Enzymatic hydrolysis of ethylene terephthalate compounds

Abstract

To provide a new method for decomposing ethylene terephthalate compounds and the like. [Solution] A method for decomposing an ethylene terephthalate compound, comprising mixing an amino acid sequence having 90% or more identity with a specific amino acid sequence and having the activity to decompose polyethylene terephthalate (PET) oligomers; a polypeptide having an amino acid sequence having 90% or more identity with another specific amino acid sequence and having the activity to decompose monohydroxyethyl terephthalate (MHET); and an ethylene terephthalate compound represented by formula (1). TIFF2026166698000017.tif35146

Description

Technical Field

[0001] Techniques relating to the enzymatic degradation of ethylene terephthalate compounds and the like are disclosed.

Background Art

[0002] Plastics are inexpensive, durable, and highly versatile materials used in a wide range of products. On the other hand, due to their excellent durability, a considerable amount of plastics accumulates in landfills and natural habitats around the world, contributing to environmental problems.

[0003] Various solutions for reducing the environmental and economic impacts associated with plastic accumulation are being investigated. For example, Patent Document 1 proposes using cutinase to degrade the polyester that constitutes plastics, and further improvements are demanded for the enzymatic degradation of plastics.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Documents

[0005]

Non-Patent Document 1

[0006] One challenge is to provide new methods for more efficiently decomposing ethylene terephthalate compounds and the like. [Means for solving the problem]

[0007] The inventions described below are provided. Section 1. A polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 1 and having the activity to degrade polyethylene terephthalate (PET) oligomers; a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 5 and having the activity to degrade monohydroxyethyl terephthalate (MHET); and formula (1): [ka] (In the formula, n is 1 to 6, A is selected from the group consisting of H, CH2CH2OH, CH2CH2OCH2CH2OH, and C6H5COOCH2CH2. B is selected from the group consisting of OH, OCH2CH2OH, OCH2CH2OCH2CH2OH, OOCC6H4COOH, and OOCC6H5. A method for decomposing an ethylene terephthalate compound, comprising mixing an ethylene terephthalate compound represented by [the formula shown]. Section 2. A polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 1 and having the activity to degrade PET oligomers, a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 5 and having the activity to degrade MHET, and formula (1): [ka] (In the formula, n is 1 to 6, A is selected from the group consisting of H, CH2CH2OH, CH2CH2OCH2CH2OH, and C6H5COOCH2CH2. B is selected from the group consisting of OH, OCH2CH2OH, OCH2CH2OCH2CH2OH, OOCC6H4COOH, and OOCC6H5. A method for producing terephthalic acid (TPA), comprising mixing an ethylene terephthalate compound represented by [formula]. Section 3. A polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 1 and having the activity to degrade PET oligomers, a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 5 and having the activity to degrade MHET, and formula (2): [ka] (In the formula, n is 1 to 6, A is selected from the group consisting of H, CH2CH2OH, and CH2CH2OCH2CH2OH. B is selected from the group consisting of OH, OCH2CH2OH, and OCH2CH2OCH2CH2OH) A method for decomposing an ethylene furanoate compound, comprising mixing the ethylene furanoate compound represented by Item 4. A polypeptide having an amino acid sequence with 90% or more identity to the amino acid sequence of SEQ ID NO: 1 and having an activity of degrading PET oligomers, a polypeptide having an amino acid sequence with 90% or more identity to the amino acid sequence of SEQ ID NO: 5 and having an activity of degrading MHET, and Formula (2):

Chemical Formula

Chemical Formula

Chemical Formula

Effects of the Invention

[0008] Efficient decomposition of ethylene terephthalate compounds and the like is enabled.

Brief Description of the Drawings

[0009] [Figure 1] The results of measuring the polyethylene terephthalate (PET) oligomer decomposition ability by the combination of the enzyme having the amino acid sequence of SEQ ID NO: 1 (IS3653) and the enzyme having the amino acid sequence of SEQ ID NO: 5 (MHETase) are shown. The vertical axis represents the production amount (concentration) of terephthalic acid (TPA). [Figure 2]This figure shows the results of measuring the PET oligomer resolution using combinations of enzymes with the amino acid sequence of SEQ ID NO: 1 (IS3653), the amino acid sequence of SEQ ID NO: 5 (MHETase), and the amino acid sequence of SEQ ID NO: 8 (IS5771). The vertical axis shows the production volume (concentration) of TPA. [Modes for carrying out the invention]

