Method for decomposing PET in a mixed substrate containing PET.

Abstract

The objective is to provide a means for efficiently decomposing polyethylene terephthalate (PET) in mixed substrates containing PET, compared to conventional methods. [Solution] A method for decomposing PET in a mixed substrate containing PET, comprising the following step (1). (1) A step of reacting an enzyme composition with a mixed substrate containing PET, The enzyme composition comprises a carboxylic acid ester hydrolase or a variant thereof, The step is that the carboxylic acid ester hydrolase or its variant is at least one selected from carboxylesterase, allylsterase, cutinase, and PETase, as well as variants thereof and lipase.

Description

Technical Field

[0001] The present disclosure relates to a method for decomposing PET in a mixed substrate containing PET and a method for producing at least one of TPA and MHET using the method.

Background Art

[0002] Polyethylene terephthalate (hereinafter also referred to as "PET") is widely used in beverage bottles, fibers, packaging materials, etc. However, since there is a problem of environmental pollution because it is not decomposed in the environment, a recycling technology for PET is required. Among the PET recycling technologies, it has been reported that PET alone can be decomposed by a PET decomposition method using an enzyme (Patent Documents 1 to 6 and Non-Patent Documents 1 to 3). As the decomposition of a mixed substrate containing PET and a substrate other than PET using a PET-degrading enzyme, the decomposition of PET in PET / cotton blended fibers has been reported (Non-Patent Document 4).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

[0005] The recycling of mixed substrates containing PET and non-PET substrates (hereinafter also referred to as mixed substrates) is difficult, and PET-degrading enzymes that can be used for industrial applications have not yet been obtained. The reasons why recycling these mixed substrates is difficult are as follows:

[0006] When a material is recycled, a single material is used as the raw material to produce a highly pure recycled product. However, it is difficult to isolate a single material from a mixture of multiple materials, such as a blended substrate (e.g., blended fibers).

[0007] Therefore, the recycling of mixed substrates containing PET has not progressed, and for example, blended fibers are currently limited to use as cleaning cloths for machinery and equipment, or in thermal recycling for energy recovery through combustion. Resource recycling from mixed substrates containing PET is needed globally, and at the industrial level, there is a need for means to decompose only the PET in mixed substrates containing PET.

[0008] Non-patent documents 1 and 2 disclose the degradation of PET alone using the substrate specificity of PET-degrading enzymes, but neither document investigates the degradation of mixed substrates including PET.

[0009] Non-patent document 4 discloses the decomposition of PET in a mixed substrate containing PET by cutinase derived from Humicola insolens (hereinafter also abbreviated as HiC), but only one type of PET-degrading enzyme and one type of mixed substrate containing PET are disclosed. Furthermore, in non-patent document 4, the enzymatic reaction by the PET-degrading enzyme requires grinding of the mixed substrate using a ball mill, i.e., the application of physical impact, making it difficult to apply to an industrial level.

[0010] Therefore, this disclosure aims to provide a means for efficiently degrading PET in a mixed substrate containing PET, compared to conventional methods. [Means for solving the problem]

[0011] As a result of diligent research into the above-mentioned problems, the present inventors have discovered that by reacting a mixed substrate containing PET with an enzyme composition containing a specific carboxylic acid ester hydrolase and its variants, PET in the mixed substrate can be efficiently degraded under milder conditions than conventional methods, thus completing the present invention.

[0012] In other words, the present invention is as follows: 1. A method for decomposing polyethylene terephthalate (PET) in a mixed substrate, comprising the following step (1). (1) A step of reacting an enzyme composition with a mixed substrate containing PET, The enzyme composition comprises a carboxylic acid ester hydrolase or a variant thereof, The step is that the carboxylic acid ester hydrolase or its variant is at least one selected from carboxylesterase, allylsterase, cutinase, and PETase, as well as variants thereof and lipase. 2. The method according to claim 1, wherein the lipase variant is a variant of PET2. 3. The variant of the PET2 is a protein consisting of an amino acid sequence in which at least one modification selected from the group consisting of the following [1] to [7] and at least one modification selected from the group consisting of [8] to

[24] is introduced into the amino acid sequence represented by SEQ ID NO: 1 (however, excluding the protein into which at least one combination of modifications selected from the group consisting of the combination of [8] and

[16] , the combination of

[11] and

[19] , and the combination of

[17] and

[23] is introduced), according to the method described in 2 above. [1] Modification to substitute the 47th amino acid residue with a cysteine residue [2] Modification to substitute the 89th amino acid residue with a cysteine residue [3] Modification to substitute the 105th amino acid residue with an arginine residue [4] Modification to substitute the 110th amino acid residue with a lysine residue [5] Modification to substitute the 156th amino acid residue with a proline residue [6] Modification to substitute the 180th amino acid residue with an alanine residue [7] Modification to substitute the 297th amino acid residue with a proline residue [8] Modification to substitute the 73rd amino acid residue with a glutamine residue [9] Modification to substitute the 76th amino acid residue with a cysteine residue

[10] Modification to substitute the 103rd amino acid residue with a lysine residue

[11] Modification to substitute the 134th amino acid residue with a glycine residue

[12] Modification to substitute the 144th amino acid residue with a cysteine residue

[13] Modification to substitute the 203rd amino acid residue with a threonine residue

[14] Modification to substitute the 222nd amino acid residue with a methionine residue

[15] Modification to substitute the 55th amino acid residue with a serine residue

[16] Modification to substitute the 73rd amino acid residue with a serine residue

[17] Modification to substitute the 228th amino acid residue with a lysine residue

[18] Modification to substitute the 264th amino acid residue with an arginine residue

[19] Modification of substituting the 134th amino acid residue with a lysine residue, a methionine residue, or a threonine residue

[20] Modification of substituting the 136th amino acid residue with a glutamic acid residue

[21] Modification of substituting the 200th amino acid residue with a histidine residue, an aspartic acid residue, or an alanine residue

[22] Modification of substituting the 227th amino acid residue with an arginine residue, a lysine residue, a glutamic acid residue, or a histidine residue

[23] Modification of substituting the 228th amino acid residue with a glutamic acid residue, a histidine residue, a lysine residue, a glutamine residue, or a tryptophan residue

[24] Modification of substituting the 229th amino acid residue with a tyrosine residue, an asparagine residue, or a phenylalanine residue 4. The method according to item 2, wherein at least one of the mutants of PET2 is selected from [C4] to [C6]. [C4] A protein consisting of the amino acid sequence represented by SEQ ID NO: 5 or 17 [C5] A protein consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by SEQ ID NO: 5 or 17, and having a PET degradation rate of 0.5% or more in a mixed substrate containing PET [C6] A protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 5 or 17, and having a PET degradation rate of 0.5% or more in a mixed substrate containing PET 5. The method according to item 1, wherein the cutinase or its mutant is LC-cutinase or its mutant 6. The method according to item 5, wherein at least one of the LC-cutinase or its mutant is selected from the following [C1] to [C3]. [C1] A protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 7 [C2] A protein consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by SEQ ID NO: 3 or 7, and having a PET degradation rate of 0.5% or more in a mixed substrate containing PET [C3] A protein having an amino acid sequence that is 80% or more identical to the amino acid sequence represented by SEQ ID NO: 3 or 7, and having a PET degradation rate of 0.5% or more in a mixed substrate containing PET. 7. The method according to 1, wherein the carboxylic acid ester hydrolase or its variant is at least one of (E3) and (E4) below. (E3) Protein consisting of the amino acid sequence represented by Sequence ID No. 5 or 17 (E4) Protein consisting of the amino acid sequence represented by Sequence ID No. 3 or 7. 8. The method according to any one of 1 to 7, wherein the mixed substrate comprises at least one of polyurethane and cotton, and PET. 9. The method according to any one of 1 to 7, wherein the PET contained in the mixed substrate is derived from at least one selected from PET fibers, PET bottles, PET films, PET flakes, and PET resin powder. 10. The method according to any one of 1 to 7, wherein step (1) is carried out in the presence of sodium carbonate. 11. The method according to any one of 1 to 7, wherein step (1) is performed at a temperature of 40 to 70°C. 12. A method for producing at least one of terephthalic acid (TPA) and monohydroxyethyl terephthalate (MHET) using the method described in any one of 1 to 7 above. 13. The method according to any one of 1 to 7 above, wherein the mixed substrate comprises polyurethane and PET. 14. The method according to 13, wherein the PET content in the mixed substrate is 50 to 99% by mass. 15. The method according to any one of 1 to 7 above, wherein the mixed substrate comprises cotton and PET. 16. The method according to 15, wherein the PET content in the mixed substrate is 50 to 99% by mass. 17. A method for producing monoethylene glycol (MEG) using the method for decomposing PET described in any one of items 1 to 7 above. [Effects of the Invention]

[0013] According to the method disclosed herein, by reacting a mixed substrate containing PET with an enzyme composition containing a specific carboxylic acid ester hydrolase and its variants, the PET in the mixed substrate can be efficiently decomposed under milder conditions compared to conventional methods, thus enabling a wide range of applications and facilitating practical application on an industrial scale. [Modes for carrying out the invention]

[0014] 1. Method for decomposing PET in a mixed substrate containing PET. A method for degrading PET in a mixed substrate containing PET according to the present disclosure (hereinafter also referred to as the "method") is characterized by comprising the following step (1). (1) A step of reacting an enzyme composition with a mixed substrate containing PET, The enzyme composition comprises a carboxylic acid ester hydrolase or a variant thereof, The step is that the carboxylic acid ester hydrolase or its variant is at least one selected from carboxylesterase, allylsterase, cutinase, and PETase, as well as variants thereof and lipase.

[0015] <Enzyme composition> (Carboxylate ester hydrolase) The enzyme composition in this disclosure comprises a carboxylic acid ester hydrolase (EC.3.1.1-) or a variant thereof. The carboxylic acid ester hydrolase or its variant has the ability to hydrolyze carboxylic acid esters, and the hydrolysis of the carboxylic acid ester produces a carboxylic acid and an alcohol. By reacting the enzyme composition with a mixed substrate containing PET, the carboxylic acid ester hydrolase or its variant can come into contact with the mixed substrate containing PET and selectively decompose the PET in the mixed substrate.

[0016] In this disclosure, the carboxylic acid esterification enzyme or its variant is at least one selected from carboxylesterase (EC 3.1.1.1), allylesterase (EC 3.1.1.2), cutinase (EC 3.1.1.74), and PETase (EC 3.1.1.101) and their variants, as well as a variant of lipase (EC 3.1.1.3). Among these, from the viewpoint of selective degradation of PET contained in a mixed substrate including PET, a variant of lipase and a variant of cutinase or its variant are preferred. These may be used individually or in combination of two or more.

[0017] Examples of lipase variants include PET2 variants (hereinafter also abbreviated as PET2 variants). Examples of cutinases include LC-cutinase or its variants. Of PET2 variants and LC-cutinase or its variants, PET2 variants are preferred from the viewpoint of PET degradation activity. PET degradation activity can be evaluated by the PET degradation rate, the amount of BHET, MHET, TPA, etc. produced by PET degradation, and the amount of PET reduced by PET degradation, as described later.

[0018] The enzyme composition in this disclosure preferably specifically contains, for example, at least one of (E3) and (E4) below. The amino acid sequences described in (E3) and (E4) below will be described later. From the viewpoint of selective degradation of PET in a mixed substrate containing PET, it is particularly preferable to include (E3) among (E3) and (E4) below. (E3) Protein consisting of the amino acid sequence represented by Sequence ID No. 5 or 17 (E4) Protein consisting of the amino acid sequence represented by Sequence ID No. 3 or 7. "Selective degradability" refers to the property of efficiently degrading PET in a mixed substrate containing PET. It is preferable that the PET degradation rate in the mixed substrate containing PET is high, while the degradation rate of substrates other than PET is low. The method for calculating the PET degradation rate will be described later.

[0019] The enzyme composition in this disclosure may contain other enzymes in addition to carboxylic acid ester hydrolase or its variants, as long as it achieves the effects of the present invention. The content of carboxylic acid ester hydrolase or its variant in the enzyme composition can be adjusted according to the type of mixed substrate containing PET and other conditions, and is not particularly limited as long as it achieves the effects of the present invention, but for example, it is preferably 0.01 to 1.00% by mass, more preferably 0.025 to 0.5% by mass, and even more preferably 0.05 to 0.25% by mass.

[0020] (Lipase) Lipases are enzymes that hydrolyze ester bonds that make up lipids, and they perform hydrolysis using triglycerides, which consist of fatty acids and glycerol, as substrates. Examples of lipases include PET2 and Tcur_1278(D1A9G5), with PET2 being preferred. These lipases are enzymes that can hydrolyze PET into monohydroxyethyl terephthalate (hereinafter also referred to as "MHET") or monoethylene glycol (MEG) and terephthalic acid (TPA).

[0021] As lipase variants, PET2 variants are preferred. PET2 is a protein (also abbreviated as "protein (A)") consisting of the amino acid sequence represented by SEQ ID NO: 1 (nucleotide sequence is SEQ ID NO: 2) (Appl. Environ. Microbiol, 2018, 84, 8, p.e02773-17), and PET2 variants can be created by modifying PET2. The PET2 variants described in this disclosure are described below.

[0022] ((Modifications in protein (A))) Examples of PET2 variants in this disclosure include proteins having an amino acid sequence in which at least one modification selected from the group consisting of [1] to [7] and at least one modification selected from the group consisting of [8] to

[24] have been introduced in the amino acid sequence represented by Sequence ID No. 1 (excluding proteins having at least one combination of modifications selected from the group consisting of the combination of [8] and

[16] , the combination of

[11] and

[19] , and the combination of

[17] and

[23] ).

[0023] [1] Modification by substituting the 47th amino acid residue with a cysteine ​​residue. [2] Modification by substituting the 89th amino acid residue with a cysteine ​​residue. [3] Modification by substituting the 105th amino acid residue with an arginine residue. [4] Modification by substituting the 110th amino acid residue with a lysine residue. [5] Modification by substituting the 156th amino acid residue with a proline residue. [6] Modification by substituting the 180th amino acid residue with an alanine residue. [7] Modification by substituting the 297th amino acid residue with a proline residue. [8] Modification by substituting the 73rd amino acid residue with a glutamine residue. [9] Modification by substituting the 76th amino acid residue with a cysteine ​​residue.

[10] Modification by substituting the 103rd amino acid residue with a lysine residue.

[11] Modification by substituting the 134th amino acid residue with a glycine residue.

[12] Modification by substituting the 144th amino acid residue with a cysteine ​​residue.

[13] Modification by substituting the 203rd amino acid residue with a threonine residue.

[14] Modification by substituting the 222nd amino acid residue with a methionine residue.

[15] Modification by substituting the 55th amino acid residue with a serine residue.

[16] Modification by substituting the 73rd amino acid residue with a serine residue

[17] Modification by substituting the 228th amino acid residue with a lysine residue.

[18] Modification by substituting the 264th amino acid residue with an arginine residue.

[19] Modifications by substituting the 134th amino acid residue with a lysine residue, a methionine residue, or a threonine residue.

[20] Modification by substituting the 136th amino acid residue with a glutamic acid residue.

[21] Modifications by substituting the 200th amino acid residue with a histidine residue, an aspartic acid residue, or an alanine residue.

[22] Modifications by substituting the 227th amino acid residue with an arginine residue, a lysine residue, a glutamic acid residue, or a histidine residue.

[23] Modifications by substituting the 228th amino acid residue with a glutamic acid residue, histidine residue, lysine residue, glutamine residue, or tryptophan residue.

[24] Modifications by substituting the 229th amino acid residue with a tyrosine residue, an asparagine residue, or a phenylalanine residue.

[0024] By creating a protein with an amino acid sequence in which at least one modification selected from the group consisting of [1] to [7] above and at least one modification selected from the group consisting of [8] to

[24] above has been introduced in the amino acid sequence represented by Sequence ID No. 1 (excluding proteins in which at least one combination of modifications selected from the group consisting of the combination of [8] and

[16] , the combination of

[11] and

[19] , and the combination of

[17] and

[23] has been introduced), at least one of the heat resistance and substrate binding ability is improved, and therefore it is thought that the protein (A) has a higher PET degradation activity compared to the protein having the amino acid sequence represented by Sequence ID No. 1.

[0025] If the modification described in [8] above is introduced, the modification described in

[16] above, which modifies the amino acid residue at the same position in SEQ ID NO: 1, cannot be introduced. If the modification described in

[11] above is introduced, the modification described in

[19] above, which modifies the amino acid residue at the same position in SEQ ID NO: 1, cannot be introduced. If the modification described in

[17] above is introduced, the modification described in

[23] above, which modifies the amino acid residue at the same position in SEQ ID NO: 1, cannot be introduced.

