Polyethylene terephthalate (PET) degrading enzyme having improved thermostability

Abstract

To provide a PETase having improved thermostability.SOLUTION: A polypeptide contains a specific amino acid substitution and has aromatic polyester degradation activity and improved thermostability.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a polyethylene terephthalate (PET) degrading enzyme with improved thermal stability, uses thereof, and a method for producing the polyethylene terephthalate (PET) degrading enzyme. [Background technology]

[0002] PET resin has been widely used for bottles and other products due to its high transparency and excellent strength. However, due to its stability, it does not decompose in nature, resulting in waste problems. To solve this problem, a search for PET-degrading bacteria was conducted, and a strain (Ideonella sakaiensis No. 201-F6 strain) that completely decomposes PET into carbon dioxide and water was isolated (Patent Document 1). Furthermore, a PET-degrading enzyme (PETase) was isolated from this strain (Patent Document 2 and Non-Patent Document 1). PETase is an enzyme that hydrolyzes aromatic polyester resins such as PET resin. However, the PET degradation rate of wild-type PETase is insufficient, and further improvement in its efficiency is desired. To improve the efficiency of PET degradation by PETase, it is preferable to perform the enzymatic reaction at high temperatures. However, the enzymatic activity of wild-type PETase gradually decreases when the reaction temperature is 35°C or higher, resulting in a decrease in the PET degradation rate. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2008-199957 [Patent Document 2] International Publication No. W02015 / 025861 [Non-patent literature]

[0004] [Non-Patent Document 1] Yoshida et al., Science, Vol. 351, Issue 6278, pp1196-1199 (2016) [Non-patent document 2] Son et al., ACS Catalysis, Vol. 9, Issue 2, pp3519-3526 (2019) Summary of the Invention [Problem to be solved by the invention]

[0005] Under the circumstances described above, an object of the present invention is to provide a PETase with superior thermostability. [Means for solving the problem]

[0006] As a result of extensive research aimed at solving the above problems, the present inventors have discovered that by substituting specific amino acids in the amino acid sequence of PETase derived from a bacterial strain (Ideonella sakaiensis No. 201-F6 strain), it is possible to significantly improve the thermostability while maintaining the enzymatic activity. Based on this finding, the present inventors have conducted further research and have completed the present invention. The present invention is described below. [Section 1] Mutations (A) and / or (B) in the amino acid sequence shown in SEQ ID NO: 1: (A) one or more amino acid substitutions selected from the group consisting of S121D, I168R, N233C, A226P, F261W, T279P, and S282C; (B) substitution of the amino acid sequence of positions 222 to 226 of SEQ ID NO: 1 with the amino acid sequence shown in LPSTTP (SEQ ID NO: 2); and a polypeptide having aromatic polyester decomposition activity and improved thermal stability; [Section 2] A polynucleotide encoding the polypeptide according to Item 1; [Section 3] A vector comprising the polynucleotide according to Item 2; [Section 4] A transformant comprising the vector according to item 3; [Section 5] A method for producing the polypeptide according to Item 1, comprising culturing the transformant according to Item 4; and [Section 6] Mutations (A) and / or (B) in the amino acid sequence shown in SEQ ID NO: 1: (A) one or more amino acid substitutions selected from the group consisting of S121D, I168R, N233C, A226P, F261W, T279P, and S282C; (B) substitution of the amino acid sequence of positions 222 to 226 of SEQ ID NO: 1 with the amino acid sequence shown in LPSTTP (SEQ ID NO: 2); A method for producing a polypeptide having aromatic polyester decomposition activity, comprising introducing the following into wild-type PETase. [Effects of the Invention]

[0007] According to the present invention, a PETase with excellent thermal stability is provided, and therefore, by utilizing the present invention, aromatic polyester resins can be efficiently hydrolyzed. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows the results of measuring the residual activity of mutant and wild-type PETase after heat treatment. DETAILED DESCRIPTION OF THE INVENTION

[0009] The amino acid sequence constituting PETase derived from the strain (No. 201-F6 strain) is known and is shown in SEQ ID NO: 1. [ka]

[0010] PETases with specific mutations have improved thermostability compared to wild-type PETase. Wild-type PETase refers to a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 or a mutant amino acid sequence thereof, which does not contain mutation (A) or (B). Even if a polypeptide has the mutant amino acid sequence shown in SEQ ID NO: 1, it is included in the wild-type PETase if it has PETase activity.