[0010] A method for degrading ethylene terephthalate compounds and the like is provided, by combining an enzyme having the amino acid sequence of SEQ ID NO: 1 and an enzyme having the amino acid sequence of SEQ ID NO: 5. The amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 5 are amino acid sequences that encode polypeptides derived from Ideonella sakaiensis. The polypeptide encoded by the amino acid sequence of SEQ ID NO: 1 has the activity to degrade PET oligomers (Non-Patent Literature 1). The polypeptide encoded by the amino acid sequence of SEQ ID NO: 5 has the activity to degrade monohydroxyethyl terephthalate (MHET) (Non-Patent Literature 2).

[0011] In one embodiment, the enzyme used in the method for degrading ethylene terephthalate compounds, etc., may be a combination of an enzyme that is a polypeptide having an amino acid sequence having a certain degree of identity with the amino acid sequence of SEQ ID NO: 1 and having the activity to degrade PET oligomers, and an enzyme that is a polypeptide having an amino acid sequence having a certain degree of identity with the amino acid sequence of SEQ ID NO: 5 and having the activity to degrade MHET. "A certain degree 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, 99% or more, or 100%.

[0012] The identity of amino acid sequences can be evaluated by any means known in the field. For example, it can be calculated using commercially available analytical tools or tools available via telecommunication lines (the Internet). As an example, the identity of amino acid sequences can be calculated by using the default parameters of the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) http: / / www.ncbi.nlm.nih.gov / BLAST / .

[0013] In one embodiment, if the amino acid sequence of the enzyme has amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 1 or 5, the type of amino acid residue substitution is not particularly limited, but it is preferable that it be 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-chain amino acids (V, L, I), and substitutions between amino acids (A, G) having linear alkyl or hydrogen side chains.

[0014] A polypeptide having an amino acid sequence that has a certain degree of identity with the amino acid sequence of SEQ ID NO: 1 preferably has the following amino acid residues preserved: S217, L249, Y382, I383, D450, H488. In one embodiment, it is preferable that the polypeptide also preserves several amino acid residues before and after these amino acid residues. Several amino acid residues are, for example, 3 amino acid residues, 2 amino acid residues, or 1 amino acid residue. The amino acid residues that should be preserved in order to maintain the enzymatic properties of the 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 activity.

[0015] A polypeptide having an amino acid sequence that has a certain degree of identity with the amino acid sequence of SEQ ID NO: 5 preferably has the following amino acid residues preserved: S225, R411, S416, D429, H528. In one embodiment, it is preferable that the polypeptide also preserves several amino acid residues before and after these amino acid residues. These several amino acid residues are, for example, 3 amino acid residues, 2 amino acid residues, or 1 amino acid residue. The amino acid residues that should be preserved in order to maintain the enzymatic properties of the polypeptide having the amino acid sequence of SEQ ID NO: 5 can be identified by comparing the amino acid sequence of SEQ ID NO: 5 with the amino acid sequences of known enzymes having similar activity.

[0016] A polypeptide having an amino acid sequence that has a certain degree of identity with the amino acid sequence of SEQ ID NO: 1 or 5 may have an arbitrary amino acid sequence at the C-terminus and / or N-terminus of the amino acid sequence having a certain degree of identity with SEQ ID NO: 1 or 5, insofar as the properties of the enzyme having the amino acid sequence of SEQ ID NO: 1 or 5 are maintained. Such 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, a polypeptide having an amino acid sequence that has a certain degree of identity with the amino acid sequence of SEQ ID NO: 1 may have a tag sequence (e.g., a Maltose binding protein (MBP) tag sequence and an HRV3C protease cleavage site sequence) at its N-terminus. In one embodiment, a polypeptide having an amino acid sequence that has a certain degree of identity with the amino acid sequence of SEQ ID NO: 5 may have a tag sequence (e.g., a His tag) at its N-terminus.