[0026] Non-limiting specific embodiments of the PET2 variants in this disclosure include, for example, a protein consisting of an amino acid sequence having one of the following modifications (A1) to (A49) in the amino acid sequence represented by Sequence ID No. 1. (A1) Modifications to [1] to [7] above and modifications to

[19] to

[24] above. (A2) Modifications to [1] to [7] above and modifications to [8] to

[14] above. (A3) Modifications of [1] to [7] above, modifications of [8] to

[14] above, and at least one modification selected from the group consisting of

[15] ,

[17] and

[18] above. (A4) Modifications of [1] to [7] above, modifications of [8] to

[14] above, and at least one modification selected from the group consisting of

[15] to

[18] above. (A5) Modifications to [1] to [7] above, modifications to [8] to

[14] above, and modifications to

[15] above. (A6) Modifications of [1] to [7] above, modifications of [8] to

[14] above, and modifications of

[16] above. (A7) Modifications of [1] to [7] above, modifications of [8] to

[14] above, and modifications of

[17] above. (A8) Modifications of [1] to [7] above, modifications of [8] to

[14] above, and modifications of

[18] above. (A9) Modifications of [1] to [7] above, modifications of [8] to

[14] above, and at least one modification selected from the group consisting of

[20] to

[24] above. (A10) Modifications of [1] to [7] above, modifications of [8] to

[14] above, and modifications of

[20] to

[24] above. (A11) Modifications of [1] to [7] above, modifications of [8] to

[10] and

[12] to

[14] above, and at least one modification selected from the group consisting of

[19] to

[24] above. (A12) Modifications to [1]-[7] above, modifications to [8]-

[10] and

[12] -

[14] above, and modifications to

[19] -

[24] above. [(A PET2 with the modifications in (A12) introduced is also abbreviated as "PET2-K7S7Hotlike".] (A13) Modifications of [1] to [7] above, modifications of [8] to

[14] above, at least one modification selected from the group consisting of

[20] to

[24] above, and modifications of

[15] and

[18] above. (A14) Modifications of [1] to [7] above, modifications of [8] to

[14] above, modifications of

[20] to

[24] above, and at least one modification selected from the group consisting of

[15] and

[18] above. (A15) Modifications of [1] to [7] above, modifications of [8] to

[10] and

[12] to

[14] above, at least one modification selected from the group consisting of

[19] to

[24] above, and modifications of

[15] and

[18] above. (A16) Modifications of [1] to [7] above, modifications of [8] to

[10] and

[12] to

[14] above, modifications of

[19] to

[24] above, and at least one modification selected from the group consisting of

[15] and

[18] above. (A17) Modifications of [1] to [7] above, modifications of [8] to

[10] and

[12] to

[14] above, modifications of

[19] to

[24] above, and modifications of

[15] above, wherein in

[19] the 134th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, and in

[22] the 227th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue. (A18) Modifications of [1] to [7] above, modifications of [8] to

[10] and

[12] to

[14] above, modifications of

[19] to

[24] above, and modifications of

[18] above, wherein in

[19] the 134th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, and in

[22] the 227th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue. (A19) Modifications of [1] to [7] above, modifications of [8] to

[10] and

[12] to

[14] above, modifications of

[19] to

[24] above, and modifications of

[15] and

[18] above, wherein in

[19] the 134th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, and in

[22] the 227th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue. (A20) Modifications of [1] to [7] above, and modifications of

[19] to

[24] above, wherein in

[19] the 134th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, and in

[22] the 227th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue. (A21) Modifications of [1] to [7] above, and modifications of

[19] to

[24] above, wherein in

[19] the 134th amino acid residue of SEQ ID NO: 1 is replaced with a methionine residue, and in

[22] the 227th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue. (A22) Modifications of [1] to [7] above, and modifications of

[19] to

[24] above, wherein in

[19] the 134th amino acid residue of SEQ ID NO: 1 is replaced with a threonine group, and in

[22] the 227th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue. (A23) Modifications of [1] to [7] above, and modifications of

[19] to

[24] above, wherein in

[19] the 134th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, and in

[22] the 227th amino acid residue of SEQ ID NO: 1 is replaced with a glutamic acid residue or a histidine residue. (A24) Modifications of [1] to [7] above, and modifications of

[19] to

[24] above, wherein in

[19] the 134th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, and in

[22] the 227th amino acid residue of SEQ ID NO: 1 is replaced with a histidine residue. (A25) A modification having the modifications of [1] to [7] above and the modifications of

[20] to

[24] above, wherein in

[22] the 227th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, and further the 134th amino acid residue of SEQ ID NO: 1 is replaced with an arginine residue. (A26) A modification having the modifications of [1] to [7] above, and the modifications of

[19] and

[21] to

[24] above, wherein in

[19] the 134th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, in

[22] the 227th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, and further the 136th amino acid residue of SEQ ID NO: 1 is replaced with a glycine residue. (A27) A modification having the modifications of [1] to [7] above, and the modifications of

[19] to

[21] ,

[23] , and

[24] above, wherein in

[19] the 134th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, and the 227th amino acid residue of SEQ ID NO: 1 is further replaced with a valine residue. (A28) A modification comprising the modifications of [1] to [7] above, the modifications of [8] to

[14] above, and the modification of

[21] above, wherein the 200th amino acid residue of SEQ ID NO: 1 is replaced with an alanine residue or an aspartic acid residue in

[21] above. (A29) A modification comprising the modifications of [1] to [7] above, the modifications of [8] to

[14] above, and the modification of

[22] above, wherein the 227th amino acid residue of Sequence ID No. 1 is replaced with an arginine residue or a histidine residue in the modification of

[22] above. (A30) A modification comprising the modifications of [1] to [7] above, the modifications of [8] to

[14] above, and the modification of

[23] above, wherein the modification of

[23] above replaces the 228th amino acid residue of SEQ ID NO: 1 with a histidine residue, a lysine residue, a glutamine residue, or a tryptophan residue. (A31) A modification having the modifications of [1] to [7] above, the modifications of [8] to

[14] above, and the modification of

[24] above, wherein the 229th amino acid residue of SEQ ID NO: 1 is replaced with an asparagine residue or a phenylalanine residue in the modification of

[24] above. (A32) A modification comprising the modifications of [1] to [7] above, the modifications of [8] to

[14] above, the modification of

[15] above, and the modification of

[22] above, wherein the modification of

[22] above replaces the 227th amino acid residue of SEQ ID NO: 1 with an arginine residue. (A33) A modification comprising the modifications of [1] to [7] above, the modifications of [8] to

[14] above, the modification of

[16] above, and the modification of

[24] above, wherein the modification of

[24] above replaces the 229th amino acid residue of SEQ ID NO: 1 with a phenylalanine residue. (A34) A modification comprising the modifications of [1] to [7] above, the modifications of [8] to

[14] above, the modification of

[21] above, and the modification of

[22] above, wherein the modification of

[21] above replaces the 200th amino acid residue of SEQ ID NO: 1 with an alanine residue, and the modification of

[22] above replaces the 227th amino acid residue of SEQ ID NO: 1 with a histidine residue. (A35) A modification comprising the modifications of [1] to [7] above, the modifications of [8] to

[14] above, the modification of

[18] above, and the modification of

[24] above, wherein the 229th amino acid residue of SEQ ID NO: 1 is replaced with a phenylalanine residue in the modification of

[24] above. (A36) A modification comprising the modifications of [1] to [7] above, the modifications of [8] to

[14] above, the modifications of

[15] above, the modifications of

[18] above, and the modifications of

[22] above, wherein the modification of

[22] above replaces the 227th amino acid residue of SEQ ID NO: 1 with an arginine residue. (A37) A modification comprising the modifications of [1] to [7] above, the modifications of [8] to

[14] above, the modifications of

[18] above, the modifications of

[21] above, and the modifications of

[22] above, wherein the modification of

[21] above replaces the 200th amino acid residue of SEQ ID NO: 1 with an alanine residue, and the modification of

[22] above replaces the 227th amino acid residue of SEQ ID NO: 1 with a histidine residue. (A38) A modification comprising the modifications of [1] to [7] above, the modifications of [8] to

[14] above, the modification of

[16] above, the modification of

[21] above, and the modification of

[22] above, wherein the modification of

[21] above replaces the 200th amino acid residue of SEQ ID NO: 1 with an alanine residue, and the modification of

[22] above replaces the 227th amino acid residue of SEQ ID NO: 1 with a histidine residue. (A39) A modification comprising the modifications of [1] to [7] above, the modifications of [8] to

[14] above, the modifications of

[15] above, the modifications of

[18] above, the modifications of

[21] above, the modifications of

[21] above, the modification of the 200th amino acid residue of SEQ ID NO: 1 with an alanine residue, and the modification of the 227th amino acid residue of SEQ ID NO: 1 with an arginine residue, wherein the modification of

[22] above. (A40) A modification comprising the modifications of [1] to [7] above, the modifications of [8] to

[14] above, the modification of

[22] above, and the modification of

[23] above, wherein the modification of

[22] above replaces the 227th amino acid residue of SEQ ID NO: 1 with a glutamic acid residue, and the modification of

[23] above replaces the 228th amino acid residue of SEQ ID NO: 1 with a histidine residue. (A41) A modification comprising the modifications of [1] to [7] above, the modifications of [8] to

[14] above, the modifications of

[18] above, the modifications of

[22] above, and the modifications of

[23] above, wherein the modification of

[22] above replaces the 227th amino acid residue of SEQ ID NO: 1 with a glutamic acid residue, and the modification of

[23] above replaces the 228th amino acid residue of SEQ ID NO: 1 with a histidine residue. (A42) A modification comprising the modifications of [1] to [7] above, the modifications of [8] to

[14] above, the modifications of

[21] above, the modifications of

[22] above, and the modifications of

[24] above, wherein the modification of

[21] above replaces the 200th amino acid residue of SEQ ID NO: 1 with an alanine residue, the modification of

[22] above replaces the 227th amino acid residue of SEQ ID NO: 1 with a glutamic acid residue, and the modification of

[24] above replaces the 229th amino acid residue of SEQ ID NO: 1 with a phenylalanine residue. (A43) A modification comprising the modifications of [1] to [7] above, the modifications of [8] to

[14] above, the modification of

[16] above, and the modification of

[24] above, wherein the 229th amino acid residue of SEQ ID NO: 1 is replaced with a phenylalanine residue in the modification of

[24] above. (A44) A modification comprising the modifications of [1] to [7] above, the modifications of [8] to

[14] above, the modification of

[17] above, and the modification of

[24] above, wherein the 229th amino acid residue of SEQ ID NO: 1 is replaced with a phenylalanine residue in the modification of

[24] above. (A45) A modification comprising the modifications of [1] to [7] above, the modifications of [8] to

[14] above, the modification of

[17] above, the modification of

[21] above, and the modification of

[22] above, wherein the modification of

[21] above replaces the 200th amino acid residue of SEQ ID NO: 1 with an alanine residue, and the modification of

[22] above replaces the 227th amino acid residue of SEQ ID NO: 1 with a glutamic acid residue. (A46) A modification comprising the modifications of [1] to [7] above, the modifications of [8] to

[14] above, the modifications of

[16] above, the modifications of

[18] above, the modifications of

[21] above, the modifications of

[22] above, wherein the modification of

[21] above replaces the 200th amino acid residue of SEQ ID NO: 1 with an alanine residue, and the modification of

[22] above replaces the 227th amino acid residue of SEQ ID NO: 1 with a glutamic acid residue. (A47) A modification comprising the modifications of [1] to [7] above, the modifications of [8] to

[14] above, the modification of

[21] above, and the modification of

[24] above, wherein the modification of

[21] above replaces the 200th amino acid residue of SEQ ID NO: 1 with an alanine residue, and the modification of

[24] above replaces the 229th amino acid residue of SEQ ID NO: 1 with a phenylalanine residue. (A48) A modification comprising the modifications of [1] to [7] above, the modifications of [8] to

[14] above, the modifications of

[16] above, the modifications of

[18] above, and the modifications of

[24] above, wherein the modification in

[21] above replaces the 200th amino acid residue of SEQ ID NO: 1 with an alanine residue, and the modification in

[24] above replaces the 229th amino acid residue of SEQ ID NO: 1 with a phenylalanine residue. (A49) A modification comprising the modifications of [1] to [7] above, the modifications of [8] to

[14] above, the modification of

[22] above, and the modification of

[22] above, wherein the 227th amino acid residue of Sequence ID No. 1 is replaced with an arginine residue in

[22] above.

[0027] In each of the embodiments described above (A1) to (A49), (A1), (A12), (A17) to (A19), (A21) to (A23), and (A28) to (A49) are preferred, (A12), (A19), (A22), (A29), (A32), (A36), (A38), and (A39) are more preferred, (A12), (A19), (A36), and (A39) are particularly preferred, and (A12) is the most preferred. Examples of amino acid sequences including a modification of (A12) include SEQ ID NO: 17 (nucleotide sequence SEQ ID NO: 18) or SEQ ID NO: 5 (nucleotide sequence SEQ ID NO: 6). SEQ ID NO: 5 is a sequence obtained by deleting the N-terminus of SEQ ID NO: 17, as will be described later.

[0028] In the above

[19] , it is preferable to substitute the 134th amino acid residue of SEQ ID NO: 1 with a methionine residue or a threonine residue, and more preferably with a threonine residue. In the above

[21] , it is preferable to substitute the 200th amino acid residue of SEQ ID NO: 1 with an aspartic acid residue or an alanine residue, and more preferably with an alanine residue. In the above

[22] , it is preferable to substitute the 227th amino acid residue of Sequence ID No. 1 with an arginine residue, a glutamic acid residue, or a histidine residue, and more preferably with an arginine residue. In the above

[23] , it is preferable to substitute the 228th amino acid residue of Sequence ID No. 1 with a histidine residue or a lysine residue. In the above

[24] , it is preferable to substitute the 229th amino acid residue of SEQ ID NO: 1 with an asparagine residue or a phenylalanine residue.

[0029] Furthermore, non-limiting specific embodiments of the PET2 variant in this disclosure include proteins consisting of amino acid sequences in which at least the modifications described in [1] to [7] and [8] to

[14] above have been introduced in the amino acid sequence represented by Sequence ID No. 1.

[0030] Examples of PET2 variants in this disclosure include proteins having an amino acid sequence in which at least one modification selected from the group consisting of

[15] to

[24] above has been introduced in the amino acid sequence represented by Sequence ID No. 1 (excluding proteins having the modification of the combination of

[17] and

[23] introduced).

[0031] Furthermore, as PET2 variants in this disclosure, there are also proteins consisting of an amino acid sequence in which at least one modification selected from the group consisting of [8] to

[14] and at least one modification selected from the group consisting of

[15] to

[24] have been introduced into the amino acid sequence represented by SEQ ID NO: 1 (excluding proteins in which at least one combination of modifications selected from the group consisting of the combination of [8] and

[16] , the combination of

[11] and

[19] , and the combination of

[17] and

[23] has been introduced). As PET2 variants, proteins consisting of the amino acid sequence represented by SEQ ID NO: 17 are preferred. Depending on the host cell used when preparing the enzyme composition containing the protein, modifications to the amino acid sequence may be made to improve the expression of the protein. Modifications for improving expression include the deletion of a signal peptide.

[0032] ((Modifications in mutant protein (B))) The PET2 variants in this disclosure include proteins having a higher PET degradation activity than the mutant protein (B), which are proteins having a modification in the amino acid sequence of a protein (also abbreviated as "mutant protein (B)") in which at least one of the following modifications is introduced: at least one modification selected from the group consisting of [1'] to [7'] and at least one modification selected from the group consisting of [8'] to [24']. (However, proteins having at least one combination of modifications selected from the group consisting of the combination of [8'] and [16'], the combination of [11'] and [19'], and the combination of [17'] and [23'] are excluded.) [1'] Modification in which the amino acid residue corresponding to the 47th amino acid residue of Sequence ID No. 1 is replaced with a cysteine ​​residue. [2'] Modification in which the amino acid residue corresponding to the 89th amino acid residue in Sequence ID No. 1 is replaced with a cysteine ​​residue. [3'] Modification in which the amino acid residue corresponding to the 105th amino acid residue of Sequence ID No. 1 is replaced with an arginine residue. [4'] Modification in which the amino acid residue corresponding to the 110th amino acid residue of Sequence ID No. 1 is replaced with a lysine residue. [5'] Modification by substituting the amino acid residue corresponding to the 156th amino acid residue of Sequence ID No. 1 with a proline residue. [6'] Modification in which the amino acid residue corresponding to the 180th amino acid residue of Sequence ID No. 1 is replaced with an alanine residue. [7'] Modification by substituting the amino acid residue corresponding to amino acid residue 297 of Sequence ID No. 1 with a proline residue. [8'] Modification that replaces the amino acid residue corresponding to the 73rd amino acid residue of Sequence ID No. 1 with a glutamine residue. [9'] Modification in which the amino acid residue corresponding to the 76th amino acid residue of Sequence ID No. 1 is replaced with a cysteine ​​residue. [10'] Modification in which the amino acid residue corresponding to the 103rd amino acid residue of Sequence ID No. 1 is replaced with a lysine residue. [11'] Modification in which the amino acid residue corresponding to the 134th amino acid residue of Sequence ID No. 1 is replaced with a glycine residue. [12'] Modification in which the amino acid residue corresponding to the 144th amino acid residue of Sequence ID No. 1 is replaced with a cysteine ​​residue. [13'] Modification in which the amino acid residue corresponding to the 203rd amino acid residue of Sequence ID No. 1 is replaced with a threonine residue. [14'] Modification in which the amino acid residue corresponding to the 222nd amino acid residue of Sequence ID No. 1 is replaced with a methionine residue. [15'] Modification in which the amino acid residue corresponding to the 55th amino acid residue of Sequence ID No. 1 is replaced with a serine residue. [16'] Modification in which the amino acid residue corresponding to the 73rd amino acid residue of Sequence ID No. 1 is replaced with a serine residue. [17'] Modification in which the amino acid residue corresponding to amino acid residue 228 of sequence number 1 is replaced with a lysine residue. [18'] Modification in which the amino acid residue corresponding to the 264th amino acid residue of Sequence ID No. 1 is replaced with an arginine residue. [19'] Modification in which the amino acid residue corresponding to the 134th amino acid residue of Sequence ID No. 1 is replaced with a lysine residue, a methionine residue, or a threonine residue. [20'] Modification in which the amino acid residue corresponding to the 136th amino acid residue of Sequence ID No. 1 is replaced with a glutamic acid residue. [21'] Modification by substituting the amino acid residue corresponding to the 200th amino acid residue of Sequence ID No. 1 with a histidine residue, an aspartic acid residue, or an alanine residue. [22'] Modification by substituting the amino acid residue corresponding to amino acid residue 227 of Sequence ID No. 1 with an arginine residue, a lysine residue, a glutamic acid residue, or a histidine residue. [23'] Modification by substituting the amino acid residue corresponding to amino acid residue 228 of Sequence ID No. 1 with a glutamic acid residue, histidine residue, lysine residue, glutamine residue, or tryptophan residue. [24'] Modification by substituting the amino acid residue corresponding to the 229th amino acid residue of Sequence ID No. 1 with a tyrosine residue, an asparagine residue, or a phenylalanine residue.

