Esterase, polyester decomposition product, method for treating polyester, and method for producing polyester

Abstract

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

Description

[0001] Related applications

[0002] This application claims priority to Japanese Patent Application No. 2023-139358, filed on August 29, 2023, and incorporates all the contents described in that Japanese application. Technical Field

[0003] This disclosure relates to enzymes (esterases) that hydrolyze ester compounds into acids and alcohols. This disclosure relates to polyester degradation products. This disclosure relates to methods for treating polyesters with esterases. Additionally, this disclosure relates to methods for manufacturing polyesters using esterases. This disclosure relates to polynucleotides, carriers, and host cells for manufacturing esterases. Background Technology

[0004] Polyester resins such as polyethylene terephthalate (PET), as general-purpose plastics, are among the most commonly used plastic materials in recycling systems. However, in current recycling systems, so-called one-way recycling is the mainstream. One-way recycling refers to plastic products not being reused directly in their original shape after use, but rather being reused as raw materials for other products. For example, plastic bottles, one of the uses of polyester resin, are washed, melted, and reshaped after use to be used for clothing fibers, industrial sheets, etc. This type of reuse is called material recycling. In material recycling, the quality of recycled plastic products decreases with each regeneration, thus making it impossible to build a circular recycling system that truly recycles plastic resources.

[0005] Therefore, chemical recycling is being researched as a method to realize a plastic resource recycling system. Chemical recycling refers to the chemical decomposition of waste plastics, using the decomposition products as raw materials for reuse. Chemical recycling decomposes plastics to the compound level, thus stabilizing the quality of recycled products and allowing the decomposition products to be used as raw materials for products other than plastics. Therefore, it is a useful recycling method for building resource recycling systems. However, for example, in chemical recycling using thermal decomposition, high-temperature plastic depolymerization and distillation purification of depolymerization products are required, consuming a large amount of heat energy for recycling.

[0006] Therefore, the chemical reuse of polyesters by enzymatic decomposition has recently attracted attention (see, for example, Patent Document 1 and Patent Document 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.

[0007] Existing technical documents

[0008] Patent documents

[0009] Patent Document 1: International Publication No. 2012 / 099018

[0010] Patent Document 2: Japanese Patent Publication No. 2019-520833

[0011] Non-patent literature

[0012] Non-patent literature 1: Tournier et al., “An engineered PET depolymerase to break down and recycle plastic bottles” Nature, Vol. 580, pp 216-219 (2020)

[0013] Non-patent literature 2: Yoshida et al., “A bacterium that degrades and assimilates poly(ethylene terephthalate)” Sciencemag.org, Vol. 351, pp 1196 (2016) Summary of the Invention

[0014] The problem the invention aims to solve

[0015] According to the esterase described in Patent Document 1, even under conditions of maximum decomposition activity, only about 30% is decomposed in one week and about 54% in two weeks, which is insufficient for industrial-scale decomposition.

[0016] To increase the rate of polyester decomposition by enzymes, it is thought that improving the enzyme's heat resistance would allow for the decomposition of polyester at high temperatures. However, if the polyester is continuously placed in a temperature environment near its glass transition temperature, crystallization occurs. For example, Non-Patent Document 1 discloses the crystallization of polyethylene terephthalate at 70°C, which is lower than its glass transition temperature of approximately 75°C. Generally, the rate of enzymatic decomposition of highly crystalline polyesters is significantly lower than that of poorly crystalline polyesters (see, for example, Non-Patent Document 2).

[0017] Therefore, it is desirable to decompose highly active esterases at a temperature lower than the crystallization temperature of the polyester, which is the product to be decomposed.

[0018] In addition, from the perspective of energy cost, it is also desirable to decompose highly active esterases at lower temperatures.

[0019] Solution for solving the problem

[0020] According to a first aspect of this disclosure, an esterase is provided that has at least 80% sequence identity with the amino acid sequence shown in Serial No. 1, wherein A172 and A209 in the amino acid sequence are substituted.

[0021] According to a second aspect of this disclosure, a polyester processing method is provided, comprising a first step of decomposing the polyester using an esterase according to a first aspect.

[0022] According to a third aspect of this disclosure, a method for manufacturing a polyester is provided, comprising a fourth step of manufacturing the polyester by using a compound separated in a polyester processing method of the second aspect as at least a portion of a monomer.

[0023] According to the fourth aspect of this disclosure, a polyester decomposition product obtained by decomposing a polyester using the esterase of the first aspect is provided.

[0024] According to the fifth aspect of this disclosure, a method for manufacturing polyester is provided, which uses the polyester decomposition products of the fourth aspect to manufacture polyester.

[0025] According to the sixth aspect of this disclosure, a polynucleotide encoding the esterase of the first aspect is provided.

[0026] According to the seventh point of this disclosure, a vector containing the polynucleotide of the sixth point is provided.

[0027] According to the eighth point of this disclosure, a host cell incorporating the polynucleotides of the sixth point is provided.

[0028] The effects of the invention

[0029] According to the esterase and polyester treatment method disclosed herein, polyester can be enzymatically decomposed into acids and alcohols at a high decomposition rate.

[0030] According to the esterase and polyester treatment method disclosed herein, polyester can be decomposed in an environment that inhibits the crystallization of polyester.

[0031] According to the esterase and polyester treatment method disclosed herein, the energy cost of polyester decomposition treatment can be suppressed.

[0032] The esterase disclosed herein has low substrate specificity.

[0033] According to the polyester manufacturing method disclosed herein, high-quality recycled polyester can be manufactured.

[0034] According to the polyester manufacturing method disclosed herein, the resource recycling of polyester can be realized. Attached Figure Description

[0035] [ Figure 1 ] Figure 1 This is a chart showing the recovery of terephthalic acid in test examples 1, 3, 5 and 7.

[0036] [ Figure 2 ] Figure 2 This is a graph showing the increase in the recovery rate of terephthalic acid in test examples 1, 3, 5 and 7.

[0037] [ Figure 3 ] Figure 3 This is a graph showing the recovery of isophthalic acid in test examples 1, 3, 5 and 7.

[0038] [ Figure 4 ] Figure 4 This is a graph showing the increase in the recovery rate of isophthalic acid in test examples 1, 3, 5 and 7.

[0039] [ Figure 5 ] Figure 5 This is a chart showing the recovery rates of all components in test examples 1, 3, 5, and 7.

[0040] [ Figure 6 ] Figure 6 This is a graph showing the increase in the recovery rate of all components in test examples 1, 3, 5 and 7.

[0041] [ Figure 7 ] Figure 7 This is a chart showing the recovery of terephthalic acid in test examples 2, 4, 6 and 8.

