Enzymatic degradation of pef

Abstract

The present invention provides a novel means for degrading PEF. Provided is a method for degrading polyethylene furanoate that includes causing an enzyme comprising a polypeptide which has an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1 and has one or more amino acid substitutions selected from the group consisting of H129W, F208S, A172C, and A209C to act on polyethylene furanoate.

Description

Enzymatic degradation of PEF

[0001] A technique for the enzymatic degradation of polyethylene furanoate (PEF) 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.

[0004] Polyethylene furanoate (hereinafter also referred to as "PEF") has properties similar to those of PET (polyethylene terephthalate), but when molded into films or bottles, it has higher barrier properties than PET. Furthermore, PEF can be produced from 100% bio-based raw materials. It can be produced using furandicarboxylic acid (FDCA), which is produced from bio-based carbohydrate raw materials, and bio-based ethylene glycol. Due to these characteristics, PEF is attracting attention as a replacement for PET.

[0005] WO2012 / 099018US10584320US11072784US11414651

[0006] Shosuke Yoshida, IFO Res. Commun. 36, p. 170, 2022; Joo et al., Nature Communications, 2018, 9: 382, ​​1-12; Austin et al., PNAS, vol. 115 no. 19, E4350-E4357

[0007] One challenge is to provide a new means for decomposing PEF.

[0008] The following representative inventions are provided: Item 1: A method for degrading polyethylene furanoate, comprising allowing an enzyme consisting of a polypeptide having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 1 and having one or more amino acid substitutions selected from the group consisting of H129W, F208S, A172C, and A209C to act on polyethylene furanoate. Item 2: The method of Item 1, in which the enzyme is allowed to act on polyethylene furanoate under conditions in which polyethylene furanoate and polyethylene terephthalate coexist. Item 3: The method of Item 1 or 2, in which the amino acid substitutions include at least H129W. Item 4: The method of any of Items 1 to 3, in which the enzyme is allowed to act on polyethylene furanoate at a temperature of 30°C or higher and lower than 60°C. Item 5: The method of any of Items 1 to 4, further comprising recovering one or more selected from the group consisting of 2,5-furandicarboxylic acid (FDCA), monohydroxyethyl 2,5-furandicarboxylate (MHEF), and bishydroxyethyl 2,5-furandicarboxylate (BHEF). Item 6. The method according to any one of Items 1 to 5, wherein the polyethylene furanoate is a plastic containing polyethylene furanoate and polyethylene terephthalate.

[0009] This allows for efficient decomposition of polyethylene furanoate.

[0010] The results of investigating the decomposition ability of wild-type and various mutant LCC enzymes on PET and PEF films are shown. The results of investigating the decomposition ability of wild-type and various mutant LCC enzymes on PEF films at 30°C ( FIG. 2( a) ) and 50°C ( FIG. 2( b) ) are shown. The results of investigating the decomposition ability of wild-type and mutant LCC enzymes on PET and PEF films at 50°C, 55°C, and 60°C over time are shown. (a) shows the result at 50°C, (b) shows the result at 55°C, and (c) shows the result at 60°C. The results of investigating the decomposition ability of mutant LCC enzymes on PET and PEF films under conditions in which both films coexist are shown. The results of investigating the decomposition ability of wild-type LCC, wild-type BhrPETase, and mutant BhrPETase on PET and PEF films are shown.

[0011] In one embodiment, the enzyme used in the method for degrading polyethylene furanoate (also referred to as "cutinase") has an amino acid sequence that has a certain level of identity to the amino acid sequence of SEQ ID NO: 1, and preferably has one or more amino acid substitutions selected from the group consisting of H129W, F208S, A172C, and A209C, from the perspective of "having improved degradation activity for PEF" and / or "having improved degradation activity for PEF compared to the degradation activity for PET (having high specificity for PEF)" compared to an enzyme (wild-type) that is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the substitution is preferably one or more selected from the group consisting of H129W, A172C, and F208S, preferably H129W and / or F208S, and preferably includes H129W.

[0012] The PEF degradation activity and PET degradation activity of the enzyme can be measured by the methods described in the Examples below. Degradation products of PEF include 2,5-furandicarboxylic acid (FDCA), monohydroxyethyl 2,5-furandicarboxylate (MHEF), and bishydroxyethyl 2,5-furandicarboxylate (BHEF). Degradation products of PET include terephthalic acid (TPA), monohydroxyethyl terephthalate (MHET), and bis-2-hydroxyethyl terephthalate (BHET).