[0033] Furthermore, if the modification in [8'] above is introduced, the modification in [16'] above, which modifies the amino acid residue corresponding to the amino acid residue at the same position in SEQ ID NO: 1, cannot be introduced. If the modification in [11'] above is introduced, the modification in [19'] above, which modifies the amino acid residue corresponding to the amino acid residue at the same position in SEQ ID NO: 1, cannot be introduced. If the modification in [17'] above is introduced, the modification in [23'] above, which modifies the amino acid residue corresponding to the amino acid residue at the same position in SEQ ID NO: 1, cannot be introduced.

[0034] In this specification, a mutant protein refers to a protein obtained by artificially deleting or substituting amino acid residues in an original protein, or by inserting or adding amino acid residues to said protein.

[0035] In the above mutant protein, having a modification consisting of at least one of deletion, substitution, insertion, and addition of amino acids means that at any position in the amino acid sequence represented by Sequence ID No. 1, 1 to 20 amino acids may have been modified by at least one of deletion, substitution, insertion, and addition. The number of amino acids that are modified by at least one of deletion, substitution, insertion, and addition is 1 to 20, preferably 1 to 10, more preferably 1 to 8, and most preferably 1 to 5.

[0036] The amino acids modified by at least one of deletion, substitution, insertion, and addition may be in their natural or non-natural form, but the natural form is preferred. Examples of natural amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and L-cysteine.

[0037] Below are examples of amino acids that are mutually substituted. Amino acids belonging to the same group are mutually substituted. Group A: Leucine, Isoleucine, Norleucine, Valine, Norvaline, Alanine, 2-Aminobutanoic acid, Methionine, O-Methylserine, t-Butylglycine, t-Butylalanine, Cyclohexylalanine Group B: Aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid Group C: Asparagine, Glutamine Group D: Lysine, Arginine, Ornithine, 2,4-Diaminobutanoic acid, 2,3-Diaminopropionic acid Group E: Proline, 3-hydroxyproline, 4-hydroxyproline Group F: Serine, Threonine, Homoserine Group G: Phenylalanine, tyrosine Group H: Aspartic acid, glutamic acid, tyrosine, tryptophan, arginine Group I: Serine, Threonine Group J: Leucine, Isoleucine, Valine, Alanine K group: Phenylanine, tyrosine, histidine, tryptophan Group L: Cysteine, Methionine Group M: Asparagine, aspartic acid, glutamine, glutamic acid, lysine, serine, threonine, cysteine, tyrosine, tryptophan N group: Asparagine, glutamine, methionine

[0038] A non-limiting specific embodiment of the PET2 variant in this disclosure is, for example, a protein having an amino acid sequence in which any of the following (B1) to (B27) is introduced into the amino acid sequence of a mutant protein (B) having a modification consisting of at least one deletion, substitution, insertion, and addition of 1 to 20 amino acid residues to the amino acid sequence represented by SEQ ID NO: 1.

[0039] (B1) Modifications to the above [1']~[7'] and modifications to the above [19']~[24']. (B2) Modifications to [1']~[7'] above and modifications to [8']~[14'] above. (B3) Modifications of [1'] to [7'] above, modifications of [8'] to [14'] above, and at least one modification selected from the group consisting of [15'], [17'] and [18'] above. (B4) Modifications of [1'] to [7'] above, modifications of [8'] to [14'] above, and at least one modification selected from the group consisting of [15'] to [18'] above. (B5) Modifications to [1']~[7'] above, modifications to [8']~[14'] above, and modifications to [15'] above. (B6) Modifications to [1']~[7'] above, modifications to [8']~[14'] above, and modifications to [16'] above. (B7) Modifications to [1']~[7'] above, modifications to [8']~[14'] above, and modifications to [17'] above. (B8) Modifications to [1']~[7'] above, modifications to [8']~[14'] above, and modifications to [18'] above. (B9) Modifications of [1'] to [7'] above, modifications of [8'] to [14'] above, and at least one modification selected from the group consisting of [20'] to [24'] above. (B10) Modifications to [1']~[7'] above, modifications to [8']~[14'] above, and modifications to [20']~[24'] above. (B11) Modifications of [1'] to [7'] above, modifications of [8'] to [10'] and [12'] to [14'] above, and at least one modification selected from the group consisting of [19'] to [24'] above. (B12) Modifications of [1'] to [7'] above, modifications of [8'] to [10'] and [12'] to [14'] above, and modifications of [19'] to [24'] above. (B13) Modifications of [1'] to [7'] above, modifications of [8'] to [14'] above, at least one modification selected from the group consisting of [20'] to [24'] above, and modifications of [15'] and [18'] above. (B14) Modifications of [1'] to [7'] above, modifications of [8'] to [14'] above, modifications of [20'] to [24'] above, and at least one modification selected from the group consisting of [15'] and [18'] above. (B15) Modifications of [1'] to [7'] above, modifications of [8'] to [10'] and [12'] to [14'] above, at least one modification selected from the group consisting of [19'] to [24'] above, and modifications of [15'] and [18'] above. (B16) Modifications of [1'] to [7'] above, modifications of [8'] to [10'] and [12'] to [14'] above, modifications of [19'] to [24'] above, and at least one modification selected from the group consisting of [15'] and [18'] above. (B17) Modifications of [1'] to [7'] above, modifications of [8'] to [10'] and [12'] to [14'] above, modifications of [19'] to [24'] above, and modifications of [15'] above, wherein in [19'] the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, and in [22'] the amino acid residue of the 227th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue. (B18) Modifications of [1'] to [7'] above, modifications of [8'] to [10'] and [12'] to [14'] above, modifications of [19'] to [24'] above, and modifications of [18'] above, wherein in [19'] above, the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, and in [22'] above, the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue. (B19) Modifications of [1'] to [7'] above, modifications of [8'] to [10'] and [12'] to [14'] above, modifications of [19'] to [24'] above, and modifications of [15'] and [18'] above, wherein in [19'] the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, and in [22'] the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue. (B20) Modifications of [1'] to [7'] above, and modifications of [19'] to [24'] above, wherein in [19'] the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, and in [22'] the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue. (B21) Modifications of [1'] to [7'] above, and modifications of [19'] to [24'] above, wherein in [19'] the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 is replaced with a methionine residue, and in [22'] the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue. (B22) Modifications of [1'] to [7'] above, and modifications of [19'] to [24'] above, wherein in [19'] the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 is replaced with a threonine group, and in [22'] the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue. (B23) Modifications of [1'] to [7'] above, and modifications of [19'] to [24'] above, wherein in [19'] the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, and in [22'] the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 is replaced with a glutamic acid residue or a histidine residue. (B24) Modifications of [1'] to [7'] above, and modifications of [19'] to [24'] above, wherein in [19'] the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, and in [22'] the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 is replaced with a histidine residue. (B25) A modification having the modifications described in [1'] to [7'] above, and the modifications described in [20'] to [24'] above, wherein in [22'] the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, and further the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 is replaced with an arginine residue. (B26) A modification having the modifications described in [1'] to [7'] above, and the modifications described in [19'] and [21'] to [24'] above, wherein in [19'] the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, in [22'] the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, and further, the amino acid residue corresponding to the 136th amino acid residue of SEQ ID NO: 1 is replaced with a glycine residue. (B27) A modification having the modifications described in [1'] to [7'] above, and the modifications described in [19'] to [21'], [23'], and [24'] above, wherein in [19'] the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, and further the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 is replaced with a valine residue. (B28) A modification having the modifications described in [1'] to [7'] above, the modifications described in [8'] to [14'] above, and the modification described in [21'] above, wherein the amino acid residue corresponding to the 200th amino acid residue of Sequence ID No. 1 is replaced with an alanine residue or an aspartic acid residue. (B29) A modification having the modifications described in [1'] to [7'] above, the modifications described in [8'] to [14'] above, and the modification described in [22'] above, wherein the amino acid residue corresponding to the 227th amino acid residue of Sequence ID No. 1 is replaced with an arginine residue or a histidine residue. (B30) A modification having the modifications described in [1'] to [7'] above, the modifications described in [8'] to [14'] above, and the modification described in [23'] above, wherein the amino acid residue corresponding to the 228th amino acid residue of Sequence ID No. 1 is replaced with a histidine residue, a lysine residue, a glutamine residue, or a tryptophan residue. (B31) A modification having the modifications described in [1'] to [7'] above, the modifications described in [8'] to [14'] above, and the modification described in [24'] above, wherein the amino acid residue corresponding to the 229th amino acid residue of SEQ ID NO: 1 is replaced with an asparagine residue or a phenylalanine residue. (B32) A modification having the modifications described in [1'] to [7'] above, the modifications described in [8'] to [14'] above, the modification described in [15'] above, and the modification described in [22'] above, wherein the amino acid residue corresponding to the 227th amino acid residue of Sequence ID No. 1 is replaced with an arginine residue. (B33) A modification comprising the modifications of [1'] to [7'] above, the modifications of [8'] to [14'] above, the modification of [16'] above, and the modification of [24'] above, wherein the amino acid residue corresponding to the 229th amino acid residue of SEQ ID NO: 1 is replaced with a phenylalanine residue in the modification of [24'] above. (B34) A modification of [1'] to [7'] above, a modification of [8'] to [14'] above, a modification of [21'] above, and a modification of [22'] above, wherein the modification of [21'] above replaces the amino acid residue corresponding to the 200th amino acid residue of SEQ ID NO: 1 with an alanine residue, and the modification of [22'] above replaces the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 with a histidine residue. (B35) A modification having the modifications described in [1'] to [7'] above, the modifications described in [8'] to [14'] above, the modification described in [18'] above, and the modification described in [24'] above, wherein the amino acid residue corresponding to the 229th amino acid residue of SEQ ID NO: 1 is replaced with a phenylalanine residue. (B36) A modification comprising the modifications of [1'] to [7'] above, the modifications of [8'] to [14'] above, the modifications of [15'] above, the modifications of [18'] above, and the modifications of [22'] above, wherein the modification of [22'] above replaces the amino acid residue corresponding to the 227th amino acid residue of Sequence ID No. 1 with an arginine residue. (B37) A modification of [1'] to [7'] above, a modification of [8'] to [14'] above, a modification of [18'] above, a modification of [21'] above, and a modification of [22'] above, wherein the modification of [21'] above replaces the amino acid residue corresponding to the 200th amino acid residue of SEQ ID NO: 1 with an alanine residue, and the modification of [22'] above replaces the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 with a histidine residue. (B38) A modification of the above [1']~[7'], the above [8']~[14'], the above [16'], the above [21'], and the above [22'], wherein the above [21'] is a modification in which the amino acid residue corresponding to the 200th amino acid residue of SEQ ID NO: 1 is replaced with an alanine residue, and the above [22'] is a modification in which the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 is replaced with a histidine residue. (B39) The modification of [1'] to [7'] above, the modification of [8'] to [14'] above, the modification of [15'] above, the modification of [18'] above, the modification of [21'] above, and the modification of [22'] above, wherein the modification of [21'] above replaces the amino acid residue corresponding to the 200th amino acid residue of SEQ ID NO: 1 with an alanine residue, and the modification of [22'] above replaces the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 with an arginine residue. (B40) A modification of the above [1']~[7'], the above [8']~[14'], the above [22'], and the above [23'], wherein the above [22'] is a modification in which the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 is replaced with a glutamic acid residue, and the above [23'] is a modification in which the amino acid residue corresponding to the 228th amino acid residue of SEQ ID NO: 1 is replaced with a histidine residue. (B41) The modification of [1'] to [7'] above, the modification of [8'] to [14'] above, the modification of [18'] above, the modification of [22'] above, and the modification of [23'] above, wherein the modification of [22'] above replaces the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 with a glutamic acid residue, and the modification of [23'] above replaces the amino acid residue corresponding to the 228th amino acid residue of SEQ ID NO: 1 with a histidine residue. (B42) A modification having the modifications of [1'] to [7'] above, the modifications of [8'] to [14'] above, the modifications of [21'] above, the modifications of [22'] above, and the modifications of [24'] above, wherein the modification of [21'] above replaces the amino acid residue corresponding to the 200th amino acid residue of SEQ ID NO: 1 with an alanine residue, the modification of [22'] above replaces the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 with a glutamic acid residue, and the modification of [24'] above replaces the amino acid residue corresponding to the 229th amino acid residue of SEQ ID NO: 1 with a phenylalanine residue. (B43) A modification having the modifications described in [1'] to [7'] above, the modifications described in [8'] to [14'] above, the modification described in [16'] above, and the modification described in [24'] above, wherein the amino acid residue corresponding to the 229th amino acid residue of SEQ ID NO: 1 is replaced with a phenylalanine residue. (B44) A modification having the modifications described in [1'] to [7'] above, the modifications described in [8'] to [14'] above, the modification described in [17'] above, and the modification described in [24'] above, wherein the amino acid residue corresponding to the 229th amino acid residue of SEQ ID NO: 1 is replaced with a phenylalanine residue. (B45) A modification comprising the modifications of [1'] to [7'] above, the modifications of [8'] to [14'] above, the modifications of [17'] above, the modifications of [21'] above, and the modifications of [22'] above, wherein the modification of [21'] above replaces the amino acid residue corresponding to the 200th amino acid residue of SEQ ID NO: 1 with an alanine residue, and the modification of [22'] above replaces the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 with a glutamic acid residue. (B46) The modification of [1'] to [7'] above, the modification of [8'] to [14'] above, the modification of [16'] above, the modification of [18'] above, the modification of [21'] above, and the modification of [22'] above, wherein the modification of [21'] above replaces the amino acid residue corresponding to the 200th amino acid residue of SEQ ID NO: 1 with an alanine residue, and the modification of [22'] above replaces the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 with a glutamic acid residue. (B47) A modification comprising the modifications of [1'] to [7'] above, the modifications of [8'] to [14'] above, the modification of [21'] above, and the modification of [24'] above, wherein the modification of [21'] above replaces the amino acid residue corresponding to the 200th amino acid residue of SEQ ID NO: 1 with an alanine residue, and the modification of [24'] above replaces the amino acid residue corresponding to the 229th amino acid residue of SEQ ID NO: 1 with a phenylalanine residue. (B48) A modification comprising the modifications of [1'] to [7'] above, the modifications of [8'] to [14'] above, the modifications of [16'] above, the modifications of [18'] above, and the modifications of [24'] above, wherein the modification in [21'] above replaces the amino acid residue corresponding to the 200th amino acid residue of SEQ ID NO: 1 with an alanine residue, and the modification in [24'] above replaces the amino acid residue corresponding to the 229th amino acid residue of SEQ ID NO: 1 with a phenylalanine residue. (B49) A modification comprising the above modifications [1'] to [7'], the above modifications [8'] to [14'], the above modification [22'], and the above modification [22'], wherein the above modification [22'] replaces the amino acid residue corresponding to the 227th amino acid residue of Sequence ID No. 1 with an arginine residue.

[0040] In each of the embodiments described above (B1) to (B49), (B1), (B12), (B17) to (B19), (B21) to (B23), and (B28) to (B49) are preferred, (B19), (B22), (B29), (B32), (B36), (B38), and (B39) are more preferred, and (B19), (B36), and (B39) are particularly preferred.

[0041] In the above [19'], it is preferable to substitute the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 with a methionine residue or a threonine residue, and more preferably with a threonine residue. In the above [22'], it is preferable to substitute the amino acid residue corresponding to the 227th amino acid residue of Sequence ID No. 1 with an arginine residue, a glutamic acid residue, or a histidine residue, and more preferably with an arginine residue. In the above [21'], it is preferable to substitute the amino acid residue corresponding to the 200th amino acid residue of SEQ ID NO: 1 with an aspartic acid residue or an alanine residue, and more preferably an alanine residue. In the above [23'], it is preferable to substitute the amino acid residue corresponding to the 228th amino acid residue of Sequence ID No. 1 with a histidine residue or a lysine residue. In the above [24'], it is preferable to substitute the amino acid residue corresponding to the 229th amino acid residue of SEQ ID NO: 1 with an asparagine residue or a phenylalanine residue.

[0042] Furthermore, non-limiting specific embodiments of the PET2 variant in this disclosure include proteins having an amino acid sequence in which at least the modifications [1']~[7'] and [8']~[14'] described above have been introduced into the amino acid sequence of the mutant protein (B), and which have higher PET degradation activity than the mutant protein (B) (excluding proteins in which the modification of the combination of [17'] and [23'] has been introduced).

[0043] Examples of PET2 variants in this disclosure include mutant protein (B), which has a modification consisting of at least one deletion, substitution, insertion, and addition of 1 to 20 amino acid residues to the amino acid sequence represented by Sequence ID No. 1, and which has an amino acid sequence in which at least one modification selected from the group consisting of [15'] to [24'] above is introduced, and which has higher PET degradation activity than mutant protein (B) (except for proteins in which the modification of the combination of [17'] and [23'] is introduced).

[0044] Furthermore, the PET2 variant in this disclosure is a mutant protein (B) whose amino acid sequence has at least one modification consisting of deletion, substitution, insertion, and addition of 1 to 20 amino acid residues to the amino acid sequence represented by SEQ ID NO: 1, and which has an amino acid sequence in which at least one modification selected from the group consisting of [8'] to [14'] and at least one modification selected from the group consisting of [15'] to [24'] have been introduced. Other examples include proteins that have higher PET degradation activity than the aforementioned mutant protein (B), excluding proteins that have been modified by at least one combination selected from the group consisting of the combinations of [8'] and [16'], [11'] and [19'], and [17'] and [23'].

