.

[0143] The kit according to the present invention may optionally include at least one other reagent (e.g., buffers, salts, enzyme cofactors, and the like) required for enzymatic degradation. The kit may further include one or more controls.

[0144] This kit may also include other components. The various components of this kit are optionally provided in suitable containers. This kit may further include containers for holding or storing polymers. If necessary, this kit may also optionally include reaction vessels, mixing vessels, and other components that facilitate the preparation of reagents and / or polymers.

[0145] Other features and advantages of the present invention will become apparent from the following detailed description with reference to the accompanying drawings. [Brief explanation of the drawing]

[0146] [Figure 1]Determination of the degradation activity of several of the commercially available enzymes claimed in this invention against substrate A. Determination of the degradation activity of enzymes 1, 2, 3, 4(1), 4(2), 5, 6, and 7 against substrate A by bromothymol blue (BTB) high-throughput assay. Substrate control (SC) means that only the optical density (OD) of the substrate is measured (i.e., measured in the absence of any enzyme). All reactions and spectrophotometric measurements were carried out under identical experimental conditions (i.e., solvent, buffer, BTB, temperature, etc.) as detailed in the Materials and Methods section. In all cases, OD was measured immediately after mixing all reagents under appropriate conditions (t=0h) and 24 hours later (t=24h). [Figure 2] Determination of the degradation activity of several of the commercially available enzymes claimed in this invention against substrate B. Determination of the degradation activity of enzymes 1, 2, 3, 4(1), 4(2), 5, 6, and 7 against substrate B by bromothymol blue (BTB) high-throughput assay. Substrate control (SC) means that only the optical density (OD) of the substrate is measured (i.e., measured in the absence of any enzyme). All reactions and spectrophotometric measurements were carried out under identical experimental conditions (i.e., solvent, buffer, BTB, temperature, etc.) as detailed in the Materials and Methods section. In all cases, OD was measured immediately after mixing all reagents (t=0h) and 24 hours later (t=24h) under appropriate conditions. [Figure 3]Determination of the degradation activity of the claimed "Inhau" enzymes of the present invention against substrate A. Determination of the degradation activity of enzymes 8, 9, 10, and 11 against substrate A by bromothymol blue (BTB) high-throughput assay. Substrate control (SC) means that only the optical density (OD) of the substrate is measured (i.e., measured in the absence of any enzymes). As a positive control (PC), the degradation activity of enzyme 4(2) against the substrate is measured. All reactions and spectrophotometric measurements were carried out under identical experimental conditions (i.e., solvent, buffer, BTB, temperature, etc.) as detailed in the Materials and Methods section. In all cases, OD was measured immediately after mixing all reagents under appropriate conditions (t=0h) and 24 hours later (t=24h). [Figure 4] Determination of the degradation activity of the claimed "Inhau" enzymes of the present invention against substrate B. Determination of the degradation activity of enzymes 8, 9, 10, and 11 against substrate B by bromothymol blue (BTB) high-throughput assay. A substrate control (SC) means that only the optical density (OD) of the substrate is measured (i.e., measured in the absence of any enzymes). As a positive control (PC), the degradation activity of enzyme 4(2) against the substrate is measured. All reactions and spectrophotometric measurements were carried out under identical experimental conditions (i.e., solvent, buffer, BTB, temperature, etc.) as detailed in the Materials and Methods section. In all cases, OD was measured immediately after mixing all reagents (t=0h) and 24 hours later (t=24h) under appropriate conditions. [Modes for carrying out the invention]

[0147] Materials and methods Bromothymol blue (BTB) high-throughput assay for screening and selection of the enzyme with the highest degradation activity, using two model substrates referred to as "Substrate A" and "Substrate B". A high-throughput BTB assay was used to determine the enzyme with the highest degradation activity. This assay utilizes bromothymol blue, a pH indicator, and therefore the color of bromothymol blue changes depending on the pH of the reaction mixture. In the presence of the degrading enzyme, substrates containing CO2 and 3-mercaptopropanoic acid (released after hydrolysis of ester bonds in substrates A and B) are degraded in various products, which have the effect of lowering the pH of the reaction mixture. With respect to CO2, this is explained by the formation of carbonic acid, shown in the following formula. Therefore, this assay makes it possible to easily detect CO2 and 3-mercaptopropanoic acid released during enzymatic degradation. [ka]

[0148] Since the enzymatic degradation of PTUR is a slow process, a large number of enzymes were screened in a short period of time using model substrates that are structurally and functionally similar to PTUR. The selected model substrates are substrate A and substrate B. The chemical formula of substrate A is: [ka] The chemical formula of substrate B is, [ka] That is the case.

[0149] Various parameters of the BTB assay, such as maximum absorption, BTB concentration range, and buffer concentration, were optimized (Shimada and Hasegawa, 2017). After optimizing these parameters, a bicarbonate calibration curve was obtained (Ocal et al., 2021). Using this calibration curve obtained with the optimized parameters (5 mM sodium phosphate buffer, pH=7.0, 135 μM BTB concentration), the enzyme's degradation activity against substrates A and B was evaluated.