[0011] When the amino acid sequence of SEQ ID NO: 1 contains substitutions of one to several amino acid residues, substitutions of amino acid residues with similar properties are thought to be more likely to maintain the function of the original polypeptide. Substitutions of one to several amino acid residues are preferably substitutions of 1 to 29, 1 to 15, 1 to 8, or 1 to 4 amino acid residues, specifically substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acid residues. Examples of wild-type PETase include polypeptides with conservative substitutions in the amino acid sequence of SEQ ID NO: 1 that have PETase activity. Conservative amino acid substitutions are well known to those skilled in the art. Conservative amino acid substitutions include, for example, polar acidic amino acids such as aspartic acid and glutamic acid; polar basic amino acids such as lysine, arginine, and histidine; nonpolar or hydrophobic amino acids such as leucine, isoleucine, methionine, valine, alanine, glycine, and proline; and polar or uncharged hydrophilic amino acids such as serine and threonine. Conservative amino acid substitutions also include classifications based on side chains. For example, amino acids with aliphatic side chains include glycine, alanine, valine, leucine, and isoleucine; amino acids with aliphatic-hydroxyl side chains include serine and threonine; amino acids with amide-containing side chains include asparagine and glutamine; amino acids with aromatic side chains include phenylalanine, tyrosine, and tryptophan; amino acids with basic side chains include lysine, arginine, and histidine; and amino acids with sulfur-containing side chains include cysteine ​​and methionine. For example, it is reasonable to expect that substitution of leucine with isoleucine or valine, substitution of aspartic acid with glutamic acid, substitution of threonine with serine, or similar substitution of an amino acid with a structurally related amino acid will not significantly affect the properties of the resulting polypeptide. Whether an amino acid exchange results in a functional polypeptide can be readily determined by assaying the PETase activity of the polypeptide.

[0012] Mutations such as amino acid substitutions, deletions, insertions, and / or additions are preferably made in regions that do not significantly affect the higher-order structure of the polypeptide or that are not directly involved in catalytic activity (e.g., regions other than the active center). Such regions include, for example, regions exposed on the surface of the protein.

[0013] Techniques for making mutations such as substitutions, deletions, insertions, and / or additions of amino acid residues into a specific amino acid sequence are known in the art and can be performed using any method, including, but not limited to, methods using homologous recombination, mutagenesis, point mutagenesis, site-directed mutagenesis, and genome editing.

[0014] The substitutions made to improve the thermostability of PETase are (A) one or more amino acid substitutions selected from the group consisting of S121D, I168R, N233C, A226P, F261W, T279P, and S282C in SEQ ID NO: 1; and / or (B) a substitution of the amino acid sequence at positions 222 to 226 in SEQ ID NO: 1 with the amino acid sequence represented by LPSTTP (SEQ ID NO: 2). Regarding the symbols representing each position in (A), the numbers refer to the position of the amino acid in the wild-type amino acid sequence. The alphabet before the number indicates the type of amino acid originally present at that position. The alphabet after the number indicates the type of amino acid substituting for the originally present amino acid. For example, "S121D" means that the serine (S) at position 121 in the wild-type amino acid sequence is substituted with aspartic acid (D). The same applies to the symbols representing other substitutions.

[0015] With regard to the substitution (B) above, the amino acid sequence at positions 222 to 226 of SEQ ID NO: 1 is MSRNA (SEQ ID NO: 3), and the amino acid sequence shown in SEQ ID NO: 2 that replaces this region is LPSTTP. That is, PETase having an amino acid sequence with the substitution (B) has LPSTTP in the amino acid sequence at positions 222 to 227 of SEQ ID NO: 1 in wild-type PETase, or positions corresponding thereto.