[0045] ((Modifications in homologous protein (C))) The PET2 variants in this disclosure include proteins having an amino acid sequence in which at least 80% identity with the amino acid sequence represented by Sequence ID No. 1 (also abbreviated as "homologous protein (C)") is introduced, and which includes at least one modification selected from the group consisting of [1''] to [7''] and at least one modification selected from the group consisting of [8''] to [24''], and which have higher PET degradation activity than the homologous protein (C) (excluding proteins in which at least one combination of modifications selected from the group consisting of the combination of [8''] and [16''], the combination of [11''] and [19''], and the combination of [17''] and [23''] is introduced). [1''] Modification that replaces the amino acid residue corresponding to the 47th amino acid residue in Sequence ID No. 1 with a cysteine ​​residue. [2''] Modification that replaces the amino acid residue corresponding to the 89th amino acid residue in Sequence ID No. 1 with a cysteine ​​residue. [3''] Modification in which the amino acid residue corresponding to the 105th amino acid residue of Sequence ID No. 1 is replaced with an arginine residue. [4''] Modification in which the amino acid residue corresponding to the 110th amino acid residue of Sequence ID No. 1 is replaced with a lysine residue. [5''] Modification that replaces the amino acid residue corresponding to the 156th amino acid residue of Sequence ID No. 1 with a proline residue. [6''] Modification that replaces the amino acid residue corresponding to the 180th amino acid residue in Sequence ID No. 1 with an alanine residue. [7''] Modification that replaces the amino acid residue corresponding to amino acid residue 297 of Sequence ID No. 1 with a proline residue. [8''] Modification that replaces the amino acid residue corresponding to the 73rd amino acid residue of Sequence ID No. 1 with a glutamine residue. [9''] Modification that replaces the amino acid residue corresponding to the 76th amino acid residue in Sequence ID No. 1 with a cysteine ​​residue. [10''] Modification that replaces the amino acid residue corresponding to the 103rd amino acid residue in Sequence ID No. 1 with a lysine residue. [11''] Modification that replaces the amino acid residue corresponding to the 134th amino acid residue of Sequence ID No. 1 with a glycine residue. [12''] Modification that replaces the amino acid residue corresponding to the 144th amino acid residue in Sequence ID No. 1 with a cysteine ​​residue. [13''] Modification that replaces the amino acid residue corresponding to the 203rd amino acid residue in Sequence ID No. 1 with a threonine residue. [14''] Modification that replaces the amino acid residue corresponding to the 222nd amino acid residue of Sequence ID No. 1 with a methionine residue. [15''] Modification that replaces the amino acid residue corresponding to the 55th amino acid residue of Sequence ID No. 1 with a serine residue. [16''] Modification that replaces the amino acid residue corresponding to the 73rd amino acid residue of Sequence ID No. 1 with a serine residue. [17''] Modification that replaces the amino acid residue corresponding to amino acid residue 228 of Sequence ID No. 1 with a lysine residue. [18''] Modification that replaces the amino acid residue corresponding to the 264th amino acid residue of Sequence ID No. 1 with an arginine residue. [19''] Modification by substituting the amino acid residue corresponding to the 134th amino acid residue of Sequence ID No. 1 with a lysine residue, a methionine residue, or a threonine residue. [20''] Modification that replaces the amino acid residue corresponding to the 136th amino acid residue of Sequence ID No. 1 with a glutamic acid residue. [21''] Modification by substituting the amino acid residue corresponding to the 200th amino acid residue of Sequence ID No. 1 with a histidine residue, an aspartic acid residue, or an alanine residue. [22''] Modification by substituting the amino acid residue corresponding to amino acid residue 227 of Sequence ID No. 1 with an arginine residue, lysine residue, glutamic acid residue, or histidine residue. [23''] Modification by substituting the amino acid residue corresponding to amino acid residue 228 of Sequence ID No. 1 with a glutamic acid residue, histidine residue, lysine residue, glutamine residue, or tryptophan residue. [24''] Modification by substituting the amino acid residue corresponding to the 229th amino acid residue of Sequence ID No. 1 with a tyrosine residue, an asparagine residue, or a phenylalanine residue. Furthermore, if the modification in [8''] above is introduced, the modification in [16''] above, which modifies the amino acid residue corresponding to the amino acid residue at the same position in SEQ ID NO: 1, cannot be introduced. If the modification in [11''] above is introduced, the modification in [19''] above, which modifies the amino acid residue corresponding to the amino acid residue at the same position in SEQ ID NO: 1, cannot be introduced. If the modification in [17''] above is introduced, the modification in [23''] above, which modifies the amino acid residue corresponding to the amino acid residue at the same position in SEQ ID NO: 1, cannot be introduced.

[0046] In this specification, homologous protein refers to a protein that is similar in structure and function to the original protein. Examples of homologous proteins include amino acid sequences that have 80% or more, preferably 90% or more, and particularly preferably 95% or more, identity with the amino acid sequence of the target protein.

[0047] In this disclosure, the percentage of sequence identity between two amino acid sequences or two nucleotide sequences is calculated as the ratio of matching residues when the two sequences are aligned to the extent that they are most identical. For example, the percentage of sequence identity can be determined using a mathematical algorithm.

[0048] Examples of such mathematical algorithms include the local homology algorithm by Smith et al (1981) Adv. Appl. Math. 2:482, the homology alignment algorithm by Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, the similarity search method by Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448, and improved versions such as the algorithm by Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, as described in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. However, mathematical algorithms are not limited to the examples above.

[0049] These mathematical algorithms can be used to perform alignment to determine the percentage of sequence identity. Such programs can be executed on a computer as appropriate. Examples of such programs, though not limited to them, include the PC / Gene programs CLUSTAL (available from Intelligenetics, Mountain View, Calif.), MAFFT (Katoh, K., Misawa, K., Kuma, K., & Miyata, T. (2002), 30(14), 3059-3066., http: / / mafft.cbrc.jp / alignment / server / ), MUSCLE (Edgar RC (2004). Nucleic acids research, 32(5), 1792-1797., http: / / www.ebi.ac.uk / Tools / msa / muscle / ), BLAST, FASTA, and TFASTA.

[0050] Alignment using these programs can be performed, for example, using initial parameters. CLUSTAL programs are described in Higgins et al. (1988) Gene 73:237-244, Higgins et al. (1989) CABIOS 5:151-153, Corpet et al. (1988) Nucleic Acids Res. 16:10881-90, Huang et al. (1992) CABIOS 8:155-65, and Pearson et al. (1994) Meth. Mol.Biol. 24:307-331.

[0051] BLAST is described in Altschul, SF, Gish, W., Miller, W., Myers, EW, & Lipman, DJ (1990). 215(3), 403-410., Mount DW (2007). CSH protocols, 2007, pdb.top17., etc. Based on BLAST, programs called BLASTP and BLASTN have been developed, and the percentage of sequence identity can be calculated using these programs with default settings.

[0052] Non-limiting specific embodiments of the PET2 variant in this disclosure include, for example, a protein consisting of an amino acid sequence in which any of the following (C1) to (C27) is introduced into the amino acid sequence of a homologous protein (C) that has 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1.

[0053] (C1) Modifications to the above [1'']~[7''] and modifications to the above [19'']~[24'']. (C2) Modifications to the above [1'']~[7''] and modifications to the above [8'']~[14'']. (C3) Modifications of [1''] to [7''] above, modifications of [8''] to [14''] above, and at least one modification selected from the group consisting of [15''], [17''] and [18''] above. (C4) Modifications of [1''] to [7''] above, modifications of [8''] to [14''] above, and at least one modification selected from the group consisting of [15''] to [18''] above. (C5) Modifications to [1'']~[7''] above, modifications to [8'']~[14''] above, and modifications to [15''] above. (C6) Modifications to [1'']~[7''] above, modifications to [8'']~[14''] above, and modifications to [16''] above. (C7) Modifications to [1'']~[7''] above, modifications to [8'']~[14''] above, and modifications to [17''] above. (C8) Modifications to [1'']~[7''] above, modifications to [8'']~[14''] above, and modifications to [18''] above. (C9) Modifications of [1''] to [7''] above, modifications of [8''] to [14''] above, and at least one modification selected from the group consisting of [20''] to [24''] above. (C10) Modifications to [1'']~[7''] above, modifications to [8'']~[14''] above, and modifications to [20'']~[24''] above. (C11) Modifications of [1''] to [7''] above, modifications of [8''] to [10''] and [12''] to [14''] above, and at least one modification selected from the group consisting of [19''] to [24''] above. (C12) Modifications of [1'']~[7''] above, modifications of [8'']~[10''] and [12'']~[14''] above, and modifications of [19'']~[24''] above. (C13) Modifications of [1''] to [7''] above, modifications of [8''] to [14''] above, at least one modification selected from the group consisting of [20''] to [24''] above, and modifications of [15''] and [18''] above. (C14) Modifications of [1''] to [7''] above, modifications of [8''] to [14''] above, modifications of [20''] to [24''] above, and at least one modification selected from the group consisting of [15''] and [18''] above. (C15) Modifications of [1''] to [7''] above, modifications of [8''] to [10''] and [12''] to [14''] above, at least one modification selected from the group consisting of [19''] to [24''] above, and modifications of [15''] and [18''] above. (C16) Modifications of [1''] to [7''] above, modifications of [8''] to [10''] and [12''] to [14''] above, modifications of [19''] to [24''] above, and at least one modification selected from the group consisting of [15''] and [18''] above. (C17) Modifications of [1''] to [7''] above, modifications of [8''] to [10''] and [12''] to [14''] above, modifications of [19''] to [24''] above, and modifications of [15''] above, wherein in [19''] above, the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, and in [22''] above, the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue. (C18) Modifications of [1'']~[7''] above, modifications of [8'']~[10''] and [12'']~[14''] above, modifications of [19'']~[24''] above, and modifications of [18''] above, wherein in [19''] above, the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, and in [22''] above, the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue. (C19) Modifications of [1''] to [7''] above, modifications of [8''] to [10''] and [12''] to [14''] above, modifications of [19''] to [24''] above, and modifications of [15''] and [18''] above, wherein in [19''] above, the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, and in [22''] above, the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue. (C20) Modifications of [1''] to [7''] above, and modifications of [19''] to [24''] above, wherein in [19''] the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, and in [22''] the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue. (C21) Modifications of [1''] to [7''] above, and modifications of [19''] to [24''] above, wherein in [19''] the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 is replaced with a methionine residue, and in [22''] the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue. (C22) Modifications of [1''] to [7''] above, and modifications of [19''] to [24''] above, wherein in [19''] the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 is replaced with a threonine group, and in [22''] the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue. (C23) Modifications of [1''] to [7''] above, and modifications of [19''] to [24''] above, wherein in [19''] the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, and in [22''] the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 is replaced with a glutamic acid residue or a histidine residue. (C24) Modifications of [1''] to [7''] above, and modifications of [19''] to [24''] above, wherein in [19''] the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, and in [22''] the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 is replaced with a histidine residue. (C25) A modification having the modifications described in [1''] to [7''] above, and the modifications described in [20''] to [24''] above, wherein in [22''] the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, and further the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 is replaced with an arginine residue. (C26) A modification having the modifications described in [1''] to [7''] above, and the modifications described in [19''] and [21''] to [24''] above, wherein in [19''] the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, in [22''] the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, and further, the amino acid residue corresponding to the 136th amino acid residue of SEQ ID NO: 1 is replaced with a glycine residue. (C27) A modification having the modifications described in [1''] to [7''] above, and the modifications described in [19''] to [21''], [23''], and [24''] above, wherein in [19''] the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue, and further the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 is replaced with a valine residue. (C28) A modification having the modifications described in [1''] to [7''] above, the modifications described in [8''] to [14''] above, and the modification described in [21''] above, wherein the amino acid residue corresponding to the 200th amino acid residue of Sequence ID No. 1 is replaced with an alanine residue or an aspartic acid residue. (C29) A modification having the modifications described in [1''] to [7''] above, the modifications described in [8''] to [14''] above, and the modification described in [22''] above, wherein the amino acid residue corresponding to the 227th amino acid residue of Sequence ID No. 1 is replaced with an arginine residue or a histidine residue. (C30) A modification having the modifications described in [1''] to [7''] above, the modifications described in [8''] to [14''] above, and the modification described in [23''] above, wherein the amino acid residue corresponding to the 228th amino acid residue of Sequence ID No. 1 is replaced with a histidine residue, a lysine residue, a glutamine residue, or a tryptophan residue. (C31) A modification having the modifications described in [1''] to [7''] above, the modifications described in [8''] to [14''] above, and the modification described in [24''] above, wherein the amino acid residue corresponding to the 229th amino acid residue of SEQ ID NO: 1 is replaced with an asparagine residue or a phenylalanine residue. (C32) A modification having the modifications described in [1''] to [7''] above, the modifications described in [8''] to [14''] above, the modification described in [15''] above, and the modification described in [22''] above, wherein the amino acid residue corresponding to the 227th amino acid residue of Sequence ID No. 1 is replaced with an arginine residue. (C33) A modification having the modifications described in [1''] to [7''] above, the modifications described in [8''] to [14''] above, the modification described in [16''] above, and the modification described in [24''] above, wherein the amino acid residue corresponding to the 229th amino acid residue of SEQ ID NO: 1 is replaced with a phenylalanine residue. (C34) Modifications of [1''] to [7''] above, modifications of [8''] to [14''] above, modifications of [21''] above, and modifications of [22''] above, wherein modification [21''] above replaces the amino acid residue corresponding to the 200th amino acid residue of SEQ ID NO: 1 with an alanine residue, and modification [22''] above replaces the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 with a histidine residue. (C35) A modification having the modifications described in [1''] to [7''] above, the modifications described in [8''] to [14''] above, the modification described in [18''] above, and the modification described in [24''] above, wherein the amino acid residue corresponding to the 229th amino acid residue of Sequence ID No. 1 is replaced with a phenylalanine residue. (C36) A modification comprising the modifications of [1''] to [7''] above, the modifications of [8''] to [14''] above, the modifications of [15''] above, the modifications of [18''] above, and the modifications of [22''] above, wherein the modification of [22''] above replaces the amino acid residue corresponding to the 227th amino acid residue of Sequence ID No. 1 with an arginine residue. (C37) Modifications of [1''] to [7''] above, modifications of [8''] to [14''] above, modifications of [18''] above, modifications of [21''] above, and modifications of [22''] above, wherein modification [21''] above replaces the amino acid residue corresponding to the 200th amino acid residue of SEQ ID NO: 1 with an alanine residue, and modification [22''] above replaces the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 with a histidine residue. (C38) Modifications of [1''] to [7''] above, modifications of [8''] to [14''] above, modifications of [16''] above, modifications of [21''] above, and modifications of [22''] above, wherein the modification of [21''] above replaces the amino acid residue corresponding to the 200th amino acid residue of SEQ ID NO: 1 with an alanine residue, and the modification of [22''] above replaces the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 with a histidine residue. (C39) Modifications of [1''] to [7''] above, modifications of [8''] to [14''] above, modifications of [15''] above, modifications of [18''] above, modifications of [21''] above, and modifications of [22''] above, wherein the modification of [21''] above replaces the amino acid residue corresponding to the 200th amino acid residue of SEQ ID NO: 1 with an alanine residue, and the modification of [22''] above replaces the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 with an arginine residue. (C40) The modification of [1''] to [7''] above, the modification of [8''] to [14''] above, the modification of [22''] above, and the modification of [23''] above, wherein the modification of [22''] above replaces the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 with a glutamic acid residue, and the modification of [23''] above replaces the amino acid residue corresponding to the 228th amino acid residue of SEQ ID NO: 1 with a histidine residue. (C41) Modifications of [1''] to [7''] above, modifications of [8''] to [14''] above, modifications of [18''] above, modifications of [22''] above, and modifications of [23''] above, wherein modification [22''] above replaces the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 with a glutamic acid residue, and modification [23''] above replaces the amino acid residue corresponding to the 228th amino acid residue of SEQ ID NO: 1 with a histidine residue. (C42) The modification of [1''] to [7''] above, the modification of [8''] to [14''] above, the modification of [21''] above, the modification of [22''] above, and the modification of [24''] above, wherein the modification of [21''] above replaces the 200th amino acid residue of SEQ ID NO: 1 with an alanine residue, the modification of [22''] above replaces the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 with a glutamic acid residue, and the modification of [24''] above replaces the amino acid residue corresponding to the 229th amino acid residue of SEQ ID NO: 1 with a phenylalanine residue. (C43) A modification having the modifications described in [1''] to [7''] above, the modifications described in [8''] to [14''] above, the modification described in [16''] above, and the modification described in [24''] above, wherein the amino acid residue corresponding to the 229th amino acid residue of Sequence ID No. 1 is replaced with a phenylalanine residue. (C44) A modification having the modifications described in [1''] to [7''] above, the modifications described in [8''] to [14''] above, the modification described in [17''] above, and the modification described in [24''] above, wherein the amino acid residue corresponding to the 229th amino acid residue of Sequence ID No. 1 is replaced with a phenylalanine residue. (C45) A modification of the above [1'']~[7''], the above [8'']~[14''], the above [17''], the above [21''], and the above [22''], wherein the above [21''] is a modification in which the amino acid residue corresponding to the 200th amino acid residue of SEQ ID NO: 1 is replaced with an alanine residue, and the above [22''] is a modification in which the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 is replaced with a glutamic acid residue. (C46) Modifications of [1''] to [7''] above, modifications of [8''] to [14''] above, modifications of [16''] above, modifications of [18''] above, modifications of [21''] above, and modifications of [22''] above, wherein [21''] is a modification in which the amino acid residue corresponding to the 200th amino acid residue of SEQ ID NO: 1 is replaced with an alanine residue, and [22''] is a modification in which the amino acid residue corresponding to the 227th amino acid residue of SEQ ID NO: 1 is replaced with a glutamic acid residue. (C47) A modification of the above [1'']~[7''], the above [8'']~[14''], the above [21''], and the above [24''], wherein the above [21''] is a modification in which the amino acid residue corresponding to the 200th amino acid residue of SEQ ID NO: 1 is replaced with an alanine residue, and the above [24''] is a modification in which the amino acid residue corresponding to the 229th amino acid residue of SEQ ID NO: 1 is replaced with a phenylalanine residue. (C48) Modifications of [1''] to [7''] above, modifications of [8''] to [14''] above, modifications of [16''] above, modifications of [18''] above, and modifications of [24''] above, wherein modification [21''] above replaces the amino acid residue corresponding to the 200th amino acid residue of SEQ ID NO: 1 with an alanine residue, and modification [24''] above replaces the amino acid residue corresponding to the 229th amino acid residue of SEQ ID NO: 1 with a phenylalanine residue. (C49) A modification comprising the above modifications [1''] to [7''], the above modifications [8''] to [14''], the above modification [22''], and the above modification [22''], wherein the above modification [22''] replaces the amino acid residue corresponding to the 227th amino acid residue of Sequence ID No. 1 with an arginine residue.