[0017] Examples of ethylene terephthalate compounds, etc., that are decomposed by the method for decomposing ethylene terephthalate compounds, etc., include ethylene terephthalate compounds or ethylene furanoate compounds. The ethylene terephthalate compounds, etc., used in the method for decomposing ethylene terephthalate compounds, etc., may be one type or a combination of two or more types. The ethylene terephthalate compound is not particularly limited and may be PET or a PET oligomer. The PET oligomer is not particularly limited, but for example, it can be a polymer formed by the polymerization of about 2 to 20 monomers obtained by the dehydration condensation of TPA and ethylene glycol (EG). In one embodiment, the ethylene terephthalate compound is preferably of formula (1): [ka] (In the formula, n is 1 to 6, A is selected from the group consisting of H, CH2CH2OH, CH2CH2OCH2CH2OH, and C6H5COOCH2CH2. B is an ethylene terephthalate compound represented by (selected from the group consisting of OH, OCH2CH2OH, OCH2CH2OCH2CH2OH, OOCC6H4COOH, and OOCC6H5).

[0018] The ethylene furanoate compound is not particularly limited and may be polyethylene furanoate (PEF) or a PEF oligomer. The PEF oligomer is not particularly limited, but for example, it can be a polymer formed by the dehydration condensation of 2,5-franzicarboxylic acid (FDCA) and EG, with approximately 2 to 20 monomers polymerized. The ethylene furanoate compound preferably has formula (2): [ka] (In the formula, n is 1 to 6, A is selected from the group consisting of H, CH2CH2OH, and CH2CH2OCH2CH2OH. B is an ethylene furanoate compound represented by (selected from the group consisting of OH, OCH2CH2OH, and OCH2CH2OCH2CH2OH).

[0019] In one embodiment, it is preferable that a polypeptide having an amino acid sequence having a certain degree of identity with the amino acid sequence of SEQ ID NO: 1 possesses not only the activity to degrade PET oligomers but also the activity to degrade polyethylene furanoate (PEF) oligomers.

[0020] The ability of a polypeptide having an amino acid sequence that is more than or equal to the amino acid sequence of SEQ ID NO: 1 to degrade PET oligomers and / or PEF oligomers can be confirmed or measured by any method. For example, the polypeptide can be reacted with a PET oligomer or PEF oligomer, the amount of PET oligomer or PEF oligomer before and after the reaction can be measured, and the activity can be confirmed or measured based on the decrease in the amount of PET oligomer or PEF oligomer.

[0021] In one embodiment, a polypeptide having an amino acid sequence that has a certain degree of identity with the amino acid sequence of Sequence ID No. 5 is preferably equipped with the activity to degrade monohydroxyethyl 2,5-franzicarboxylate (MHEF) in addition to the activity to degrade MHET.

[0022] The ability of a polypeptide having an amino acid sequence that is more than or equal to the amino acid sequence of Sequence ID No. 5 to degrade MHET and / or MHEF can be confirmed or measured by any method. For example, the polypeptide can be reacted with MHET or MHEF, the amount of MHET or MHEF before and after the reaction can be measured, and the activity can be confirmed or measured based on the decrease in MHET or MHEF.

[0023] In one embodiment, the polypeptide having an amino acid sequence that has a certain degree of identity with the amino acid sequence of SEQ ID NO: 1 or 5 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 it is separated from other substances that would coexist with it in nature. In one embodiment, an isolated polypeptide means a purified polypeptide.

[0024] Polypeptides having an amino acid sequence that has a certain degree of identity with the amino acid sequence of SEQ ID NO: 1 or 5 can be obtained by any method. For example, polypeptides can be prepared by genetic engineering. Specifically, they can be prepared by transforming a suitable host cell (e.g., E. 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 purified as needed. An example of the DNA encoding the enzyme of SEQ ID NO: 1 is the base sequence of SEQ ID NO: 2, and an example of the DNA encoding the enzyme of SEQ ID NO: 5 is the base sequence of SEQ ID NO: 6. An example of DNA encoding a polypeptide having an amino acid sequence that has a certain degree of identity with the amino acid sequence of SEQ ID NO: 1 or 5 is DNA having a base sequence that has a certain degree of identity with the base sequence of SEQ ID NO: 2 or 6. "Above a certain level" 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, 99% or more, or 100%.

[0025] By combining the above enzymes and mixing them with ethylene terephthalate compounds, etc., they can be decomposed to obtain decomposition products. The decomposition products of ethylene terephthalate compounds are not particularly limited, but examples include bis(benzoyloxytyl) terephthalate (BETEB), bis-2-hydroxyethyl terephthalate (BHET), monohydroxyethyl terephthalate (MHET), 2-hydroxyethyl benzoate (HEB), TPA, and ethylene glycol. The decomposition product of ethylene terephthalate compounds is preferably TPA. The decomposition products of ethylene furanoate compounds are not particularly limited, but examples include monohydroxyethyl 2,5-fransilcarboxylate (MHEF), bishydroxyethyl 2,5-fransilcarboxylate (BHEF), 2,5-fransilcarboxylic acid (FDCA), and ethylene glycol. The decomposition product of ethylene furanoate compounds is preferably FDCA. In other words, in one embodiment, a method is provided for producing a decomposition product of an ethylene terephthalate compound or an ethylene furanoate compound, preferably TPA or FDCA, which includes combining the above enzymes and mixing them with an ethylene terephthalate compound or the like.

