Esterase, polyester decomposition product, polyester treatment method, and polyester production method

Abstract

The present invention addresses the problem of obtaining an esterase having a high decomposition activity at a low temperature. In order to solve this problem, provided is an esterase which has at least 80% sequence identity with the amino acid sequence represented by SEQ ID NO: 1 and in which A172 and A209 in the amino acid sequence are substituted.

Description

Esterase, polyester degradation product, polyester treatment method, and polyester production method Related Applications

[0001] This application claims priority from Japanese Patent Application No. 2023-139358, filed on August 29, 2023, and incorporates all of the contents of that Japanese application by reference.

[0002] The present disclosure relates to enzymes (esterases) that hydrolyze ester compounds into acids and alcohols. The present disclosure relates to polyester degradation products. The present disclosure relates to methods for treating polyesters with esterases. The present disclosure also relates to methods for producing polyesters using esterases. The present disclosure relates to polynucleotides, vectors, and host cells for producing esterases.

[0003] Polyester resins, such as polyethylene terephthalate (PET), a commodity plastic, are one of the plastic materials for which recycling systems have been established. However, the current recycling system is dominated by so-called one-way recycling. One-way recycling refers to the process in which a plastic product is reused as a raw material for another product, rather than being reused in its original form after use. For example, PET bottles, one of the uses of polyester resin, are washed, melted, remolded, and otherwise transformed into fibers for clothing, industrial sheets, and other uses. This type of recycling is called material recycling. However, with material recycling, the quality of recycled plastic products deteriorates with each recycle, making it impossible to establish a circular recycling system that circulates plastic resources.

[0004] Therefore, chemical recycling has been considered as a recycling method that can enable a plastic resource circulation recycling system. Chemical recycling involves chemically decomposing waste plastics and reusing the decomposition products as raw materials. Because chemical recycling decomposes plastics down to the compound level, it is possible to stabilize the quality of the recycled products and the decomposition products can also be used as raw materials for products other than plastics, making it a useful recycling method for building a resource circulation recycling system. However, for example, chemical recycling, which uses thermal decomposition, requires depolymerization of plastics at high temperatures and purification of the depolymerization products by distillation, which requires a large amount of thermal energy for recycling.

[0005] Therefore, chemical recycling in which polyesters are decomposed with enzymes has recently attracted attention (see, for example, Patent Documents 1 and 2). Patent Document 1 describes a cutinase-like esterase as an enzyme capable of decomposing polyesters. Patent Document 2 describes a mutant of the esterase described in Patent Document 1.

[0006] International Publication No. 2012 / 099018 Special Publication No. 2019-520833

[0007] Tournier et al., “An engineered PET depolymerase to break down and recycle plastic bottles” Nature, Vol. 580, p. p. 216-219 (2020) Shosuke Yoshida et al., “A bacterium that degrades and assimilates poly (ethylene terephthalate)” Sciencemag. org, Vol. 351, p. p. 1196 (2016)

[0008] According to the esterase described in Patent Document 1, even under conditions of maximum decomposition activity, decomposition is only about 30% in one week and about 54% in two weeks, which is not a sufficient decomposition rate at an industrial level.

[0009] To increase the rate of enzymatic decomposition of polyesters, it seems possible to increase the enzyme's heat resistance and decompose the polyester under high-temperature conditions. However, if polyesters are left in a temperature environment close to their glass transition temperature, crystallization of the polyesters will progress. For example, Non-Patent Document 1 shows that crystallization of polyethylene terephthalate progresses at 70°C, which is lower than its glass transition temperature of approximately 75°C. In general, the rate of enzymatic decomposition of highly crystalline polyesters is significantly lower than the rate of enzymatic decomposition of less crystalline polyesters (see, for example, Non-Patent Document 2). Therefore, there is a need for esterases that have high decomposition activity at temperatures below the temperature at which crystallization of the polyesters to be decomposed progresses.

[0010] Also, from the viewpoint of energy costs, an esterase with high decomposition activity at lower temperatures is desired.

[0011] According to a first aspect of the present disclosure, there is provided an esterase having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1, wherein A172 and A209 in the amino acid sequence have been substituted.

[0012] According to a second aspect of the present disclosure, there is provided a method for treating polyester, comprising a first step of degrading polyester using the esterase according to the first aspect.

[0013] According to a third aspect of the present disclosure, there is provided a method for producing a polyester, comprising a fourth step of producing a polyester using the compound isolated in the method for treating a polyester according to the second aspect as at least a part of the monomers.

[0014] According to a fourth aspect of the present disclosure, there is provided a polyester degradation product obtained by decomposing a polyester with the esterase according to the first aspect.

[0015] According to a fifth aspect of the present disclosure, there is provided a method for producing a polyester, in which a polyester is produced using the polyester decomposition product according to the fourth aspect.

[0016] According to a sixth aspect of the present disclosure, there is provided a polynucleotide encoding the esterase according to the first aspect.

[0017] According to a seventh aspect of the present disclosure, there is provided a vector comprising the polynucleotide according to the sixth aspect.

[0018] According to an eighth aspect of the present disclosure, there is provided a host cell into which a polynucleotide according to the sixth aspect has been introduced.

[0019] According to the esterase and polyester treatment method of the present disclosure, polyester can be enzymatically decomposed into acid and alcohol at a high decomposition rate.

[0020] According to the esterase and polyester treatment method of the present disclosure, polyester can be decomposed in an environment in which the progress of polyester crystallization can be suppressed.

[0021] According to the esterase and polyester treatment method of the present disclosure, the energy costs associated with polyester decomposition treatment can be reduced.

[0022] The esterases of the present disclosure have low substrate specificity.

[0023] According to the polyester production method of the present disclosure, high-quality recycled polyester can be produced.

[0024] According to the method for producing polyester of the present disclosure, resource recycling of polyester can be realized.

[0025] FIG. 1 is a graph showing the amount of terephthalic acid recovered in Test Examples 1, 3, 5, and 7. FIG. 2 is a graph showing the increase in the amount of terephthalic acid recovered in Test Examples 1, 3, 5, and 7. FIG. 3 is a graph showing the amount of isophthalic acid recovered in Test Examples 1, 3, 5, and 7. FIG. 4 is a graph showing the increase in the amount of isophthalic acid recovered in Test Examples 1, 3, 5, and 7. FIG. 5 is a graph showing the amount of all components recovered in Test Examples 1, 3, 5, and 7. FIG. 6 is a graph showing the increase in the amount of all components recovered in Test Examples 1, 3, 5, and 7. FIG. 7 is a graph showing the amount of terephthalic acid recovered in Test Examples 2, 4, 6, and 8. FIG. 8 is a graph showing the increase in the amount of terephthalic acid recovered in Test Examples 2, 4, 6, and 8. FIG. 9 is a graph showing the amount of isophthalic acid recovered in Test Examples 2, 4, 6, and 8. FIG. 10 is a graph showing the increase in the recovery amount of isophthalic acid in Test Examples 2, 4, 6, and 8. FIG. 11 is a graph showing the recovery amounts of all components in Test Examples 2, 4, 6, and 8. FIG. 12 is a graph showing the increase in the recovery amount of all components in Test Examples 2, 4, 6, and 8. FIG. 13 is a graph showing the change in the recovery amount of decomposition products in Test Example 9. FIG. 14 is a graph showing the change in the recovery amount of decomposition products in Test Example 10. FIG. 15 is a graph showing the change in the recovery amount of decomposition products in Test Example 11. FIG. 16 is a graph showing the change in the recovery amount of decomposition products in Test Example 12. FIG. 17 is a graph showing the recovery amount of decomposition products in Test Example 13. FIG. 18 is a graph showing the recovery amount of decomposition products in Test Example 14.

