Enzyme for degrading ethylene terephthalate compounds

Abstract

Provided is a novel means capable of degrading an ethylene terephthalate oligomer. Specifically, provided is an isolated polypeptide having an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 1 and possessing MHET degradation activity.

Description

Ethylene terephthalate compound decomposing enzyme

[0001] A technology relating to an enzyme capable of decomposing ethylene terephthalate oligomers and the like is disclosed.

[0002] Plastics are inexpensive, durable, and versatile materials used in a wide range of products. However, their durability also contributes to environmental problems, with significant amounts of plastic accumulating in landfills and natural habitats around the world.

[0003] Various solutions have been investigated to reduce the environmental and economic impacts associated with the accumulation of plastics. For example, Patent Document 1 proposes using cutinase to decompose polyesters that make up plastics, but further improvements in the decomposition of plastics using enzymes are needed.

[0004] WO2012 / 099018

[0005] One challenge is to provide a new means for decomposing ethylene terephthalate oligomers.

[0006] The following representative means are provided: Item 1: An isolated polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 1 and having MHET decomposition activity. Item 2: An enzyme preparation comprising the polypeptide of Item 1. Item 3: An enzyme preparation comprising a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 1. Item 4: An enzyme preparation comprising an ethylene terephthalate compound represented by formula (1) (wherein n is 1 to 6, and A is H, CH ２ CH ２ OH, CH ２ CH ２ OCH ２ CH ２ OH, C ６ H ５ COOCH ２ CH ２ , C.H. ２ C ６ H ５ B is selected from the group consisting of CH ２ OH, CH ２ OCH２ CH ２ OH, CH ２ OCH ２ CH ２ OCH ２ CH ２ OH, CH ２ OOCC ６ H ４ COOH, CH ２ OOCC ６ H ５ , and C ６ H ５ Item 5: An enzyme preparation according to Item 2 or 3 for decomposing an ethylene terephthalate compound represented by the formula (1), wherein the enzyme preparation is selected from the group consisting of: (wherein n is 1 to 6, and A is H, CH ２ CH ２ OH, CH ２ CH ２ OCH ２ CH ２ OH, C ６ H ５ COOCH ２ CH ２ , C.H. ２ C ６ H ５ B is selected from the group consisting of CH ２ OH, CH ２ OCH ２ CH ２ OH, CH ２ OCH ２ CH ２ OCH ２ CH ２ OH, CH ２ OOCC ６ H ４ COOH, CH ２ OOCC ６ H ５ , and C ６ H ５ Item 6. A method for decomposing an ethylene terephthalate compound, comprising treating a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 1 with a compound represented by formula (1): (wherein n is 1 to 6, and A is H, CH ２ CH ２ OH, CH ２ CH ２ OCH ２ CH ２ OH, C ６ H ５ COOCH ２ CH ２ , C.H. ２ C ６ H ５ B is selected from the group consisting of CH ２ OH, CH ２ OCH ２ CH ２ OH, CH ２ OCH ２ CH ２ OCH ２ CH ２ OH, CH ２ OOCC ６ H ４ COOH, CH ２ OOCC ６ H ５ , and C ６ H ５ Item 7. A method for producing a degradation product of an ethylene terephthalate compound, comprising allowing an enzyme having polyethylene terephthalate decomposition activity to act on polyethylene terephthalate in the presence of the polypeptide of Item 1, thereby decomposing polyethylene terephthalate. Item 8. A method for decomposing polyethylene furanoate in the presence of the polypeptide of Item 1, thereby allowing an enzyme having polyethylene terephthalate decomposition activity to act on polyethylene furanoate. Item 9. A method for producing a degradation product of polyethylene terephthalate, by allowing an enzyme having polyethylene terephthalate decomposition activity to act on polyethylene terephthalate in the presence of the polypeptide of Item 1. Item 10. A method for producing a degradation product of polyethylene furanoate by allowing an enzyme having polyethylene furanoate decomposition activity to act on polyethylene furanoate in the presence of the polypeptide of Item 1.

[0007] A means is provided for enabling decomposition of ethylene terephthalate oligomers.

[0008] The figures show the results of investigating the relationship between monohydroxyethyl terephthalate (MHET) decomposition activity and reaction temperature for an enzyme having the amino acid sequence of SEQ ID NO: 3. The figures show the results of investigating the relationship between MHET decomposition activity and reaction pH for an enzyme having the amino acid sequence of SEQ ID NO: 3. The figures show the results of measuring the substrate specificity of three types of enzymes. The figures show the results of measuring the substrate specificity of IS5771. The figures show the results of measuring the decomposition ability of lipase, PETase, and other enzymes to decompose bis-2-hydroxyethyl terephthalate (BHET). (a) 1: Blank (control, i.e., no enzyme added); 2: Lipase PS Amano SD; 3: Lipase AK Amano; 4: Lipozyme CALB L; 5: Lipozyme TL 100 L; 6: Novocor AD L; 7: Palatase 20000 L; 8: Resinase HT; and 9: enzyme having the amino acid sequence of SEQ ID NO: 3. The figures show the results of measuring the decomposition ability of polyethylene furanoate (PEF) by combinations of enzymes. PETase represents IsPETase, and MHETase represents IsMHETase. The results are shown for the measurement of the ability of an enzyme having the amino acid sequence of SEQ ID NO: 1 to decompose monohydroxyethyl 2,5-furandicarboxylate (MHEF). NEC represents blank (conditions in which no enzyme was added). The results are shown for the measurement of the ability of an enzyme having the amino acid sequence of SEQ ID NO: 3 to decompose PET in combination with IsPETase. PETase represents IsPETase.