[0150] In addition to the BTB assay, high-throughput enzyme screening can be performed using an Elmann reagent-based high-throughput assay for free thiol estimation, and HPLC-UV based detection after BzCl derivatization for primary amine estimation.

[0151] Table 1 lists the 11 enzymes that exhibit the highest degradation activity against substrates A and B, as identified using the BTB assay.

[0152] [Table 1]

[0153] Conclusion: Regarding the screening of commercially available enzymes for their ability to degrade substrates A and B, the tests showed that all commercially available enzymes (enzymes 1-7) possess degrading activity against substrates A and B (Figures 1 and 2).

[0154] Regarding the screening of in-house enzymes for their ability to degrade substrates A and B, tests have shown that all in-house enzymes (enzymes 8-11) possess degrading activity against substrates A and B (Figures 3 and 4).

[0155] Cloning, overexpression, and purification of the "in-house" enzyme of the present invention As previously described, primers were selected, the genes were cloned, and the proteins were overexpressed in Escherichia coli (E. coli) (Vergne-Vaxelaire et al., Adv.Synth.Catal.2013,355,1763-1779). Each expression plasmid was transformed into Escherichia coli (E. coli) (e.g., E. coli BL21-CodonPlus(DE3)-RIPL). Cell culture, induction of isopropyl bD-thiogalactopyranoside (IPTG) for protein production, and cell lysis were performed as previously published (C.Guerard-Helaine et al. ChemCatChem 2015,7,1871-1879).

[0156] The enzyme was purified by loading cell-free extracts onto a Ni-NTA column (QIAGEN) according to the manufacturer's instructions. The elution buffer consisted of 50 mM phosphate (pH 7.5), 50 mM NaCl, 250 mM imidazole, and 10% glycerol, while the desalting buffer consisted of 50 mM phosphate (pH 7.5), 50 mM NaCl, and 10% glycerol.

[0157] The following examples refer to the 11 enzymes of the present invention listed in Table 1 above, which have been identified by a BTB high-throughput assay. [Examples]

[0158] Degradation activity of the enzyme of the present invention against MR-7 and MR-8 polymer cubes. Enzymes selected in BTB assays against substrates A and B (commercial crude lysates and purified proteins) were tested for their ability to degrade MR-7 and MR-8 polymer cubes (Hou et al., 2019). Several experiments were performed in which MR-7 or MR-8 polymer cubes were mixed with one of the selected enzymes in a 50 mM sodium phosphate buffer solution at pH 7. Two enzyme concentrations, either 200 or 2000 mU, were tested. The reaction mixture was incubated at 50°C with constant stirring (150 rpm). Every 7 days, the reaction mixture was removed and fresh buffer and enzyme were added. The reaction was stopped after 30 days.

[0159] The degradation activity of each enzyme was determined by calculating the weight loss of the polymer cube at the end of the reaction. This weight loss was expressed using the following formula:

number

[0160] The most significant weight loss, and therefore the most significant polymer degradation, is obtained by enzymes 4(2) and 9 for MR-7 polymers, and by enzyme 9 for MR-8 polymers.

[0161] References S. Tsushima, Y. Matsushita, Technical Report on the PCR-DGGE Analysis of Bacterial and Fungal Soil Communities, National Institute for Agro-Environmental Sciences, Tsukuba (2010). A. Amobonye, ​​P. Bhagwat, S. Singh, S. Pillai, Plastic biodegradation: frontline microbes and their enzymes, Sci. Total Environ., 759(2021), p.143536. AKUrbanek, AMMironczuk, A. Garcia-Martin, A. Saborido, I. Mata, M. Arroyo, Biochemical properties and biotechnological applications of microbial enzymes involved in the degradation of polyester-type plastics, BBA - Proteins and Proteomics, 1868(2020), p.140315. K. Mukai, Y. Doi, Y. Sema, K. Tomita, Substrate specificities in hydrolysis of polyhydroxyalkanoates by microbial esterases, Biotechnol. Lett., 15(1993), pp.601-604. Cristina Ferris et al.,”Dithiothreitol-based polyurethanes.Synthesis and degradation studies”,POLYMER DEGRADATION AND STABILlTY,vol.95,no.9,1 September 2010,pages 1480-1487. Toru Shimada and Takeshi Hasegawa,Determination of equilibrium structures of bromothymol blue revealed by using quantum chemistry with an aid of multivariate analysis of electronic absorption spectra,Spectrochimica Acta Part A:Mol.Biomol.Spectr.,185,2017,104-110. Nazim Ocal,Auraelie Lagarde,Melanie L’enfant,Franck Charmantray,and Laurence Hecquet,High-Throughput Solid-Phase Assay for Substrate Profiling and Directed Evolution of Transketolase,ChemBioChem,2021,22,1-8. Zhipeng Hou,Wei Zhang,Jing Guob,Zhangpei Chena,Jianshe Hua,Liqun Yang,The in vitro enzymatic degradation of poly(trimethylene carbonate-co-2,2’-dimethyltrimethylene carbonate),Eur.Polym.J.,2019,112,51-59. Vergne-Vaxelaire et al.,Nitrilase Activity Screening on Structurally Diverse Substrates:Providing Biocatalytic Tools for Organic Synthesis,Adv.Synth.Catal.2013,355,1763-1779. Christine Guerard-Helaine et al.,Genome Mining for Innovative Biocatalysts:New Dihydroxyacetone Aldolases for the Chemist’s Toolbox, ChemCatChem,2015,7,1871-1879.