[0013] In one embodiment, the enzyme used in the method for degrading polyethylene furanoate is a Type I enzyme having PET degradation activity (also referred to as "PETase"), preferably an enzyme having one or more amino acid substitutions selected from the group consisting of H129W, F208S, A172C, and A209C based on the amino acid sequence of SEQ ID NO: 1. Type I PETases are characterized by the absence of the additional disulfide bond found in Type II enzymes and the absence of the extended loop found in IsPETase. Type I PETases also have His and Phe / Tyr residues at positions corresponding to Trp159 and Ser238, respectively, of IsPETase (positions 129 and 208 in the case of SEQ ID NO: 1 without the signal peptide). Such enzymes preferably have an amino acid sequence that is at least somewhat identical to the amino acid sequence of a wild-type enzyme. In one embodiment, the substitution is preferably one or more selected from the group consisting of H129W, A172C, and F208S, preferably H129W and / or F208S, and preferably includes H129W.

[0014] Enzymes having PET decomposition activity classified as Type I include, for example, PETases derived from any of the following microorganisms: Micromonospora chokoriensis, Micromonospora lupini, Micromonospora citrea, Micromonospora halophytica, Micromonospora echinospora, Asanoa ishikariensis, Xiangella phaseoli, Verrucosispora maris, Nonomuraea jiangxiensis, Nonomuraea solani, Nonomuraea candida, Actinoplanes philippinensis, Actinoplanes globisporus, Cryptosporangium aurantiacum, Herbidospora sakaeratensis, Herbidospora daliensis, Actinoplanes echinospora, Thermomonospora curvata, Actinoplanes rubrobrunea, Hamadaea tsunoensis, and Amycolatopsis. sacchari, Amycolatopsis thermoflava, Prauserella rugosa, Rhodococcus rhodnii, Amycolatopsis regifaucium, Amycolatopsis orientalis, Amycolatopsis decaplanina, Actinodura hibisca, Actinodura latina, Streptosporangium amethystogenes, Streptosporangium roseum, Amycolatopsis pretoriensis, Amycolatopsis rifamycinica, Amycolatopsis mediterranei, Amycolatopsis vancoresmycina, Actinoalloteichus spitiensis, Kribbella flavida, Saccharothrix syringae, Lechevalieriaxinjiangensis, 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. (Non-patent document 2)

[0015] "H129W" means that the amino acid residue corresponding to position 129 in the amino acid sequence of SEQ ID NO: 1 (histidine "H" in SEQ ID NO: 1) has been substituted with tryptophan "W", and the amino acid residue before substitution does not necessarily have to be H. The same applies to other amino acid substitutions.

[0016] "A certain level or higher" means, for example, 60% or higher, 70% or higher, 80% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher.

[0017] In one embodiment, the amino acid sequence having a certain level of identity to the amino acid sequence of SEQ ID NO: 1 may be the amino acid sequence of SEQ ID NO: 15. In one embodiment, the enzyme may have a certain level of identity to the amino acid sequence of SEQ ID NO: 15 and the above-mentioned specific amino acid substitution.

[0018] 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.

[0019] When the amino acid sequence of the enzyme has one or more amino acid substitutions in addition to the above-mentioned specific amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1, the type of 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 with linear alkyl or hydrogen side chains (A, G).

[0020] The enzyme preferably has the activity to degrade PET in addition to the activity to degrade polyethylene furanoate.

[0021] The enzyme may have any amino acid sequence at the C-terminus and / or N-terminus of the amino acid sequence having a certain level of identity to SEQ ID NO: 1, as long as the enzyme retains its improved ability to degrade polyethylene furanoate. The any 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.

[0022] Enzymes can be obtained by any method. For example, they can be prepared by genetic engineering techniques. Specifically, enzymes can be prepared by transforming suitable host cells (e.g., Escherichia coli or yeast) with DNA encoding the enzyme and recovering the protein expressed in the transformant (or the protein secreted outside the transformant). The recovered protein is appropriately purified as needed. Examples of DNA encoding the enzyme include DNA having a certain level of identity to the base sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18. "A certain level or higher" means, for example, 60% or higher, 70% or higher, 80% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher.