[0009] The polypeptide preferably has a certain level of identity to the amino acid sequence of SEQ ID NO: 1. "A certain level or more" means, for example, 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The amino acid sequence of SEQ ID NO: 1 is an amino acid sequence encoding a polypeptide derived from Ideonella sakaiensis, and this polypeptide has the activity of degrading various ethylene terephthalate compounds, etc., as shown in the Examples described below.

[0010] The identity of amino acid sequences can be evaluated by any means known in the art. For example, it can be calculated using an analysis tool that is commercially available or available via an electric communication line (Internet). For example, the identity of amino acid sequences can be calculated using the homology algorithm BLAST (Basic local alignment search tool) of the National Center for Biotechnology Information (NCBI) at http: / / www.ncbi.nlm.nih.gov / BLAST / , using default (initial setting) parameters.

[0011] The polypeptide preferably conserves the following amino acid residues of the amino acid sequence of SEQ ID NO: 1: G9, A10, W11, H76, S77, Y78, D101, F103, L111, N115, M123, K125, I137, F142, F164, F188, H190, F193, H215, and D216. These amino acid residues are thought to constitute the active site or substrate-binding site. In one embodiment, the polypeptide preferably also conserves several amino acid residues before and after these amino acid residues. The several amino acid residues may be, for example, three, two, or one amino acid residue. The amino acid residues to be conserved in order to maintain the enzymatic properties of a polypeptide having the amino acid sequence of SEQ ID NO: 1 can be identified by comparing the amino acid sequence of SEQ ID NO: 1 with the amino acid sequences of known enzymes having similar activities.

[0012] In one embodiment, when the amino acid sequence of the polypeptide has an amino acid residue substitution compared to the amino acid sequence of SEQ ID NO: 1, the type of the amino acid substitution is not particularly limited, but is preferably a conservative amino acid substitution. Examples of conservative substitutions include, but are not limited to, substitutions between basic amino acids (H, K, R), substitutions between acidic amino acids (D, E), substitutions between neutral nonpolar amino acids (A, V, L, I, P, F, M, W), substitutions between neutral polar amino acids (G, N, Q, S, T, V, C), substitutions between aromatic amino acids (W, F, H, Y), substitutions between nitrogen-containing amino acids (K, R, N, Q, P), substitutions between sulfur-containing amino acids (C, M), substitutions between oxygen-containing amino acids (S, T), substitutions between β-branched amino acids (V, L, I), and substitutions between amino acids (A, G) having a linear alkyl or hydrogen side chain.

[0013] The polypeptide has the formula (1) (wherein n is 1 to 6, and A is H, CH ２ CH ２ OH, CH ２ CH ２ OCH ２ CH ２ OH, C ６ H ５ COOCH ２ CH ２ , C.H. ２ C ６ H ５ B is selected from the group consisting of CH ２ OH, CH ２ CH ２ CH ２ OH, CH ２ CH ２ CH ２ OCH ２ CH ２ OH, CH ２ OOCC ６ H ４ COOH, CH ２ OOCC ６ H ５ , and C ６ H ５In one embodiment, n in formula (1) is preferably 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1. In one embodiment, the ethylene terephthalate compound is preferably an esterification reaction product of ethylene glycol and terephthalic acid (TPA), and is a compound having a number average degree of polymerization of ethylene terephthalate of 2 to 5. In one embodiment, the degree of polymerization is preferably 3 or 4.

[0014] In one embodiment, the polypeptide preferably has activity to decompose a compound other than the ethylene terephthalate compound represented by formula (1). Examples of such compounds include 2-hydroxyethyl benzoate and dibenzyl terephthalate (DBZT). In one embodiment, the polypeptide preferably has activity to further decompose PET degradation products, and the degradation products are preferably enzymatic degradation products. Examples of PET degradation products include, but are not limited to, bis(benzoyloxyethyl) terephthalate (BETEB), bis-2-hydroxyethyl terephthalate (BHET), monohydroxyethyl terephthalate (MHET), and 2-hydroxyethyl benzoate (HEB).

[0015] In another embodiment, the polypeptide preferably has activity to decompose a compound in which the structure corresponding to terephthalic acid in the above formula (1) is the structure of isophthalic acid, and n is 1 to 5, 1 to 4, 1 to 3, or 1 or 2.

[0016] In other embodiments, the polypeptide may have activity to degrade degradation products of other polyesters, including, but not limited to, polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoic acid (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), poly(ethylene adipate) (PEA), polyethylene naphthalate (PEN), and mixtures of these polymers.

[0017] In one embodiment, the polypeptide has excellent decomposition efficiency for BHET and / or MHET. In one embodiment, the polypeptide preferably has an optimum temperature for MHET decomposition activity of around 50°C. An optimum temperature of around 50°C means that the activity at 50°C is higher than both the activity at 40°C and the activity at 60°C. In one embodiment, the polypeptide preferably has an optimum pH for MHET decomposition activity of around 9. An optimum pH of 9 means that the activity at pH 9 is higher than both the activity at pH 8 and the activity at pH 10.

[0018] The polypeptide preferably further has an activity to further decompose a degradation product of polyethylene furanoate (PEF), and the degradation product is preferably an enzymatic degradation product. The degradation product of PEF is not particularly limited, but examples thereof include monohydroxyethyl 2,5-furandicarboxylate (MHEF) and bishydroxyethyl 2,5-furandicarboxylate (BHEF).