[0026] According to a preferred embodiment of the above aspect, A172 and A209 are substituted with cysteine ​​residues (C).

[0027] According to a preferred embodiment of the above aspect, D203 and S248 in the amino acid sequence are further substituted.

[0028] According to a preferred embodiment of the above aspect, D203 and S248 are substituted with cysteine ​​residues (C).

[0029] In a preferred embodiment of the above aspect, D203C and S248C form a disulfide bond.

[0030] According to a preferred embodiment of the above aspect, in the first step, the polyester is decomposed in a treatment solvent at a temperature of 40° C. or higher and lower than the glass transition temperature of the polyester, and at a pH in the range of 7 to 10.

[0031] According to a preferred embodiment of the above aspect, in the first step, the polyester is decomposed in a treatment solvent at a temperature of 65° C. or less.

[0032] According to a preferred embodiment of the above aspect, the polyester contains 50 mol % or more of a terephthalic acid component based on the total amount of acid components, and 65 mol % or more of an ethylene glycol component based on the total amount of alcohol components.

[0033] According to a preferred embodiment of the above aspect, the polyester further contains 2 mol % or more of an isophthalic acid component based on the total amount of the acid components.

[0034] According to a preferred embodiment of the above aspect, the polyester is at least one of a post-consumer product and a pre-consumer product.

[0035] According to a preferred embodiment of the above aspect, the method for treating a polyester further includes a second step of isolating at least one compound selected from the group consisting of terephthalic acid, mono(2-hydroxyethyl) terephthalate, bis(2-hydroxyethyl) terephthalate, and salts thereof from the decomposition product obtained in the first step.

[0036] According to a preferred embodiment of the above aspect, in the second step, at least one compound selected from the group consisting of isophthalic acid, mono(2-hydroxyethyl)isophthalate, bis(2-hydroxyethyl)isophthalate, and salts thereof is also isolated.

[0037] According to a preferred embodiment of the above aspect, the method for treating a polyester further comprises a third step of isolating a decomposition product derived from the alcohol component of the polyester from the decomposition product obtained in the first step.

[0038] According to a preferred embodiment of the above aspect, in the fourth step of the method for producing a polyester, the compound includes at least one compound selected from the group consisting of terephthalic acid, mono(2-hydroxyethyl) terephthalate, and bis(2-hydroxyethyl) terephthalate.

[0039] According to a preferred embodiment of the above aspect, in the fourth step of the method for producing a polyester, the compound includes at least one selected from the group consisting of isophthalic acid, mono(2-hydroxyethyl)isophthalate, and bis(2-hydroxyethyl)isophthalate.

[0040] According to a preferred embodiment of the above aspect, in the method for producing a polyester, in the fourth step, the compound contains ethylene glycol.

[0041] In this disclosure, amino acid substitution positions are based on the amino acid numbering shown in SEQ ID NO:1.

[0042] In the present disclosure, "sequence identity" is a parameter indicating the relatedness of two amino acid sequences and represents the degree of identity between the two amino acid sequences. Identity can be calculated using the homology search program BLAST (Basic Local Alignment Search Tool). For example, sequence identity can be calculated using the website provided by the National Center for Biotechnology Information (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE=Proteins).

[0043] In the present disclosure, for example, "A172" indicates the alanine residue at position 172 in the amino acid sequence shown in SEQ ID NO: 1. "A172C" indicates that the alanine residue at position 172 has been substituted with a cysteine ​​residue.

[0044] In the present disclosure, a "substitution" with respect to an amino acid sequence indicates that an amino acid residue in the amino acid sequence has been replaced with another amino acid residue.

[0045] In the present disclosure, the amino acid sequence can be determined using, for example, the Edman degradation method, mass spectrometry (e.g., MS / MS method), or the like.

[0046] The esterases of the present disclosure may contain disulfide bonds.

[0047] First Embodiment An esterase according to a first embodiment of the present disclosure will be described.

[0048] The esterase according to the first embodiment has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. The amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence of the esterase (LCC; leaf-branch compost cutinase) described in WO 2012 / 099018 (Patent Document 1). The esterase according to the first embodiment has preferably 85% or more, more preferably 90% or more, sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[0049] In the esterase according to the first embodiment, A172 and A209 in the amino acid sequence shown in SEQ ID NO: 1 have been substituted.

[0050] In the esterase according to the first embodiment, A172 is preferably substituted with a cysteine ​​residue (C).

[0051] In the esterase according to the first embodiment, A209 is preferably substituted with a cysteine ​​residue (C).

[0052] In the esterase according to the first embodiment, it is more preferable that A172 and A209 are each substituted with a cysteine ​​residue (C).

[0053] The esterase according to the first embodiment can be produced using known methods. For example, the esterase according to the first embodiment can be produced by genetic engineering techniques. For example, a vector containing a polynucleotide encoding the esterase according to the first embodiment is introduced into a microorganism and / or a host cell and allowed to grow. As the host cell, for example, Escherichia coli can be used. The esterase expressed in the microorganism and / or the host cell can be collected and purified to obtain the esterase.

[0054] The esterase according to the first embodiment can suitably decompose polyesters, as will be described later in the description of the polyester treatment method. For example, the esterase according to the first embodiment can have high activity at temperatures below the glass transition temperature of polyesters whose main backbone is composed of terephthalic acid components and ethylene glycol components. The esterase according to the first embodiment can efficiently decompose polyesters, for example, at temperatures of 65°C or lower, preferably 60°C or lower, and more preferably in the range of 50°C to 60°C. Furthermore, the esterase according to the first embodiment is active in environments of 40°C or higher, preferably 45°C or higher, and more preferably 50°C or higher, and can therefore more efficiently decompose polyesters.

[0055] The esterase according to the first embodiment can decompose polyester at a temperature at which the progress of crystallization of the polyester is suppressed, thereby preventing the decomposition rate from slowing down.

[0056] According to the esterase of the first embodiment, the decomposition temperature can be lowered, and therefore energy costs can be reduced.