[0016] In the present disclosure, a "corresponding position" in an amino acid sequence or a nucleotide sequence can be determined by aligning a target sequence with a reference sequence (e.g., the amino acid sequence of SEQ ID NO: 1) to maximize homology. Alignment of amino acid sequences or nucleotide sequences can be performed using known algorithms, and procedures for doing so are well known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Thompson, J.D. et al., 1994, Nucleic Acids Res. 22:4673-4680) with default settings. Clustal W is available, for example, on the websites of the European Bioinformatics Institute (EBI) [www.ebi.ac.uk / index.html] and the DNA Data Bank of Japan (DDBJ) [www.ddbj.nig.ac.jp / searches-j.html], operated by the National Institute of Genetics. For example, a position in a target sequence that is aligned to an arbitrary position in a reference sequence by the above-described alignment is a "position corresponding to" that arbitrary position.

[0017] The above-mentioned specific amino acid sequence substitutions (A) and / or (B) may be made in a single type to the wild-type or corresponding amino acid sequence, or in a combination of two or more, three or more, four or more, five or more, six or more, or seven or more types to the wild-type or corresponding amino acid sequence. When a combination of two or more amino acid substitutions is made to the wild-type or corresponding amino acid sequence, the combination is arbitrary.

[0018] In one embodiment, a preferred combination of amino acid substitutions in the amino acid sequence of SEQ ID NO: 1 is: (i) N233C and S282C, (ii) F261W, T279P, N233C, S282C, and LPSTTP at positions 222 to 227 of SEQ ID NO: 1 in wild-type PETase or positions corresponding thereto; (iii) F261W, T279P, N233C, S282C, I168R, and LPSTTP at positions 222 to 227 of SEQ ID NO: 1 in wild-type PETase or positions corresponding thereto; (iv) F261W, T279P, N233C, S282C, I168R, S121D, and LPSTTP at positions 222 to 227 of SEQ ID NO: 1 in wild-type PETase or positions corresponding thereto; (v) A226P, F261W, T279P, N233C and S282C, (vi) N233C, S282C, and LPSTTP at positions 222 to 227 of SEQ ID NO: 1 or corresponding positions in wild-type PETase; (vii) F261W, N233C and S282C, and (viii) an amino acid substitution selected from the group consisting of T279P, N233C, and S282C.

[0019] The thermostability-improved PETase of the present disclosure may have mutations other than those shown in the specific amino acid substitutions (A) and (B), as long as it retains aromatic polyester-degrading activity. The PETase may have an amino acid sequence that includes substitutions, deletions, insertions, and / or additions of one to several amino acid residues in addition to the amino acid substitutions (A) and (B). In the present disclosure, substitutions, deletions, insertions, and / or additions of one to several amino acid residues refer to, for example, substitutions, deletions, insertions, and / or additions of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 30 or more, 40 or more, or 50 or more amino acid residues, preferably 1 to 2, 1 to 5, 1 to 8, 1 to 11, 1 to 14, 1 to 29, 1 to 43, 1 to 58, or any number therebetween. Alternatively, the PETase may have a mutation other than those shown in amino acid substitutions (A) and (B), resulting in an amino acid sequence that has 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to the original amino acid sequence shown in SEQ ID NO: 1. As a result of such mutations, the positions of the mutations (A) and (B) in the polypeptide may be shifted. Thus, for example, S121D (substitution of the serine residue at the 121st position from the N-terminus of SEQ ID NO: 1 with an aspartic acid residue) may include substitution of the serine residue at the position corresponding to the 121st position from the N-terminus with an aspartic acid residue. This applies to all mutations shown in (A) and (B) of the present disclosure.

[0020] The present disclosure also provides a method for producing a polypeptide having aromatic polyester degrading activity, which comprises introducing the mutations (A) and / or (B) of the present disclosure into a wild-type PETase. When the mutations are introduced into a wild-type PETase, the wild-type PETase is a polypeptide comprising any of the amino acid sequences (i) to (iii) below: (i) the amino acid sequence shown in SEQ ID NO: 1; (ii) an amino acid sequence containing substitution, deletion, insertion, and / or addition of one to several amino acid residues in the amino acid sequence shown in SEQ ID NO: 1, or (iii) An amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.

[0021] By having the above-mentioned specific amino acid substitutions in the wild-type amino acid sequence or an amino acid sequence corresponding thereto, the polypeptide has improved thermostability. Improved thermostability means having higher thermostability compared to PETase having the wild-type amino acid sequence (i.e., being less susceptible to denaturation or inactivation than wild-type PETase under the same temperature conditions, or having higher residual activity).