[0019] In one embodiment, the degradation product of PEF is represented by the following formula (2): (wherein n is 1 to 6, and A is H, CH ２ CH ２ OH, CH ２ CH ２ OCH ２CH ２ OH, C ６ H ５ COOCH ２ CH ２ , C.H. ２ C ６ H ５ B is selected from the group consisting of CH ２ OH, CH ２ OCH ２ CH ２ OH, CH ２ OCH ２ CH ２ OCH ２ CH ２ OH, CH ２ OOCC ６ H ４ COOH, CH ２ OOCC ６ H ５ , and C ６ H ５ In one embodiment, n in formula (2) is preferably 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1. In one embodiment, the ethylene furanoate compound is preferably an esterification reaction product of ethylene glycol and furandicarboxylic acid, and the number average degree of polymerization of ethylene furanoate is preferably 2 to 5. In one embodiment, the degree of polymerization is preferably 3 or 4.

[0020] The decomposition activity of a polypeptide for ethylene terephthalate compounds or ethylene furanoate compounds can be confirmed or measured by any method. For example, the polypeptide can be allowed to act on an ethylene terephthalate compound, the amount of ethylene terephthalate compound measured before and after the act, and the activity can be confirmed and measured based on the amount of ethylene terephthalate compound reduced. Alternatively, the polypeptide can be allowed to act on an ethylene terephthalate compound, the amount of degradation product measured before and after the act, and the activity can be confirmed and measured based on the increase in the amount of degradation product. More specifically, the activity can be confirmed and measured according to the methods employed in the Examples described below.

[0021] In one embodiment, the polypeptide is preferably an isolated polypeptide. An isolated polypeptide means that the polypeptide exists in a state different from the state in which it exists in nature. For example, it means that the polypeptide is separated from other substances that coexist when it exists in nature. In one embodiment, an isolated polypeptide means a purified polypeptide.

[0022] In one embodiment, the enzyme preparation preferably contains the polypeptide. The enzyme preparation may contain only the polypeptide, or may contain other components. Examples of other components include, but are not limited to, sugars, sugar alcohols, organic acid salts, amino acids, oligopeptides, proteins, surfactants, and polymers. Examples of sugars include glucose, sucrose, fructose, lactose, galactose, arabinose, ribose, melibiose, melezitose, dextrin, α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin. Examples of sugar alcohols include inositol, sorbitol, arabitol, xylitol, glucitol, ribitol, D-mannitol, and trehalose. Examples of organic acid salts include gluconate, citrate, and maleate. Examples of amino acids include alanine, serine, threonine, asparagine, glutamine, valine, leucine, isoleucine, alanine, serine, sodium L-glutamate, glycine, lysine, and arginine. Examples of oligopeptides include glycylglycine, alanylglutamine, glycylglutamine, and glutathione. Examples of proteins include bovine serum albumin (BSA), sericin, and proteins derived from HSP70 family proteins. Examples of surfactants include sodium cholate, Triton X-100, and Tween 20. Examples of polymers include polyvinylpyrrolidone. One or more of these other ingredients can be used in any combination. The enzyme preparation can be in the form of a liquid, powder, granules, or the like.

[0023] In one embodiment, the enzyme preparation is preferably an enzyme preparation for degrading at least one ethylene terephthalate compound represented by the above formula (1) or at least one ethylene furanoate compound represented by the above formula (2). Here, the at least one ethylene terephthalate compound or at least one ethylene furanoate compound is preferably a compound for which the above polypeptide has decomposition activity. In one embodiment, the enzyme preparation is preferably an enzyme preparation for degrading a compound other than the above ethylene terephthalate compound represented by the above formula (1).

[0024] The polypeptide may have an arbitrary amino acid sequence at the C-terminus and / or N-terminus of an amino acid sequence having a certain level of identity to SEQ ID NO: 1. The arbitrary amino acid sequence may be, for example, 30 amino acid residues or less, 25 amino acid residues or less, 20 amino acid residues or less, 15 amino acid residues or less, 10 amino acid residues or less, or 5 amino acid residues or less. In one embodiment, the enzyme may have a tag sequence (e.g., a His tag) at its C-terminus. In one embodiment, the polypeptide preferably has the oligopeptide CAASSPKNAEKK (SEQ ID NO: 5) at the N-terminus of the amino acid sequence of SEQ ID NO: 1. In another embodiment, the polypeptide preferably does not have the amino acid sequence of SEQ ID NO: 5 at the N-terminus of the amino acid sequence of SEQ ID NO: 1. An amino acid sequence having the amino acid sequence of SEQ ID NO: 5 at the N-terminus of the amino acid sequence of SEQ ID NO: 1 is represented by SEQ ID NO: 3. The amino acid sequence of SEQ ID NO: 3 is the amino acid sequence of a wild-type polypeptide derived from Ideonella sakaiensis. In one embodiment, the polypeptide preferably has an amino acid sequence having a certain level of identity to the amino acid sequence of SEQ ID NO: 3. Here, "a certain level or more" is the same as that described for the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 4 is one of the base sequences encoding the amino acid sequence of SEQ ID NO: 3.

[0025] The above-mentioned polypeptides can be obtained by any method. For example, they can be prepared by genetic engineering techniques. Specifically, they can be prepared by transforming a suitable host cell (e.g., Escherichia coli or yeast) with DNA encoding the polypeptide and recovering the protein expressed in the transformant (or the protein secreted outside the transformant). The recovered protein can be appropriately purified as needed. Examples of DNA encoding the polypeptide include the nucleotide sequence of SEQ ID NO: 2 or 4, or DNA having a certain level of identity thereto. "A certain level or higher" means, for example, 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0026] The polypeptide can be allowed to act on the ethylene terephthalate compound represented by formula (1) or the like, to decompose the compound and obtain a degradation product. The manner in which the polypeptide is allowed to act on the ethylene terephthalate compound or the like is not particularly limited, and can be carried out, for example, by adding the polypeptide and the polyethylene terephthalate compound or the like to an appropriate solvent (e.g., a buffer solution) and appropriately stirring the mixture. The buffer solution is not particularly limited, and examples thereof include bicine, glycine, MES, Bis-Tris, Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, HEPES, DIPSO, TAPSO, POPSO, HEPPSO, EPPS, Tricine, K ２ HPO ４ and Na ２ HPO ４ Examples of the suitable solution include a buffer solution, preferably a bicine or glycine buffer solution. The polypeptide can also be allowed to act on the ethylene terephthalate compound or the like by adding the polypeptide to the ethylene terephthalate compound or the like, or by adding the polypeptide to the material to be decomposed that has been buried in the ground.