[0057] The esterase according to the first embodiment has low substrate specificity, and the substrate is not limited to polyethylene terephthalate. For example, the esterase according to the first embodiment can decompose not only a polyethylene terephthalate homopolymer but also a polyester in which polyethylene terephthalate is copolymerized with a component other than a terephthalic acid component and an ethylene glycol component (for example, isophthalic acid) with high efficiency.

[0058] Second Embodiment An esterase according to a second embodiment of the present disclosure will be described.

[0059] The esterase according to the second embodiment is preferably the esterase according to the first embodiment, wherein at least one selected from the group consisting of D203 and S248 in the amino acid sequence is further substituted, and more preferably D203 and S248 are further substituted. In the esterase according to the second embodiment, it is preferable that at least one selected from the group consisting of D203 and S248 is substituted with a cysteine ​​residue (C), and more preferably D203 and S248 are substituted with a cysteine ​​residue (C). It is even more preferable that D203C and S248C form a disulfide bond. Formation of a disulfide bond can be confirmed by electrophoresis after reduction.

[0060] The esterase according to the second embodiment can be produced by the same method as that for the esterase according to the first embodiment.

[0061] The esterase according to the second embodiment can have the same effect as the esterase according to the first embodiment.

[0062] The esterase according to the second embodiment has higher heat resistance than the esterase according to the first embodiment. The esterase according to the second embodiment can have higher decomposition activity than the esterase according to the first embodiment at temperatures closer to the glass transition temperature. The esterase according to the second embodiment can efficiently decompose polyesters at temperatures in the range of 55°C to 65°C, for example. Therefore, by making the decomposition environment closer to the glass transition temperature, the esterase according to the second embodiment can decompose polyesters more efficiently than the esterase according to the first embodiment.

[0063] Third Embodiment A polyester treatment method according to a third embodiment of the present disclosure will be described. The polyester treatment method according to the third embodiment of the present disclosure is also a method for producing polyester monomers and / or oligomers.

[0064] The method for treating polyester according to the third embodiment includes a first step of decomposing polyester using at least one selected from the group consisting of the esterase according to the first embodiment and the esterase according to the second embodiment.

[0065] The treatment solvent may be, for example, water.

[0066] The concentration of the esterase in the treatment solution can be, for example, 30 nM or more, 50 nM or more, 80 nM or more, 100 nM or more, or 200 nM or more. The concentration of the esterase can be 1000 nM or less, 500 nM or less, or 200 nM or less.

[0067] The esterase is used in an amount of, for example, 0.5 × 10 per 1 g of polyester, which is the product to be decomposed. －９ More than mol, 1×10 －９ More than mol, 0.5×10 －８ More than mol, 1×10 －８ More than mol, 0.5×10 －８ mol or more, or 1 x 10 －７ The amount of esterase can be 1×10 mol or more relative to 1 g of polyester, which is the product to be decomposed. －６ Less than mol, 0.5×10 －６ Less than mol, 1×10 －７ Less than mol, 0.5×10 －７ Less than mol, 1×10 －８ mol or less, or 0.5 x 10 －８ mol or less.

[0068] The decomposition temperature, i.e., the temperature of the treatment solvent, can be 40°C or higher, preferably 45°C or higher, and more preferably 50°C or higher. By increasing the decomposition temperature, the decomposition rate can be increased. The decomposition temperature is preferably lower than the glass transition temperature of the polyester to be decomposed. If the decomposition temperature is higher than the glass transition temperature, crystallization of the polyester proceeds during treatment, resulting in a decrease in the decomposition rate. The decomposition temperature can be, for example, lower than 70°C, 65°C or lower, 60°C or lower, or 55°C or lower.

[0069] The pH of the treatment solvent can be in the range of 7 to 11, preferably in the range of 7 to 10. Since the pH decreases during decomposition, it is preferable to maintain the pH (for example, at pH 9) using an alkali (for example, aqueous sodium hydroxide solution).

[0070] The crystallinity of the polyester to be decomposed is 7% or less, preferably 6% or less, more preferably 5% or less, and even more preferably 3% or less. The polyester is preferably amorphous.

[0071] The crystallinity can be calculated using the following formula: Crystallinity (%) = ((ΔHm-ΔHc) / ΔHm) × 100. The crystallization temperature (Tc), crystallization enthalpy (ΔHc), melting point (Tm), and heat of fusion (ΔHm) are measured using a differential scanning calorimetry (DSC) device by heating a polyester in nitrogen from 30°C to 300°C at a heating rate of 10°C / min.

[0072] In the polyester that is the decomposition product, the acid component may contain 50 mol% or more of a terephthalic acid component. The terephthalic acid component may be, for example, 55 mol% or more, 60 mol% or more, 65 mol% or more, 70 mol% or more, 75 mol% or more, 80 mol% or more, 85 mol% or more, 90 mol% or more, 95 mol% or more, or 100 mol% relative to the total amount of the acid components. The terephthalic acid component may be, for example, 98 mol% or less, 95 mol% or less, 90 mol% or less, 85 mol% or less, 80 mol% or less, 75 mol% or less, 70 mol% or less, or 65 mol% or less relative to the total amount of the acid components.

[0073] In the polyester that is the decomposition product, the acid component may further contain 2 mol% or more of an isophthalic acid component relative to the acid component. When the acid component contains an isophthalic acid component as a copolymerization component, the isophthalic acid component may be, for example, 5 mol% or more, 10 mol% or more, 15 mol% or more, or 20 mol% or more relative to the total amount of the acid components. The isophthalic acid component may be, for example, 25 mol% or less, 20 mol% or less, 15 mol% or less, 10 mol% or less, or 5 mol% or less relative to the total amount of the acid components. The acid component may consist of a terephthalic acid component and an isophthalic acid component (total 100 mol%).

[0074] The acid component in the polyester to be decomposed may contain other acid components. Examples of the other acid components include orthophthalic acid, 2,6-naphthalenedicarboxylic acid, 2,4-furandicarboxylic acid, 2,5-furandicarboxylic acid, adipic acid, sebacic acid, succinic acid, dimer acid, 1,4-cyclohexadicarboxylic acid, dimethyl terephthalate, and dimethyl isophthalate. These other acid components may be added either alone or in combination of two or more in any desired ratio.

[0075] In the polyester to be decomposed, the alcohol component may contain 65 mol% or more of ethylene glycol components. The ethylene glycol component may be, for example, 70 mol% or more, 75 mol% or more, 80 mol% or more, 85 mol% or more, 90 mol% or more, 95 mol% or more, or 100 mol% relative to the total amount of the alcohol component. The ethylene glycol component may be, for example, 95 mol% or less, 90 mol% or less, 85 mol% or less, 80 mol% or less, or 75 mol% or less relative to the total amount of the alcohol component. The alcohol component may consist of ethylene glycol components (total 100 mol%).