[0023] SEQ ID NO: 2 is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 4 is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 having H129W (SEQ ID NO: 3). SEQ ID NO: 6 is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 having F208S (SEQ ID NO: 5). SEQ ID NO: 8 is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 having H129W and F208S (SEQ ID NO: 7). SEQ ID NO: 10 is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 having A172C and A209C (SEQ ID NO: 9). SEQ ID NO: 12 is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 having A172C, F208S, and A209C (SEQ ID NO: 11). SEQ ID NO: 14 is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 having H129W / F208S / A172C / A209C (SEQ ID NO: 13). SEQ ID NO: 16 is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 15. SEQ ID NO: 18 is a base sequence encoding the amino acid sequence of SEQ ID NO: 15 (SEQ ID NO: 17) having H129W.

[0024] Various modifications are possible using techniques for obtaining enzymes as recombinant proteins. For example, by inserting DNA encoding cutinase and other appropriate DNA into the same vector and using that vector to produce recombinant proteins, modifications such as the addition of sugar chains and / or lipids, or N- or C-terminal processing can be performed. Furthermore, the enzyme can also be produced using general protein chemical synthesis methods (e.g., liquid-phase and solid-phase methods) based on the amino acid sequence information shown in SEQ ID NO: 1. The enzyme can also be obtained according to the method described in Patent Document 1.

[0025] Polyethylene furanoate can be decomposed by allowing an enzyme to act on it. Thus, plastics containing polyethylene furanoate as a constituent material can be decomposed by allowing an enzyme to act on them. Allowing an enzyme to act on polyethylene furanoate or plastic can be achieved, for example, by bringing cutinase into contact with polyethylene furanoate or plastic.

[0026] The mode of allowing the enzyme to act on polyethylene furanoate (or plastic) is not particularly limited, and can be carried out, for example, by adding the enzyme and polyethylene furanoate to an appropriate solvent (e.g., a buffer solution) and appropriately stirring, etc. 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, and Na ２ HPO ４ Examples of suitable solutions include buffer solutions, preferably bicine or glycine buffer solutions. The action of an enzyme on polyethylene furanoate can also be carried out by adding the enzyme to polyethylene furanoate (or plastic) or to an object to be decomposed that has been buried in the ground.

[0027] The degradation of PEF using an enzyme is preferably carried out in the presence of PET (under conditions in which PEF and PET coexist). The enzyme having the above-mentioned specific amino acid substitution has improved PEF degradation activity, and can therefore efficiently decompose PEF in an environment in which PEF and PET coexist, and can efficiently supply PEF degradation products. In one embodiment, when the enzyme is allowed to act on equal amounts of coexisting PEF and PET for a certain period of time (e.g., 24 hours), the specificity for PEF, as calculated by the following formula (1), is preferably 2 or more, 3 or more, 4 or more, or 5 or more. There is no particular upper limit, but it may be, for example, 15 or less or 10 or less. [PEF] / [PET] (1) [PEF]: Sum (μmol) of PEF degradation products (FDCA, MHEF, BHEF) [PET]: Sum (μmol) of PET degradation products (TPA, MHET, BHET)

[0028] In one embodiment, it is preferable to recover one or more degradation products of PEF following (or during) the enzymatic degradation of PEF. In one embodiment, it is preferable to recover one or more degradation products of PET following (or during) the enzymatic degradation of PEF. In one embodiment, it is preferable to recover one or more degradation products of PEF and one or more degradation products of PET following (or during) the enzymatic degradation of PEF. The recovered degradation products can be used for any purpose. For example, the degradation products can be used as raw materials for recycling PEF or PET.

[0029] The reaction conditions when the enzyme is allowed to act on polyethylene furanoate 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 90°C. In one embodiment, the reaction temperature is preferably 30°C or higher, 35°C or higher, 40°C or higher, 45°C or higher, 50°C or higher, 55°C or higher, 60°C or higher, or 65°C or higher, and is preferably 90°C or lower, 85°C or lower, 80°C or lower, or 75°C or lower, 70°C or lower, 65°C or lower, or 60°C or lower. These lower and upper temperature limits can be arbitrarily combined. In one embodiment, by setting the reaction temperature high (e.g., 50°C or higher, 55°C or higher, 60°C or higher, 65°C or higher, 70°C or higher, or 75°C or higher), the substrate specificity of cutinase for polyethylene furanoate (compared to, for example, the reactivity for polyethylene terephthalate) can be increased.

[0030] 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.

[0031] The reaction time can be appropriately set depending on the purpose. 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. These lower and upper limits of the reaction time can be appropriately combined to form a range. In one embodiment, the substrate specificity of the enzyme for polyethylene furanoate can be increased by combining a longer reaction time (e.g., 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, or 24 hours or more) with a high reaction temperature.