[0027] The reaction conditions when reacting a polypeptide with an ethylene terephthalate compound or the like are not particularly limited and can be set appropriately depending on the purpose. The reaction temperature can be set, for example, in the range of 10°C to 70°C. In one embodiment, the reaction temperature is preferably 30°C or higher, 35°C or higher, 40°C or higher, or 45°C or higher, and is preferably 70°C or lower, 65°C or lower, or 60°C or lower. These lower and upper temperature limits can be combined in any desired manner.

[0028] The pH during the reaction can be appropriately set, for example, in the range of 4 to 12. In one embodiment, the pH during the reaction can be set to 5 or more, 6 or more, 7 or more, or 8 or more, and can be set to 11 or less, or 10 or less. These lower and upper limits of the pH can be appropriately combined to form a range.

[0029] The reaction time can be set appropriately depending on the purpose, reaction conditions, etc. For example, the reaction time can be set in the range of 1 hour or more to 1 week or less. In one embodiment, the reaction time can be 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 11 hours or more, 12 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, 3 days or more, 4 days or more, 5 days or more, or 6 days or more, and can be 7 days or less, 6 days or less, 5 days or less, 4 days or less, or 3 days or less. The lower and upper limits of these reaction times can be appropriately combined to form a range.

[0030] The concentration or amount of the polypeptide during the reaction can be appropriately set depending on the purpose. For example, the concentration of the polypeptide in the solvent can be appropriately set in the range of 10 nM to 5000 nM.

[0031] In one embodiment, the polypeptide is preferably allowed to act on PET or PEF or a plastic or resin containing them together with another enzyme capable of degrading PET or PEF. The other enzyme generates intermediate products from PET or PEF that can be degraded by the polypeptide, and the intermediate products can be further degraded by the polypeptide.

[0032] The other enzyme is optional, but examples include enzymes belonging to EC enzyme numbers 3.1.1.1 to 3.1.1.122. In one embodiment, the other enzyme is preferably a PET-degrading enzyme (PETase) belonging to Type I and a mutant enzyme thereof. Examples of Type I PETases having PET degradation activity include PETases derived from any of the following microorganisms: Micromonospora chokoriensis, Micromonospora lupini, Micromonospora citrea, Micromonospora halophilica, Micromonospora echinospora, Asanoa ishikariensis, Xiangella phaseoli, Verrucosispora maris, Nonomuraea jiangxiensis, Nonomuraea solani, Nonomuraea candida, and Actinoplanes. philippinensis, Actinoplanes globisporus, Cryptosporangium aurantiacum, Herbidospora sakaeratensis, Herbidospora daliensis, Actinomadura echinospora, Thermomonospora curvata, Actinomadura rubrobrunea, Hamadaea tsunoensis, Amycolatopsis sacchari, Amycolatopsis thermoflava, Prauserella rugosa, Rhodococcus rhodnii, Amycolatopsis regifaucium, Amycolatopsis orientalis, Amycolatopsis decaplanina, Actinomadura hibisca, Actinomadura latina, Streptosporangium amethystogenes, Streptosporangium roseum, Amycolatopsis pretoriensis, Amycolatopsis rifamycinica,Amycolatopsis mediterranei、Amycolatopsis vancoresmycina、Actinoalloteichus spitiensis、Kribbella flavida、Saccharothrix syringae、Lechevalieria xinjiangensis、Planomonospora sphaerica、Saccharomonospora azurea、Saccharomonospora cyanea、Saccharomonospora viridis、Amycolatopsis halophila、Actinoalloteichus hymeniacidonis、Nocardiopsis chromatogenes、Nocardiopsis alkaliphila、Nocardiopsis listeri、Nocardiopsis kunsanensis、Nocardiopsis halotolerans、Nocardiopsis dassonvillei、Actinopolyspora iraqiensis、Thermobifida halotolerans、Thermobifida alba、Thermobifida fusca。、

[0033] In one embodiment, the other enzyme may be a PETase classified as Type II. Examples of PETase classified as Type II include enzymes derived from any of the following microorganisms: Pseudomonas pseudoalcaligenes, Pseudomonas litoralis, Pseudomonas pachastrellae, Pseudomonas sabulinigri, Marinobacter segnicrescens, Oleispira antarctica, Marinobacter nanhaiticus, Pseudomonas alcaligenes, Vibrio gazogenes, Vibrio sp. CECT 9026, Burkholderiales bacterium, Polyangium brachysporum, Acidovorax delafieldii, Ideonella sakaiensis. Further examples include enzymes listed in the PET-degrading enzyme database (https: / / pazy.eu / doku.php?id=pet).

[0034] In a preferred embodiment, the other enzyme is selected from Ideonella sakaiensis-derived PETase (IsPETase), branch and leaf compost metagenomic-derived cutinase LCC, metagenomic-derived cutinase BhrPETase, and Humicola insolens-derived HiC, and mutant enzymes thereof.