[0076] The alcohol component in the polyester to be decomposed may contain other alcohol components. Examples of other alcohol components include neopentyl glycol (2,2-dimethyl-1,3-propanediol), 1,3-propanediol, 2-methyl-1,3-propanediol, 2-butyl-2-ethyl-1,3-propanediol, 1,2-propanediol, 1,4-butanediol, 1,3-butanediol, diethylene glycol, 1,4-cyclohexanediol, and 1,4-cyclohexanedimethanol. These other alcohol components may be added either alone or in combination of two or more in any desired ratio.

[0077] The polyester to be decomposed may be, for example, polyethylene terephthalate (PET) or a polyester having polyethylene terephthalate as the main skeleton and having an isophthalic acid component as the acid component. The polyester to be decomposed may be a mixture (blend, polymer alloy) of multiple polyesters. The polyester to be decomposed may also be a laminate (laminated film) of multiple polyesters.

[0078] The monomer component of the polyester to be decomposed may be a component derived from petroleum, a component derived from biomass, or a component derived from recycled materials.

[0079] Biomass-derived components can be detected, for example, by measuring the number of radioactive carbon atoms using accelerator mass spectrometry (AMS). １２ It is an isotope of C １４ C and １３ Measure the amount of C, １４ C and １３ The amount of biomass-derived raw material used can be determined from the amount of C.

[0080] The polyester to be decomposed may be at least one of a post-consumer product and a pre-consumer product. Post-consumer products refer to used polyester products. Used polyester products include, but are not limited to, products recovered after use as cosmetic containers, bottles, sheets, and other products. Pre-consumer products refer to polyesters that are not treated as products, and include, but are not limited to, waste generated during the manufacturing process of polyester products, non-compliant products, oligomers generated during the polymerization process, and scraps and waste generated during molding. The shape of the polyester to be decomposed is not particularly limited, and it may be processed into powder, flakes, sheets, films, or pellets as needed. These may be used alone or in a mixture.

[0081] The method for treating polyester further comprises a second step of isolating a polyester degradation product from the degradation product obtained in the first step, the degradation product comprising a mixture of polyester monomers and / or oligomers.

[0082] In the second step, when the polyester contains a terephthalic acid component and an ethylene glycol component, it is preferable to isolate at least one compound selected from the group consisting of terephthalic acid, mono(2-hydroxyethyl)terephthalate, bis(2-hydroxyethyl)terephthalate, and salts thereof as a decomposition product of the polyester. In the second step, when the polyester contains a terephthalic acid component, an isophthalic acid component, and an ethylene glycol component, it is preferable to isolate at least one compound selected from the group consisting of terephthalic acid, mono(2-hydroxyethyl)terephthalate, bis(2-hydroxyethyl)terephthalate, and salts thereof as a decomposition product of the polyester, and it is more preferable to also isolate at least one compound selected from the group consisting of isophthalic acid, mono(2-hydroxyethyl)isophthalate, bis(2-hydroxyethyl)isophthalate, and salts thereof.

[0083] Among the decomposition products, water-insoluble compounds derived from the acid components of the polyester can be separated and recovered from the treatment solvent by, for example, acidifying the treatment solvent. Examples of acids that can be used for neutralization include sulfuric acid and hydrochloric acid. The acid components of the decomposition products may be subjected to processes such as distillation and separation. When the polyester contains a terephthalic acid component and an ethylene glycol component, examples of compounds containing the acid components of the decomposition products include at least one compound selected from the group consisting of terephthalic acid, mono(2-hydroxyethyl)terephthalate, and bis(2-hydroxyethyl)terephthalate. When the polyester contains an isophthalic acid component and an ethylene glycol component, examples of water-insoluble compounds derived from the acid components include at least one compound selected from the group consisting of isophthalic acid, mono(2-hydroxyethyl)isophthalate, and bis(2-hydroxyethyl)isophthalate.

[0084] The method for treating polyester preferably further comprises a third step of isolating decomposition products derived from the alcohol component of the polyester from the decomposition products obtained in the first step.

[0085] In the third step, examples of the compound to be isolated include ethylene glycol, neopentyl glycol (2,2-dimethyl-1,3-propanediol), 1,3-propanediol, 2-methyl-1,3-propanediol, 2-butyl-2-ethyl-1,3-propanediol, 1,2-propanediol, 1,4-butanediol, 1,3-butanediol, diethylene glycol, 1,4-cyclohexanediol, 1,4-cyclohexanedimethanol, mono(2-hydroxyethyl)terephthalate, bis(2-hydroxyethyl)terephthalate, etc. In the third step, when the polyester contains an ethylene glycol component, the decomposition product derived from the alcohol component of the polyester to be isolated preferably contains ethylene glycol, but may consist solely of ethylene glycol.

[0086] Among the decomposition products, the alcohol component of the polyester decomposition product can be separated from the treatment solvent by, for example, distillation. When the polyester contains an ethylene glycol component, the compound containing the alcohol component of the decomposition product can be at least one compound selected from the group consisting of ethylene glycol, diethylene glycol, mono(2-hydroxyethyl)terephthalate, and bis(2-hydroxyethyl)terephthalate.

[0087] When it is not possible to isolate a single compound from the acid component and the alcohol component separated from the treatment solvent, or when isolation is not required, the decomposition products can be recovered as a mixture. The content ratio of each component in the mixture can be analyzed, for example, by nuclear magnetic resonance (NMR). The decomposition product can be applied as a mixture to the method for producing a polyester according to the fourth embodiment.

[0088] According to the polyester treatment method of the third embodiment, polyester can be decomposed with high efficiency. Furthermore, the decomposition products obtained by the polyester treatment method of the third embodiment can be used as raw materials. For example, the decomposition products can be used to regenerate new polyester.

[0089] According to the method for treating polyester according to the third embodiment, the decomposition temperature can be lowered, so that the polyester can be decomposed while reducing energy costs.

[0090] According to the method for treating polyesters of the third embodiment, not only a homopolymer of polyethylene terephthalate but also a polyester in which polyethylene terephthalate is copolymerized with a component other than a terephthalic acid component and an ethylene glycol component (for example, isophthalic acid) can be decomposed with high efficiency.

[0091] Fourth Embodiment A method for producing a polyester according to a fourth embodiment of the present disclosure will be described.

[0092] A method for producing a polyester according to a fourth embodiment of the present disclosure includes a fourth step of producing a polyester using, as at least a part of the monomers, the decomposition products obtained in the method for treating a polyester according to the third embodiment. Examples of the decomposition products include the compounds isolated in the third embodiment.

[0093] In the method for producing a polyester according to the present disclosure, the raw material monomers other than the decomposition products can be appropriately adjusted according to the properties of the desired polyester, and are not particularly limited.

[0094] In the method for producing a polyester according to the present disclosure, raw material monomers other than the decomposition products may or may not be used. When raw material monomers other than the decomposition products are used, the raw material monomers may be petroleum-derived components, biomass-derived components, or recycled materials-derived components.

