Enzymatic degradation of pef

Abstract

The present invention provides a novel means for efficiently degrading PEF. Provided is a method for degrading polyethylene furanoate and polyethylene terephthalate that includes causing an enzyme comprising a polypeptide that has an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1 to act on polyethylene furanoate and polyethylene terephthalate in a buffer solution having a pH of 8.5 to 11.

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 polyethylene terephthalate (PET), 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. These characteristics have made PEF a popular alternative to PET.

[0005] WO2012 / 099018US10584320US11072784US11414651 JP2022-155487

[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 efficiently decomposing PEF.

[0008] The following representative inventions are provided: Item 1. A method for degrading polyethylene furanoate and polyethylene terephthalate, 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 to act on polyethylene furanoate and polyethylene terephthalate in a buffer solution of pH 8.5 to 11. Item 2. The method of Item 1, 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 3. The method of Item 1 or 2, wherein the polypeptide has the amino acid sequence of SEQ ID NO: 1.

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

[0010] The results of investigating the decomposition ability of wild-type IsPETase enzyme on PET and PEF films are shown.

[0011] The enzyme (also referred to as "PETase") used in the method for degrading polyethylene furanoate and polyethylene terephthalate preferably has an amino acid sequence having a certain level of identity to the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of SEQ ID NO: 1 is the amino acid sequence of a PET-degrading enzyme (IsPETase) derived from Ideonella sakaiensis. This enzyme has degrading activity for PEF and PET with high specificity for PEF (particularly, high specificity or directionality compared to reactivity for PET). Therefore, it enables efficient (or preferential) decomposition of PEF under conditions in which PEF and PET coexist.

[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, 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 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more, or 60 or more. There is no particular upper limit, but it may be, for example, 100 or less or 80 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)

[0014] "A certain level or higher" refers to, 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. In one embodiment, the enzyme is preferably a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0015] In one embodiment, the amino acid sequence of the enzyme preferably conserves (is not mutated) the amino acid residues at positions 132, 158, 176, 211, and / or 212 in the amino acid sequence of SEQ ID NO: 1.

[0016] 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.

[0017] In one embodiment, when the amino acid sequence of the enzyme has an amino acid sequence in which one or several amino acids are substituted with respect 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 with linear alkyl or hydrogen side chains (A, G).

[0018] The enzyme can be obtained by any method. For example, the enzyme can be prepared by genetic engineering methods. Specifically, the enzyme can be prepared by transforming a suitable host cell (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. An example of the DNA encoding the enzyme is DNA having the base sequence of SEQ ID NO: 2.

[0019] Various modifications are possible using techniques for obtaining enzymes as recombinant proteins. For example, by inserting the DNA encoding the enzyme and other appropriate DNA into the same vector and using that vector to produce a recombinant protein, 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.

[0020] By allowing an enzyme to act on polyethylene furanoate (and polyethylene terephthalate), polyethylene furanoate (and polyethylene terephthalate) can be decomposed. Thus, by allowing an enzyme to act on plastics containing polyethylene furanoate (and polyethylene terephthalate) as a constituent material, the plastics can be decomposed. Allowing an enzyme to act on polyethylene furanoate (and polyethylene terephthalate) or plastics can be achieved, for example, by bringing the enzyme into contact with polyethylene furanoate (and polyethylene terephthalate) or plastics.

[0021] The manner in which the enzyme is allowed to act on polyethylene furanoate, polyethylene terephthalate, 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 ４Examples of suitable solutions include a buffer solution, preferably a bicine or glycine buffer solution. The enzyme can also be allowed to act on polyethylene furanoate or the like by adding the enzyme to polyethylene furanoate or the like, or by adding the enzyme to the material to be decomposed that has been buried in the ground.

[0022] 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.

[0023] 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 20°C or higher, 25°C or higher, 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. These lower and upper temperature limits can be combined in any manner.

[0024] 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 pH can be appropriately combined to set the range. In one embodiment, the pH during the reaction is preferably 8.5 or more and 11 or less.

[0025] The reaction time can be set appropriately 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. The lower and upper limits of these reaction times can be appropriately combined to form a range.

[0026] 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.

[0027] 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.

[0028] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0029] 1. Preparation of IsPETase Enzyme: E. coli BL21(DE3) CodonPlus RIPL (Agilent Technologies) was transformed with an expression vector containing the polynucleotide of SEQ ID NO:2 (base sequence of IsPETase) inserted into the NdeI (5' end) / XhoI (3' end) sites of the pET-21b vector (Novagen, San Diego, CA). The enzyme was expressed by induction with 0.1 mM IPTG (16°C). The cells were 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 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 unbound proteins with the lysis buffer, 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 then purified using a PD-10 gel filtration column (GE Healthcare, Piscataway, NJ) containing 50 mM NaCl. ２ The buffer was exchanged into HPO4-HCl (pH 7.0) solution, and the concentration of the purified enzyme was determined based on the molar extinction coefficient at 280 nm.

[0030] 2. Measurement of PET and PEF decomposition ability One or both of PET or PEF films, each 0.2 mm thick and 6 mm in diameter, were added to 200 mM Bicine-NaOH (pH 9.0) or 200 mM Glycine-NaOH (pH 10.0), and the enzyme obtained in 1 above was added to a final concentration of 100 nM or 200 nM, followed by reaction at 30°C for 24 or 48 hours. 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. 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%

[0031] 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 shown in Figure 1, the results confirmed that the degradation of PEF proceeded significantly faster than the degradation of the substrate PET under all conditions of reaction pH and enzyme concentration. This was also true when PET and PEF were simultaneously used as substrates. The total amount (moles) of PEF degradation products was approximately 7 to 61 times the total amount (moles) of PET degradation products. This indicates that IsPETase has a much higher substrate specificity for PEF than reported in Patent Documents 1 and 5, etc. These results confirmed that the use of this enzyme can efficiently decompose PEF in the presence of a mixture of PET and PEF.

Claims

1. A method for degrading polyethylene furanoate and polyethylene terephthalate, which comprises 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 to act on polyethylene furanoate and polyethylene terephthalate in a buffer solution of pH 8.5 to 11.

2. The method of claim 1, 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).

3. The method of claim 1 or 2, wherein the polypeptide has the amino acid sequence of SEQ ID NO:1.

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