[0032] The concentration or amount of the enzyme during the reaction can be appropriately set depending on the purpose. For example, the concentration of cutinase in the solvent can be appropriately set in the range of 10 nM to 5000 nM. In one embodiment, the concentration of the enzyme in the solvent during the reaction is preferably 50 nM to 200 nM, 75 nM to 150 nM, or 80 nM to 130 nM, from the viewpoint of exhibiting high substrate specificity for polyethylene furanoate.

[0033] The enzyme exhibits high substrate specificity for polyethylene furanoate. Therefore, in one embodiment, the PEF-containing plastic, which is the product to be decomposed by the enzyme, preferably includes a polymer or resin other than PEF. The other resin is not particularly limited, but may be, for example, a resin composed of one or more polymers selected from the group consisting of polyethylene terephthalate (PET), poly-ε-caprolactone (PCL), poly-L-lactic acid (PLA), polybutylene succinate (PBS), polyethylene succinate (PES), polyethylene adipate (PEA), and polybutylene succinate adipate polymer (PBSA). In one embodiment, the other resin preferably includes at least polyethylene terephthalate. In one embodiment, the polyethylene furanoate-containing plastic is preferably a post-consumer plastic.

[0034] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0035] 1. Preparation of mutant LCC enzymes. A polynucleotide having any of the nucleotide sequences set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14 was inserted into the NdeI (5' end) / XhoI (3' end) sites of the pET-21b vector (Novagen, San Diego, CA) to form an expression vector. E. coli BL21(DE3) CodonPlus RIPL (Agilent Technologies) was transformed with the vector. The enzyme was expressed by induction with 0.1 mM IPTG (16°C), and the cells were then collected by centrifugation (5,000 x g, 5 min, 4°C) and resuspended in lysis buffer (50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5 mM imidazole). The resuspended solution 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 the lysis buffer described above, the bound protein was eluted with elution buffer (50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 150 mM imidazole) and buffer-exchanged to 50 mM NaHPO-HCl (pH 7.0) using a PD-10 gel filtration column (GE Healthcare, Piscataway, NJ). The concentration of the purified enzyme thus obtained was determined based on the molar extinction coefficient at 280 nm.

[0036] 2. Measurement of PET and PEF resolution A 0.2 mm thick, 6 mm diameter PET or PEF film was added to 200 mM Bicine-NaOH (pH 9.0), and then each enzyme obtained in 1 above was added to a final concentration of 100 nM, followed by a reaction at 40°C for 24 hours (a control was set in which no enzyme was added to the PEF film). A portion of the reaction solution was diluted with 80% (v / v) 20 mM NaH ２ P.O. ４ -H ３ P.O. ４The solution was diluted with 20% (v / v) DMSO (pH 2.5) and heated at 90°C for 10 minutes, after which a portion of the supernatant was subjected to HPLC. Known concentrations of TPA, MHET, BHET, FDCA, MHEF, and BHEF solutions were used as standard solutions. 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 NaH ２ P.O. ４ -H ３ P.O. ４ (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%

[0037] The peak areas of PET degradation products (TPA, MHET, BHET) and PEF degradation products (FDCA, MHEF, BHEF) separated and detected by HPLC were compared with the peak areas of the standard solutions to quantify the degradation products under each condition. As a result, as shown in Figure 1, it was confirmed that the introduction of mutations improved PET degradation activity and PEF degradation activity, and in particular, H129W and F208S improved PEF degradation activity. Among them, H129W improved PEF degradation activity without causing any change in PET degradation ability, and therefore was confirmed to specifically improve PEF degradation.

[0038] 3. PEF resolution at 30°C or 50°C A PEF film with a thickness of 0.2 mm and a diameter of 6 mm was added to 50 mM Bicine-NaOH (pH 9.0), and then the enzyme was added to a final concentration of 100 nM, followed by reaction at 30°C or 50°C for 24 hours. A portion of the reaction solution was diluted with 80% (v / v) 20 mM NaH ２ P.O. ４ -H ３ P.O. ４The solution was diluted with 20% (v / v) DMSO (pH 2.5) and heated at 90°C for 10 minutes. A portion of the supernatant was then subjected to HPLC. FDCA and MHEF solutions of known concentrations were used as standard solutions. The HPLC conditions were the same as those in 2 above.

[0039] The peak areas of the PEF degradation products (FDCA and MHEF) separated and detected by HPLC were compared with those of the standard solution to quantify the degradation products under each condition. As shown in Figure 2, although an overall decrease in activity was observed at 30°C (Figure 2a), the tendency for PEF degradation ability to improve with the introduction of mutations was maintained, and at 50°C (Figure 2b), the results were almost identical to those at 40°C shown in Figure 1.