[0095] Examples of acid components of raw material monomers other than decomposition products in the production method for polyester according to the present disclosure include terephthalic acid, isophthalic acid, orthophthalic acid, 2,6-naphthalenedicarboxylic acid, 2,4-furandicarboxylic acid, 2,5-furandicarboxylic acid, adipic acid, sebacic acid, succinic acid, dimer acid, 1,4-cyclohexadicarboxylic acid, dimethyl terephthalate, dimethyl isophthalate, bis-2-hydroxyethyl terephthalate, bis-2-hydroxyethyl isophthalate, etc. Any of these acid components may be added alone, or two or more may be added in any ratio.

[0096] Examples of alcohol components of raw material monomers other than decomposition products in the polyester production method of the present disclosure include ethylene glycol, neopentyl glycol (2,2-dimethyl-1,3-propanediol), 1,3-propanediol, 2-methyl-1,3-propanediol, 2-butyl-2-ethyl-1,3-propanediol, 1,2-propanediol, 1,4-butanediol, 1,3-butanediol, diethylene glycol, triethylene glycol, 1,4-cyclohexanediol, 1,4-cyclohexanedimethanol, polyethylene glycol, polytrimethylene glycol, polytetramethylene glycol, etc. Any of these alcohol components may be added alone, or two or more may be added in any ratio.

[0097] The blending ratio of the decomposition products in the polyester production method of the present disclosure is the mass of the components derived from the decomposition products in the recycled polyester divided by the mass of the entire recycled polyester. The blending ratio of the decomposition products can be, for example, 10% by mass or more, 20% by mass or more, 30% by mass or more, 50% by mass or more, 70% by mass or more, 90% by mass or more, 95% by mass or more, or 100% by mass. The blending ratio of the decomposition products can be, for example, 95% by mass or less, 90% by mass or less, 70% by mass or less, 50% by mass or less, 30% by mass or less, or 20% by mass or less.

[0098] In the polyester production method of the present disclosure, the blending ratio of components derived from recycled materials, including decomposition products, is calculated by adding the mass of components derived from decomposition products in the recycled polyester and the mass of monomer components other than the decomposition products in the recycled polyester that are derived from recycled materials, and dividing the sum by the total mass of the recycled polyester. The blending ratio of components derived from recycled materials, including decomposition products, can be, for example, 10% by mass or more, 20% by mass or more, 30% by mass or more, 50% by mass or more, 70% by mass or more, 90% by mass or more, 95% by mass or more, or 100% by mass. The blending ratio of components derived from recycled materials, including decomposition products, can be, for example, 95% by mass or less, 90% by mass or less, 70% by mass or less, 50% by mass or less, 30% by mass or less, or 20% by mass or less.

[0099] The polymerization step in the polyester production method of the present disclosure is not particularly limited and can be carried out by a known polymerization method using a known catalyst. The polyester production method may be either a method of direct esterification using an unsubstituted polycarboxylic acid as a starting material, or a method of carrying out a transesterification reaction using an esterified product such as a dimethyl ester as a starting material. In producing the polyester, in order to obtain a sufficient reaction rate, it is desirable to carry out a transesterification reaction under normal pressure using a known catalyst in the first step, followed by a polycondensation reaction under reduced pressure using a known catalyst in the second step. A solid-state polymerization reaction may be carried out after the polycondensation reaction. The intrinsic viscosity of the polyester can be increased by the solid-state polymerization reaction.

[0100] According to the polyester production method according to the fourth embodiment of the present disclosure, polyester can be regenerated from the decomposition products of polyester decomposed using esterase.

[0101] Fifth Embodiment A polyester decomposition product according to a fifth embodiment of the present disclosure will be described.

[0102] The polyester degradation product according to the fifth embodiment of the present disclosure is obtained by decomposing a polyester with the esterase according to the first embodiment of the present disclosure and / or the esterase according to the second embodiment. The polyester degradation product according to the fifth embodiment of the present disclosure is similar to the degradation product obtained in the first step of the polyester treatment method according to the third embodiment, and contains a mixture of polyester monomers and / or oligomers. The polyester degradation product according to the fifth embodiment of the present disclosure is distinguishable from conventional polyester degradation products because it contains the esterase according to the first embodiment and / or the esterase according to the second embodiment. A polyester can be produced using the polyester degradation product according to the fifth embodiment of the present disclosure. In this case, the polyester can be produced in the same manner as the polyester production method according to the fourth embodiment of the present disclosure.

[0103] Sixth Embodiment A polyester according to a sixth embodiment of the present disclosure will be described.

[0104] The polyester according to the sixth embodiment of the present disclosure is a polyester produced by the production method according to the fourth embodiment. That is, the polyester according to the sixth embodiment of the present disclosure is a polyester produced using a decomposition product of a polyester as a raw material.

[0105] Seventh Embodiment A polynucleotide according to a seventh embodiment of the present disclosure encodes the esterase according to the first and second embodiments. A vector contains the polynucleotide. Furthermore, a host cell is a host cell into which the polynucleotide and / or vector has been introduced. An example of the host cell is Escherichia coli.

[0106] In some cases, it may be impossible or simply impractical to directly identify the esterases of the present disclosure by composition, structure, properties, etc. In such cases, it should be permitted to identify the esterases of the present disclosure by their method of manufacture.

[0107] The esterase, polyester treatment method, and polyester production method of the present disclosure will be described below using examples, but the esterase, polyester treatment method, and polyester production method of the present disclosure are not limited to the following examples.

[0108] [Production of Esterases] The vector pET21b(+) (Novagen, Germany) was used for expression of cutinase LCC and its mutants in Escherichia coli BL21 (DE3) CodonPlus RIPL (Agilent Technologies GmbH, Germany).

[0109] The codon usage was optimized for expression in Escherichia coli of genes encoding sequences obtained by removing the N-terminal signal peptide from LCC and its mutants, and sequences were designed to add sequences at both ends for incorporation into the NdeI and XhoI restriction enzyme sites of the pET21b(+) vector by In-Fusion cloning, followed by total synthesis. The synthesized DNA fragment was incorporated into the NdeI and XhoI restriction enzyme sites of the pET21b(+) vector by In-Fusion cloning, so that a 6xHis-Tag was introduced at the C-terminus. The length of the DNA fragment incorporated into the plasmid vector was confirmed by colony PCR. PCR was performed using Gotaq Green Master Mix (Promega) and the T7 promoter (5'-CGCGAAATTAATACGACTCACTATAGGG-3') and T7 terminator (5'-GCTAGTTATTGCTCAGCGGTGG-3') as primers. The gene sequence was determined by DNA sequencing using the T7 promoter and T7 terminator as primers. The plasmid vector was introduced into E. coli BL21 (DE3) CodonPlus RIPL.