[0026] The manner in which the enzyme is mixed with ethylene terephthalate compounds, etc., is not particularly limited, but can be carried out, for example, by adding the enzyme or polypeptide and polyethylene terephthalate compounds, etc., to a suitable solvent (e.g., a buffer solution) and stirring as appropriate. The buffer solution is not particularly limited, but examples include Bicine, Glycine, MES, Bis-Tris, Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, HEPES, DIPSO, TAPSO, POPSO, HEPPSO, EPPS, Tricine, K2HPO4, and Na2HPO4 buffer solutions, and Bicine or Glycine buffer solution is preferred. Furthermore, mixing the enzyme with ethylene terephthalate compounds, etc., can also be carried out by adding the enzyme to ethylene terephthalate compounds, etc., or by adding the enzyme or polypeptide to the decomposition target buried in the ground. The order in which the enzyme having the amino acid sequence of SEQ ID NO: 1, the enzyme having the amino acid sequence of SEQ ID NO: 5, and the ethylene terephthalate compounds, etc., are mixed is not limited. For example, the enzyme and the ethylene terephthalate compound may all be mixed at the same time. In one embodiment, from the viewpoint of decomposition efficiency, it is preferable to mix the enzyme having the amino acid sequence of SEQ ID NO: 1 with the ethylene terephthalate compound first.

[0027] The reaction conditions when mixing the enzyme with an ethylene terephthalate compound, etc., are not particularly limited and can be set as appropriate depending on the purpose. The reaction temperature can be set, for example, in the range of 10°C to less than 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 preferably 65°C or lower, 60°C or lower, or 50°C or lower. These lower and upper temperature limits can be combined arbitrarily.

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

[0029] The reaction time can be set appropriately depending on the purpose and reaction conditions. For example, the reaction time can be set within a range of 1 hour to 1 week. 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 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 reaction time can be combined as appropriate to form a range.

[0030] The concentration or amount of each enzyme during the reaction can be appropriately set according to the purpose. For example, the concentration of each enzyme in the solvent can be appropriately set within the range of 10 nM to 5000 nM. In one embodiment, the concentration or amount of each enzyme can be 10 nM or more, 20 nM or more, 50 nM or more, 100 nM or more, 200 nM or more, 300 nM or more, or 500 nM or more, and 5000 nM or less, 4000 nM or less, 3000 nM or less, 2000 nM or less, 1000 nM or less, 800 nM or less, or 600 nM or less. These lower and upper limits of concentration can be appropriately combined to form a range.

[0031] In one embodiment, the above enzyme and ethylene terephthalate compounds, etc., can be mixed with another enzyme capable of degrading ethylene terephthalate compounds, etc., including PET or PEF. The other enzyme is arbitrary, but examples include cutinase, PET-degrading enzyme (PETase), MHET-degrading enzyme, etc. In one embodiment, the other enzyme is preferably an enzyme derived from Ideonella sakaiensis.

[0032] In one embodiment, another enzyme preferred is a polypeptide having an amino acid sequence that has a certain degree of identity with the amino acid sequence of SEQ ID NO: 8 and has the activity to degrade PET oligomers. "A certain degree of identity" 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, 99% or more, or 100%. The amino acid sequence of SEQ ID NO: 8 is an amino acid sequence that encodes a polypeptide derived from Ideonella sakaiensis. The polypeptide encoded by the amino acid sequence of SEQ ID NO: 8 has the activity to degrade PET oligomers, as shown in the examples described later. In one embodiment, it is preferable that the polypeptide having an amino acid sequence that has a certain degree of identity with the amino acid sequence of SEQ ID NO: 8 has the activity to degrade PEF oligomers in addition to the activity to degrade PET oligomers. The ability of a polypeptide having an amino acid sequence that is more than or equal to the amino acid sequence of SEQ ID NO: 8 to degrade PET oligomers or PEF oligomers can be confirmed or measured by any method. For example, it can be measured by the method described in the examples below.