[0022] Thermal stability can be measured by any method. For example, the temperature at which a protein changes its conformation can be measured and thermal stability determined using the protein thermal shift assay (see Non-Patent Document 2; Son et al., ACS Catalysis, Vol. 9, Issue 2, pp. 3519-3526 (2019)) used in the Examples described below. When using this measurement method, since the denaturation temperature of wild-type PETase is 48°C, it is preferable that PETase with improved thermal stability has a denaturation temperature higher than 48°C. PETase with improved thermostability has a denaturation temperature of, for example, 49°C or higher, 50°C or higher, 51°C or higher, 52°C or higher, 53°C or higher, 54°C or higher, 55°C or higher, 56°C or higher, 57°C or higher, 58°C or higher, 59°C or higher, 60°C or higher, 61°C or higher, 62°C or higher, 63°C or higher, 64°C or higher, 65°C or higher, 66°C or higher, 67°C or higher, 68°C or higher, 69°C or higher, 70°C or higher, 71°C or higher, 72°C or higher, 73°C or higher, 74°C or higher, or 75°C or higher.

[0023] Thermostability can also be evaluated by measuring the residual activity of PETase before and after heat treatment at a certain temperature for a predetermined time (heat treatment). If the residual activity of PETase having a substituted polypeptide is higher than that of PETase having a polypeptide before substitution, it can be evaluated as having improved thermostability. For example, as shown in the Examples below, PETase exhibits residual activity of 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more after 10 minutes at 60°C; residual activity of 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more after 20 minutes at 60°C; and residual activity of 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more after 30 minutes at 60°C. %, or 60% or more, 70% or more, 80% or more, or 90% or more, and when maintained at 60°C for 60 minutes, the residual activity is preferably 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, and when maintained at 60°C for 90 minutes, the residual activity is preferably 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.

[0024] In the present disclosure, "activity" and "enzyme activity" refer to esterase activity unless otherwise specified. Methods for measuring esterase activity are known, and any method can be selected for measurement. For example, a method using the synthetic substrate PNP (p-nitrophenyl)-butyrate as a substrate and measuring free PNP, as employed in the Examples described below, can be mentioned. The terms PETase activity and aromatic polyester degradation activity are used interchangeably herein. PETase activity can be measured by methods known to those skilled in the art, including, for example, methods for measuring esterase activity. For example, PETase activity can be assessed by degrading PET as a substrate with PETase, followed by quantitative analysis of the degradation products, terephthalic acid, monohydroxyethyl terephthalate, or bishydroxyethyl terephthalate, by reverse-phase HPLC.

[0025] The PETase variants of the present invention can be produced, for example, by introducing a nucleic acid sequence encoding the amino acid sequence of each variant, or a plasmid vector incorporating a mutated PETase DNA fragment, into Escherichia coli (host) or the like to obtain the desired transformant. There are no particular limitations on the type of recombinant vector used, and any vector commonly used in the art may be used. Furthermore, the PETase-producing transformant may be Escherichia coli, Bacillus bacteria, or yeast.

[0026] The polypeptide can be produced using the transformant by culturing the transformant and recovering the polypeptide from the culture. The culture can be performed by subculture or batch culture using a medium suitable for the host. The "culture" includes, for example, culture supernatant, cultured cells, cultured bacterial cells, or cell or bacterial lysate.

[0027] Culture conditions can also be appropriately set depending on the type of host. Typically, culture is carried out at 10 to 45°C, preferably 15 to 37°C, for 8 hours or more, 12 hours or more, 16 hours or more, 24 hours or more, 48 hours or more, 72 hours or more, 96 hours or more, or 120 hours or more, preferably 8 to 24 hours. When an inducible promoter is used, a promoter inducer can also be added to the medium before culture.

[0028] Purification or isolation of a polypeptide from a culture can be carried out by appropriately combining known techniques. Examples include techniques using various types of chromatography (e.g., gel filtration chromatography, cation exchange chromatography, affinity chromatography). For example, when a tag is added to the polypeptide, a support to which a substance having affinity for the tag is bound can be used as a support for affinity chromatography.