[0054] In each of the above embodiments (C1) to (C49), (C1), (C12), (C17) to (C19), (C21) to (C23), and (C28) to (C49) are preferred, (C19), (C22), (C29), (C32), (C36), (C38), and (C39) are more preferred, and (C19), (C36), and (C39) are particularly preferred.

[0055] In the above [19''], it is preferable to substitute the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 with a methionine residue or a threonine residue, and more preferably with a threonine residue. In the above [22''], it is preferable that the amino acid residue corresponding to the 227th amino acid residue of Sequence ID No. 1 be an arginine residue, a glutamic acid residue, or a histidine residue, and more preferably an arginine residue. In the above [21''], it is preferable that the amino acid residue corresponding to the 200th amino acid residue of Sequence ID No. 1 be an aspartic acid residue or an alanine residue, and more preferably an alanine residue. In the above [23''], it is preferable that the amino acid residue corresponding to the 228th amino acid residue of Sequence ID No. 1 be a histidine residue or a lysine residue. In the above [24''], it is preferable that the amino acid residue corresponding to the 229th amino acid residue of SEQ ID NO: 1 be an asparagine residue or a phenylalanine residue.

[0056] Furthermore, non-limiting specific embodiments of the PET2 variant in this disclosure include proteins having an amino acid sequence in which at least one modification selected from the group consisting of [15''] to [24''] is introduced in the amino acid sequence of the homologous protein (C), and which have higher PET degradation activity than the homologous protein (C) (excluding proteins in which the combination of [17''] and [23''] is introduced).

[0057] Examples of PET2 variants in this disclosure include proteins having amino acid sequences in which at least one modification selected from the group consisting of [15''] to [24''] is introduced into the amino acid sequence of homologous protein (C) which has 80% or more identity with the amino acid sequence represented by Sequence ID No. 1, and which have higher PET degradation activity than homologous protein (C) (excluding proteins in which the modification combination of [17''] and [23''] is introduced).

[0058] Furthermore, the PET2 variant in this disclosure consists of an amino acid sequence in which at least one modification selected from the group consisting of [8''] to [14''] and at least one modification selected from the group consisting of [15''] to [24''] are introduced into the amino acid sequence of a homologous protein (C) that has 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1. Other examples include proteins that have higher PET degradation activity than the homologous protein (C) mentioned above (excluding proteins into which at least one combination of modifications selected from the group consisting of the combinations of [8''] and [16''], [11''] and [19''], and [17''] and [23''] has been introduced).

[0059] The PET2 variant in this disclosure is a protein consisting of an amino acid sequence in which a predetermined modification has been introduced in a protein consisting of an amino acid sequence of homologous protein (C) having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1. Preferably, it is a mutant protein (B) in which the predetermined modification has been introduced in an amino acid sequence in which the predetermined modification has been introduced in a protein consisting of an amino acid sequence (A) in which the predetermined modification has been introduced in an amino acid sequence of protein consisting of the amino acid sequence represented by SEQ ID NO: 1. More preferably, it is a protein consisting of an amino acid sequence in which a predetermined modification has been introduced in a protein consisting of an amino acid sequence of amino acid sequence represented by SEQ ID NO: 1.

[0060] (cutinase) Cutinase is a hydrolytic enzyme classified under EC 3.1.1.74.

[0061] Examples of cutinases include cutinases derived from metagenomic libraries, such as the LC-cutinase described in Sulaiman et al., 2012, as well as cutinases from Thermobifida cellulosityca, Thermobifida halotolerans, Thermobifida fusca, Thermobifida alba, Bacillus subtilis, Fusarium solani pisi, Humicola insolens (e.g., registration number A0A075B5G4 in the UniProt database), Sirococcus conigenus, Pseudomonas mendocina, and Thielavia terrestris, and their variants.

[0062] In this disclosure, LC-cutinase or its variants are preferred as cutinase or its variants. An example of LC-cutinase is the amino acid sequence represented by SEQ ID NO: 3 (nucleotide sequence is SEQ ID NO: 4). A preferred example of an LC-cutinase variant (hereinafter also abbreviated as LC-cutinase variant) is a protein consisting of the amino acid sequence represented by SEQ ID NO: 7 (nucleotide sequence is SEQ ID NO: 8).

[0063] Examples of LC-cutinase or its variants in this disclosure include at least one selected from the following [C1] to [C3]. [C1] A protein consisting of the amino acid sequence represented by sequence number 3 or 7. [C2] A protein having an amino acid sequence in which, preferably, 1 to 20 amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by Sequence ID No. 3 or 7, and having a PET degradation rate of 0.5% or more in a mixed substrate containing PET. [C3] A protein having an amino acid sequence that preferably has 80% or more identity with the amino acid sequence represented by SEQ ID NO: 3 or 7, and having a PET degradation rate of 0.5% or more in a mixed substrate containing PET.

[0064] In [C2] above, the number of deleted, substituted, inserted and / or added amino acid residues in the amino acid sequence represented by SEQ ID NO: 3 or 7 is preferably 1 to 20, more preferably 1 to 15, more preferably 1 to 10, and even more preferably 1 to 5. The amino acid residue mutation may be introduced into one region of the amino acid sequence, or into multiple different regions.

[0065] In the above [C3], the identity with the amino acid sequence represented by SEQ ID NO: 3 or 7 is preferably 80% or more, and more preferably, in order below, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0066] The PET degradation rate in the mixed substrate containing PET in [C2] and [C3] above is determined by the following method. (Method) The following enzyme composition was brought into contact with a mixed substrate containing the following PET, and the amounts of TPA and MHET produced were measured by high-performance liquid chromatography (hereinafter also referred to as "HPLC"), and the PET degradation rate was determined from the following formula (1). Mixed substrate: A mixed substrate containing PET and other substrates, with a PET content of 50-99% by mass. Average particle size of mixed substrate: 50 μm to 150 μm Enzyme composition: An enzyme composition containing 0.01 to 1.00% by mass of a carboxylic acid ester hydrolase or its variant. Temperature: 40~70℃ Reaction time: 24 hours pH: 6~11

[0067] PET decomposition rate = (TPA concentration (mM) + MHET concentration (mM)) / theoretical TPA concentration (mM) × 100(%)...Equation (1) In equation (1), the following applies: TPA concentration (mM): Quantitative value analyzed by HPLC MHET concentration (mM): Quantitative value analyzed by HPLC Theoretical TPA concentration (mM): TPA concentration assuming that the PET used as a substrate is 100% decomposed.

[0068] Furthermore, in equation (1), the calculation of the PET degradation rate may use TPA concentration (mM) and MHET concentration (mM), or at least one of TPA concentration (mM), MHET concentration (mM), or BHET concentration (mM). The BHET concentration (mM) can also be a quantitative value analyzed by HPLC.

[0069] Furthermore, the PET degradation rate can be calculated not only using equation (1) but also using equation (2) below. PET decomposition rate = (PET weight before decomposition reaction - PET weight after decomposition reaction) / (PET weight before decomposition reaction) × 100 (%) ... Equation (2)

[0070] ((Function of carboxylic acid ester hydrolase or its variants)) The carboxylic acid ester hydrolase or its variants in this disclosure can degrade the main chain of PET. Therefore, the carboxylic acid ester hydrolase or its variants in this disclosure can degrade bis(2-hydroxyethyl) terephthalate (hereinafter also referred to as "BHET"), an intermediate product of PET degradation, to MHET, and further degrade MHET to terephthalic acid (hereinafter also referred to as "TPA") and monoethylene glycol (hereinafter also referred to as "MEG"). In other words, a protein in one aspect of the present invention can hydrolyze PET or BHET, a substructure of PET, as a substrate to produce MHET, and further produce TPA and MEG. Alternatively, a protein in one aspect of the present invention can hydrolyze PET or BHET, a substructure of PET, as a substrate to produce TPA and MEG, without necessarily requiring the production of MHET.

[0071] The carboxylic acid ester hydrolase or its variant in this disclosure preferably has a PET degradation rate of 0.5% or more in a mixed substrate containing PET obtained by the method described above, more preferably 10% or more, even more preferably 20% or more, 30% or more, 50% or more, and particularly preferably 80% or more.

[0072] (Method for preparing carboxylic acid ester hydrolase or its variants) A carboxylic acid ester hydrolase or its variant (hereinafter also referred to as a protein) is prepared by preparing a DNA fragment of an appropriate length containing the portion encoding the protein, as needed, based on the DNA encoding the protein. By introducing the recombinant DNA containing the DNA fragment into a host cell compatible with the expression vector, a transformant that produces the protein is obtained. Any host cell capable of expressing the target gene can be used, such as bacteria, yeast, animal cells, insect cells, or plant cells. When using prokaryotes such as bacteria as host cells, it is preferable that the recombinant DNA is capable of autonomous replication in the prokaryote and is composed of a promoter, a ribosome-binding sequence, the DNA of the present invention, and a transcription termination sequence.

[0073] Examples of expression vectors include pET28a (Sigma-Aldrich), pColdI (Takara Bio Inc.), pCDF-1b, pRSF-1b (both from Novagen), pMAL-c2x (New England Biolabs), pGEX-4T-1 (GE Healthcare Biosciences), pTrcHis (Invitrogen), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-30 (Qiagen), pQE-60 (Qiagen), pET-3 (Novagen), pKYP10 (Japanese Patent Publication No. 58-110600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci., USA, 82, 4306 (1985)], pBluescriptII SK(+), pBluescript II KS(-) (manufactured by Stratagene), pTrS30 [prepared from Escherichia Koli JM109 / pTrS30 (FERM BP-5407)], pTrS32 [prepared from Escherichia Koli JM109 / pTrS32 (FERM BP-5408)], pPAC31 (International Publication No. 98 / 12343), pUC19 [Gene, 33, 103 (1985)], pSTV28 (manufactured by Takara Bio Inc.), pUC118 (manufactured by Takara Bio Inc.), pPA1 (Japanese Patent Publication No. 63-233798), pGHA2 [prepared from Escherichia coli IGHA2 (FERM B-400), Japanese Patent Publication No. 60-221091], pGKA2 [prepared from Escherichia coli IGKA2 (FERM BP-6798), Japanese Patent Publication No. 60-221091], pTerm2 (US Patent No. 4686191, US Patent No. 4939094, US Patent No. 5160735), pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol.Examples include pCG1 (JP-A-57-134500), pCG2 (JP-A-58-35197), pCG4 (JP-A-57-183799), pCG11 (JP-A-57-134500), pCG116, pCE54, pCB101 (both JP-A-58-105999), pRI109 (International Publication No. 00 / 044886), pCE51, pCE52, pCE53 [all from Molecular and General Genetics, 196, 175 (1984)].

[0074] Any promoter that functions in host cells such as E. coli is acceptable. Examples include promoters derived from E. coli or phages, such as the trp promoter (Ptrp), lac promoter (Plac), PL promoter, PR promoter, and PSE promoter, as well as the SpO1 promoter, SpO2 promoter, and penP promoter. Additionally, artificially designed and modified promoters such as a promoter with two Ptrps in series (Ptrp×2), the tac promoter, the lacT7 promoter, and the let I promoter are also acceptable.

[0075] It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome-binding sequence, and the start codon is adjusted to an appropriate distance (e.g., 6 to 18 bases). While a transcription termination sequence is not necessarily required for the expression of DNA in one aspect of the present invention, it is preferable to place the transcription termination sequence directly below the structural gene in recombinant DNA.

[0076] Examples of prokaryotes include microorganisms belonging to genera such as Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, and Pseudomonas.

[0077] Any method for introducing recombinant vectors into host cells can be used, including, for example, the calcium ion method [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], the protoplast method (Japanese Patent Publication No. 57-186492, Japanese Patent Publication No. 57-18649), electroporation [e.g., Journal of Bacteriology, 175, 4096 (1993), Appl. Microbiol. Biotechnol., 52, 541 (1999)], and the methods described in Gene, 17, 107 (1982) and Molecular & General Genetics, 168, 111 (1979).

[0078] When using yeast strains as host cells, expression vectors such as AOX-Dasher GFP (ATUM), YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15 can be used.

[0079] Any promoter that functions in the yeast strain can be used, such as the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock polypeptide promoter, MFα1 promoter, and CUP 1 promoter.

[0080] When using yeast strains as host cells, suitable host cells include yeast strains belonging to genera such as Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Schwanniomyces, Pichia, and Candida. Specifically, examples include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichia pastoris, and Candida utilis.

[0081] Any method for introducing recombinant DNA into yeast can be used, such as electroporation (Methods Enzymol., 194, 182 (1990)), spheroplast (Proc. Natl. Acad. Sci., USA, 81, 4889 (1984)), and lithium acetate (J. Bacteriol., 153, 163 (1983)).

[0082] The above-mentioned transformants can be cultured in a culture medium to generate and accumulate a protein according to one aspect of the present invention in the culture, and the protein can be produced by harvesting it from the culture. Methods for producing the protein according to one aspect of the present invention include producing it inside a host cell, secreting it outside a host cell, or producing it on the outer membrane of a host cell, and the structure of the protein produced can be changed depending on the selected method.

[0083] The carboxylic acid ester hydrolase or its variant used in this method may be 1) the protein itself extracted and purified from a microorganism, or 2) a processed product or extract of a transformant.

[0084] (When using proteins extracted and purified from microorganisms as carboxylic acid ester hydrolases or their variants) To isolate and purify the proteins produced by the transformants in this disclosure, conventional enzyme isolation and purification methods can be used. Culture media for the transformants are well known and can be used, for example, nutrient media such as LB medium, BMGY medium, BSM medium, or minimal media such as M9 medium, to which carbon sources, nitrogen sources, vitamin sources, etc., can be added. Depending on the host, the transformants in this disclosure are usually cultured at 16 to 42°C, preferably 25 to 37°C, for 5 to 168 hours, preferably 8 to 72 hours. Depending on the host, either shaking culture or static culture is possible, but stirring or aeration may be performed as needed. When actinomycetes are selected as the expression host, conditions that can be used to produce the protein can be used as appropriate. In addition, when an inducible promoter is used for protein expression, a promoter inducer can be added to the culture medium before cultivation.

[0085] The produced protein can be purified and isolated from the transformant extract by known precipitation methods such as salting out, isoelectric focusing, or solvent precipitation; methods utilizing molecular weight differences such as dialysis, ultrafiltration, or gel filtration; methods utilizing specific affinity such as ion exchange chromatography; methods utilizing hydrophobicity differences such as hydrophobic chromatography or reverse-phase chromatography; and other methods such as affinity chromatography, SDS polyacrylamide electrophoresis, isoelectric focusing, or a combination thereof. When the target protein is secreted and expressed, the culture supernatant containing the protein can be obtained by removing the bacterial cells from the culture medium obtained by culturing the microorganisms, etc. The protein can also be purified and isolated from this culture supernatant.

[0086] ((Processed product of transformed organisms)) In this disclosure, a transformed organism refers to a bacterial cell whose plasma membrane is permeable to substances. In this disclosure, a permeable plasma membrane means that various molecules, both small (ions, etc.) and large (proteins, etc.), can freely enter and exit the cell membrane by diffusion. In this disclosure, the transformed organism is preferably a dormant bacterial cell that has lost its ability to proliferate due to treatment to impart membrane permeability.

[0087] Examples of processed products of the transformed organism include surfactant-treated products of the transformed organism, solvent-treated products of the organism, enzyme-treated products of the organism, immobilized products of the organism that contain live cells that have the same function as the culture of the organism as an enzyme source, ultrasonically treated products of the organism, and mechanically ground products of the organism.

[0088] Methods for making the cell plasma membrane permeable to substances include, for example, chemical treatment and mechanical treatment. The timing for making the cell plasma membrane of the transformants permeable to substances is not particularly limited, as long as the effects of the present invention are achieved, the cell plasma membrane of each transformant may be made permeable to substances in advance, or it may be done when the transformants used in the reaction are brought into contact with each other and reacted.

[0089] Chemical treatments include, for example, methods using surfactants, methods using organic solvents, and methods using enzymes. As surfactants, nonionic surfactants are preferred because they have a milder effect on proteins (compared to ionic surfactants). Examples of such surfactants include digitonin, saponin, TritonX100 (hereinafter, Triton is a registered trademark), TritonX114, Tween20 (hereinafter, Tween is a registered trademark), Tween80, N,N-Bis(3-D-gluconamidopropyl)cholamide [BIGCHAP], N,N-Bis(3-D-gluconamidopropyl)deoxycholamide [Deoxy-BIGCHAP], NIKKOLBL-9EX [Polyoxyethylene(9)LaurylEther], Octanoyl-N-methylglucamide [MEGA-8], and benzalkonium chloride.

[0090] Examples of organic solvents include Benzene, Toruene, Xylene, and other alcohols. Examples of enzymes include lysozyme and achromopeptidase.

[0091] The conditions for treatment with the above-mentioned substance, such as concentration, temperature, and time, vary depending on the cell type, and appropriate conditions must be set to perform the desired analysis. However, typical treatment concentrations are 10-1000 μg / ml, more commonly 20-200 μg / ml, with temperatures of 2-37°C and treatment times of 1-30 minutes.

[0092] Examples of mechanical treatments include ultrasonic treatment and mechanical grinding.

[0093] ((Extract from the transformed organism)) Examples of the extract of the transformant in the present disclosure include, for example, a crude enzyme extract obtained from the cells of the transformant, a purified enzyme obtained from the cells treated as described above (for example, chemical treatment or mechanical treatment), a concentrate of a culture obtained by culturing the transformant or its treated product as described above, and a dried product of the culture. In addition, the extract of the transformant includes cells obtained by centrifuging or filtering the culture, a dried product of the cells, and a freeze-dried product of the cells.