[0042] [ Figure 8 ] Figure 8 This is a graph showing the increase in the recovery rate of terephthalic acid in test examples 2, 4, 6 and 8.

[0043] [ Figure 9 ] Figure 9 This is a graph showing the recovery of isophthalic acid in test examples 2, 4, 6 and 8.

[0044] [ Figure 10 ] Figure 10 This is a graph showing the increase in the recovery rate of isophthalic acid in test examples 2, 4, 6 and 8.

[0045] [ Figure 11 ] Figure 11 This is a chart showing the recovery rates of all components in test examples 2, 4, 6, and 8.

[0046] [ Figure 12 ] Figure 12 This is a graph showing the increase in the recovery rate of all components in test examples 2, 4, 6 and 8.

[0047] [ Figure 13 ] Figure 13 This is a graph showing the trend of the recovery amount of decomposition products in Experiment Example 9.

[0048] [ Figure 14 ] Figure 14 This is a graph showing the trend of the recovery amount of decomposition products in Experiment Example 10.

[0049] [ Figure 15 ] Figure 15 This is a graph showing the trend of the recovery amount of decomposition products in Experiment Example 11.

[0050] [ Figure 16 ] Figure 16 This is a graph showing the trend of the recovery amount of decomposition products in Experiment Example 12.

[0051] [ Figure 17 ] Figure 17 This is a chart showing the recovery amount of decomposition products in Experiment Example 13.

[0052] [ Figure 18 ] Figure 18 This is a graph showing the recovery amount of decomposition products in Test Example 14. Detailed Implementation

[0053] According to the preferred approach described above, A172 and A209 are replaced by cysteine ​​residues (C).

[0054] According to the preferred approach described above, D203 and S248 in the amino acid sequence are further substituted.

[0055] According to the preferred form of the above viewpoint, D203 and S248 are replaced by cysteine ​​residues (C).

[0056] According to the preferred method described above, D203C and S248C form a disulfide bond.

[0057] According to the preferred embodiment of the above viewpoint, in the first step, the polyester is decomposed in a processing solvent with a temperature above 40°C and below the glass transition temperature of the polyester, and a pH range of 7 to 10.

[0058] According to the preferred embodiment of the above viewpoint, in the first step, the polyester is decomposed in a processing solvent at a temperature below 65°C.

[0059] According to the preferred embodiment of the above viewpoint, the polyester contains terephthalic acid content of 50 mol% or more relative to the total amount of acid content, and contains ethylene glycol content of 65 mol% or more relative to the total amount of alcohol content.

[0060] According to the preferred embodiment of the above viewpoint, the polyester also contains isophthalic acid content of 2 mol% or more relative to the total amount of acid content.

[0061] According to the preferred approach of the above viewpoint, polyester is at least one of post-consumer products and pre-consumer products.

[0062] According to the preferred embodiment of the above viewpoint, the polyester processing method further includes a second step, wherein the second step separates at least one compound selected from the group consisting of terephthalic acid, mono(2-hydroxyethyl) terephthalate and bis(2-hydroxyethyl) terephthalate, and their salts from the decomposition products obtained in the first step.

[0063] According to the preferred embodiment of the above viewpoint, in the second step, at least one compound selected from the group consisting of isophthalic acid, mono(2-hydroxyethyl) isophthalic acid and bis(2-hydroxyethyl) isophthalic acid, and their salts are also separated.

[0064] According to the preferred embodiment of the above viewpoint, the polyester processing method further includes a third step: separating the decomposition products of alcohol components derived from the polyester from the decomposition products obtained in the first step.

[0065] According to the preferred embodiment of the above viewpoint, in the fourth step of the polyester manufacturing method, the compound comprises at least one compound selected from the group consisting of terephthalic acid, mono(2-hydroxyethyl) terephthalate and bis(2-hydroxyethyl) terephthalate.

[0066] According to the preferred embodiment of the above viewpoint, in the fourth step of the polyester manufacturing method, the compound includes at least one selected from the group consisting of isophthalic acid, mono(2-hydroxyethyl) isophthalate, and bis(2-hydroxyethyl) isophthalate.

[0067] According to the preferred embodiment of the above viewpoint, in the fourth step of the polyester manufacturing process, the compound comprises ethylene glycol.

[0068] In this disclosure, the amino acid substitution positions are based on the amino acid number indicated by sequence number 1.

[0069] In this disclosure, "sequence identity" is a parameter representing the correlation between two amino acid sequences, indicating the degree of similarity between them. 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).

[0070] In this disclosure, for example, "A172" represents the alanine residue at position 172 in the amino acid sequence shown in Serial No. 1. "A172C" indicates that the alanine residue at position 172 is replaced by a cysteine ​​residue.

[0071] In this disclosure, the term "substitution" in relation to an amino acid sequence means that an amino acid residue in the amino acid sequence is replaced by another amino acid residue.

[0072] In this disclosure, the amino acid sequence can be determined, for example, using Edman degradation, mass spectrometry (e.g., MS / MS).

[0073] The esterase disclosed herein may contain disulfide bonds.

[0074] <First Implementation>

[0075] The esterase of the first embodiment of this disclosure will be described.

[0076] The esterase of the first embodiment has at least 80% sequence identity with the amino acid sequence shown in Serial No. 1. The amino acid sequence shown in Serial No. 1 is the amino acid sequence of the esterase (LCC; leaf-branch compost cutinase) described in International Patent Publication No. 2012 / 099018 (Patent Document 1). The esterase of the first embodiment preferably has at least 85% and more preferably at least 90% sequence identity with the amino acid sequence shown in Serial No. 1.

[0077] In the esterase of the first embodiment, A172 and A209 in the amino acid sequence shown in sequence number 1 are replaced.

[0078] In the esterase of the first embodiment, A172 is preferably replaced by a cysteine ​​residue (C).

[0079] In the esterase of the first embodiment, A209 is preferably replaced by a cysteine ​​residue (C).

[0080] In the esterase of the first embodiment, it is more preferable that A172 and A209 are replaced by cysteine ​​residues (C), respectively.

[0081] The esterase of the first embodiment can be manufactured using known methods. For example, the esterase of the first embodiment can be manufactured using genetic engineering methods. For example, a vector containing a polynucleotide encoding the esterase of the first embodiment is introduced into a microorganism and / or a host cell and allowed to proliferate. For example, *Escherichia coli* can be used as a host cell. The esterase can be obtained by recovering and purifying the esterase found in the microorganism and / or the host cell.