[0035] The use of the above polypeptide in combination with another enzyme, PETase, can synergistically enhance the decomposition efficiency of plastics including PET or PEF. The conditions for combining the above polypeptide with another enzyme to decompose PET or PEF or plastics or resins containing them can be set based on the above-mentioned reaction conditions and the properties of the other enzyme.

[0036] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0037] 1. Preparation of Enzyme A polynucleotide having the nucleotide sequence of SEQ ID NO:4 was inserted into the NdeI (5' end) / XhoI (3' end) site of the pET-21b vector (Novagen, San Diego, CA) to prepare an enzyme expression vector. This vector was transformed into E. coli BL21(DE3) CodonPlus RIPL (Agilent Technologies), and the enzyme was expressed in the transformant by induction with 0.1 mM IPTG (16°C). The expressed enzyme has an amino acid sequence in which a tag sequence (LEHHHHHH (SEQ ID NO:6)) has been added to the C-terminus of the amino acid sequence of SEQ ID NO:3. The cells were collected by centrifugation (5,000 × g, 10 min, 4°C) and resuspended in binding buffer (50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5 mM imidazole). The resuspension was sonicated on ice and then centrifuged (10,000 × g, 20 min, 4°C) to collect the supernatant, which was then applied to TALON Metal affinity resin (Takara Bio). After washing off unbound proteins with washing buffer (50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 15 mM imidazole), the proteins bound to the column were eluted with elution buffer (50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 150 mM imidazole) and buffer exchanged into 50 mM NaHPO-HCl (pH 7.0) solution using a PD-10 gel filtration column (GE Healthcare, Piscataway, NJ). After concentration using an Amicon Ultra 10 kDa centrifugal filter (Merck Millipore), the concentration of the purified enzyme (also referred to as "IS5771") was determined based on the molar extinction coefficient at 280 nm.

[0038] 2. Temperature dependence 50mM Na ２ HPO ４ MHET at a final concentration of 300 μM and the enzyme (IS5771) at a final concentration of 2 nM were added to HCl (pH 7) (containing a final 10% (v / v) DMSO), and the mixture was allowed to react for 30 minutes at 20°C, 30°C, 40°C, 50°C, 60°C, or 70°C. A portion of the reaction solution after the reaction was diluted with 80% (v / v) 20 mM NaH ２ P.O.４ -H ３ P.O. ４ The solution was diluted with 20% (v / v) DMSO (pH 2.5) and heated at 85°C for 10 minutes. After centrifugation, a portion of the supernatant was subjected to HPLC. Standard solutions of known concentrations of TPA and MHET were used. The HPLC conditions were as follows: Apparatus: LC-2010A HT (Shimadzu Corporation) Column: Cosmosil 5C18-AR-II guard column, Cosmosil 5C18-AR-II column (Nacalai Tesque) Mobile phase: Methanol / 20 mM NaH2PO4-H3PO4 (pH 2.5) Flow rate: 1.0 mL / min Detection wavelength: 240 nm Elution conditions: 0-15 min: 25% (v / v) methanol, 15-25 min: 25-100% methanol gradient. The peak area of ​​the degradation product (TPA) separated and detected by HPLC was compared with that of the standard solution to quantify the degradation product under each condition. The results are shown in Figure 1. The enzyme was able to decompose MHET between 30°C and 60°C, with the optimal reaction temperature being around 50°C. The fact that this MHETase retains sufficient activity even at 60°C is in contrast to other MHETases derived from Ideonella sakaiensis, which lose their activity at 60°C (Palm, GJ, Reisky, L., Bottcher, D. et al. (2019) Nature Communications, 10, 1717).

[0039] 3. pH Dependence 300 μM MHET and a final concentration of 2 nM enzyme (IS5771) were added to buffer solutions of pH 5 to 10 (containing 10% (v / v) DMSO), and the mixture was incubated at 30°C for 30 minutes. The buffer solution consisted of 50 mM sodium citrate (pH 5.0), 50 mM MES-NaOH (pH 6.0), 50 mM Na ２ HPO ４The enzymes used were 50 mM Tris-HCl (pH 7.0), 50 mM Tris-HCl (pH 8.0), 50 mM glycine-NaOH (pH 9.0), and 50 mM CHES-NaOH (pH 10.0). Quantitation of the degradation products was carried out in the same manner as in 2 above. The results are shown in Figure 2. As a result, the enzyme can decompose MHET in the pH range of 6 to 10, and the optimum reaction pH is thought to be around 9.

[0040] The decomposition ability of PET film, BETEB, BHET, MHET, and HEB was measured. PET film (0.2 mm thick, 6 mm diameter), 1 mM BETEB, 1 mM BHET, 1 mM MHET, or 1 mM HEB was dissolved in 50 mM Na ２ HPO ４ After the reaction, a portion of the reaction solution was added to 80% (v / v) 20 mM NaHCO3. ２ P.O. ４ -H ３ P.O. ４ The enzyme was diluted with 20% (v / v) DMSO (pH 2.5) and heated at 85°C for 10 minutes. The supernatant was centrifuged and the degradation products were quantified by HPLC to evaluate the substrate specificity of the enzyme. HPLC was performed under the same conditions as in 2 above. In addition to the enzyme obtained in 1 above, the substrate specificity of IsPETase and IsMHETase derived from Ideonella sakaiensis was also evaluated in the same manner.