[0094] <Mixed substrate containing PET> The mixed substrate containing PET in the present disclosure is not particularly limited as long as it contains PET and a substrate other than PET. The content of PET in the mixed substrate is, for example, preferably 50% by mass or more, 60% by mass or more, more preferably at least 65% by mass or more, 70% by mass or more, 75% by mass or more, 80% by mass or more, 85% by mass or more, 90% by mass or more, 95% by mass or more, based on the total mass of the mixed substrate.

[0095] The origin of PET contained in the mixed substrate is not particularly limited, but for example, it is preferably derived from at least one selected from PET fibers, PET bottles, PET films, PET flakes, and PET resin powders. From the viewpoint of more selectively decomposing PET contained in the mixed substrate, the mixed substrate is preferably one obtained by subjecting at least one selected from PET fibers, PET bottles, PET films, PET flakes, and PET resin powders to heat quenching treatment to make PET into amorphous PET. Further, for pulverization, for example, pulverization treatment, freeze pulverization treatment, etc. can also be carried out.

[0096] The average particle size of the mixed substrate in the present disclosure is preferably 50 μm to 150 μm. In the present disclosure, the "average particle size" means the particle size at the integrated value of 50% in the particle size distribution determined by the laser diffraction / scattering method.

[0097] The crystallinity of amorphous PET is preferably 0% to 25%, more preferably 0% to 16%. The form of the mixed substrate is not limited and includes, for example, fibrous, granular, flake, pellet, film, lump, and bottle forms.

[0098] Examples of mixed substrates containing PET in this disclosure include those containing PET and at least one selected from the group consisting of [s1] to [s34] below. However, the mixed substrate may contain other substrates along with PET, not limited to [s1] to [s34] below. [s1] Polyurethane (PU) [s2] Cotton [s3] Polylactic acid (PLA) [s4] Polyethylene (PE) [s5] Polyamide (PA) [s6] Polybutylene adipate terephthalate (PBAT) [s7] Polybutylene succinate (PBS) [s8] Polycaprolactone (PCL) [s9] Polystyrene (PS) [s10] Polytrimethylene terephthalate (PBT) [s11] Polytrimethylene terephthalate (PTT) [s12] Polyethylene naphthalate (PEN) [s13] Polybutylene naphthalate (PBN) [s14] Wool [s15] Linen [s16] Silk [s17] Rayon [s18] Polynosic [s19] Cupro [s20] Lyocell [s21] Promix Fiber [s22] Triacetate [s23] Polyvinyl alcohol (PVAL) [s24] Acrylic [s25] Cashmere [s26] Alpaca [s27] Angola [s28] Mohair [s29] Camel [s30] Acetate [s31] Polyvinylidene chloride [s32] Polyvinyl chloride (PVC) [s33] Polypropylene (PP) [s34] Polyclar (a copolymer of vinyl chloride and vinyl alcohol)

[0099] The mixed substrate in this disclosure preferably includes at least one of [s1] polyurethane and [s2] cotton, and PET.

[0100] In this embodiment, the mixed substrate contains PET and at least one selected from the group consisting of [s1] to [s34] below. The content of PET in the mixed substrate is not particularly limited as long as PET is included, but for example, 50 to 99% by mass is possible. In this embodiment, the PET content is preferably 50% by mass or more, more preferably 65% ​​by mass or more, even more preferably 80% by mass or more, and also preferably 99% by mass or less.

[0101] In embodiments where the mixed substrate contains PET and polyurethane, the PET content in the mixed substrate can be, for example, 50 to 99% by mass. In such embodiments, the PET content is preferably 50% by mass or more, more preferably 65% ​​by mass or more, even more preferably 85% by mass or more, and preferably 99% by mass or less.

[0102] In embodiments in which the mixed substrate contains PET and cotton, the PET content in the mixed substrate can be, for example, 50 to 99% by mass. In such embodiments, the PET content is preferably 50% by mass or more, more preferably 60% by mass or more, even more preferably 65% ​​by mass or more, and preferably 99% by mass or less.

[0103] In embodiments in which the mixed substrate contains PET and at least one selected from the group consisting of [s1] to [s34] below, the ratio (mass ratio) of the content of PET to the non-PET substrate in the mixed substrate is not particularly limited, but for example, it can be 1:1 to 99:1, preferably 13:7 to 99:1, and particularly preferably 4:1 to 99:1.

[0104] In embodiments in which PET and polyurethane are included in the mixed substrate, the ratio (mass ratio) of PET to polyurethane is not particularly limited, but examples include 1:1 to 99:1, preferably 13:7 to 99:1, and particularly preferably 17:3 to 99:1.

[0105] In embodiments in which the mixed substrate contains PET and cotton, the ratio (mass ratio) of PET to cotton is not particularly limited, but examples include 1:1 to 99:1, preferably 3:2 to 99:1, and particularly preferably 13:7 to 99:1.

[0106] (Decomposition of PET) The reaction conditions in step (1) of this method, in which an enzyme composition containing a carboxylic acid ester hydrolase or a variant thereof is reacted with the mixed substrate, are not particularly limited as long as a hydrolysis reaction by the carboxylic acid ester hydrolase or a variant thereof occurs. In one embodiment, step (1) is preferably performed by decomposing PET in a reaction solution containing sodium carbonate, but is not limited thereto. One specific embodiment is a method in which PET is decomposed in a reaction solution containing the enzyme composition, the mixed substrate, and sodium carbonate.

[0107] The concentration of sodium carbonate in the reaction solution when decomposing PET is preferably 0.05 to 1 M, and more preferably 0.1 to 0.3 M. Sodium carbonate is an alkaline substance and can raise the pH of the aqueous solution. When decomposing PET, raising the pH to the alkaline side has the advantage of promoting the alkaline hydrolysis reaction of the ester bonds of PET.

[0108] In one embodiment, the concentration of carboxylic acid ester hydrolase or its variant in the reaction solution of step (1) is preferably 1 to 100 μg / mL, more preferably 5 to 20 μg / mL, from the viewpoint of efficiency of PET degradation. However, the concentration is not limited to this, and can be appropriately set depending on the type and state of the mixed substrate to be degraded, the amount of PET contained in the mixed substrate, etc.

[0109] The reaction temperature in step (1) is preferably 40 to 70°C, more preferably 55 to 70°C, and even more preferably 55 to 65°C, from the viewpoint of the heat resistance or PET degradation activity of the carboxylic acid ester hydrolase or its variant.

[0110] The pH during the reaction in step (1) is preferably 6 to 11, more preferably 7.5 to 9.5, and even more preferably 8.0 to 9.5, from the viewpoint of the PET degradation activity of the carboxylic acid ester hydrolase or its variant.

[0111] The reaction time in step (1) can be appropriately set depending on the amount of PET contained in the mixed substrate to be decomposed, and is preferably 1 to 48 hours. If processing is to be performed for a long time, the enzyme composition may be added periodically.

[0112] According to this method, PET contained in a mixed substrate containing PET can be selectively decomposed. In one embodiment of this method, in the reaction solution after step (1) in which the enzyme composition is reacted with the mixed substrate, the content of decomposition products of substances other than PET contained in the mixed substrate is preferably, for example, 0.001 to 1.000 mM, more preferably 0.001 to 0.700 mM, and even more preferably 0.001 to 0.400 mM. Also, for example, after step (1), the decomposition rate of PET is preferably 0.5% or more, more preferably 10% or more, and even more preferably 20% or more.

[0113] In one embodiment of this method, specifically, for example, when the mixed substrate containing PET is a mixed substrate containing PET and PU, the content of 4,4′-diaminodiphenylmethane, which is a degradation product of PU, in the reaction solution after step (1) of reacting the enzyme composition with the mixed substrate is preferably, for example, 0.001 to 0.100 mM, and more preferably 0.001 to 0.030 mM. In this embodiment, the PET degradation rate after step (1) is preferably 0.5% or more, more preferably 10% or more, and even more preferably 20% or more. The PET degradation rate can be determined by the method described later in the examples.

[0114] In one embodiment of this method, when the mixed substrate containing PET is a mixed substrate containing PET and cotton, the content of glucose, which is a decomposition product of cotton, in the reaction solution after step (1) of reacting the enzyme composition with the mixed substrate is preferably, for example, 0.010 to 1.000 mM, and more preferably 0.010 to 0.400 mM. In this embodiment, the PET decomposition rate after step (1) is preferably 20% or more, more preferably 50% or more, and even more preferably 80% or more.

[0115] In one embodiment of the method of the present disclosure, specific examples of combinations of carboxylic acid ester hydrolase or a variant thereof with a mixed substrate include the following: x1) A carboxylic acid ester hydrolase or its variant is a lipase variant, The mixed substrate is a mixed substrate containing PET, polyurethane, and / or cotton. x2) The carboxylic acid ester hydrolase or its variant is cutinase or its variant, and the cutinase is excluding HiC, The mixed substrate is a mixed substrate containing PET, polyurethane, and at least one of cotton. x3) ​​Carboxylate ester hydrolase or its variant is cutinase or its variant, The mixed substrate contains PET and a non-PET substrate, and the non-PET substrate excludes cotton.

[0116] x4) At least one carboxylic acid ester hydrolase or its variant is selected from PET2 variants and LC-cutinase or its variants, The mixed substrate is a mixed substrate containing PET. x5) At least one carboxylic acid ester hydrolase or its variant is selected from PET2 variants and LC-cutinase or its variants. The mixed substrate is a mixed substrate containing PET, polyurethane, and / or cotton.

[0117] x6) A carboxylic acid ester hydrolase or a variant thereof is a variant of PET2, and the variant of PET2 is a protein having an amino acid sequence in which at least one modification selected from the group consisting of [1] to [7] and at least one modification selected from the group consisting of [8] to

[24] have been introduced in the amino acid sequence represented by Sequence ID No. 1 (excluding proteins in which at least one combination of modifications selected from the group consisting of the combination of [8] and

[16] , the combination of

[11] and

[19] , and the combination of

[17] and

[23] has been introduced), The mixed substrate is a mixed substrate containing PET. [1] Modification by substituting the 47th amino acid residue with a cysteine ​​residue. [2] Modification by substituting the 89th amino acid residue with a cysteine ​​residue. [3] Modification by substituting the 105th amino acid residue with an arginine residue. [4] Modification by substituting the 110th amino acid residue with a lysine residue. [5] Modification by substituting the 156th amino acid residue with a proline residue. [6] Modification by substituting the 180th amino acid residue with an alanine residue. [7] Modification by substituting the 297th amino acid residue with a proline residue. [8] Modification by substituting the 73rd amino acid residue with a glutamine residue. [9] Modification by substituting the 76th amino acid residue with a cysteine ​​residue.

[10] Modification by substituting the 103rd amino acid residue with a lysine residue.

[11] Modification by substituting the 134th amino acid residue with a glycine residue.

[12] Modification by substituting the 144th amino acid residue with a cysteine ​​residue.

[13] Modification by substituting the 203rd amino acid residue with a threonine residue.

[14] Modification by substituting the 222nd amino acid residue with a methionine residue.

[15] Modification by substituting the 55th amino acid residue with a serine residue.

[16] Modification by substituting the 73rd amino acid residue with a serine residue

[17] Modification by substituting the 228th amino acid residue with a lysine residue.

[18] Modification by substituting the 264th amino acid residue with an arginine residue.

[19] Modifications by substituting the 134th amino acid residue with a lysine residue, a methionine residue, or a threonine residue.

[20] Modification by substituting the 136th amino acid residue with a glutamic acid residue.

[21] Modifications by substituting the 200th amino acid residue with a histidine residue, an aspartic acid residue, or an alanine residue.

[22] Modifications by substituting the 227th amino acid residue with an arginine residue, a lysine residue, a glutamic acid residue, or a histidine residue.

[23] Modifications by substituting the 228th amino acid residue with a glutamic acid residue, histidine residue, lysine residue, glutamine residue, or tryptophan residue.

[24] Modifications by substituting the 229th amino acid residue with a tyrosine residue, an asparagine residue, or a phenylalanine residue. x7) A carboxylic acid ester hydrolase or a variant thereof is a variant of PET2, and the variant of PET2 is a protein in which the amino acid sequence represented by Sequence ID No. 1 is modified by the modifications described in [1] to [7] and the modifications described in

[19] to

[24] , The mixed substrate is a mixed substrate containing PET. x8) A carboxylic acid ester hydrolase or a variant thereof is a variant of PET2, and the variant of PET2 is a protein in which the modifications described in [1] to [7] and the modifications described in [8] to

[14] are introduced into the amino acid sequence represented by Sequence ID No. 1. The mixed substrate is a mixed substrate containing PET. x9) A carboxylic acid ester hydrolase or a variant thereof is a variant of PET2, and the variant of PET2 is a protein in which the amino acid sequence represented by Sequence ID No. 1 has been modified by the modifications described in [1] to [7] and the modifications described in [8] to

[10] and

[12] to

[14] , The mixed substrate is a mixed substrate containing PET. x10) A carboxylic acid ester hydrolase or a variant thereof is a variant of PET2, and the variant of PET2 is a protein in which at least one modification selected from the group consisting of

[15] to

[18] is introduced in the amino acid sequence represented by Sequence ID No. 1. The mixed substrate is a mixed substrate containing PET. x11) A carboxylic acid ester hydrolase or a variant thereof is a variant of PET2, and the variant of PET2 is a protein in which at least one modification selected from the group consisting of

[19] to

[24] is introduced into the amino acid sequence represented by Sequence ID No. 1. The mixed substrate is a mixed substrate containing PET. x12) A carboxylic acid ester hydrolase or a variant thereof is a variant of PET2, and the variant of PET2 is a protein in which the modifications of

[15] and

[18] are further introduced in the amino acid sequence represented by Sequence ID No. 1. The mixed substrate is a mixed substrate containing PET. x13) A carboxylic acid ester hydrolase or a variant thereof is a variant of PET2, and the variant of PET2 is a protein having an amino acid sequence in which at least one modification selected from the group consisting of

[15] to

[24] has been introduced in the amino acid sequence represented by Sequence ID No. 1 (excluding proteins having a modification in which the combination of

[17] and

[23] has been introduced), The mixed substrate is a mixed substrate containing PET. x14) A carboxylic acid ester hydrolase or a variant thereof is a variant of PET2, and the variant of PET2 is a protein consisting of an amino acid sequence in which the following modifications are introduced in the amino acid sequence represented by Sequence ID No. 1: modifications of [1] to [7], modifications of [8] to

[10] and

[12] to

[14] , and modifications of

[19] to

[24] . The mixed substrate is a mixed substrate containing PET.

[0118] x15) A carboxylic acid ester hydrolase or a variant thereof is a variant of PET2, and the variant of PET2 is a protein having an amino acid sequence in which at least one modification selected from the group consisting of [1] to [7] and at least one modification selected from the group consisting of [8] to

[24] has been introduced in the amino acid sequence represented by Sequence ID No. 1 (excluding proteins having at least one combination of modifications selected from the group consisting of the combination of [8] and

[16] , the combination of

[11] and

[19] , and the combination of

[17] and

[23] ), The mixed substrate is a mixed substrate containing PET, polyurethane, and / or cotton. x16) A carboxylic acid ester hydrolase or a variant thereof is a variant of PET2, and the variant of PET2 is a protein in which the amino acid sequence represented by Sequence ID No. 1 is modified by the modifications described in [1] to [7] and the modifications described in

[19] to

[24] , The mixed substrate is a mixed substrate containing PET, polyurethane, and / or cotton. x17) A carboxylic acid ester hydrolase or a variant thereof is a variant of PET2, and the variant of PET2 is a protein in which the modifications described in [1] to [7] and the modifications described in [8] to

[14] are introduced into the amino acid sequence represented by Sequence ID No. 1. The mixed substrate is a mixed substrate containing PET, polyurethane, and / or cotton. x18) A carboxylic acid ester hydrolase or a variant thereof is a variant of PET2, and the variant of PET2 is a protein in which the amino acid sequence represented by Sequence ID No. 1 has been modified by the modifications described in [1] to [7] and the modifications described in [8] to

[10] and

[12] to

[14] , The mixed substrate is a mixed substrate containing PET, polyurethane, and / or cotton. x19) A carboxylic acid ester hydrolase or a variant thereof is a variant of PET2, and the variant of PET2 is a protein in which at least one modification selected from the group consisting of

[15] to

[18] is introduced in the amino acid sequence represented by Sequence ID No. 1. The mixed substrate is a mixed substrate containing PET, polyurethane, and / or cotton. x20) A carboxylic acid ester hydrolase or a variant thereof is a variant of PET2, and the variant of PET2 is a protein in which at least one modification selected from the group consisting of

[19] to

[24] is introduced into the amino acid sequence represented by Sequence ID No. 1. The mixed substrate is a mixed substrate containing PET, polyurethane, and / or cotton. x21) A carboxylic acid ester hydrolase or a variant thereof is a variant of PET2, and the variant of PET2 is a protein in which the modifications of

[15] and

[18] are further introduced in the amino acid sequence represented by Sequence ID No. 1. The mixed substrate is a mixed substrate containing PET, polyurethane, and / or cotton. x22) A carboxylic acid ester hydrolase or a variant thereof is a variant of PET2, and the variant of PET2 is a protein having an amino acid sequence in which at least one modification selected from the group consisting of

[15] to

[24] has been introduced in the amino acid sequence represented by Sequence ID No. 1 (excluding proteins having a modification of the combination of

[17] and

[23] introduced), The mixed substrate is a mixed substrate containing PET, polyurethane, and / or cotton. x23) A carboxylic acid ester hydrolase or a variant thereof is a variant of PET2, and the variant of PET2 is a protein consisting of an amino acid sequence in which the following modifications are introduced in the amino acid sequence represented by Sequence ID No. 1: modifications of [1] to [7], modifications of [8] to

[10] and

[12] to

[14] , and modifications of

[19] to

[24] . The mixed substrate is a mixed substrate containing PET, polyurethane, and / or cotton.