[0110] Freshly transformed E. coli BL21 (DE3) CodonPlus RIPL cells were inoculated into LB medium containing ampicillin and chloramphenicol, and protein expression was induced using IPTG as a protein expression inducer. The cells were collected, disrupted using an ultrasonic disrupter, and centrifuged at 14,000 rpm at 4°C for 20 minutes. The supernatant was used as a crude enzyme solution.

[0111] The crude enzyme solution was subjected to affinity chromatography using TALON resin (manufactured by TAKARA BIO). Bands in each eluate were confirmed by SDS-PAGE to confirm whether the target protein had been purified. The purified protein was desalted using a PD-10 desalting column (manufactured by Cytiva) with 50 mM NaCl. ２ HPO ４The buffer was exchanged with HCl, pH 7.0. The protein concentration was calculated using the wavelength of 280 nm. The molar extinction coefficient ε of the protein was calculated using the ProtParam tool (http: / / web.expasy.org / protparam / ).

[0112] All chemicals were of analytical grade from Sigma (Germany).

[0113] [Method of treating polyester] (1) Degraded polyesters The polyesters of Samples 1 to 4 shown in Table 2 were treated with the enzymes A to D shown in Table 1, respectively. Sample 1 is polyethylene terephthalate (PET). Samples 2 to 4 are polyesters in which isophthalic acid is copolymerized with PET as a third component. The copolymerization rate of isophthalic acid in each sample was measured by proton NMR spectroscopy using a Bruker DPX-400 FT-NMR apparatus by dissolving each sample in a mixed solution of trifluoroacetic acid-d and deuterated chloroform, mixing with tetramethylsilane as a standard.

[0114] (2) Crystallinity, Glass Transition Temperature, Crystallization Temperature, and Melting Point Table 2 shows the crystallinity, glass transition temperature, crystallization temperature, and melting point of each sample. 10 mg of polyester resin was weighed and heated from 30°C to 300°C at a heating rate of 10°C / min in a nitrogen atmosphere using a differential scanning calorimeter DSC (TA Instruments, DSC2500), and the crystallization temperature (Tc), crystallization enthalpy (ΔHc), melting point (Tm), and heat of fusion (ΔHm) were measured. The crystallization temperature (Tc) was calculated using the following formula: Crystallinity (Tc) (%) = ((ΔHm-ΔHc) / ΔHm) x 100

[0115]

[0116]

[0117] (3) Film Formation Each of Samples 1 to 4 was formed into a film shape with a thickness of 0.2 mm in order to increase the contact area with the esterase.

[0118] (4) Decomposition of Polyester A polyester film having a thickness of 0.2 mm and a diameter of 6 mm was added to 200 mM Bicine-NaOH (pH 9.0), and the purified enzyme obtained by the above-mentioned method was added to a final concentration of 100 nM. The mixture was heated to 50 to 65°C and allowed to stand for 3, 6, 24, or 48 hours to react.

[0119] (5) Measurement of decomposition products A portion of the reaction solution after the reaction was taken and diluted with a buffer solution (80% (v / v) 20 mM NaH ２ P.O. ４ -H ３ P.O. ４ The resulting solution was diluted with 20% (v / v) DMSO (pH 2.5) and left to stand at 90°C for 10 minutes for heat treatment, and a portion of the supernatant was analyzed by high performance liquid chromatography (HPLC). Solutions of known concentrations of terephthalic acid, mono(2-hydroxyethyl)terephthalate, bis(2-hydroxyethyl)terephthalate, and isophthalic acid were used as standard solutions.

[0120] 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, Inc.) 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%

[0121] The peak areas of the decomposition products (terephthalic acid, mono(2-hydroxyethyl) terephthalate, bis(2-hydroxyethyl) terephthalate, and isophthalic acid) separated and detected by HPLC were compared with the peak areas of the standard solutions to quantify the decomposition products under each condition.

[0122] (Test Examples 1 to 8) Decomposition tests using the esterases shown in Table 1 were conducted using polyester samples 1 to 4 shown in Table 2 as the decomposition target. The esterases used were LCC (Enzyme D) having the amino acid sequence shown in SEQ ID NO: 1, and its variants A172C_A209C (Enzyme A), A172C_A209C_D203C_S248C (Enzyme B), and D203C_S248C (Enzyme C). The symbols A to D are used in the following table. After decomposition of each sample at 50°C or 60°C for 24 hours, the recovered amounts of terephthalic acid, isophthalic acid, and all components (terephthalic acid, mono(2-hydroxyethyl)terephthalate, bis(2-hydroxyethyl)terephthalate, and isophthalic acid) were measured. Tables 3 to 10 show the recovered amounts of terephthalic acid, isophthalic acid, and all components. The total amount of recovered components is the total amount of terephthalic acid, isophthalic acid, mono(2-hydroxyethyl) terephthalate, and bis(2-hydroxyethyl) terephthalate.

[0123] The recovery rates of the degradation products obtained by enzymes A, B, and C were calculated as the percentage increase relative to the recovery rate of the degradation products obtained by enzyme D. The percentage increase was calculated based on the following formula. The recovery rates shown in each table are rounded to three digits. However, since the percentage increase shown in the table is calculated based on the recovery amounts before rounding, the percentage increase calculated based on the recovery amounts shown in the table may not completely match the percentage increase shown in the table.

[0124] Increase rate (%) = (amount recovered of degradation product by target esterase (µmol) - amount recovered of degradation product by LCC (µmol)) / amount recovered of degradation product by LCC (µmol) × 100

[0125] Figures 1 to 8 show graphs comparing the recovery amounts of decomposition products of Samples 1 to 4 using each esterase. Figures 1 and 2 are graphs showing the amount of terephthalic acid recovered and its rate of increase when Samples 1 to 4 are decomposed at a decomposition temperature of 50°C, respectively. Figures 3 and 4 are graphs showing the amount of isophthalic acid recovered and its rate of increase when Samples 1 to 4 are decomposed at a decomposition temperature of 50°C, respectively. Figures 5 and 6 are graphs showing the amount of all components recovered and its rate of increase when Samples 1 to 4 are decomposed at a decomposition temperature of 50°C, respectively. Figures 7 and 8 are graphs showing the amount of terephthalic acid recovered and its rate of increase when Samples 1 to 4 are decomposed at a decomposition temperature of 60°C, respectively. Figures 9 and 10 are graphs showing the amount of isophthalic acid recovered and its rate of increase when Samples 1 to 4 are decomposed at a decomposition temperature of 60°C, respectively. 11 and 12 are graphs showing the total component recovery amounts and their increasing rates when Samples 1 to 4 are decomposed at a decomposition temperature of 60° C. The horizontal axes shown in FIGS. 1 and 2, 5 to 8, and 11 to 12 represent the content of terephthalic acid contained in the acid components of Samples 1 to 4. The horizontal axes shown in FIGS. 3 and 4 and 9 to 10 represent the content of isophthalic acid contained in the acid components of Samples 1 to 4.