[0082] The esterase of the first embodiment can effectively decompose the polyester described in the polyester processing method described later. For example, the esterase of the first embodiment can exhibit high activity at temperatures below the glass transition temperature of polyesters whose main backbone is composed of terephthalic acid and ethylene glycol. The esterase of the first embodiment can efficiently decompose polyesters at temperatures, for example, below 65°C, preferably below 60°C, and more preferably between 50°C and 60°C. Furthermore, the esterase of the first embodiment is active at temperatures above 40°C, preferably above 45°C, and more preferably above 50°C, thus enabling even more efficient decomposition of polyesters.

[0083] According to the esterase of the first embodiment, the polyester can be decomposed at a temperature that inhibits the crystallization of the polyester. Therefore, the slowing down of the decomposition rate can be suppressed.

[0084] The esterase according to the first embodiment can lower the decomposition temperature, thereby reducing energy costs.

[0085] The esterase of the first embodiment has low substrate specificity, and the substrate is not limited to polyethylene terephthalate. For example, the esterase of the first embodiment can efficiently decompose not only homopolymers of polyethylene terephthalate, but also polyesters copolymerized from polyethylene terephthalate and components other than terephthalic acid and ethylene glycol (e.g., isophthalic acid).

[0086] <Second Implementation Method>

[0087] The esterase of the second embodiment of this disclosure will be described.

[0088] In the esterase of the second embodiment, preferably in the esterase of the first embodiment, at least one of the amino acid sequences selected from the group consisting of D203 and S248 is further substituted, more preferably D203 and S248 are further substituted.

[0089] In the esterase of the second embodiment, it is preferable that at least one of the group consisting of D203 and S248 is replaced by a cysteine ​​residue (C), and more preferably that D203 and S248 are replaced by a cysteine ​​residue (C).

[0090] More preferably, D2O3C and S248C form disulfide bonds. The formation of disulfide bonds can be confirmed by electrophoresis after reduction.

[0091] The esterase of the second embodiment can be manufactured using the same method as the esterase of the first embodiment.

[0092] The esterase of the second embodiment can have the same effect as the esterase of the first embodiment.

[0093] The esterase of the second embodiment has higher heat resistance than the esterase of the first embodiment. The esterase of the second embodiment exhibits higher decomposition activity than the esterase of the first embodiment at temperatures closer to the glass transition temperature. For example, the esterase of the second embodiment can efficiently decompose polyesters at temperatures ranging from 55°C to 65°C. Therefore, by bringing the decomposition environment closer to the glass transition temperature, the esterase of the second embodiment can decompose polyesters more efficiently than the esterase of the first embodiment.

[0094] <Third Implementation Method>

[0095] The polyester processing method of the third embodiment of this disclosure will be described. The polyester processing method of the third embodiment of this disclosure is also a method for manufacturing polyester monomers and / or oligomers.

[0096] The polyester processing method of the third embodiment includes a first step of decomposing the polyester using at least one esterase selected from the group consisting of the esterases of the first embodiment and the esterases of the second embodiment.

[0097] Water can be used as a processing solvent, for example.

[0098] The concentration of esterase in the treatment solution can be, for example, 30 nM or higher, 50 nM or higher, 80 nM or higher, 100 nM or higher, or 200 nM or higher. The concentration of esterase can be below 1000 nM, below 500 nM, or below 200 nM.

[0099] The esterase relative to 1g of polyester as the product to be decomposed can be, for example, 0.5 × 10⁻⁶. -9 mol or more, 1×10 -9 Above mol, 0.5 × 10 -8 mol or more, 1×10 -8 Above mol, 0.5 × 10 -8 mol or more, or 1×10 -7 More than mol. The esterase, relative to 1 g of polyester as the product, can produce 1 × 10⁻⁶ mol / L. -6 Below mol, 0.5 × 10 -6 Below mol, 1×10 -7 Below mol, 0.5 × 10 -7 Below mol, 1×10 -8 Below mol, or 0.5 × 10 -8 Below mol.

[0100] The decomposition temperature, i.e., the temperature of the processing solvent, can be 40°C or higher, preferably 45°C or higher, and more preferably 50°C or higher. Increasing the decomposition temperature can increase the decomposition rate. The decomposition temperature is preferably lower than the glass transition temperature of the polyester being decomposed. When the decomposition temperature is above the glass transition temperature, the polyester crystallizes during the process, and the decomposition rate decreases. For example, the decomposition temperature can be set to less than 70°C, less than 65°C, less than 60°C, or less than 55°C.

[0101] The pH of the solvent being processed can be set in the range of 7 to 11, preferably in the range of 7 to 10. During decomposition, the pH decreases, so it is preferable to use an alkali (e.g., an aqueous solution of sodium hydroxide) to maintain the pH (e.g., maintain it at pH 9).

[0102] The polyester, as the product of decomposition, has a crystallinity of 7% or less, preferably 6% or less, more preferably 5% or less, and even more preferably 3% or less. The polyester is preferably amorphous.

[0103] Crystallinity can be determined using a differential scanning calorimetry (DSC) apparatus. The polyester is heated from 30°C to 300°C in nitrogen at a heating rate of 10°C / min. The crystallization temperature (Tc), crystallization enthalpy (ΔHc), melting point (Tm), and heat of fusion (ΔHm) are measured and calculated using the following formula.

[0104] Crystallinity (%) = ((ΔHm - ΔHc) / ΔHm) × 100

[0105] In the polyester that is being decomposed, the acid component may include 50 mol% or more of terephthalic acid. The total amount of terephthalic acid relative to the total 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%. The terephthalic acid content relative to the total 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.

[0106] In the polyester that is being decomposed, the acid component may further include isophthalic acid at a concentration of 2 mol% or more relative to the acid component. When the acid component includes isophthalic acid as a copolymer component, the total amount of isophthalic acid relative to the acid component may be, for example, 5 mol% or more, 10 mol% or more, 15 mol% or more, or 20 mol% or more. The total amount of isophthalic acid relative to the 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. The acid component may consist of terephthalic acid and isophthalic acid (total 100 mol%).

[0107] In the polyester that is being decomposed, the acid component may contain other acid components. Examples of other acid components include phthalic acid, 2,6-naphthalenedicarboxylic acid, 2,4-furandicarboxylic acid, 2,5-furandicarboxylic acid, adipic acid, sebacic acid, succinic acid, dimer acid, 1,4-cyclohexanedicarboxylic acid, dimethyl terephthalate, and dimethyl isophthalate. Any one of these other acid components may be added alone, or two or more may be added in any proportion.