[0040] 4. Reaction Time and PET and PEF Resolution Abilities The PET and PEF resolution abilities of the wild-type enzyme and the mutant enzymes (H129W and H129W / F208S) were measured in the same manner as in Section 2 above, except that the reaction time was changed to 3, 24, or 48 hours and the reaction temperature to 50° C., 55° C., or 60° C. As a result, as shown in Figure 3, it was confirmed that the wild-type enzyme had similar PET and PEF resolution abilities at all temperatures, whereas the mutant enzymes had higher PEF resolution ability than PET resolution ability (specifically decomposed PEF).

[0041] 5. Ability to decompose coexisting PET and PEF One or both of the aforementioned PET or PEF films was added to 200 mM Bicine-NaOH (pH 9.0), and then the enzyme (H129W) was added to a final concentration of 100 nM or 200 nM, followed by reaction at 50°C for 24 or 48 hours. The decomposition products after the reaction were quantified in the same manner as in 2 above. As a result, as shown in Figure 4, it was confirmed that the ability to specifically decompose PEF was maintained even when PET and PEF coexisted.

[0042] 6. Preparation of mutant BhrPETase To examine the PET and PEF decomposition ability of the BhrPETase_H129W mutant enzyme, in which the conserved His in subsite II of BhrPETase, which belongs to the same Type I as LCC, was changed to Trp, E. coli BL21(DE3) (Nippon Gene) was transformed with an expression vector in which a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 16 or 18 was inserted into the NdeI (5' end) / XhoI (3' end) site of the pET-24b vector (Novagen, San Diego, CA). After expressing the enzyme by induction with 0.1 mM IPTG (18°C), the cells were collected by centrifugation (5,000 x g, 10 min, 4°C), suspended and dissolved in BugBuster Master Mix, and then centrifuged (16,000 x g, 20 min, 4°C) to collect the supernatant, which was then applied to HiTrap TALON crude (Cytiva). After washing off unbound proteins with washing buffer (50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 20 mM imidazole), the proteins bound to the column were eluted with elution buffer (50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 250 mM imidazole) and then purified using a PD-10 gel filtration column (GE Healthcare, Piscataway, NJ) with 50 mM NaCl. ２ The buffer was exchanged into a HPO4-HCl (pH 7.0) solution containing 100 mM NaCl. The concentration of the purified enzyme was determined based on the molar extinction coefficient at 280 nm.

[0043] 7. PET and PEF Decomposition Abilities of Mutant BhrPETase One or both of the aforementioned PET or PEF films were added to 200 mM Bicine-NaOH (pH 9.0), and then wild-type LCC, wild-type BhrPETase, and the mutant BhrPETase prepared in 6 above were added to a final concentration of 100 nM or 200 nM, followed by reaction at 50°C for 24 or 48 hours. The degradation products after the reaction were quantified in the same manner as in 2 above. However, the HPLC conditions were as follows: Apparatus: Nexera XR (Shimadzu Corporation) 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 As a result, as shown in Figure 5, it was confirmed that the decomposition activity and specificity for PEF were also enhanced by introducing the H129W mutation into BhrPETase. From these results, it is predicted that enzymes belonging to Type I other than LCC and BhrPETase will also exhibit similar PEF decomposition specificity by introducing a mutation that replaces the His conserved in subsite II with Trp.

Claims

1. A method for degrading polyethylene furanoate, comprising allowing an enzyme consisting of a polypeptide having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1 and having one or more amino acid substitutions selected from the group consisting of H129W, F208S, A172C, and A209C to act on polyethylene furanoate.

2. The method according to claim 1, wherein the enzyme is allowed to act on polyethylene furanoate in the presence of polyethylene furanoate and polyethylene terephthalate.

3. The method of claim 1 or 2, wherein the amino acid substitution includes at least H129W.

4. The method according to any one of claims 1 to 3, wherein the enzyme is allowed to act on the polyethylene furanoate at a temperature of 30°C or higher and lower than 60°C.

5. The method of any one of claims 1 to 4, further comprising recovering one or more selected from the group consisting of 2,5-furandicarboxylic acid (FDCA), monohydroxyethyl 2,5-furandicarboxylate (MHEF), and bishydroxyethyl 2,5-furandicarboxylate (BHEF).

6. The method according to any one of claims 1 to 5, wherein the polyethylene furanoate is a plastic comprising polyethylene furanoate and polyethylene terephthalate.

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