Claims

1. A method for enzymatically degrading polythiourethane (PTUR), in which a thiourethane bond of the formula -S-C(=O)-N(H)- is contained in all repeating units of the polymer chain, comprising the molecule and a substance capable of degrading the PTUR and the following: - This is enzyme 1, and its amino acid sequence is as follows: 【Chemistry 1】 (Sequence ID 1 in the accompanying sequence listing) Enzyme 1, which exhibits at least 60% identity with, - This is enzyme 2, and its amino acid sequence is as follows: 【Chemistry 2】 (Sequence ID 2 in the accompanying sequence listing) Enzyme 2, which exhibits at least 60% identity with, - This is enzyme 3, and its amino acid sequence is as follows: 【Transformation 3】 (Sequence ID 3 in the accompanying sequence listing) Enzyme 3, which exhibits at least 60% identity with, - Enzyme 4, which corresponds to lipase derived from Aspergillus oryzae. - Enzyme 5, which corresponds to the lipase derived from Thermomyces lanuginosus. - Enzyme 6, which corresponds to serine endopeptidase, - Enzyme 7, which corresponds to the protease derived from Bacillus licheniformis. - This is enzyme 8, and its amino acid sequence is as follows: 【Chemistry 4】 (Sequence ID 4 in the accompanying sequence listing) Enzyme 8, which exhibits at least 60% identity with, - This is enzyme 9, and its amino acid sequence is as follows: 【Transformation 5】 (Sequence number 5 in the accompanying sequence listing) Enzyme 9, which exhibits at least 60% identity with, - This is enzyme 10, and its amino acid sequence is as follows: 【Transformation 6】 (Sequence number 6 in the accompanying sequence listing) Enzyme 10, which exhibits at least 60% identity with, - This is enzyme 11, and its amino acid sequence is as follows: 【Transformation 7】 (Sequence ID 7 in the accompanying sequence listing) Enzyme 11 exhibits at least 60% identity with A method comprising contacting at least one enzyme selected from the group consisting of the following.

2. The method for enzymatically degrading PTUR according to claim 1, wherein the enzyme 1 is lipase A derived from Candida antarctica.

3. The method for enzymatically degrading PTUR according to claim 1 or 2, wherein the enzyme 1 is a commercially available lipase Novocor® AD-L.

4. The method for enzymatically degrading PTUR according to any one of claims 1 to 3, wherein the enzyme 2 is porcine pancreatic lipase, preferably a commercially available porcine pancreatic lipase.

5. The method for enzymatically degrading PTUR according to any one of claims 1 to 4, wherein the enzyme 3 is an esterase derived from pig liver, preferably a commercially available esterase derived from pig liver.

6. The method for enzymatically degrading PTUR according to any one of claims 1 to 5, wherein the enzyme 4 is a commercially available lipase derived from Aspergillus oryzae.

7. The method for enzymatically degrading PTUR according to any one of claims 1 to 6, wherein the enzyme 4 is Novozym® 51032, a commercially available lipase derived from Aspergillus oryzae.

8. The method for enzymatically degrading PTUR according to any one of claims 1 to 7, wherein the enzyme 5 is a commercially available lipase, Lipozyme (registered trademark) TL 100L.

9. The method for enzymatically degrading PTUR according to any one of claims 1 to 8, wherein the enzyme 6 is commercially available Savinase® 16L.

10. The method for enzymatically degrading PTUR according to any one of claims 1 to 9, wherein the enzyme 7 is a commercially available protease derived from B. licheniformis.

11. The enzymatic degradation method for PTUR according to any one of claims 1 to 10, wherein the at least one enzyme is used in the form of a pure protein, a crude lysate, or the at least one enzyme is immobilized on a carrier.

12. The enzymatic decomposition method for PTUR according to any one of claims 1 to 11, wherein the polythiourethane is either MR-7 or MR-8.

13. Use of the method according to claim 12 for generating fragments, oligomers, monomers, or repeating unit derivatives derived from the polythiourethane that can be recovered or reused to obtain a polythiourethane.

14. A kit for enzymatic degradation of a polythiourethane having a thiourethane bond of the formula -S-C(=O)-N(H)- in all repeating units of the polymer chain, comprising at least one enzyme according to any one of claims 1 to 11.

15. The kit according to claim 14, further comprising at least one reagent for the decomposition reaction.

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