[0033] A polypeptide having an amino acid sequence that is more or less identical to the amino acid sequence of SEQ ID NO: 8 is preferably one in which the following amino acid residues of the amino acid sequence of SEQ ID NO: 8 are preserved: A46, S113, Y114, D137, H251. For matters not described below regarding this polypeptide, refer to the description of the polypeptide having an amino acid sequence that is more or less identical to the amino acid sequence of SEQ ID NO: 1 described above.

[0034] By using the above enzyme in combination with other enzymes capable of decomposing ethylene terephthalate compounds, including PET or PEF, the decomposition efficiency of ethylene terephthalate compounds, including PET or PEF, or plastics or resins containing them, can be synergistically increased. The conditions for decomposing ethylene terephthalate compounds, such as PET or PEF, or plastics or resins containing them, in combination with the above enzyme can be set based on the above reaction conditions and the characteristics of the other enzymes. There are no particular restrictions on the order in which other enzymes are mixed with the above enzyme and ethylene terephthalate compounds. For example, the additional enzymes may be mixed simultaneously with the above enzyme and ethylene terephthalate compounds, or they may be added after the above enzyme and ethylene terephthalate compounds have been mixed.

[0035] In one embodiment, an enzyme preparation is provided that includes an enzyme having the amino acid sequence of SEQ ID NO: 1 and an enzyme having the amino acid sequence of SEQ ID NO: 5. In another embodiment, the enzymes included in the enzyme preparation may be a combination of an enzyme that is a polypeptide having an amino acid sequence having a certain degree of identity with the amino acid sequence of SEQ ID NO: 1 and having the activity to degrade PET oligomers, and an enzyme that is a polypeptide having an amino acid sequence having a certain degree of identity with the amino acid sequence of SEQ ID NO: 5 and having the activity to degrade MHET. "A certain degree of identity" 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, 99% or more, or 100%.

[0036] In one embodiment, the enzyme preparation may contain only the above-mentioned enzyme, or it may contain other components. Other components are not particularly limited, but examples include 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, melegitose, 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 gluconates, citrates, and maleates. 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, TritonX-100, and Tween20. Examples of polymers include polyvinylpyrrolidone. One or more of these other components can be used in any combination. The enzyme preparation may be in the form of a liquid, powder, or granules.

[0037] The content of each of the above enzymes contained in the enzyme preparation depends on the application, manner of use, and target compound, and is not limited to these, but can be, for example, 0.0001 to 100% by mass, preferably 0.001 to 50% by mass.

[0038] In one embodiment, the enzyme preparation is preferably an enzyme preparation for degrading ethylene terephthalate compounds, such as at least one ethylene terephthalate compound represented by formula (1) above, or ethylene furanoate compounds, such as at least one ethylene furanoate compound represented by formula (2) above. Here, the at least one ethylene terephthalate compound or ethylene furanoate compound is preferably a compound that the polypeptide described above has degrading activity over.

[0039] In one embodiment, the enzyme preparation preferably further comprises an enzyme which is a polypeptide having an amino acid sequence having a certain degree of identity with the amino acid sequence of SEQ ID NO: 8 and having an amino acid sequence having the activity to degrade PET oligomers. The enzyme and its content shall be as described above. [Examples]

[0040] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto.