[0041] The decomposition ability of DBZT (dibenzyl terephthalate) was measured. ２ HPO ４ After the reaction, a portion of the reaction solution was added to 80% (v / v) 20 mM NaHCO3. ２ P.O. ４ -H ３ P.O. ４The enzyme's substrate specificity was evaluated by diluting the sample with 20% (v / v) DMSO (pH 2.5) and filtering the digested product, which was then quantified by HPLC. HPLC was performed under the following conditions: Apparatus: LC-2010A HT (Shimadzu Corporation); Column: Cosmosil 5C18-AR-II column (Nacalai Tesque); Mobile phase: 0.1% formic acid / acetonitrile; Flow rate: 1.0 mL / min; Detection wavelength: 240 nm; Elution conditions: 0-10 min: 5-90% (v / v) acetonitrile gradient, 10-15 min: 90% (v / v) acetonitrile; 15-15.1 min: 90-5% (v / v) acetonitrile gradient, 15.1-20 min: 5% (v / v) acetonitrile.

[0042] As shown in Figure 3, IsPETase was able to degrade PET and relatively large small substrates such as BETEB and BHET, but did not degrade oligomers smaller than MHET. On the other hand, IsMHETase degraded relatively small oligomers such as MHET and HEB, but not substrates larger than BHET. In contrast to these enzymes, the enzyme obtained in 1 above was found to have no degrading ability for PET, but completely degraded the small substrates BETEB, BHET, MHET, and HEB down to the monomer. Since it degraded BETEB, which has two benzoic acid groups attached to both ends of BHET via ester bonds, it is believed to be able to degrade longer PET oligomers as well. As shown in Figure 4, IS5771 degraded DBZT, producing monobenzyl terephthalate (MBZT) and TPA as degradation products. Its ability to degrade MBZT was also confirmed.

[0043] 5. Comparison of BHET Degradation Ability with Lipases and PET-Degrading Enzymes Commercially available lipases were used: Lipase PS Amano SD (Amano Enzyme), Lipase AK Amano (Amano Enzyme), Lipozyme CALB L (Novozymes), Lipozyme TL 100 L, NovoCor AD L (Novozymes), Palatase 20000 L (Novozymes), and Resinase HT (Novozymes). The PET-degrading enzymes used were FASTPETase, a mutant enzyme of IsPETase, HiC derived from Humicola insolens, and ICCG, a mutant enzyme of LCC.

[0044] BHET at a final concentration of 10 mM and either enzyme were added to 50 mM MES-NaOH (pH 7.0) to a final concentration of 1 μM (Resinase HT only: 30 μg / mL), and the reaction was carried out by inverting the solution (a) at 30°C for 30 minutes or (b) at 50°C for 30 minutes. The reaction solution contained 10% (v / v) DMSO. The solution after the reaction was collected, diluted 100-fold with DMF, and subjected to HPLC analysis under the following conditions. The 100-fold DMF dilution of the reaction solution before measurement was stored refrigerated and filtered through a 0.2 μm filter immediately before measurement. Standard samples of TPA, MHET, and BHET with known concentrations were also measured, and the degradation products were quantified by comparing the results. Apparatus: Chromaster (Hitachi) Column: Imtakt Cadenza CD-C18 3 μm 2 × 150 mm Mobile phase: 0.1% formic acid / acetonitrile Flow rate: 0.4 mL / min Detection wavelength: 258 nm Elution conditions: 0 to 30 min; 15% (v / v) acetonitrile

[0045] The results are shown in Figure 5. It was confirmed that all lipases hardly decomposed BHET (Figure 5(a)). It was confirmed that FASTPETase, HiC, and ICCG all decomposed BHET, but preferentially produced MHET as a degradation product (Figure 5(b)). In contrast, it was confirmed that the enzyme obtained in Example 1 (IS5771) rapidly and completely decomposed BHET to TPA. This enzyme has a particularly fast rate of decomposing MHET to TPA and is considered to be an excellent MHETase.

[0046] 6. Oligomer resolution 6-1. Preparation of PET oligomer 432 g of terephthalic acid (TPA) and 323 g of ethylene glycol (EG) were mixed and subjected to an esterification reaction at 250°C under pressure (0.25 MPa) for 108 minutes. ２ O ３ A PET oligomer mixture was prepared by adding 6.5 mL of a 12 g / L EG solution. 0.01 g of the resulting PET oligomer mixture was dissolved in 1 mL of HFIP, and the resulting sample was further diluted 100 times with DMF. HPLC analysis and LC / MS analysis were performed on the resulting sample. The resulting PET oligomer mixture had the composition shown in Table 1 below.

[0047]

[0048] In Table 1, "L2" is a molecule containing two TPA units, "L3" is a molecule containing three TPA units, and the same applies to "L4" to "L7". "L2" contains [TPA-EG] １ -TPA, [TPA-EG] ２ , EG-[TPA-EG] ２ , EG-[TPA-EG] ２ -EG, DEG- [TPA-EG] ２ -EG (DEG stands for diethylene glycol), etc.

[0049] 6-2. Obtaining Polypeptide (Enzyme) 6-2-1. Enzyme Expression The nucleotide sequence of SEQ ID NO: 2 was fused to a vector-derived Glutathione S-transferase (GST) tag. Specifically, the polypeptide of SEQ ID NO: 1 was cloned into the SmaI / NotI site of the pGEXM plasmid, transformed into Rosetta 2 DE3 competent cells by heat shock (42°C, 90 seconds), spread on an LB-Amp-Cam plate (100 mg / mL ampicillin, 35 μg / mL chloramphenicol), and incubated overnight at 37°C. pGEXM is described in a non-patent document (https: / / www.nature.com / articles / s41598-021-81409-y#Sec12). After growing the culture in 10 mL of LB-Amp-Cam medium at 37°C and 180 rpm until turbid, 1 mL of the seed culture was added to 100 mL x 2 LB-Amp-Cam medium and cultured overnight at 30°C and 140 rpm. A batch culture was prepared by adding 30 mL of the overnight culture to 2 L x 6 LB-Amp-Cam medium and culturing until the OD600nm reached 0.6-0.8. Expression of the target polypeptide (IS5771) was induced with 0.1 M IPTG, and the culture was incubated at 18°C ​​and 90 rpm for 21 hours. The cells were harvested and centrifuged at 4000 rpm at 20°C for 15 minutes. The supernatant was removed, and the pellet was stored at -30°C.