[0108] In the polyester that is being decomposed, the alcohol component may contain 65 mol% or more of ethylene glycol. The ethylene glycol content, relative to the total alcohol content, 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%. The ethylene glycol content, relative to the total alcohol content, may be, for example, less than 95 mol%, less than 90 mol%, less than 85 mol%, less than 80 mol%, or less than 75 mol%. The alcohol component may consist entirely of ethylene glycol (totaling 100 mol%).

[0109] In polyesters that are being decomposed, the alcohol component 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-cyclohexanediol. These other alcohol components can be added individually or in any proportion of two or more.

[0110] The polyester to be decomposed can be, for example, polyethylene terephthalate (PET), and a polyester having polyethylene terephthalate as the main backbone and isophthalic acid as the acid component. The polyester to be decomposed can be a mixture of multiple polyesters (blends, polymer alloys). Furthermore, the polyester to be decomposed can be a laminate of multiple polyesters (laminated film).

[0111] The monomer components of polyester, which are decomposed products, can be derived from petroleum, biomass, or recycled materials.

[0112] Components derived from biomass can be detected, for example, by measuring the number of radioactive carbon atoms using accelerator mass spectrometry (AMS). AMS determination can identify components that are... 12 C isotopes 14 C and 13 The quantity of C is determined by 14 C and 13 The determination of C is based on the amount of biomass raw materials used.

[0113] The polyester that is being decomposed can be at least one of post-consumer products and pre-consumer products.

[0114] Post-consumer products refer to used polyester products. The definition of used polyester products is not specifically limited; for example, it includes products recycled after use, such as cosmetic containers, bottles, and sheets.

[0115] Pre-consumer products refer to polyester that is not treated as a product. There are no special limitations. Examples include debris generated during the manufacturing process of polyester products, mismatched products, oligomers generated during the polymerization process, and residues and debris generated during molding.

[0116] The shape of the polyester as a product is not particularly limited; it can be processed into powder, flakes, sheets, films, or granules as needed. They can be used alone or in combination with other products.

[0117] The polyester processing method also includes a second step of separating the decomposition products of the polyester from the decomposition products obtained in the first step. The decomposition products comprise a mixture of polyester monomers and / or oligomers.

[0118] In the second step, when the polyester contains terephthalic acid and ethylene glycol, it is preferable to separate at least one compound selected from the group consisting of terephthalic acid, mono(2-hydroxyethyl) terephthalate, bis(2-hydroxyethyl) terephthalate, and their salts as a decomposition product of the polyester.

[0119] In the second step, when the polyester contains terephthalic acid, isophthalic acid and ethylene glycol, it is preferable to separate at least one compound selected from the group consisting of terephthalic acid, mono(2-hydroxyethyl) terephthalate, bis(2-hydroxyethyl) terephthalate and their salts as a decomposition product of the polyester. More preferably, at least one compound selected from the group consisting of isophthalic acid, mono(2-hydroxyethyl) terephthalate and bis(2-hydroxyethyl) terephthalate and their salts is also separated.

[0120] In the decomposition products, the non-water-soluble compounds of the acidic component derived from the polyester can be separated and recovered from the treatment solvent, for example, by making the treatment solvent acidic. Sulfuric acid, hydrochloric acid, etc., can be used as the acid for neutralization. The acidic component of the decomposition products can be subjected to processes such as distillation and separation. When the polyester contains terephthalic acid and ethylene glycol, at least one compound selected from the group consisting of terephthalic acid, mono(2-hydroxyethyl) terephthalate, and bis(2-hydroxyethyl) terephthalate can be used as the compound containing the acidic component of the decomposition products. When the polyester contains isophthalic acid and ethylene glycol, at least one non-water-soluble compound derived from the acidic component can be selected from the group consisting of isophthalic acid, mono(2-hydroxyethyl) terephthalate, and bis(2-hydroxyethyl) terephthalate.

[0121] The polyester processing method preferably further includes a third step of separating the decomposition products derived from the polyester alcohol components from the decomposition products obtained in the first step.

[0122] In the third step, the compounds that can be separated include, for example, 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-cyclohexanediol, mono(2-hydroxyethyl) terephthalate, bis(2-hydroxyethyl) terephthalate, etc.

[0123] In the third step, when the polyester contains ethylene glycol, the decomposition products of the alcohol components derived from the polyester preferably contain ethylene glycol, but may also contain only ethylene glycol.

[0124] In the decomposition products, the alcohol component of the polyester decomposition products can be separated from the processing solvent, for example, by distillation. When the polyester contains an ethylene glycol component, at least one compound selected from the group consisting of ethylene glycol, diethylene glycol, mono(2-hydroxyethyl) terephthalate, and bis(2-hydroxyethyl) terephthalate can be cited as a compound containing the alcohol component of the decomposition products.

[0125] In cases where a single compound cannot be separated from the acid and alcohol components isolated from the processing solvent, or where separation is not required, the decomposition products can be recovered in the form of a mixture. The proportion of each component in the mixture can be analyzed, for example, by nuclear magnetic resonance (NMR) analysis. The decomposition products can be directly applied in the form of a mixture to the polyester manufacturing method of Embodiment 4.

[0126] The polyester processing method according to the third embodiment can efficiently decompose polyester. Furthermore, the decomposition products obtained by the polyester processing method of the third embodiment can be used as raw materials. For example, the decomposition products can be used to regenerate new polyester.

[0127] According to the polyester processing method of the third embodiment, the decomposition temperature can be reduced, thus enabling the decomposition of polyester to suppress energy costs.

[0128] According to the polyester processing method of the third embodiment, not only can the homopolymer of polyethylene terephthalate be decomposed efficiently, but also the polyester copolymerized with polyethylene terephthalate and other components (such as isophthalic acid) other than terephthalic acid and ethylene glycol components can be decomposed efficiently.

[0129] <Fourth Implementation>

[0130] The method for manufacturing polyester according to the fourth embodiment of this disclosure will be described.

[0131] The polyester manufacturing method of the fourth embodiment of this disclosure includes a fourth step of manufacturing polyester by using the decomposition products from the polyester processing method of the third embodiment as at least a portion of the monomer. Examples of decomposition products include the aforementioned compounds isolated in the third embodiment.

[0132] In the polyester manufacturing method disclosed herein, the raw material monomers other than the decomposition products can be appropriately adjusted according to the properties of the target polyester, without particular limitation.

[0133] In the polyester manufacturing method disclosed herein, raw material monomers other than decomposition products may or may not be used. When raw material monomers other than decomposition products are used, they may be derived from petroleum, biomass, or recycled materials.