[0041] 1. Preparation of enzyme IS3653 An expression vector was created by commissioning Integrated DNA Technologies (US) to fused a polynucleotide, which was obtained by concatenating the sequence of sequence number 4 to the 5' end of the sequence of sequence number 2 (optimized for expression in E. coli), into the AvaI (5' end) / XmnI (3' end) site of a modified pMAL-p5X vector. E. coli Rosetta 2 (DE3) was transformed with this vector. The transformed E. coli were cultured at 37°C in LB medium supplemented with 100 μg / ml ampicillin and 35 μg / ml chloramphenicol. The enzyme was expressed in these transformants by induction of 0.1 mM IPTG (15°C, 24 hours). The expressed enzyme has an amino acid sequence (sequence number 3) in which the N-terminus of the polypeptide sequence number 1 (with the N-terminal region from 1 to 47 of wild-type IS3653 removed) is sequentially fused with vector-derived maltose binding protein (MBP), a spacer sequence, and an HRV3C cutting site polypeptide. Cells were collected by centrifugation (10,000 g, 20 min) and resuspended in lysis buffer (10 mM Tris-HCl (pH 8), 100 mM NaCl, 0.5% Tween®-20). The resuspended cells were sonicated on ice, then ultracentrifuged (30,000 rpm, 4°C, 40 min), and the supernatant was collected and loaded onto an amylose column (New England Biolabs). Proteins bound to the column were eluted with elution buffer (20 mM Tris-HCl (pH 8.5), 100 mM NaCl, 20 mM maltose). The eluate was loaded onto a HiTrap® Q anion-exchange column (GE Healthcare) and fractionated using elution buffer (20 mM Tris-HCl (pH 8.5), buffer with a concentration gradient of 50-400 mM NaCl). The fraction containing the IS3653 enzyme was loaded onto a Superdex® 200 gel filtration column (GE Healthcare) and buffered with 10 mM HEPES-OH (pH 7) and 300 mM NaCl solution.After concentration using a 30kDa cutoff Amicon centrifugal filter (Merck Millipore), the concentration of the purified enzyme (also referred to as "IS3653") was determined based on the molar extinction coefficient at 280 nm.

[0042] 2. Preparation of the enzyme MHETase The gene sequence of MHETase (GenBank accession number, GAP38911.1) was optimized for expression in E. coli by optimizing the codons. The signal peptide was predicted using the LipoP 1.0 server (http: / / www.cbs.dtu.dk / services / LipoP), and the corresponding nucleotide sequence was removed to obtain the nucleotide sequence described in SEQ ID NO: 6. E. coli Rosetta-gami B (DE3) (Merck Millipore) was transformed using an expression vector in which the polynucleotide consisting of the aforementioned nucleotide sequence was inserted into the NdeI (5′ terminal) / XbaI (3′ terminal) site of a pCold II vector (Takara Bio). The enzyme was expressed in these transformants by induction of 0.1 mM IPTG (15°C). The expressed enzyme has an amino acid sequence in which the N-terminus of the polypeptide of SEQ ID NO: 5 (with the signal peptide of wild-type MHETase (GenBank accession number, GAP38911.1) removed) is fused with a 6×His tag sequence (SEQ ID NO: 7) derived from the vector. Cells were collected by centrifugation (5000g, 5 min, 4°C) and suspended in lysis buffer (50mM Tris-HCl (pH 7.5), 300mM NaCl, 20mM imidazole-HCl). The suspension was sonicated on ice, then centrifuged (15,000g, 20 min, 4°C), and the supernatant was collected and applied to a Ni-NTA column. Free proteins were removed by washing with lysis buffer, and bound proteins were eluted with elution buffer (50mM Tris-HCl (pH 7.5), 300mM NaCl, 250mM imidazole-HCl). This eluate was loaded onto a Superdex® 200 gel filtration column (GE Healthcare) and the buffer was changed to 10mM HEPES-OH (pH 7), 300mM NaCl solution. The fraction containing MHETase was concentrated using an Amicon centrifugal filter (Merck Millipore) with a 30 kDa cutoff, and the concentration of the purified enzyme (also referred to as "MHETase") was determined based on the molar extinction coefficient at 280 nm.

[0043] 3. Degradation of PET oligomers by enzyme combinations 3-1. Preparation of PET oligomers 432 g of terephthalic acid (TPA) and 323 g of ethylene glycol (EG) were mixed, and an esterification reaction was carried out at 250°C under pressure (0.25 MPa) for 108 minutes. Subsequently, 6.5 mL of Sb2O3 (12 g / L-EG solution), a catalyst for PET polymerization, was added to prepare a PET oligomer mixture. 0.01 g of the obtained PET oligomer mixture was dissolved in 1 mL of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), and the sample was further diluted 100-fold with dimethyl sulfoxide (DMF). HPLC and LC / MS analyses were performed on the sample. As a result, the UV258nm peak areas of each component contained in the PET oligomer mixture were as follows: L7 component 2.1%, L6 component 4.1%, L5 component 8.2%, L4 component 14.0%, L3 component 20.9%, L2 component 25.9%, BHET 18.7%, MHET 1.5%, TPA 0.0%, and L1 component other than BHET, MHET, and TPA 4.6%. "L2" is a molecule containing two TPA units, "L3" is a molecule containing three TPA units, and the same applies to "L4" to "L7".