[0029] When the polypeptide accumulates within the host cells, the transformed cells can be disrupted and purified or isolated from the centrifuged supernatant in the same manner as described above. For example, after the culture is completed, the cells are collected by centrifugation, suspended in a cell disruption buffer (20 to 50 mM Tris-HCl (pH 8.0)), disrupted by sonication, and the disrupted solution is centrifuged at 10,000 to 15,000 rpm for 30 minutes to obtain the supernatant.

[0030] In one embodiment, a method is provided for improving the thermostability of PETase while maintaining its activity by introducing specific amino acid substitutions in the wild-type amino acid sequence. [Example]

[0031] The present invention will be specifically described below with reference to examples, although the present invention is not limited to these examples.

[0032] The three-dimensional structure of wild-type PETase derived from the strain No. 201-F6 is publicly known. Therefore, we searched for and extracted positions likely to affect PETase stability from the three-dimensional structure of wild-type PETase, and then performed protein engineering to modify amino acids that are likely to physicochemically stabilize the enzyme's three-dimensional structure. We selected specific amino acid sequences predicted to improve thermostability, and substituted them with specific amino acid sequences. Conversion to specific amino acid sequences included substitutions with amino acid sequences predicted to be effective, for example, in improving hydrophobic interactions, stabilizing the loop structure of the peptide backbone, introducing covalent bonds between amino acids via disulfide bonds, and spatial density orientation.

[0033] The nucleic acid (SEQ ID NO: 3) encoding wild-type PETase derived from the No. 201-F6 strain was chemically synthesized by a gene synthesis service (Eurofins Genomics) and ligated downstream of the promoter of the commercially available pCold II vector to construct an expression plasmid vector. Using this as a template, mutations were introduced into the gene by the Quick Change method to create mutants 1-21 in Table 1. The mutation sites were confirmed by sequence analysis.Specifically, variant 1 has an I218F substitution in the amino acid sequence set forth in SEQ ID NO:1; variant 2 has an S223P substitution in the amino acid sequence set forth in SEQ ID NO:1; variant 3 has an A226P substitution in the amino acid sequence set forth in SEQ ID NO:1; variant 4 has an F261W substitution in the amino acid sequence set forth in SEQ ID NO:1; variant 5 has a T279P substitution in the amino acid sequence set forth in SEQ ID NO:1; variant 6 has a combination of N233C / S282C substitutions in the amino acid sequence set forth in SEQ ID NO:1; variant 7 has a combination of N233C / S282C substitutions in the amino acid sequence set forth in SEQ ID NO:1 Mutant 8 has a combination of Mutant 4+5+6+7 substitutions in the amino acid sequence set forth in SEQ ID NO:1; Mutant 9 has a combination of I168R+Mutant 8 substitutions in the amino acid sequence set forth in SEQ ID NO:1; Mutant 10 has a combination of S121D+Mutant 9 substitutions in the amino acid sequence set forth in SEQ ID NO:1; Mutant 11 has a combination of I124C / I168C substitutions in the amino acid sequence set forth in SEQ ID NO:1; Mutant 12 has a combination of I124C / I168C substitutions in the amino acid sequence set forth in SEQ ID NO:1; Mutant 13 has a combination of T77C / D150C substitutions in the amino acid sequence set forth in SEQ ID NO: 1; Mutant 14 has a combination of RQVASLNGTSSSPIYGK(132-148) (SEQ ID NO: 4) → NYLRTSSPSAVRAR (SEQ ID NO: 5) substitution in the amino acid sequence set forth in SEQ ID NO: 1; Mutant 15 has a combination of S141 deletion + Mutant 8 substitution in the amino acid sequence set forth in SEQ ID NO: 1; Mutant 16 has a combination of S141 deletion + Mutant 8 substitution in the amino acid sequence set forth in SEQ ID NO: 1 Mutant 17 has a combination of S143 deletion and mutant 16 substitution in the amino acid sequence set forth in SEQ ID NO:1; Mutant 18 has a combination of mutants 3, 4, 5, and 6 substitution in the amino acid sequence set forth in SEQ ID NO:1; Mutant 19 has a combination of mutants 6, 7, and 7 substitution in the amino acid sequence set forth in SEQ ID NO:1; Mutant 20 has a combination of mutants 4, 4, and 6 substitution in the amino acid sequence set forth in SEQ ID NO:1; Mutant 21 has a combination of mutants 5, 5, and 6 substitution in the amino acid sequence set forth in SEQ ID NO:1.