[0119] x24) The carboxylic acid ester hydrolase or its variant is LC-cutinase or its variant, and the LC-cutinase or its variant is at least one selected from the following [C1] to [C3], The mixed substrate is a mixed substrate containing PET. [C1] A protein consisting of the amino acid sequence represented by sequence number 3 or 7. [C2] A protein having an amino acid sequence in which, preferably, 1 to 20 amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by Sequence ID No. 3 or 7, and having a PET degradation rate of 0.5% or more in a mixed substrate containing PET. [C3] A protein having an amino acid sequence that preferably has 80% or more identity with the amino acid sequence represented by SEQ ID NO: 3 or 7, and having a PET degradation rate of 0.5% or more in a mixed substrate containing PET. Of the LC-cutinase or its variants, [C1] is preferred among [C1] to [C3]. x25) The carboxylic acid ester hydrolase or its variant is LC-cutinase or its variant, and the LC-cutinase or its variant is at least one selected from [C1] to [C3], The mixed substrate is a mixed substrate containing PET, polyurethane, and / or cotton. Of the LC-cutinase or its variants, [C1] is preferred among [C1] to [C3].

[0120] x26) The carboxylic acid ester hydrolase or its variant is a variant of PET2, and the variant of PET2 is at least one selected from [C4] to [C6], The mixed substrate is a mixed substrate containing PET. [C4] A protein consisting of an amino acid sequence represented by sequence number 5 or 17. [C5] A protein body comprising an amino acid sequence in which, preferably, 1 to 20 amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by Sequence ID No. 5 or 17, and in which the PET degradation rate in a mixed substrate containing PET is 0.5% or more. A protein consisting of an amino acid sequence having preferably 80% or more identity with the amino acid sequence represented by SEQ ID NO: 5 or 17, and having a PET degradation rate of 0.5% or more in a mixed substrate containing PET. As a variant of PET2, among [C4] to [C6], [C4] is preferred. x27) The carboxylic acid ester hydrolase or a variant thereof is at least one of the following (E3) and (E4), The mixed substrate is a mixed substrate containing PET. (E3) A protein consisting of the amino acid sequence represented by SEQ ID NO: 5 or 17 (E4) A protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 7

[0121] Regarding x1) to x27), examples of the content of PET contained in the mixed substrate include 50 to 99% by mass, preferably 65 to 99% by mass, and particularly preferably 80 to 99% by mass.

[0122] <Method for producing at least one of TPA and MHET> As one embodiment of the present disclosure, there is provided a method for producing at least one of TPA and MHET (hereinafter also abbreviated as the present production method) including the method for decomposing PET of the present disclosure described above. The carboxylic acid ester hydrolase or a variant thereof in the present disclosure can decompose the main chain of PET and decompose bis(2-hydroxyethyl) terephthalate (hereinafter also referred to as "BHET"), an intermediate product of PET decomposition, into MHET, and further decompose MHET into TPA and monoethylene glycol (hereinafter also referred to as "MEG").

[0123] That is, the carboxylic acid ester hydrolase or a variant thereof in one embodiment in the present disclosure can hydrolyze PET or BHET, which is a partial structure of PET, as a substrate to generate MHET, and further generate TPA and MEG.

[0124] The concentration of the carboxylic acid ester hydrolase or its variant in the reaction solution in one embodiment of this manufacturing method, the concentration of sodium carbonate in the reaction solution in step (1) of reacting the enzymatically degraded composition with a mixed substrate containing PET, the reaction temperature, the pH during the reaction, the reaction time, and the form of PET contained in the mixed substrate to be degraded are as described above (Degradation of PET).

[0125] As described above, the following configurations are disclosed in this specification. 1. A method for decomposing polyethylene terephthalate (PET) in a mixed substrate, comprising the following step (1). (1) A step of reacting an enzyme composition with a mixed substrate containing PET, The enzyme composition comprises a carboxylic acid ester hydrolase or a variant thereof, The step is that the carboxylic acid ester hydrolase or its variant is at least one selected from carboxylesterase, allylsterase, cutinase, and PETase, as well as variants thereof and lipase. 2. The method according to claim 1, wherein the lipase variant is a variant of PET2. 3. The method according to 2, wherein the variant of PET2 is a protein comprising an amino acid sequence in which at least one modification selected from the group consisting of [1] to [7] and at least one modification selected from the group consisting of [8] to

[24] are introduced in the amino acid sequence represented by Sequence ID No. 1 (excluding proteins in which at least one combination of modifications selected from the group consisting of the combination of [8] and

[16] , the combination of

[11] and

[19] , and the combination of

[17] and

[23] are introduced). [1] Modification by substituting the 47th amino acid residue with a cysteine ​​residue. [2] Modification by substituting the 89th amino acid residue with a cysteine ​​residue. [3] Modification by substituting the 105th amino acid residue with an arginine residue. [4] Modification by substituting the 110th amino acid residue with a lysine residue. [5] Modification by substituting the 156th amino acid residue with a proline residue. [6] Modification by substituting the 180th amino acid residue with an alanine residue. [7] Modification by substituting the 297th amino acid residue with a proline residue. [8] Modification by substituting the 73rd amino acid residue with a glutamine residue. [9] Modification by substituting the 76th amino acid residue with a cysteine ​​residue.

[10] Modification by substituting the 103rd amino acid residue with a lysine residue.

[11] Modification by substituting the 134th amino acid residue with a glycine residue.

[12] Modification by substituting the 144th amino acid residue with a cysteine ​​residue.

[13] Modification by substituting the 203rd amino acid residue with a threonine residue.

[14] Modification by substituting the 222nd amino acid residue with a methionine residue.

[15] Modification by substituting the 55th amino acid residue with a serine residue.

[16] Modification by substituting the 73rd amino acid residue with a serine residue

[17] Modification by substituting the 228th amino acid residue with a lysine residue.

[18] Modification by substituting the 264th amino acid residue with an arginine residue.

[19] Modifications by substituting the 134th amino acid residue with a lysine residue, a methionine residue, or a threonine residue.

[20] Modification by substituting the 136th amino acid residue with a glutamic acid residue.

[21] Modifications by substituting the 200th amino acid residue with a histidine residue, an aspartic acid residue, or an alanine residue.

[22] Modifications by substituting the 227th amino acid residue with an arginine residue, a lysine residue, a glutamic acid residue, or a histidine residue.

[23] Modifications by substituting the 228th amino acid residue with a glutamic acid residue, histidine residue, lysine residue, glutamine residue, or tryptophan residue.

[24] Modifications by substituting the 229th amino acid residue with a tyrosine residue, an asparagine residue, or a phenylalanine residue. 4. The method according to 2 or 3, wherein the variant of PET2 is at least one selected from [C4] to [C6]. [C4]A protein consisting of the amino acid sequence represented by SEQ ID NO: 5 or 17. [C5]A protein consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 5 or 17, and having a PET degradation rate of 0.5% or more in a mixed substrate containing PET. [C6]A protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 5 or 17, and having a PET degradation rate of 0.5% or more in a mixed substrate containing PET. 5. The method according to any one of 1 to 4 above, wherein the cutinase or its variant is LC-cutinase or its variant. 6. The method according to 5 above, wherein the LC-cutinase or its variant is at least one selected from the following [C1] to [C3]. [C1]A protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 7. [C2]A protein consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 3 or 7, and having a PET degradation rate of 0.5% or more in a mixed substrate containing PET. [C3]A protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 3 or 7, and having a PET degradation rate of 0.5% or more in a mixed substrate containing PET. 7. The method according to any one of 1 to 6 above, wherein the carboxylic acid ester hydrolase or its variant is at least one of the following (E3) and (E4). (E3) A protein consisting of the amino acid sequence represented by SEQ ID NO: 5 or 17 (E4) A protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 7 8. The method according to any one of 1 to 7 above, wherein the mixed substrate contains at least one of polyurethane and cotton and PET. 9. The method according to any one of 1 to 8 above, wherein the PET contained in the mixed substrate is derived from at least one selected from PET fiber, PET bottle, PET film, PET flake and PET resin powder. 10. The method according to any one of 1 to 9, wherein step (1) is carried out in the presence of sodium carbonate. 11. The method according to any one of 1 to 10, wherein step (1) is performed at a temperature of 40 to 70°C. 12. A method for producing at least one of terephthalic acid (TPA) and monohydroxyethyl terephthalate (MHET) using the method described in any one of 1 to 11 above. 13. The method according to any one of 1 to 11, wherein the mixed substrate comprises polyurethane and PET. 14. The method according to 13, wherein the PET content in the mixed substrate is 50 to 99% by mass. 15. The method according to any one of 1 to 11, wherein the mixed substrate comprises cotton and PET. 16. The method according to 15, wherein the PET content in the mixed substrate is 50 to 99% by mass. 17. A method for producing monoethylene glycol (MEG) using the method described in any one of the above 1 to 11.

[0126] [Analysis Example 1] In the examples, the analysis and quantification of TPA and MHET were performed according to the procedure of Analytical Condition 1 shown below. The solution after the enzymatic reaction was centrifuged, the supernatant was collected, and filtered through a 0.2 μm filter. The TPA and MHET contained in the supernatant were analyzed by HPLC (Shimadzu Corporation) and quantified using standard samples. Standard data for TPA can be obtained from, for example, product number 208-08162 (Fujifilm Wako Pure Chemical Corporation), and standard data for MHET can be obtained from, for example, product number AC-2862 (Amatek Chemical).

[0127] (Analysis conditions 1) Column: Discovery® HS C18 15cm x 4.6mm 5μm (manufactured by Discovery Inc.) Column temperature: 25℃ Mobile phase: 0.1% formic acid, 20% acetonitrile (v / v) Flow rate: 0.8mL / min Detection wavelength: 240nm Analysis time: 20min

[0128] [Analysis Example 2] In the examples, the analysis and quantification of 4,4′-diaminodiphenylmethane, a PU degradation product, was performed according to the procedure of analytical condition 2 shown below. The solution after the enzymatic reaction was centrifuged, and the supernatant was collected and filtered through a 0.2 μm filter. 4,4'-diaminodiphenylmethane contained in the supernatant was analyzed by HPLC (Shimadzu Corporation) and quantified using a standard sample. The standard sample can be obtained, for example, from product number M0220 (Tokyo Chemical Industries).

[0129] (Analysis conditions 2) Column: Discovery® HS C18 15cm x 4.6mm 5μm (manufactured by Discovery Inc.) Column temperature: 40℃ Mobile phase: A: Water, B: Methanol Flow rate: 0.8mL / min Detection wavelength: 240nm Analysis time: 20min Gradient conditions: 0.0 to 2.0 minutes, A:B = 70:30 2.0~8.0 minutes A:70→5,B:30→95 8.0~12.0 minutes A:B=5:95 12.0~20.0 minutes A:B=70:30

[0130] [Analysis Example 3] In the example, the analysis and quantification of glucose, a decomposition product of cotton, was performed according to the procedure of analytical condition 3 shown below. The solution after the enzymatic reaction was centrifuged, the supernatant was collected, and filtered through a 0.2 μm filter. The glucose contained in the supernatant was analyzed using a glucose analyzer (manufactured by A&T).

[0131] (Analysis conditions 3) Measure a glucose standard sample [glucose BP standard solution, product number: 223054 (A&T), etc.] and a buffer solution, connect these two points with a straight line to create a calibration curve, and calculate the concentration of glucose in the sample based on the calibration curve. Calibration curve concentration range: 0 - 200 mg / dL

Example

[0132] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples as long as the gist thereof is not exceeded.

[0133] [Example 1] Comparison of PET degradation ability in a mixed substrate of wild-type PET2 and wild-type LCC (LCC is also called LC-chitinase) (1) Construction of wild-type PET2 expression plasmid Using DNA (SEQ ID NO: 2) consisting of the base sequence of the gene encoding the amino acid sequence of PET2 (SEQ ID NO: 1) as a template, perform PCR using DNA consisting of the base sequences represented by SEQ ID NOs: 9 and 10 as a primer set to obtain a DNA fragment of wild-type PET2. The DNA consisting of the base sequence represented by SEQ ID NO: 2 was prepared by artificial synthesis.

[0134] The DNA fragment of wild-type PET2 obtained above and the expression vector pET28a (manufactured by Sigma-Aldrich) were ligated using the In-Fusion HD Cloning Kit (manufactured by Takara Bio Inc.) to obtain the wild-type PET2 expression plasmid pET28a-PET2.

[0135] (2) Construction of wild-type LCC expression plasmid Using DNA (SEQ ID NO: 4) consisting of the base sequence of the gene encoding the amino acid sequence (SEQ ID NO: 3) as a template, perform PCR using DNA consisting of the base sequences represented by SEQ ID NOs: 11 and 12 as a primer set to obtain a DNA fragment of wild-type LCC. The DNA consisting of the base sequence represented by SEQ ID NO: 4 was prepared by artificial synthesis.

[0136] The wild-type LCC DNA fragment obtained above and the expression vector pET28a (Sigma-Aldrich) were ligated using the In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain the wild-type LCC expression plasmid pET28a-LCC.

[0137] (3) Creation of microorganisms possessing wild-type PET2 and wild-type LCC expression plasmids The wild-type PET2 expression plasmid pET28a-PET2 and the wild-type LCC expression plasmid pET28a-LCC, created in (1) and (2), were transformed into SHuffle T7 Express Component petent E. coli to obtain wild-type PET2 and wild-type LCC strains, respectively.

[0138] (4) Obtaining wild-type PET2 and wild-type LCC purified enzymes The transformants obtained in (3) were cultured on an LB plate at 30°C for 24 hours, then inoculated into a large test tube containing 3 mL of LB medium with 100 mg / L kanamycin and cultured with shaking at 30°C for 19 hours. Subsequently, 3.0 mL of the resulting culture was inoculated into a flask containing 300 mL of LB medium with 100 mg / L kanamycin and cultured with shaking at 37°C for 2 hours. IPTG was added to a final concentration of 0.5 mM, and the culture was further cultured with shaking at 17°C for 22 hours. After the culture was complete, the culture was centrifuged to remove the supernatant and the bacterial cells were collected.

[0139] Crude protein extract was obtained from the obtained bacterial cells by a standard method, and wild-type PET2 and wild-type LCC enzyme solutions were recovered from the extract using TALON® Metal Affinity Resin (manufactured by Takara Bio Inc.). The enzyme solutions were diluted with PBS (pH 7.4), concentrated and desalted using Amicon® Ultra- (manufactured by Merck Millipore), to obtain purified wild-type PET2 enzyme and purified wild-type LCC enzyme.

[0140] (5) Evaluation of PET degradation activity in mixed substrates The PET degradation activity of wild-type PET2 purified enzyme and wild-type LCC purified enzyme in a mixed substrate was measured by the following enzymatic reaction. A 0.5 mL reaction solution was prepared consisting of 25 μg of each purified enzyme, 100 mM sodium carbonate buffer (pH 9.2), and 0.01 g of mixed substrate PET powder prepared according to the following procedure. The enzymatic reaction was carried out at 50°C, 60°C, or 70°C for 24 hours.

[0141] The mixed substrate PET powder was prepared as follows: PET / cotton blended fibers with a PET content of 65% by mass (PET:cotton = 65:35, mass ratio) and PET / PU blended fibers with a PET content of 85% by mass (PET:PU = 85:15, mass ratio) were ground and prepared. Next, the ground PET / cotton blended fibers and PET / PU blended fibers were supplied to a twin-screw extruder equipped with a strand-cut (SC) granulation system. The PET flakes were melted at a barrel temperature of 280°C, and the molten resin discharged from the die was cooled and solidified in air to form strands (filamentous PET). Pelletization was then performed using a strand cutter to obtain molded articles mixed with PET.

[0142] The molded body containing the obtained PET was cut at an arbitrary position and pulverized using a freeze-grinding or shear-impact grinding device to obtain the mixed substrate PET powder used in this example. The prepared mixed substrate PET is shown in Table 1. In the table, "SC" indicates a sample prepared using a twin-screw extruder with a strand-cut (SC) granulation device.

[0143] In the table, "particle size" refers to the average particle diameter, which is the particle size at 50% of the cumulative value in the particle size distribution determined by laser diffraction and scattering. In the table, "PET / cotton" refers to a mixed substrate containing PET and cotton, and "PET / PU" refers to a mixed substrate containing PET and polyurethane.

[0144] [Table 1]

[0145] After the reaction was complete, TPA and MHET produced by PET degradation were analyzed by HPLC. Table 2 shows the amount (mM) of TPA and MHET produced by the enzymatic reaction of wild-type PET2 purified enzyme and wild-type LCC purified enzyme, as well as their total value (mM) (degradation amount) and degradation rate (%).

[0146] The PET degradation rate was calculated using the following formula. PET decomposition rate = (TPA concentration (mM) + MHET concentration (mM)) / theoretical TPA concentration (mM) × 100(%)...Equation (1) In equation (1), the following applies: TPA concentration (mM): Quantitative value analyzed by HPLC MHET concentration (mM): Quantitative value analyzed by HPLC Theoretical TPA concentration (mM): TPA concentration assuming that the PET used as a substrate is 100% decomposed.

[0147] [Table 2]

[0148] As shown in Table 2, wild-type LCCs were demonstrated to degrade PET in mixed substrates with excellent degradation rates.

[0149] [Example 2] Comparison of PET resolution of mutant PET2 and mutant LCC in mixed substrates (1) Creation of mutant PET2 expression plasmids Using DNA (SEQ ID NO: 6) consisting of the base sequence of the gene encoding the amino acid sequence (SEQ ID NO: 5) as a template, PCR was performed using DNA consisting of the base sequences represented by SEQ ID NOs: 13 and 14 as a primer set to obtain a DNA fragment of PET2-K7S7 Hotlike. The DNA represented by SEQ ID NO: 6 was prepared by artificial synthesis.