[0134] The acid components of the raw material monomers in the polyester manufacturing method disclosed herein, excluding decomposition products, may include, for example, terephthalic acid, isophthalic acid, phthalic acid, 2,6-naphthalenedicarboxylic acid, 2,4-furandicarboxylic acid, 2,5-furandicarboxylic acid, adipic acid, sebacic acid, succinic acid, dimer acid, 1,4-cyclohexanedicarboxylic acid, dimethyl terephthalate, dimethyl isophthalate, bis-2-hydroxyethyl terephthalate, and bis-2-hydroxyethyl isophthalate. Any one of these acid components may be added alone, or two or more may be added in any proportion.

[0135] In the polyester manufacturing method disclosed herein, the alcohol components of the raw material monomers other than the decomposition products may include, for example, 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-cyclohexanediol, polyethylene glycol, polytrimethylenediol, polytetramethylenediol, etc. Any one of these alcohol components may be added alone, or two or more may be added in any proportion.

[0136] In the polyester manufacturing method disclosed herein, the blending ratio of the decomposition products is obtained by dividing the mass of the component derived from the decomposition products in the recycled polyester by the total mass of the recycled polyester. The blending ratio of the decomposition products can be, for example, set to 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 also be, for example, set to 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.

[0137] The blending ratio of the component derived from recycled materials containing decomposition products in the polyester manufacturing method disclosed herein is obtained by adding the mass of the component derived from decomposition products in the recycled polyester to the mass of the substance derived from recycled materials in the monomer components other than decomposition products in the recycled polyester, and dividing by the total mass of the recycled polyester. The blending ratio of the component derived from recycled materials containing decomposition products can be, for example, set to 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 component derived from recycled materials containing decomposition products can be, for example, set to 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.

[0138] The polymerization step in the polyester manufacturing method disclosed herein is not particularly limited, and can be carried out using a known catalyst and a known polymerization method. The polyester manufacturing method can be any of the following: direct esterification using an unsubstituted polycarboxylic acid as a starting material, or transesterification using an esterified compound such as dimethyl ester as a starting material. In order to obtain a sufficient reaction rate during polyester manufacturing, it is preferable to: as a first stage, carry out a transesterification reaction using a known catalyst at atmospheric pressure, followed by a second stage, carrying out a polycondensation reaction using a known catalyst under reduced pressure. After the polycondensation reaction, a solid-state polymerization reaction can be carried out. Through solid-state polymerization, the intrinsic viscosity of the polyester can be increased.

[0139] According to the polyester manufacturing method of the fourth embodiment of this disclosure, polyester can be regenerated by using the decomposition products of polyester obtained by esterase decomposition.

[0140] <Fifth Implementation>

[0141] The polyester decomposition products of the fifth embodiment of this disclosure will be described.

[0142] The polyester decomposition product of the fifth embodiment of this disclosure is obtained by decomposing polyester using the esterase of the first embodiment and / or the esterase of the second embodiment of this disclosure.

[0143] The polyester decomposition product of the fifth embodiment of this disclosure is the same as the decomposition product obtained in the first step of the polyester processing method of the third embodiment, comprising a mixture of polyester monomers and / or oligomers.

[0144] The polyester decomposition product of the fifth embodiment of this disclosure can be distinguished from conventional polyester decomposition products in that it contains the esterase of the first embodiment and / or the esterase of the second embodiment.

[0145] Polyester can be manufactured using the polyester decomposition products of the fifth embodiment of this disclosure. In this case, polyester can be manufactured in the same manner as the polyester manufacturing method of the fourth embodiment of this disclosure.

[0146] <Sixth Implementation>

[0147] The polyester of the sixth embodiment of this disclosure will be described.

[0148] The polyester of the sixth embodiment of this disclosure is a polyester manufactured by the manufacturing method of the fourth embodiment. That is, the polyester of the sixth embodiment of this disclosure is a polyester manufactured using polyester decomposition products as raw materials.

[0149] <Seventh Implementation>

[0150] The polynucleotide of the seventh embodiment of this disclosure encodes the esterase of the first and second embodiments. The vector contains the polynucleotide. Furthermore, the host cell is a host cell to which the polynucleotide and / or the vector has been introduced. Examples of host cells include, for instance, *Escherichia coli*.

[0151] The esterases disclosed herein are sometimes impossible or practical to determine directly based on their composition, structure, properties, etc. In such cases, the esterases disclosed herein should be allowed to be determined by their manufacturing methods.

[0152] Example

[0153] The following examples illustrate the esterase, polyester treatment method, and polyester manufacturing method of this disclosure. However, the esterase, polyester treatment method, and polyester manufacturing method of this disclosure are not limited to the following examples.

[0154] [Preparation of esterase]

[0155] The vector pET21b(+) (Novagen, Germany) was used to express the keratinase LCC and its mutants from Escherichia coli BL21 (DE3) CodonPlusRIPL (Agilent Technologies GmbH, Germany).

[0156] For the gene encoding a sequence from LCC and its mutants with the N-terminal signal peptide removed, codon usage frequency optimization for expression in *E. coli* and sequence design for appending terminal sequences to the NdeI and XhoI restriction enzyme sites of the pET21b(+) vector via in-Fusion cloning were performed, and total synthesis was performed. The synthesized DNA fragment was inserted into the NdeI and XhoI restriction enzyme sites of the pET21b(+) vector via in-Fusion cloning with a 6×His-Tag at the C-terminus. The length of the DNA fragment inserted into the plasmid vector was confirmed by colony PCR. PCR was performed using Gotaq GreenMaster Mix (Promega) as primers, with T7 promoter (5'-CGCGAAATTAATACGACTCACTATAGGG-3') and T7 terminator (5'-GCTAGTTATTGCTCAGCGGTGG-3'). The gene sequence was determined by DNA sequencing using T7 promoter and T7 terminator as primers. The plasmid vector was introduced into E. coli BL21(DE3)CodonPlus RIPL.

[0157] 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. The cells were collected, disrupted using an ultrasonic homogenizer, and centrifuged at 14,000 rpm for 20 minutes at 4°C. The supernatant was used as the crude enzyme solution.

[0158] The crude enzyme solution was subjected to affinity chromatography using TALON resin (TAKARA BIO). Each eluent was analyzed by SDS-PAGE to confirm the purification of the target protein. The purified protein was then exchanged with 50 mM Na₂HPO₄-HCl, pH 7.0 buffer using a PD-10 desalting column (Cytiva). The concentration of the purified protein was calculated by measuring it at 280 nm. The molar absorptivity ε of the protein was then calculated using the ProtParam tool (http: / / web.expasy.org / protparam / ).

[0159] All chemicals were sourced from Sigma (Germany) and were of analytical grade.