[0034] The expression plasmid vector was introduced into competent cells of E. coli BL21 (DE3) by the heat shock method. The transformant was then cultured in liquid at 37°C, and the OD of the culture medium was measured. 600 When the β-actin ratio reached approximately 0.5, the culture temperature was shifted to 15°C to express the protein. The cells were collected, disrupted using an ultrasonic disrupter, and centrifuged to remove the supernatant. The supernatant was then purified by nickel affinity chromatography. SDS-PAGE of each eluate obtained from the column purification confirmed the presence of a PETase band (approximately 28 kDa) in the eluted fraction. The heat resistance and activity of the purified enzyme were measured. Heat resistance was evaluated by measuring the denaturation temperature (Tm) at which the enzyme protein changes its conformation using the protein thermal shift assay (TSA) described in Non-Patent Document 2 (Son et al., ACS Catalysis, Vol. 9, Issue 2, pp. 3519-3526 (2019)). TSA was performed using each PETase mutant enzyme dissolved in 50 mM sodium phosphate buffer (pH 7.0). The measurement results are shown in Table 1. [Table 1]

[0035] As shown in Table 1, mutants 3 to 10, 15, and 18 to 21 all had improved heat resistance (thermostability) compared to the wild-type enzyme. Mutants 6, 8 to 10, and 18 to 21, which had multiple mutations, showed particularly significant improvements in thermostability. Mutants 11 to 13 were improved in thermostability by introducing disulfide bonds, but Tm did not increase compared to wild-type PETase. Mutants 14 to 17 were improved in thermostability by restricting fluctuations in the loop region, but Tm did not increase compared to wild-type PETase. Data could not be obtained for mutants 14, 16, and 17 because they formed aggregates at room temperature.

[0036] Next, 50 mM sodium phosphate buffer (pH 7.0) containing either wild-type PETase or Mutant 10 at concentrations of 0.02–0.05 mg / ml was prepared. This solution was maintained at 60°C, and aliquots of the enzyme solution were removed after 1, 10, 20, 30, 60, and 90 minutes, and their residual activity was measured at 30°C. The substrate was 1 mM PNP-butylate, and the amount of PNP released was measured. Residual activity was calculated by setting the activity at the start of the 60°C treatment (i.e., 0 minutes) as 100%. The results are shown in Figure 1. As shown in Figure 1, the residual activity after 10 minutes of heating was 30% of that of the wild-type, but 100% for Mutant 10. Furthermore, Mutant 10 showed no decrease in activity even after 60 minutes. [Industrial Applicability]

[0037] The polyethylene terephthalate (PET)-degrading enzyme of the present invention with improved thermostability can efficiently degrade PET.

Claims

1. Mutations (A) and / or (B) in the amino acid sequence shown in SEQ ID NO: 1: (A) one or more amino acid substitutions selected from the group consisting of S121D, I168R, N233C, A226P, F261W, T279P, and S282C; (B) substitution of the amino acid sequence of positions 222 to 226 of SEQ ID NO: 1 with the amino acid sequence shown in LPSTTP (SEQ ID NO: 2); and having aromatic polyester-degrading activity and improved thermal stability.

2. A polynucleotide encoding the polypeptide of claim 1.

3. A vector comprising the polynucleotide of claim 2.

4. A transformant comprising the vector according to claim 3.

5. A method for producing the polypeptide according to claim 1, comprising the step of culturing the transformant according to claim 4.

6. Mutations (A) and / or (B) in the amino acid sequence shown in SEQ ID NO: 1: (A) one or more amino acid substitutions selected from the group consisting of S121D, I168R, N233C, A226P, F261W, T279P, and S282C; (B) substitution of the amino acid sequence of positions 222 to 226 of SEQ ID NO: 1 with the amino acid sequence shown in LPSTTP (SEQ ID NO: 2); A method for producing a polypeptide having aromatic polyester decomposition activity, comprising introducing the above into a wild-type PETase.

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