[0150] By ligating the mutant PET2 DNA fragment obtained above with the expression vector AOX-Dasher GFP (ATUM Corporation) using the In-Fusion HD Cloning Kit (Takara Bio Inc.), the amino acid sequence of PET2 represented by SEQ ID NO: 1 is modified to include L73Q (substitution of the 73rd L-leucine residue with an L-glutamine residue), G76C (substitution of the 76th L-glycine residue with an L-cysteine ​​residue), L103K (substitution of the 103rd L-leucine residue with an L-lysine residue), A144C (substitution of the 144th L-alanine residue with an L-cysteine ​​residue), and I203T (substitution of the 203rd L-isoleucine residue with an L-glutamine residue). (substitute L-threonine residue), A222M (substitute L-alanine residue at position 222 with L-methionine residue), R47C (substitute L-arginine residue at position 47 with L-cysteine ​​residue), G89C (substitute L-glycine residue at position 89 with L-cysteine ​​residue), F105R (substitute L-phenylalanine residue at position 105 with L-arginine residue), E110K (substitute L-glutamic acid residue at position 110 with L-lysine residue), S156P (substitute L-serine residue at position 156) (Residues are replaced with L-proline residues), G180A (180th L-glycine residue replaced with an L-alanine residue), T297P (297th L-threonine residue replaced with an L-proline residue), Q134K (134th L-glutamine residue replaced with an L-lysine residue), S136E (136th L-serine residue replaced with an L-glutamic acid residue), N200H (200th L-asparagine residue replaced with an L-histidine residue), A227K (227th L-alanine residue replaced with an L-proline residue) The following mutations were added: L228E (substitution of the 228th L-leucine residue with an L-lysine residue), and H229Y (substitution of the 229th L-histidine residue with an L-tyrosine residue). In addition, the N-terminal signal peptide was deleted for stable expression in Pichia cells, resulting in the expression plasmid AOX-Dasher-PET2-K7S7Hotlike, a mutant PET2 called PET2-K7S7-Hotlike (N-terminal deletion type).Furthermore, the DNA consisting of the amino acid sequence of SEQ ID NO: 5 and the base sequence of the gene encoding it (SEQ ID NO: 6) is a sequence obtained by deleting the N-terminal signal peptide from the DNA consisting of the amino acid sequence of SEQ ID NO: 17 and the base sequence of the gene encoding it (SEQ ID NO: 18), but both include the modification described in (A12) above.

[0151] (2) Creation of mutant LCC (LCC-ICCG) expression plasmids Next, using DNA (SEQ ID NO: 8) consisting of the base sequence of the gene encoding the amino acid sequence (SEQ ID NO: 7) as a template, PCR was performed using DNA consisting of the base sequences represented by SEQ ID NOs: 15 and 16 as a primer set to obtain the LCC-ICCG DNA fragment. The DNA represented by SEQ ID NO: 8 was prepared by artificial synthesis.

[0152] By ligating the mutant LCC DNA fragment obtained above with the expression vector AOX-Dasher GFP (ATUM) using the In-Fusion HD Cloning Kit (Takara Bio Inc.), an expression plasmid AOX-Dasher-LCC-ICCG was obtained, which is a mutant LCC in which the amino acid sequence of the LCC shown in Sequence ID No. 3 has been modified with the following mutations: Y102G (substitution of the 102nd L-tyrosine residue with an L-glycine residue), D213C (substitution of the 213th L-aspartic acid residue with an L-cysteine ​​residue), F218I (substitution of the 218th L-phenylalanic acid residue with an L-isoleucine residue), and S258C (substitution of the 258th L-serine residue with an L-cysteine ​​residue).

[0153] (3) Creation of microorganisms possessing mutant PET2 and mutant LCC expression plasmids The mutant PET2 expression plasmid AOX-Dasher-PET2-K7S7Hotlike and the mutant LCC expression plasmid AOX-Dasher-LCC-ICCG, created in (1) and (2), were used to transform the host cell Pichia pastoris aox1Δ (MutS) (PPS-9011), respectively, to obtain mutant PET2 and mutant LCC strains.

[0154] (4) Obtaining mutant PET2 and mutant LCC purified enzyme The transformants obtained in (3) were cultured on a YPD plate at 30°C for 48 hours, then inoculated into a flask containing 300 mL of BMGY medium and cultured with shaking at 30°C for 20 hours. Subsequently, 35 mL of the resulting culture solution was inoculated into a jar fermenter containing 350 mL of BSM medium, and cultured with aeration and stirring at 30°C and pH 5.0, ensuring that the dissolved oxygen (Dissolved Oxygen, also called DO) did not fall below 20%. Glycerol feeding was performed when the glycerol in the BSM medium was completely consumed and the DO level rose. Glycerol feeding was stopped when the cell OD660 value reached approximately 200, and methanol feeding was performed again when the DO level rose. Culture with aeration and stirring under methanol feed was performed for 40 hours.

[0155] After culturing was complete, the culture medium was centrifuged and the supernatant was collected. The obtained supernatant was concentrated using Amicon® Ultra- (Merck Millipore). The concentrated mutant PET2 solution was buffer-changed with 20 mM sodium acetate buffer (pH 5.0), purified using SP Sepharose Fast Flow (Cytiva), and the enzyme solution was collected. The collected enzyme solution was concentrated using Amicon Ultra-, purified using Superdex200 Increase (Cytiva), and the enzyme solution was collected. The collected enzyme solution was concentrated using Amicon Ultra-, buffer-changed with PBS, and purified mutant PET2 enzyme was obtained.

[0156] Next, the concentrated mutant LCC solution was buffer-exchanged with 10 mM Tris-HCl buffer (pH 7.5), purified using SP Sepharose HP (Cytiva), and the enzyme solution was recovered. The recovered enzyme solution was concentrated using Amicon Ultra-, purified using Superdex200 Increase (Cytiva), and the enzyme solution was recovered. The recovered enzyme solution was concentrated using Amicon Ultra-, buffer-exchanged with PBS, and the purified mutant LCC enzyme was obtained.

[0157] (5) Evaluation of PET degradation activity in mixed substrates The PET degradation activity of mutant PET2 purified enzyme and mutant LCC purified enzyme in a mixed substrate was measured by the following enzymatic reaction. A 0.5 mL reaction solution was prepared consisting of 5 μg or 25 μg of each purified enzyme, 100 mM sodium carbonate buffer (pH 9.2), and 0.01 g of PET powder from Table 1. The enzymatic reaction was carried out at 40°C, 50°C, 60°C, or 70°C for 24 hours.

[0158] Furthermore, the PET degradation activity of the mutant PET2 purified enzyme and the mutant LCC purified enzyme in a mixed substrate was measured by the following enzymatic reaction. A reaction solution consisting of 0.01 g of substrate was prepared by mixing 5 μg of each purified enzyme, 100 mM sodium carbonate buffer (pH 9.2), and PET powder and PU powder in the following ratios: 100:0 (100% by mass of PET in the mixed substrate), 85:15 (85% by mass of PET in the mixed substrate), and 70:30 (70% by mass of PET in the mixed substrate). The enzymatic reaction was carried out at 60°C or 70°C for 23 hours.

[0159] PET powder and PU powder were prepared as follows. Two types of PET powder were prepared: bottle-derived PET and fiber-derived PET. First, bottle-derived PET was prepared by crushing and washing used PET bottles to remove contaminants and foreign matter, resulting in PET flakes (IV: 0.8, melting point: 250°C). Next, the undried PET flakes were supplied to a twin-screw extruder equipped with an underwater cut (UWC) granulation system, and the PET flakes were melted at a barrel temperature of 280°C. At the same time, water was injected into the extruder through a plunger pump while the molten resin was flowing. The molten resin discharged from the die was pelletized using the UWC. The water temperature for the UWC was set to 80°C. The PET molded body (pellets) obtained as described above was pulverized using a freeze-milling device to obtain the PET powder used in this example.

[0160] Next, the fiber-derived PET and PU powders were obtained by pulverizing PET fibers and PU pellets using the method described in Example 1(5) to obtain the PET powder and PU powder used in this example. The prepared substrate PET is shown in Table 3. In the table, "UWC" indicates that the sample was prepared using a twin-screw extruder with an underwater cut (UWC) granulation system. In the table, "particle size" refers to the average particle size, which is the particle size at 50% of the cumulative value in the particle size distribution determined by laser diffraction and scattering.

[0161] [Table 3]

[0162] After the reaction was complete, TPA and MHET produced by PET degradation were analyzed by HPLC. The amounts (mM) of TPA and MHET produced by the enzymatic reaction of mutant PET2 purified enzyme and mutant LCC purified enzyme, as well as their total value (mM) (degradation amount) and degradation rate (%), are shown in Tables 4, 5, and 6.

[0163] Furthermore, 4,4′-diaminodiphenylmethane, a degradation product of PU, and glucose, a degradation product of cotton, were analyzed by HPLC and glucose loader, respectively. The amount (mM) and degradation rate (%) of 4,4′-diaminodiphenylmethane and the amount (mM) and degradation rate (%) of glucose produced by the enzymatic reaction of mutant PET2 purified enzyme and mutant LCC purified enzyme are shown in Tables 7 and 8, respectively.

[0164] [Table 4]

[0165] As shown in Table 4, PET2-K7S7Hotlike and LCC-ICCG were shown to be able to decompose PET in PET / PU mixed substrates. Furthermore, when using the L-2 sample (PET and / PU, freeze-milled), the highest PET decomposition rate was observed at a reaction temperature of 50°C, while when using the A-6 sample (PET / PU, shear and impact milling), the PET decomposition rate tended to be higher at reaction temperatures of 50°C or 60°C.

[0166] [Table 5]

[0167] As shown in Table 5, PET2-K7S7Hotlike and LCC-ICCG were demonstrated to decompose PET in a PET / cotton mixed substrate with excellent degradation rates. Furthermore, high PET degradation was demonstrated at reaction temperatures of 50°C, 60°C, and 70°C.

[0168] [Table 6]

[0169] As shown in Table 6, PET2-K7S7Hotlike and LCC-ICCG were demonstrated to decompose PET in a substrate mixed with PET powder and PU powder with excellent decomposition rates, regardless of the PET content in the mixed substrate.

[0170] [Table 7]

[0171] As shown in Table 7, when PET2-K7S7Hotlike and LCC-ICCG were used, the concentration of 4,4′-diaminodiphenylmethane, a degradation product of PU, was 0.021 mM or less, indicating that they hardly degraded PU in the PET / PU mixed substrate. From Tables 4 and 7, it was shown that PET2-K7S7Hotlike and LCC-ICCG can selectively degrade PET in the PET / PU mixed substrate.

[0172] [Table 8]

[0173] As shown in Table 8, when PET2-K7S7Hotlike and LCC-ICCG were used, the degradation rate of glucose, a degradation product of cotton, was 0.82% or less, indicating that they hardly degraded the cotton in the PET / cotton mixed substrate. From Tables 5 and 8, it was shown that PET2-K7S7Hotlike and LCC-ICCG can selectively degrade PET in the PET / cotton mixed substrate.

[0174] From the results described above, it was found that PET in a mixed substrate can be selectively degraded by reacting the mixed substrate with an enzyme composition containing a carboxylic acid ester-degrading enzyme. [Sequence Listing Free Text]

[0175] Sequence ID 1: Amino acid sequence of metagenomically derived PET2 WT Sequence ID 2: Base sequence of the metagenomically derived PET2 WT gene Sequence ID 3: Amino acid sequence of LCC WT derived from fallen leaf compost metagenomics Sequence ID 4: Nucleotide sequence of the LCC WT gene derived from a leaf litter compost metagenomic. Sequence ID 5: Amino acid sequence of metagenomically derived PET2-K7S7 Hotlike (N-terminal deletion type) Sequence ID 6: Base sequence of the metagenomically derived PET2-K7S7 Hotlike (N-terminal deletion) gene Sequence ID 7: Amino acid sequence of LCC-ICCG derived from fallen leaf compost metagenomics Sequence ID 8: Nucleotide sequence of the LCC-ICCG gene derived from the leaf litter compost metagenomic. Sequence ID 9: Nucleotide sequence of forward primer for PET2 WT amplification Sequence ID 10: Base sequence of reverse primer for PET2 WT amplification Sequence ID 11: Nucleotide sequence of forward primer for LCC WT amplification Sequence ID 12: Base sequence of reverse primer for LCC WT amplification Sequence ID 13: Nucleotide sequence of forward primer for K7S7 hotlike amplification Sequence ID No. 14: Nucleotide sequence of reverse primer for K7S7 Hotlike amplification. Sequence ID 15: Nucleotide sequence of forward primer for LCC-ICCG amplification Sequence ID 16: Base sequence of reverse primer for LCC-ICCG amplification Sequence ID 17: Amino acid sequence of metagenomically derived PET2-K7S7 Hotlike Sequence ID 18: Nucleotide sequence of the metagenomically derived PET2-K7S7 Hotlike gene

Claims

1. A method for decomposing polyethylene terephthalate (PET) in a mixed substrate, comprising the following step (1). (1) A step of reacting an enzyme composition with a mixed substrate containing PET, The enzyme composition comprises a carboxylic acid ester hydrolase or a variant thereof. The step is that the carboxylic acid ester hydrolase or its variant is at least one selected from carboxylesterase, allylsterase, cutinase, and PETase, as well as variants thereof and lipase.

2. The method according to claim 1, wherein the lipase variant is a variant of PET2.

3. The method according to claim 2, wherein the mutant of PET2 is a protein comprising an amino acid sequence in which at least one modification selected from the group consisting of [1] to [7] and at least one modification selected from the group consisting of [8] to [24] are introduced in the amino acid sequence represented by Sequence ID No. 1 (excluding proteins in which at least one combination of modifications selected from the group consisting of the combination of [8] and [16], the combination of [11] and [19], and the combination of [17] and [23] are introduced). [1] Modification by substituting the 47th amino acid residue with a cysteine ​​residue. [2] Modification by substituting the 89th amino acid residue with a cysteine ​​residue. [3] Modification by substituting the 105th amino acid residue with an arginine residue. [4] Modification by substituting the 110th amino acid residue with a lysine residue. [5] Modification by substituting the 156th amino acid residue with a proline residue. [6] Modification by substituting the 180th amino acid residue with an alanine residue. [7] Modification by substituting the 297th amino acid residue with a proline residue. [8] Modification by substituting the 73rd amino acid residue with a glutamine residue. [9] Modification by substituting the 76th amino acid residue with a cysteine ​​residue. [10] Modification by substituting the 103rd amino acid residue with a lysine residue. [11] Modification by substituting the 134th amino acid residue with a glycine residue. [12] Modification by substituting the 144th amino acid residue with a cysteine ​​residue. [13] Modification by substituting the 203rd amino acid residue with a threonine residue. [14] Modification by substituting the 222nd amino acid residue with a methionine residue. [15] Modification by substituting the 55th amino acid residue with a serine residue. [16] Modification by substituting the 73rd amino acid residue with a serine residue. [17] Modification by substituting the 228th amino acid residue with a lysine residue. [18] Modification by substituting the 264th amino acid residue with an arginine residue. [19] Modification by substituting the 134th amino acid residue with a lysine residue, a methionine residue, or a threonine residue. [20] Modification by substituting the 136th amino acid residue with a glutamic acid residue. [21] Modification by substituting the 200th amino acid residue with a histidine residue, an aspartic acid residue, or an alanine residue. [22] Modification by substituting the 227th amino acid residue with an arginine residue, a lysine residue, a glutamic acid residue, or a histidine residue. [23] Modification by substituting the 228th amino acid residue with a glutamic acid residue, histidine residue, lysine residue, glutamine residue, or tryptophan residue. [24] Modification by substituting the 229th amino acid residue with a tyrosine residue, an asparagine residue, or a phenylalanine residue.

4. The method according to claim 2, wherein the mutant of PET2 is at least one selected from [C4] to [C6]. [C4] A protein consisting of the amino acid sequence represented by SEQ ID NO: 5 or 17. [C5] A protein having an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by Sequence ID No. 5 or 17, and in which the PET degradation rate in a mixed substrate containing PET is 0.5% or more. [C6] A protein having an amino acid sequence that is 80% or more identical to the amino acid sequence represented by Sequence ID No. 5 or 17, and having a PET degradation rate of 0.5% or more in a mixed substrate containing PET.

5. The method according to claim 1, wherein the cutinase or its variant is LC-cutinase or its variant.

6. The method according to claim 5, wherein the LC-cutinase or its variant is at least one selected from the following [C1] to [C3]. [C1] A protein consisting of an amino acid sequence represented by SEQ ID NO: 3 or 7. [C2] A protein having an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by Sequence ID No. 3 or 7, and in which the PET degradation rate in a mixed substrate containing PET is 0.5% or more. [C3] A protein having an amino acid sequence that is 80% or more identical to the amino acid sequence represented by Sequence ID No. 3 or 7, and having a PET degradation rate of 0.5% or more in a mixed substrate containing PET.

7. The method according to claim 1, wherein the carboxylic acid ester hydrolase or its variant is at least one of the following (E3) and (E4). (E3) Protein consisting of the amino acid sequence represented by SEQ ID NO: 5 or 17 (E4) Protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 7

8. The method according to any one of claims 1 to 7, wherein the mixed substrate comprises at least one of polyurethane and cotton, and PET.

9. The method according to any one of claims 1 to 7, wherein the PET contained in the mixed substrate is derived from at least one selected from PET fibers, PET bottles, PET films, PET flakes, and PET resin powder.

10. The method according to any one of claims 1 to 7, wherein step (1) is carried out in the presence of sodium carbonate.

11. The method according to any one of claims 1 to 7, wherein step (1) is performed at a temperature of 40 to 70°C.

12. A method for producing at least one of terephthalic acid (TPA) and monohydroxyethyl terephthalate (MHET) using the method described in any one of claims 1 to 7.

13. The method according to any one of claims 1 to 7, wherein the mixed substrate comprises polyurethane and PET.

14. The method according to claim 13, wherein the PET content in the mixed substrate is 50 to 99% by mass.

15. The method according to any one of claims 1 to 7, wherein the mixed substrate comprises cotton and PET.

16. The method according to claim 15, wherein the PET content in the mixed substrate is 50 to 99% by mass.

17. A method for producing monoethylene glycol (MEG) using the method for decomposing PET described in any one of claims 1 to 7.

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