[0050] 6-2-2. Column chromatography using glutathione Sepharose 4B resin. The bacterial cell pellet from 4 mL of culture medium was dissolved in 300 mL of wash / lysis buffer consisting of 20 mM Tris-HCl (pH 7.4), 100 mM NaCl, and 1 mM DTT. The bacterial cells were sonicated (amp: 50%, pulse: 2 minutes on, 6 minutes off, time: 40 minutes), centrifuged at 30,000 rpm, 4°C, for 40 minutes, and the supernatant was collected. Protein purification was performed using affinity chromatography using Glutathione Sepharose 4B resin (GS4B). After equilibrating the column with 5x bed volume of wash / lysis buffer, the supernatant was applied to the column, and the target protein was eluted with an elution buffer containing 20 mM Tris-HCl (pH 7.4), 100 mM NaCl, 20 mM reduced glutathione, and 1 mM DTT.

[0051] 6-2-3. Ion Exchange Chromatography (IEC). Because the theoretical pI of GST-tagged IS5771 is calculated to be 6.1, a Tris-HCl buffer solution at pH 8.5 was used to ensure that the protein has a net negative charge and binds to positively charged moieties in the chromatography matrix. Anion exchange chromatography was performed using an AKTA Purifier connected to two HiTrap Q HP columns. The two columns were equilibrated with Buffer A (20 mM Tris-HCl pH 8.5, 1 mM DTT) and Buffer B (20 mM Tris-HCl pH 8.5, 1 M NaCl, 1 mM DTT), respectively. Chromatography was performed at a flow rate of 2 mL / min at 0.3 MPa, with an elution fraction volume of 5 mL. The bound target protein was eluted and collected with a salt gradient ranging from 50 mM to 400 mM.

[0052] 6-2-4. Cleavage of the GST tag from the target protein To cleave the GST tag from the target fusion protein, approximately 1 mg of PreScission Protease (Cytiva, USA) was added to the eluted fraction and incubated overnight at 8°C. The GS4B column was equilibrated with a buffer containing 10 mM HEPES-Na (pH 7.2), 100 mM NaCl, and 1 mM DTT, and the protein solution was then reapplied to remove the cleaved GST tag, and the flow-through fraction containing the target protein was collected. The resulting target protein (polypeptide) had the amino acid sequence of SEQ ID NO: 1, with two amino acid residues, GP, derived from the HRV3C protease cleavage site added to the N-terminus.

[0053] 6-2-5. Size Exclusion Chromatography (SEC) Proteins were concentrated to less than 5 mL using a 10-kDa Amicon ultracentrifugal filter. SEC was performed using an AKTA Purifier connected to a HiLoad Superdex 75-pg column (Cytiva, USA) pre-equilibrated with a buffer containing 10 mM HEPES-Na (pH 7.2), 100 mM NaCl, and 1 mM DTT. Approximately 4 mL of protein was applied, and chromatography was performed at a flow rate of 2 mL / min and 0.3 MPa, with an elution fraction volume of 5 mL.

[0054] 6-3. Evaluation of resolution ability A PET oligomer mixture with a final concentration of 15 mg / mL and IS5771 with a final concentration of 200 nM were added to 200 mM Bicine-NaOH (pH 9.0), and the mixture was allowed to react for 22 hours by inversion at 50°C. The condition without adding enzyme was used as the blank. The solution after the reaction was recovered, diluted 10-fold with HFIP, and then further diluted 100-fold with DMF, and subjected to HPLC analysis under the following conditions. Apparatus: Nexera XR (Shimadzu Corporation) Column: Imtakt Cadenza CD C18 3 μm 2 × 150 mm Mobile phase: (A) 0.1% formic acid / (B) acetonitrile 0 min (5% B) - 28 (100) - 33 (100) - 33.01 (5) - 38 (5) Flow rate: 0.4 mL / min Column temperature: 45 °C Injection volume: 5 μL Detection wavelength: 258 nm

[0055] The DMF diluted reaction solution was stored in a refrigerator and filtered through a 0.2 μm filter. Standard samples of TPA, MHET, and BHET with known concentrations were also analyzed, and the degradation products were quantified using a UV detector. PET linear dimers to heptamers (L2 to L7) were converted and quantified using BHET. The results are shown in Table 2.

[0056]

[0057] A comparison between the blank and the case where the enzyme was added confirmed that the enzyme (IS5771) significantly decomposed PET oligomers from dimer to pentamer, and was also able to decompose PET oligomers from hexamer.