[0160] [Polyester processing methods]

[0161] (1) Decomposition of polyester

[0162] The polyesters of samples 1 to 4 shown in Table 2 were treated with enzymes A to D shown in Table 1. Sample 1 was polyethylene terephthalate (PET). Samples 2 to 4 were polyesters copolymerized with isophthalic acid as the third component on PET. The copolymerization rate of isophthalic acid in each sample was determined as follows: using a Bruker DPX-400 FT-NMR apparatus, each sample was dissolved in a mixed solution of trifluoroacetic acid-d and deuterated chloroform, mixed with tetramethylsilane as a reference, and measured using proton NMR spectroscopy.

[0163] (2) Crystallinity, glass transition temperature, crystallization temperature and melting point

[0164] Table 2 shows the crystallinity, glass transition temperature, crystallization temperature and melting point of each sample.

[0165] Weigh 10 mg of polyester resin and use a differential scanning calorimeter (DSC, TA Instruments, DSC2500) in a nitrogen atmosphere to heat from 30 °C to 300 °C at a rate of 10 °C / min. Measure the crystallization temperature (Tc), enthalpy of crystallization (ΔHc), melting point (Tm), and heat of fusion (ΔHm). The crystallization temperature (Tc) is calculated using the following formula.

[0166] Crystallinity (Tc) (%) = ((ΔHm - ΔHc) / ΔHm) × 100

[0167] [Table 1]

[0168]

[0169] [Table 2]

[0170]

[0171] (3) Thin film

[0172] Samples 1-4 were shaped into thin films with a thickness of 0.2 mm to increase the contact area with the esterase.

[0173] (4) Decomposition of polyester

[0174] A polyester film with a thickness of 0.2 mm and a diameter of 6 mm was added to 200 mM Bicine-NaOH (pH 9.0), and then the purified enzyme obtained by the above method was added to a final concentration of 100 nM. The mixture was heated at 50℃ to 65℃ and allowed to stand for 3 hours, 6 hours, 24 hours or 48 hours to allow the reaction to proceed.

[0175] (5) Determination of decomposition products

[0176] A portion of the reaction solution was aliquoted and diluted with buffer (80% (v / v) 20 mM NaH2PO4-H3PO4 (pH 2.5) / 20% (v / v) DMSO). After heating at 90°C for 10 minutes, a portion of the supernatant was analyzed by high-performance liquid chromatography (HPLC). Known concentrations of terephthalic acid, mono(2-hydroxyethyl) terephthalate, bis(2-hydroxyethyl) terephthalate, and isophthalic acid solutions were used as standards.

[0177] The HPLC conditions are as follows:

[0178] Device: LC-2010A HT (manufactured by Shimadzu Corporation)

[0179] Column: Cosmosil 5C18-AR-II guard column, Cosmosil 5C18-AR-II column (manufactured by NacalaiTesque Co., Ltd.)

[0180] Mobile phase: Methanol / 20mM NaH2PO4-H3PO4 (pH 2.5)

[0181] Flow rate: 1.0 mL / min

[0182] Detection wavelength: 240nm

[0183] Elution conditions: 0–15 min; 25% (v / v) methanol, 15–25 min; methanol concentration gradient of 25–100%.

[0184] 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 solution to quantify the decomposition products under various conditions.

[0185] (Experimental Examples 1-8)

[0186] The polyesters of samples 1-4 shown in Table 2 were used as the decomposition products, and decomposition tests were performed using the esterases shown in Table 1. The esterases used were LCC (enzyme D) with the amino acid sequence shown in Serial No. 1, and its mutants A172C_A209C (enzyme A), A172C_A209C_D203C_S248C (enzyme B), and D203C_S248C (enzyme C). In the following tables, enzymes A to D are used. After each sample was decomposed at 50°C or 60°C for 24 hours, the recoveries of terephthalic acid, isophthalic acid, and all components (terephthalic acid, mono(2-hydroxyethyl) terephthalate, bis(2-hydroxyethyl) terephthalate, and isophthalic acid) were determined.

[0187] Tables 3 through 10 show the recovery amounts of terephthalic acid, isophthalic acid, and all components. The total recovery amount refers to the total amount of terephthalic acid, isophthalic acid, mono(2-hydroxyethyl) terephthalate, and bis(2-hydroxyethyl) terephthalate.

[0188] For the recovery amounts of the degradation products of enzymes A, B, and C, the increase rate relative to the recovery amount of the degradation products of enzyme D was calculated. The increase rate was calculated based on the following formula. It should be noted that the recovery amounts shown in each table are rounded to three decimal places. However, the increase rates shown in the tables are calculated based on the recovery amounts before rounding; therefore, if the increase rate is calculated based on the recovery amounts shown in the tables, it may not always be exactly the same as the increase rate shown in the tables.

[0189] Increase rate (%) = (Recovery amount of degradation products based on the esterase (μmol) - Recovery amount of degradation products based on LCC (μmol)) / Recovery amount of degradation products based on LCC (μmol) × 100

[0190] Figures 1-8 A graph showing the recovery of the degradation products of samples 1-4 for each esterase. Figure 1 and Figure 2 The graphs show the recovery of terephthalic acid and its increase rate when samples 1-4 were decomposed at a decomposition temperature of 50℃. Figure 3 and Figure 4 The graphs show the recovery of isophthalic acid and its increase rate when samples 1-4 were decomposed at a decomposition temperature of 50℃. Figure 5 and Figure 6 The graphs show the total recovery of all components and their increase rate when samples 1-4 were decomposed at a decomposition temperature of 50℃. Figure 7 and Figure 8 The graphs show the recovery of terephthalic acid and its increase rate when samples 1-4 were decomposed at a decomposition temperature of 60℃. Figure 9 and Figure 10 The graphs show the recovery of isophthalic acid and its increase rate when samples 1-4 were decomposed at a decomposition temperature of 60℃. Figure 11 and Figure 12 The graphs show the total recovery of all components and their rate of increase when samples 1-4 were decomposed at a decomposition temperature of 60℃. Figures 1-2 , Figures 5-8 and Figures 11-12 The horizontal axis shown represents the percentage of terephthalic acid contained in the acid components of samples 1-4. Figures 3-4 and Figures 9-10 The horizontal axis shown represents the percentage of isophthalic acid contained in the acid components of samples 1-4.

[0191] The decomposition efficiency of D203C_S248C at 50℃ and 60℃ is lower than that of LCC. On the other hand, A172C_A209C and A172C_A209C_D203C_S248C can both achieve decomposition efficiencies higher than LCC.