[0044] 3-2. Evaluation of PET oligomer resolution The PET oligomer mixture obtained in 3-1 above was dissolved in DMF and heated at 99°C for 5 minutes to obtain a PET oligomer solution with a final concentration of 33.3 mg / mL. The PET oligomer solution with a final concentration of 3.3 mg / mL, IS3653 with a final concentration of 100 nM, and MHETase with a final concentration of 100 nM were added to 50 mM Na2HPO4-HCl (pH 7.0), and the reaction was carried out by inverting the mixture at 30°C for 24 hours. The condition without enzyme addition was referred to as Blank. The solution after the reaction was collected, diluted 2-fold by HFIP, and then further diluted 100-fold with DMF, and HPLC analysis was performed under the following conditions. Device: Nexera XR (Shimadzu Corporation) Column: Imtakt Cadenza CD C18 3μm 2×150mm Mobile phase: (A) 0.1% formic acid / (B) acetonitrile 0min(5%B)-28(100)-33(100)-33.01(5)-38(5) Flow rate: 0.4mL / min Column temperature: 45℃ Injection volume: 5μL Detection wavelength: 258nm

[0045] The DMF dilution of the reaction solution was stored under refrigeration and filtered through a 0.2 μm filter. TPA standards of known concentrations were analyzed similarly, and degradation products were quantified using a UV detector. The results are shown in Table 1 and Figure 1.

[0046] [Table 1]

[0047] These results confirm that IS3653 and MHETase alone are insufficient to produce TPA from PET oligomers, but by combining IS3653 and MHETase and applying them to PET oligomers, the PET oligomers can be degraded to TPA more efficiently than when each is applied individually. IS3653 is an enzyme that recognizes and degrades the EG terminus of PET oligomers, and MHETase is suggested to recognize the carboxyl group of MHET and have MHET-degrading activity (Non-patent documents 1 and 2). Therefore, it is thought that similar degradation will be promoted when these enzymes are applied to PEF oligomers.

[0048] 4. Degradation of PET oligomers by a combination of 3 enzymes 4-1. Preparation of enzyme IS5771 4-1-1. Enzyme Expression The nucleotide sequence of Sequence ID No. 9 was fused to a Glutathione S-transferase (GST) tag derived from the vector. Specifically, the polypeptide of Sequence ID No. 8 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 ug / mL chloramphenicol), and incubated overnight at 37°C. pGEXM is described in non-patent literature (https: / / www.nature.com / articles / s41598-021-81409-y#Sec12). After growing in 10 mL of LB-Amp-Cam medium at 37°C and 180 rpm until turbid, 1 mL of seed culture was added to 100 mL of LB-Amp-Cam medium and incubated overnight at 30°C and 140 rpm. A batch culture was prepared by adding 30 mL of culture medium cultured overnight to 2 L of LB-Amp-Cam medium and culturing until the OD600 nm 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, centrifuged at 4000 rpm, 20°C for 15 minutes, the supernatant was removed, and the pellet was stored at -30°C.

[0049] 4-1-2. Column chromatography using glutathione Sepharose 4B resin A 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 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 with glutathione septarose 4B resin (GS4B). After washing and equilibrating the column with 5 times the total volume of 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.

[0050] 4-1-3. Ion exchange chromatography (IEC) Since the theoretical pI of GST-labeled IS5771 is calculated to be 6.1, a pH 8.5 Tris-HCl buffer was used so that the protein would have a net negative charge and bind to the positively charged portion 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 and 0.3 MPa, with an elution volume of 5 mL. The bound target protein was eluted and recovered under a salt gradient in the range of 50 mM to 400 mM.

[0051] 4-1-4. 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) was added to the elution 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. 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) has an amino acid sequence in which two amino acid residues GP derived from the HRV3C protease cleavage site are added to the N-terminus of the amino acid sequence of SEQ ID NO: 8.

[0052] 4-1-5. Size exclusion chromatography (SEC) Proteins were concentrated to less than 5 mL using a 10-kDa Amicon ultracentrifugal filter. This was performed using an AKTA Purifier connected to a HiLoad Superdex® 75-pg column (Cytiva) pre-equilibriumated 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 volume of 5 mL.

[0053] 4-2. Evaluation of PET oligomer resolution The PET oligomer mixture obtained in 3-1 was dissolved in DMF and heated at 99°C for 5 minutes to obtain a PET oligomer solution with a final concentration of 33.3 mg / mL. The reaction and analysis were carried out in the same manner as described in 3-2, except that IS5771, MHETase, and IS3653 with final concentrations of 100 nM were added as enzymes.