[0058] 7. Decomposition of PEF A PEF film (0.2 mm thick, 5 mm x 5 mm) was dissolved in 50 mM Na ２ HPO ４ After adding the solution to 80% (v / v) 20 mM NaHCO3, IsPETase, IsMHETase, and IS5771 (obtained in step 6) were added singly or in combination to a final concentration of 50 nM, and the mixture was allowed to react at 30°C for 24 hours.２ P.O. ４ -H ３ P.O. ４ The sample was diluted with 20% (v / v) DMSO (pH 2.5) and heated at 85°C for 10 minutes, and a portion of the supernatant was centrifuged and analyzed by HPLC. The HPLC conditions were as follows. The peak areas of the decomposition products (FDCA and MHEF) separated and detected by HPLC were compared with the peak areas of standard solutions of each component at known concentrations to quantify the decomposition products under each condition. Apparatus: LC-2010A HT (Shimadzu Corporation) Column: Cosmosil 5C18-AR-II guard column, Cosmosil 5C18-AR-II column (Nacalai Tesque) Mobile phase: Methanol / 20 mM NaH2PO4-H3PO4 (pH 2.5) Flow rate: 1.0 mL / min Detection wavelength: 240 nm Elution conditions: 0 to 15 min: 25% (v / v) methanol, 15 to 25 min: methanol concentration gradient change from 25 to 100%

[0059] The results are shown in Figure 6. These results indicate that IsPETase has the ability to degrade PEF, but IsMHETase and IS5771 do not. Interestingly, the combination of IsPETase and IS5771 on PEF promoted PEF degradation more than the combination of PETase and IS5771. In addition, the amount of MHEF among the degradation products decreased, resulting in a doubling of FDCA production. This suggests that IS5771 has the ability to degrade MHEF. In contrast, the co-addition of IsPETase and IsMHETase resulted in little FDCA production, suggesting that these enzymes do not synergistically promote PEF degradation.

[0060] 8. MHEF Decomposition MHEF was dissolved in 50 mM NaCl to a final concentration of 300 μM. ２ HPO ４The mixture was added to HCl (pH 7.0) and the enzyme (IS5771 obtained in 6 above) was added to a final concentration of 100 nM, and the mixture was allowed to react at 30°C for 24 hours. The reaction solution was subjected to HPLC analysis in the same manner as in 7 above. As a result, as shown in Figure 7, approximately 64% of MHEF was decomposed and FDCA was produced. This confirmed that the enzyme (IS5771) has the ability to decompose MHEF.

[0061] 9. PET Degradation by Combined Enzymes A PET film (0.2 mm thick, 6 mm diameter) was added to 50 mM NaHPO-HCl (pH 7.0), followed by the addition of IsPETase at a final concentration of 50 nM and IS5771 (obtained in 1 above) at final concentrations of 5-50 nM (300 μL reaction volume). The reaction solution was analyzed as in 7 above, and the degradation products were quantified. As a result, as shown in Figure 8, the combined use of IsPETase and IS5771 increased TPA production compared to IsPETase alone. This is thought to be due to the rapid degradation of PET to MHET by IsPETase, followed by the rapid degradation of MHET by IS5771.

[0062] The results of tests 7 to 9 above confirmed that PET or PEF can be efficiently decomposed into TPA or FDCA by combining it with a PET-degrading enzyme such as IsPETase.

Claims

1. An isolated polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 1 and having MHET decomposition activity.

2. An enzyme preparation comprising the polypeptide of claim 1.

3. The ethylene terephthalate compound is represented by the formula (1) (wherein n is 1 to 6, and A is H, CH ２ CH ２ OH, CH ２ CH ２ OCH ２ CH ２ OH, C ６ H ５ COOCH ２ CH ２ , C.H. ２ C ６ H ５ B is selected from the group consisting of CH ２ OH, CH ２ CH ２ CH ２ OH, CH ２ CH ２ CH ２ OCH ２ CH ２ OH, CH ２ OOCC ６ H ４ COOH, CH ２ OOCC ６ H ５ , and C ６ H ５ The enzyme preparation according to claim 2, for decomposing an ethylene terephthalate compound represented by the formula:

4. A polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 1 is represented by the formula (1) (wherein n is 1 to 6, and A is H, CH ２ CH ２ OH, CH ２ CH ２ OCH ２ CH ２ OH, C ６ H ５ COOCH ２ CH ２ , C.H. ２ C ６ H ５ B is selected from the group consisting of CH ２ OH, CH ２ OCH ２ CH ２ OH, CH ２ OCH ２ CH ２ OCH ２ CH ２ OH, CH ２ OOCC ６ H ４ COOH, CH ２ OOCC ６ H ５ , and C ６ H ５ A method for decomposing an ethylene terephthalate compound, comprising: subjecting an ethylene terephthalate compound represented by the formula (I) to an action of a compound represented by the formula (I) on the compound.

5. A polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 1 is represented by the formula (1) (wherein n is 1 to 6, and A is H, CH ２ CH ２ OH, CH ２ CH ２ OCH ２ CH ２ OH, C ６ H ５ COOCH ２ CH ２ , C.H. ２ C ６ H ５ B is selected from the group consisting of CH ２ OH, CH ２ OCH ２ CH ２ OH, CH ２ OCH ２ CH ２ OCH ２ CH ２ OH, CH ２ OOCC ６ H ４ COOH, CH ２ OOCC ６ H ５ , and C ６ H ５ A method for producing a decomposition product of an ethylene terephthalate compound, comprising:

6. A method for decomposing polyethylene terephthalate, which comprises allowing an enzyme having polyethylene terephthalate decomposition activity to act on polyethylene terephthalate in the presence of the polypeptide of claim 1.

7. A method for decomposing polyethylene furanoate, comprising allowing an enzyme having polyethylene furanoate decomposing activity to act on polyethylene furanoate in the presence of the polypeptide according to claim 1.

8. A method for producing a degradation product of polyethylene terephthalate, comprising allowing an enzyme having polyethylene terephthalate decomposition activity to act on polyethylene terephthalate in the presence of the polypeptide of claim 1.

9. A method for producing a degradation product of polyethylene furanoate, which comprises allowing an enzyme having polyethylene furanoate decomposition activity to act on polyethylene furanoate in the presence of the polypeptide of claim 1.

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