[0192] [Table 3]

[0193]

[0194] [Table 4]

[0195]

[0196] [Table 5]

[0197]

[0198] [Table 6]

[0199]

[0200] [Table 7]

[0201]

[0202] [Table 8]

[0203]

[0204] [Table 9]

[0205]

[0206] [Table 10]

[0207]

[0208] (Experimental Examples 9-12)

[0209] Using the polyesters of samples 1 and 4 shown in Table 2 as the decomposition products, the decomposition rates of the esterases shown in Table 1 were compared. For each esterase, the recovery of decomposition products was determined at decomposition times of 3 hours, 6 hours, and 24 hours. Tables 11-14 show the results. Figure 13 This indicates the shift in the recovery amount of decomposition products when sample 1 is decomposed at 50°C. Figure 14 This indicates the shift in the recovery amount of decomposition products when sample 1 is decomposed at 60°C. Figure 15 This indicates the shift in the recovery amount of decomposition products when sample 4 is decomposed at 50°C. Figure 16 This indicates the shift in the recovery amount of decomposition products when sample 4 is decomposed at 60℃.

[0210] Regardless of the polyester composition, enzymes A (A172C_A209C) and B (A172C_A209C_D203C_S248C) decompose the polyester faster than enzymes C (D203C_S248C) and D (LCC).

[0211] [Table 11]

[0212]

[0213] [Table 12]

[0214]

[0215] [Table 13]

[0216]

[0217] [Table 14]

[0218]

[0219] (Experimental Examples 13-14)

[0220] Using the polyesters of samples 1 and 4 shown in Table 2 as the decomposition products, decomposition tests were conducted at 65°C using enzymes B and C shown in Table 1. Tables 15 and 16 show the test results. Figure 17 This indicates the amount of decomposition products recovered when sample 1 is decomposed at 65°C. Figure 18 This indicates the amount of decomposition products recovered when sample 4 is decomposed at 65°C.

[0221] Even at a high temperature of 65°C, enzyme B decomposes rapidly. Furthermore, enzyme B is known to have heat resistance up to 65°C. Therefore, it can be concluded that it can decompose polyester at higher temperatures where polyester crystallization is inhibited.

[0222] [Table 15]

[0223]

[0224] [Table 16]

[0225]

[0226] It should be noted that although the experimental results are omitted, heating sample 1 at 70°C for 48 hours resulted in a crystallinity of 5.4%. If the crystallinity of the polyester increases, the decomposition rate of the esterase decreases. Therefore, the decomposition temperature is preferably below 65°C, and more preferably below 60°C.

[0227] (Experimental Example 15)

[0228] [Production of Recycled Polyester 1]

[0229] In a reaction vessel equipped with a stirrer, distillation tube, and vacuum distillation device, 17.3 g of terephthalic acid and isophthalic acid (derived from recycled materials), 21.3 g of terephthalic acid (derived from petroleum), 4.7 g of isophthalic acid (derived from petroleum), 66.2 g of bis-2-hydroxyethyl terephthalate (derived from recycled materials), and 8.1 g of ethylene glycol (derived from petroleum) collected under the conditions of Experimental Example 8-2 were added and heated to 250°C under a nitrogen atmosphere. Esterification was carried out at this state for 3 hours. Next, 54 ppm of orthophosphoric acid and 120 ppm of germanium dioxide were added as polymerization catalysts, and the mixture was stirred. The pressure was reduced to below 133 Pa for 1 hour, during which time the internal temperature was increased from 250°C to 285°C. The mixture was stirred under a high vacuum below 133 Pa until the specified viscosity was achieved, and polycondensation was carried out. The resulting polymer was extruded into water in filament form and cut into granules. Through the above operations, recycled polyester 1 was produced.

[0230] For the obtained recycled polyester 1, the intrinsic viscosity and color space Lab were determined as follows.

[0231] <Determination of intrinsic viscosity>

[0232] 0.5000 g ± 0.001 g of the sample was dissolved in 50 mL of a mixed solvent of phenol and tetrachloroethane (60:40 by mass). The intrinsic viscosity (IV) at 20 °C was measured using an automatic viscosity measuring device (manufactured by SUN Electronic Industries Corp., ALC-6C) equipped with an Ubbelohde viscometer. The result was an intrinsic viscosity (IV) of 0.604 dl / g.

[0233] <Measurement of Color Space Lab>

[0234] The color space Lab of the obtained recycled polyester 1 was measured using a colorimeter (manufactured by Nippon Denshoku Kogyo Co., Ltd., ZE6000), and the results were L = 65.9, a = -1.0, b = 3.9.

[0235] (Experimental Example 16)

[0236] [Production of Recycled Polyester 2]

[0237] In Experiment 15, terephthalic acid and isophthalic acid (derived from recycled materials) collected under the conditions of Experiment 8-2 were not used. Instead, 33.3 g of terephthalic acid (derived from petroleum), 10.0 g of isophthalic acid (derived from petroleum), 66.2 g of bis-2-hydroxyethyl terephthalate (derived from recycled materials), and 8.1 g of ethylene glycol (derived from petroleum) were used. Otherwise, recycled polyester 2 was prepared in the same manner as in Experiment 15.

[0238] The intrinsic viscosity and color space Lab of the obtained recycled polyester 2 were measured in the same manner as above, and the results showed that the intrinsic viscosity (IV) was 0.612 dl / g, L was 65.0, a was -1.2, and b was 3.7.

[0239] (Experimental Example 17)

[0240] [Production of Recycled Polyester 3]

[0241] In Experiment 15, 66.2 g of petroleum-derived bis-2-hydroxyethyl terephthalate was used instead of 66.2 g of bis-2-hydroxyethyl terephthalate derived from recycled materials. Otherwise, recycled polyester 3 was prepared in the same manner as in Experiment 15.

[0242] The intrinsic viscosity and color space Lab of the obtained recycled polyester 3 were measured in the same manner as above, and the results showed that the intrinsic viscosity (IV) was 0.610 dl / g, L was 64.8, a was -1.1, and b was 4.0.

[0243] (Experimental Example 18)

[0244] [Production of Recycled Polyester 4]

[0245] In Experiment 15, 43.3 g of terephthalic acid and isophthalic acid (derived from recycled materials), 66.2 g of bis-2-hydroxyethyl terephthalate (derived from recycled materials), and 8.1 g of ethylene glycol (derived from petroleum) were collected under the conditions of Experiment 8-2. Otherwise, recycled polyester 4 was prepared in the same manner as in Experiment 15.

[0246] The intrinsic viscosity and color space Lab of the obtained recycled polyester 4 were measured in the same manner as above, and the results showed that the intrinsic viscosity (IV) was 0.602 dl / g, L was 65.5, a was -1.1, and b was 3.9.