[0054] The DMF dilution of the reaction solution was stored under refrigeration and filtered through a 0.2 μm filter. TPA standards of known concentrations were analyzed similarly, and degradation products were quantified using a UV detector. The results are shown in Table 2 and Figure 2.

[0055] [Table 2]

[0056] These results indicate that applying IS3653, IS5771, and MHETase in combination to PET oligomers promotes more efficient degradation of PET oligomers to TPA than applying IS3653 and MHETase together. As mentioned above, it is thought that similar degradation will be promoted when these enzymes are applied to PEF oligomers.

Claims

1. A polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 1 and having the activity to degrade polyethylene terephthalate (PET) oligomers; a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 5 and having the activity to degrade monohydroxyethyl terephthalate (MHET); and formula (1): 【Chemistry 1】 (In the formula, n is between 1 and 6, 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 selected from the group consisting of B is OH, OCH ２ CH ２ OH, OCH ２ CH ２ OCH ２ CH ２ OH, OOCC ６ H ４ COOH and OOCC ６ H ５ (Selected from the group consisting of) A method for decomposing an ethylene terephthalate compound, comprising mixing an ethylene terephthalate compound represented by [the formula shown].

2. A polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 1 and having the activity to degrade PET oligomers, a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 5 and having the activity to degrade MHET, and formula (1): 【Chemistry 2】 (In the formula, n is between 1 and 6, 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 Selected from the group consisting of, B is OH, OCH ２ CH ２ OH, OCH ２ CH ２ OCH ２ CH ２ OH, OOCC ６ H ４ COOH and OOCC ６ H ５ (Selected from the group consisting of) A method for producing terephthalic acid (TPA), comprising mixing an ethylene terephthalate compound represented by [formula].

3. A polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 1 and having the activity to degrade PET oligomers, a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 5 and having the activity to degrade MHET, and formula (2): 【Transformation 3】 (In the formula, n is between 1 and 6, A is H, CH ２ CH ２ OH and CH ２ CH ２ OCH ２ CH ２ Selected from the group consisting of OH, B is OH, OCH ２ CH ２ OH and OCH ２ CH ２ OCH ２ CH ２ (Selected from the group consisting of OH) A method for decomposing an ethylene furanoate compound, comprising mixing it with an ethylene furanoate compound represented by [formula].

4. A polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 1 and having the activity to degrade PET oligomers, a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 5 and having the activity to degrade MHET, and formula (2): 【Chemistry 4】 (In the formula, n is between 1 and 6, A is H, CH ２ CH ２ OH and CH ２ CH ２ OCH ２ CH ２ Selected from the group consisting of OH, B is OH, OCH ２ CH ２ OH and OCH ２ CH ２ OCH ２ CH ２ (Selected from the group consisting of OH) A method for producing 2,5-franzicarboxylic acid, comprising mixing an ethylene furanoate compound represented by [formula].

5. The method according to any one of claims 1 to 4, comprising mixing an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 1 at 10 to 1000 nM and having the activity to degrade PET oligomers, a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 5 at 10 to 1000 nM and having the activity to degrade MHET, and an ethylene terephthalate compound or an ethylene furanoate compound.

6. An enzyme preparation comprising a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence of Sequence ID No. 1 and having the activity to degrade PET oligomers, and a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence of Sequence ID No. 5 and having the activity to degrade MHET.

7. Ethylene terephthalate compounds are given by formula (1): 【Transformation 5】 (In the formula, n is between 1 and 6, 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 Selected from the group consisting of, B is OH, OCH ２ CH ２ OH, OCH ２ CH ２ OCH ２ CH ２ OH, OOCC ６ H ４ COOH and OOCC ６ H ５ (Selected from the group consisting of) An enzyme preparation according to claim 6 for decomposing an ethylene terephthalate compound represented by [the specified formula].

8. Ethylene furanoate compounds are given by formula (2): 【Transformation 6】 (In the formula, n is between 1 and 6, A is H, CH ２ CH ２ OH, and CH ２ CH ２ OCH ２ CH ２ selected from the group consisting of OH, B is OH, OCH ２ CH ２ OH and OCH ２ CH ２ OCH ２ CH ２ (Selected from the group consisting of OH) An enzyme preparation according to claim 6 for decomposing an ethylene furanoate compound represented by [the specified formula].

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