[0126] D203C_S248C had lower decomposition efficiency than LCC at both 50° C. and 60° C. On the other hand, A172C_A209C and A172C_A209C_D203C_S248C were both able to increase the decomposition efficiency compared to LCC.

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135] (Test Examples 9 to 12) The decomposition rates of the esterases shown in Table 1 were compared using the polyesters of Samples 1 and 4 shown in Table 2 as the decomposition target materials. For each esterase, the amount of decomposition product recovered was measured when the decomposition time was 3 hours, 6 hours, and 24 hours. The measurement results are shown in Tables 11 to 14. Figure 13 shows the change over time in the amount of decomposition product recovered when Sample 1 was decomposed at 50°C. Figure 14 shows the change over time in the amount of decomposition product recovered when Sample 1 was decomposed at 60°C. Figure 15 shows the change over time in the amount of decomposition product recovered when Sample 4 was decomposed at 50°C. Figure 16 shows the change over time in the amount of decomposition product recovered when Sample 4 was decomposed at 60°C.

[0136] Regardless of the polyester composition, enzyme A (A172C_A209C) and enzyme B (A172C_A209C_D203C_S248C) degraded faster than enzyme C (D203C_S248C) and enzyme D (LCC).

[0137]

[0138]

[0139]

[0140]

[0141] (Test Examples 13 and 14) Using the polyesters of Samples 1 and 4 shown in Table 2 as decomposition targets, decomposition tests were carried out at 65°C using Enzyme B and Enzyme C shown in Table 1. Tables 15 and 16 show the measurement results. Figure 17 shows the amount of decomposition product recovered when Sample 1 was decomposed at 65°C. Figure 18 shows the amount of decomposition product recovered when Sample 4 was decomposed at 65°C.

[0142] The decomposition rate of Enzyme B was faster even at a high temperature of 65°C. It was also found that Enzyme B has heat resistance at 65°C. This indicates that Enzyme B can decompose polyester at higher temperatures where crystallization of polyester is suppressed.

[0143]

[0144]

[0145] Although the test results are not shown, when Sample 1 was heated at 70°C for 48 hours, the crystallinity increased to 5.4%. As the crystallinity of polyester increases, the decomposition rate by esterase decreases. Therefore, it is considered that the decomposition temperature is preferably 65°C or lower, and more preferably 60°C or lower.

[0146] (Test Example 15) [Preparation of Recycled Polyester 1] A total of 17.3 g of terephthalic acid and isophthalic acid (derived from recycled materials) collected under the conditions of Test Example 8-2 was charged into a reaction vessel equipped with a stirrer, a distillation tube, and a pressure reducing device, along with 21.3 g of terephthalic acid (petroleum-derived), 4.7 g of isophthalic acid (petroleum-derived), 66.2 g of bis-2-hydroxyethyl terephthalate (derived from recycled materials), and 8.1 g of ethylene glycol (petroleum-derived), and heated to 250 ° C. under a nitrogen flow. The mixture was then maintained for 3 hours to carry out esterification. Subsequently, 54 ppm of orthophosphoric acid and 120 ppm of germanium dioxide were added as a polymerization catalyst and stirred, after which the pressure was reduced to 133 Pa or less over 1 hour, during which the internal temperature was raised from 250 ° C. to 285 ° C., and the mixture was stirred under a high vacuum of 133 Pa or less until a predetermined viscosity was reached, thereby carrying out a polycondensation reaction. The resulting polymer was extruded into water in the form of a strand, cut, and pelletized. In this way, Recycled Polyester 1 was produced.

[0147] The intrinsic viscosity and color space Lab of the obtained recycled polyester 1 were measured as follows.

[0148] <Measurement of Intrinsic Viscosity> 0.5000 g±0.001 g of a sample was dissolved in 50 mL of a mixed solvent of phenol:tetrachloroethane = 60:40 (mass ratio), and the intrinsic viscosity (IV) at 20°C was measured using an automatic viscosity measuring device (ALC-6C, manufactured by Sun Electronics Industries Co., Ltd.) equipped with an Ubbelohde viscosity tube. The intrinsic viscosity (IV) was found to be 0.604 dL / g.

[0149] <Measurement of color space Lab> The color space Lab of the obtained recycled polyester 1 was measured using a color meter (ZE6000, manufactured by Nippon Denshoku Industries Co., Ltd.) and found to be L=65.9, a=-1.0, and b=3.9.

[0150] Test Example 16 Preparation of Recycled Polyester 2 Recycled polyester 2 was prepared in the same manner as in Test Example 15, except that the terephthalic acid and isophthalic acid (derived from recycled materials) collected under the conditions of Test Example 8-2 were not used, and instead 33.3 g of terephthalic acid (petroleum-derived), 10.0 g of isophthalic acid (petroleum-derived), 66.2 g of bis-2-hydroxyethyl terephthalate (derived from recycled materials), and 8.1 g of ethylene glycol (petroleum-derived) were used. The intrinsic viscosity and color space Lab of the obtained recycled polyester 2 were measured in the same manner as above, and the results were intrinsic viscosity (IV) = 0.612 dl / g, L = 65.0, a = -1.2, and b = 3.7.

[0151] (Test Example 17) [Preparation of Recycled Polyester 3] Recycled polyester 3 was prepared in the same manner as in Test Example 15, except that 66.2 g of petroleum-derived bis-2-hydroxyethyl terephthalate was used instead of 66.2 g of recycled material-derived bis-2-hydroxyethyl terephthalate in Test Example 15. The intrinsic viscosity and color space Lab of the obtained recycled polyester 3 were measured in the same manner as above, and the results were intrinsic viscosity (IV) = 0.610 dl / g, L = 64.8, a = -1.1, and b = 4.0.

[0152] Test Example 18 Preparation of Recycled Polyester 4 Recycled polyester 4 was prepared in the same manner as in Test Example 15, except that a total of 43.3 g of terephthalic acid and isophthalic acid (derived from recycled materials) collected under the conditions of Test Example 8-2, 66.2 g of bis-2-hydroxyethyl terephthalate (derived from recycled materials), and 8.1 g of ethylene glycol (derived from petroleum) were used. The intrinsic viscosity and color space Lab of the obtained recycled polyester 4 were measured in the same manner as above, and the results were intrinsic viscosity (IV) = 0.602 dl / g, L = 65.5, a = -1.1, and b = 3.9.

[0153] Test Example 19 Preparation of Recycled Polyester 5 Recycled polyester 5 was prepared in the same manner as in Test Example 15, except that a total of 32.5 g of terephthalic acid and isophthalic acid (derived from recycled materials) collected under the conditions of Test Example 8-2 and 82.8 g of bis-2-hydroxyethyl terephthalate (derived from recycled materials) were used. The intrinsic viscosity and color space Lab of the obtained Recycled Polyester 5 were measured in the same manner as above, and the results were intrinsic viscosity (IV) = 0.618 dl / g, L = 66.0, a = -0.9, and b = 3.4.