[0247] (Experimental Example 19)

[0248] [Production of Recycled Polyester 5]

[0249] In Test Example 15, 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 to prepare recycled polyester 5 in the same manner as in Test Example 15.

[0250] The intrinsic viscosity and color space Lab of the obtained recycled polyester 5 were measured in the same manner as above, and the results showed that the intrinsic viscosity (IV) was 0.618 dl / g, L was 66.0, a was -0.9, and b was 3.4.

[0251] (Experimental Example 20)

[0252] [Production of Recycled Polyester 6]

[0253] In a reaction vessel equipped with a stirrer, distillation tube, and pressure reducing device, 86.6 g of terephthalic acid and isophthalic acid (derived from recycled materials) collected under the conditions of Experimental Example 8-2, and 40.5 g of ethylene glycol (derived from petroleum) were added. The mixture was pressurized to 0.2 MPa with nitrogen and maintained for 8 hours for esterification. Afterward, the pressure was released to atmospheric pressure, and the water produced was distilled off. Subsequent polymerization processes and thereafter were carried out in the same manner as in Experimental Example 15 to produce recycled polyester 6.

[0254] The intrinsic viscosity and color space Lab of the obtained recycled polyester 6 were measured in the same manner as above, and the results showed that the intrinsic viscosity (IV) was 0.620 dl / g, L was 63.8, a was -1.3, and b was 4.5.

[0255] (Experimental Example 21)

[0256] [Production of virgin polyester]

[0257] In Test Example 15, the terephthalic acid and isophthalic acid collected under the conditions of Test Example 8-2 (derived from recycled materials) were not used. Instead, 33.3 g of petroleum-derived terephthalic acid, 10.0 g of isophthalic acid, 66.2 g of bis-2-hydroxyethyl terephthalate, and 8.1 g of ethylene glycol were used. Otherwise, virgin polyester was produced in the same manner as in Test Example 15.

[0258] The intrinsic viscosity and color space Lab of the obtained virgin polyester were measured in the same manner as above, and the results showed that the intrinsic viscosity (IV) was 0.609 dl / g, L was 64.7, a was -1.2, and b was 4.2.

[0259] As can be seen from the results of Test Examples 15-21, the recycled polyesters made using the polyester decomposition products of the esterase disclosed in Test Examples 15 and 17-19 (Test Example 8-2) have the same quality as the recycled polyester made without the polyester decomposition products of the esterase disclosed in Test Example 16 (Test Example 8-2) and the virgin polyester in Test Example 21.

[0260] The esterases, polyester degradation products, polyester processing methods, polyester manufacturing methods, polynucleotides, carriers, and host cells disclosed herein have been described based on the above embodiments and examples, but are not limited to the above embodiments and examples. Various modifications, alterations, and improvements can be made to each disclosed element (including the elements described in the claims, specification, and drawings) based on the basic technical concept of the present invention, within the scope of the present invention. Furthermore, within the scope of the claims of the present invention, various combinations, substitutions, or selections of each disclosed element are possible.

[0261] Further issues, objects, and methods (including modifications) of the present invention will become apparent from the full disclosure of the invention, including the claims.

[0262] The numerical ranges described in this specification should be interpreted as any numerical value or range specifically described in this specification, even if not specifically stated otherwise.

[0263] Some or all of the above embodiments may also be described as in the following appendices, but are not limited to the following descriptions. The appendices may also be combined with the claims as set forth in the claims.

[0264] [Appendix 1]

[0265] Polyester manufactured by the polyester manufacturing method disclosed herein.

[0266] [Appendix 2]

[0267] Methods for processing polyesters, and methods for manufacturing polyester monomers and / or oligomers based on the present disclosure.

[0268] [Appendix 3]

[0269] Polyester monomers and / or oligomers manufactured by the polyester processing method disclosed herein.

[0270] Industrial availability

[0271] According to the esterase disclosed herein, containers, films, sheets, fibers, clothing, etc., made of biodegradable polyester can be broken down to the compound level. By using polyester decomposition products as raw materials, a resource recycling system for polyester can be constructed.

Claims

1. An esterase having at least 80% sequence identity with the amino acid sequence shown in Serial No. 1, A172 and A209 in the amino acid sequence were replaced.

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

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

4. The esterase according to claim 3, wherein, D203 and S248 are replaced by cysteine ​​residues (C).

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

6. A polyester processing method comprising a first step of decomposing the polyester using an esterase as described in any one of claims 1 to 5.

7. The method for processing polyester according to claim 6, wherein, In the first step, the polyester is decomposed in a processing solvent with a temperature above 40°C and below the glass transition temperature of the polyester, and a pH range of 7 to 10.

8. The method for processing polyester according to claim 7, wherein, In the first step, the polyester is decomposed in the processing solvent at a temperature below 65°C.

9. The method for processing polyester according to any one of claims 6 to 8, wherein, The polyester contains terephthalic acid at a total amount of 50 mol% or more relative to the total acid content, and ethylene glycol at a total amount of 65 mol% or more relative to the total alcohol content.

10. The method for processing polyester according to claim 9, wherein, The polyester also contains isophthalic acid at a concentration of 2 mol% or more relative to the total amount of the acid component.

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

12. The method for processing polyester according to any one of claims 6 to 11, further comprising: The second step involves separating at least one compound selected from the group consisting of terephthalic acid, mono(2-hydroxyethyl) terephthalate, bis(2-hydroxyethyl) terephthalate, and their salts from the decomposition products obtained in the first step.

13. The method for processing 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 and bis(2-hydroxyethyl) isophthalate, and their salts are also separated.

14. The method for processing polyester according to any one of claims 6 to 13, further comprising: The third step involves separating the decomposition products derived from the polyester alcohol component from the decomposition products obtained in the first step.

15. A method for manufacturing polyester, comprising a fourth step of manufacturing polyester by using at least a portion of the compound separated in the processing method of the polyester according to any one of claims 12 to 14 as a monomer.

16. The method for manufacturing polyester according to claim 15, wherein, In the fourth step, the compound comprises 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 manufacturing 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 manufacturing polyester according to any one of claims 15 to 17, wherein, In the fourth step, the compound comprises ethylene glycol.

19. A polyester decomposition product obtained by decomposing polyester using any one of the esterases according to claims 1 to 5.

20. A method for manufacturing polyester, wherein the polyester decomposition product of claim 19 is used to manufacture polyester.

21. A polynucleotide encoding an esterase as described in any one of claims 1 to 5.

22. A vector comprising the polynucleotide of claim 21.

23. A host cell having the polynucleotide of claim 21 introduced therein.

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