[0154] (Test Example 20) [Preparation of Recycled Polyester 6] A total of 86.6 g of terephthalic acid and isophthalic acid (derived from recycled materials) collected under the conditions of Test Example 8-2 and 40.5 g of ethylene glycol (derived from petroleum) were charged into a reaction vessel equipped with a stirrer, a distillation tube, and a pressure reducing device, and the mixture was pressurized with nitrogen to 0.2 MPa and held for 8 hours to carry out an esterification reaction. The pressure was then released to atmospheric pressure, and the generated water was distilled off. The subsequent polymerization process and subsequent steps were carried out in the same manner as in Test Example 15 to prepare Recycled Polyester 6. The intrinsic viscosity and color space Lab of the obtained Recycled Polyester 6 were measured in the same manner as above, and found to be intrinsic viscosity (IV) = 0.620 dl / g, L = 63.8, a = -1.3, and b = 4.5.

[0155] Test Example 21 Preparation of Virgin Polyester A virgin polyester was prepared in the same manner as in Test Example 15, except that the terephthalic acid and isophthalic acid (derived from recycled materials) collected under the conditions of Test Example 8-2 were not used, and instead 33.3 g of terephthalic acid, 10.0 g of isophthalic acid, 66.2 g of bis-2-hydroxyethyl terephthalate, and 8.1 g of ethylene glycol, all of which were derived from petroleum, were used. The intrinsic viscosity and color space Lab of the obtained virgin polyester were measured in the same manner as above, and found to be intrinsic viscosity (IV) = 0.609 dl / g, L = 64.7, a = -1.2, and b = 4.2.

[0156] The results of Test Examples 15 to 21 show that the recycled polyesters produced using the polyester decomposition product (Test Example 8-2) by the esterase of the present disclosure in Test Examples 15 and 17 to 19 have the same quality as the recycled polyester produced without using the polyester decomposition product (Test Example 8-2) by the esterase of the present disclosure in Test Example 16, and the virgin polyester in Test Example 21.

[0157] The disclosed esterases, polyester degradation products, polyester treatment methods, polyester production methods, polynucleotides, vectors, and host cells have been described based on the above embodiments and examples, but are not limited to the above embodiments and examples, and can include various modifications, changes, and improvements to each disclosed element (including elements described in the claims, specification, and drawings) within the scope of the present invention and based on the basic technical concept of the present invention. Furthermore, various combinations, substitutions, and selections of each disclosed element are possible within the scope of the claims of the present invention.

[0158] Further objects, purposes and modes (including modifications) of the present invention will become apparent from the entire disclosure of the present invention including the claims.

[0159] With respect to numerical ranges set forth herein, unless otherwise specified, any numerical value or range falling within that range should be construed as being specifically set forth herein.

[0160] Some or all of the above embodiments can be described as in the following supplementary notes, but are not limited to the following descriptions. Each supplementary note can also be combined with each claim described in the claims. [Supplementary Note 1] A polyester produced by the polyester production method of the present disclosure. [Supplementary Note 2] A method for producing polyester monomers and / or oligomers by the polyester treatment method of the present disclosure. [Supplementary Note 3] A polyester monomer and / or oligomer produced by the polyester treatment method of the present disclosure.

[0161] The esterase of the present disclosure can decompose containers, films, sheets, fibers, clothing, and the like made from degradable polyesters down to the chemical compound level. By using polyester decomposition products as raw materials, a resource circulation recycling system for polyesters can be established.

Claims

1. An esterase having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:1, wherein A172 and A209 in said amino acid sequence are substituted.

2. The esterase according to claim 1, wherein A172 and A209 are substituted with cysteine ​​residues (C).

3. The esterase according to claim 1 or 2, wherein D203 and S248 in the amino acid sequence are further substituted.

4. The esterase described in claim 3, in which D203 and S248 are substituted with cysteine ​​residues (C).

5. The esterase described in claim 3 or 4, wherein D203C and S248C form a disulfide bond.

6. A method for treating polyester, comprising a first step of degrading polyester using the esterase according to any one of claims 1 to 5.

7. The method for treating polyester according to claim 6, wherein in the first step, the polyester is decomposed in a treatment solvent at a temperature of 40° C. or higher and lower than the glass transition temperature of the polyester and at a pH in the range of 7 to 10.

8. The method for treating polyester according to claim 7, wherein in the first step, the polyester is decomposed in the treatment solvent at a temperature of 65° C. or less.

9. The method for treating polyester according to any one of claims 6 to 8, wherein the polyester contains 50 mol % or more of terephthalic acid components based on the total amount of acid components, and 65 mol % or more of ethylene glycol components based on the total amount of alcohol components.

10. The method for treating polyester according to claim 9, wherein the polyester further contains 2 mol % or more of an isophthalic acid component based on the total amount of the acid components.

11. The method for treating polyester according to any one of claims 6 to 10, wherein the polyester is at least one of a post-consumer product and a pre-consumer product.

12. The method for treating the polyester according to any one of claims 6 to 11, further comprising a second step of isolating at least one compound selected from the group consisting of terephthalic acid, mono(2-hydroxyethyl)terephthalate, bis(2-hydroxyethyl)terephthalate, and salts thereof from the decomposition product obtained in the first step.

13. The method for treating polyester according to claim 12, wherein in the second step, at least one compound selected from the group consisting of isophthalic acid, mono(2-hydroxyethyl)isophthalate, bis(2-hydroxyethyl)isophthalate, and salts thereof is also isolated.

14. The method for treating polyester described in any one of claims 6 to 13, further comprising a third step of isolating a decomposition product derived from an alcohol component of the polyester from the decomposition product obtained in the first step.

15. A method for producing a polyester, comprising a fourth step of producing a polyester using the compound isolated in the method for treating a polyester according to any one of claims 12 to 14 as at least a part of the monomers.

16. The method for producing a polyester according to claim 15, wherein in the fourth step, the compound includes at least one compound selected from the group consisting of terephthalic acid, mono(2-hydroxyethyl)terephthalate, and bis(2-hydroxyethyl)terephthalate.

17. The method for producing a polyester according to claim 15 or 16, wherein in the fourth step, the compound further comprises at least one selected from the group consisting of isophthalic acid, mono(2-hydroxyethyl)isophthalate, and bis(2-hydroxyethyl)isophthalate.

18. The method for producing a polyester according to any one of claims 15 to 17, wherein in the fourth step, the compound includes ethylene glycol.

19. A polyester degradation product obtained by decomposing a polyester with the esterase according to any one of claims 1 to 5.

20. A method for producing a polyester, comprising using the polyester decomposition product according to claim 19 to produce a polyester.

21. A polynucleotide encoding an esterase according to any one of claims 1 to 5.

22. A vector comprising the polynucleotide of claim 21.

23. A host cell into which the polynucleotide of claim 21 has been introduced.

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