Protein having polyethylene terephthalate decomposition activity and method for decomposing polyethylene terephthalate

Abstract

The present invention pertains to a protein or the like obtained by introducing, into the amino acid sequence represented by SEQ ID NO: 1, at least one modified amino acid sequence selected from the group consisting of [1] to [7] described below. [1] Transformation of substitution of amino acid residue at position 103 to lysine residue, [2] Transformation of substitution of amino acid residue at position 76 to cysteine residue, [3] Transformation of substitution of amino acid residue at position 144 to cysteine residue, [4] Transformation of substitution of amino acid residue at position 134 to glycine residue, [5] Transformation of substitution of amino acid residue at position 134 to glycine residue, and [5] Transformation of substitution of amino acid residue at position 103th to lysine residue. The amino acid residue at the 222 site is modified by replacing methionine residue, the amino acid residue at the 73 site is modified by replacing glutamine residue, and the amino acid residue at the 203 site is modified by replacing threonine residue.

Description

Technical Field

[0001] The present invention relates to a protein having polyethylene terephthalate-degrading activity, a DNA encoding the protein, a transformant obtained by transforming a host cell with the DNA, a method for degrading polyethylene terephthalate, and a method for producing at least one of terephthalic acid and mono-hydroxyethyl terephthalate. Background Art

[0002] Polyethylene terephthalate (hereinafter also referred to as "PET") is used in various applications such as beverage bottles, fibers, and packaging materials. However, since it does not decompose in the environment, there is a problem of environmental pollution. Therefore, PET recycling technology is required. In PET recycling technology, chemical recycling cannot sufficiently remove impurities, so there is a problem that the use of decomposed PET is limited. On the other hand, chemical recycling can decompose and repolymerize PET in the form of monomer units, so it is easy to remove impurities and is attracting attention as a sustainable recycling technology.

[0003] In chemical recycling, the PET decomposition method using an enzyme can be said to be an environmentally friendly recycling method from the viewpoints of reacting under mild conditions and not using an organic solvent. As PET-degrading enzymes, PETase from Ideonella sakaiensis (Patent Document 1), cutinase from Thermobifida fusca (Patent Document 2), etc. have been discovered. In addition, it has been reported that LC-cutinase from a leaf litter compost metagenome has high decomposition activity and heat resistance (Patent Document 3), and more than 97% of PET can be decomposed within 24 hours by enzyme modification (Non-Patent Document 1). In addition, in a report on searching for PET-degrading enzymes from metagenomes, PET2 having relatively high heat resistance was found in the enzyme, and it was also known that only a trace amount can decompose PET (Non-Patent Document 2). Furthermore, there are reports of improving the decomposition activity and heat resistance by modifying PET2 (Non-Patent Document 3).

[0004] Prior Art Documents

[0005] Patent Documents

[0006] Patent Document 1: International Publication No. 2015 / 025861

[0007] Patent Document 2: Japanese Patent No. 6315978

[0008] Patent Document 3: International Publication No. 2012 / 099018

[0009] Non-Patent Documents

[0010] Non-Patent Document 1: Nature, 2020, 580, 9, p.216-219

[0011] Non-Patent Document 2: Appl. Environ. Microbiol, 2018, 84, 8, p.e02773-17

[0012] Non-Patent Document 3: ACS Catal. 2021, 11, p.8550-8564 Summary of the Invention

[0013] Problems to be Solved by the Invention

[0014] Although the PET-degrading enzymes described in Patent Documents 1 and 2 have been researched and developed, they have not been put into practical use yet. In addition, as a result of the research by the present inventors, both the degradation activity and the heat resistance are low. Further, as a result of the research by the present inventors, even when using the enzymes described in Patent Document 3 and Non-Patent Documents 1 to 3, it takes a long time to decompose PET by an enzymatic reaction. Therefore, in industrial applications, it is desired to develop a PET-degrading enzyme with higher activity.

[0015] Accordingly, an object of the present invention is to provide a protein having high PET-degrading activity.

[0016] Means for Solving the Problems

[0017] The present inventors conducted in-depth research on the above problems and found that a protein composed of an amino acid sequence in which at least one modification that replaces an amino acid residue at a specific position in the amino acid sequence shown in SEQ ID NO: 1 with a specific amino acid residue is introduced has higher PET-degrading activity than the protein having the amino acid sequence shown in SEQ ID NO: 1, thereby completing the present invention.

[0018] That is, the present invention is as follows.

[0019] <1> A protein composed of an amino acid sequence in which at least one modification selected from the group consisting of the following [1] to [7] is introduced into the amino acid sequence shown in SEQ ID NO: 1.

[0020] [1] Modification that replaces the amino acid residue at position 103 with a lysine residue

[0021] [2] Modification that replaces the amino acid residue at position 76 with a cysteine residue

[0022] [3] Modification that replaces the amino acid residue at position 144 with a cysteine residue

[0023] [4] Modification that replaces the amino acid residue at position 134 with a glycine residue

[0024] [5] Modification of substituting the 222nd amino acid residue with a methionine residue

[0025] [6] Modification of substituting the 73rd amino acid residue with a glutamine residue

[0026] [7] Modification of substituting the 203rd amino acid residue with a threonine residue

[0027] <2> A protein, which is composed of an amino acid sequence of a modified mutant protein (B) having at least one of deletion, substitution, insertion, and addition of 1 to 20 amino acid residues relative to the amino acid sequence shown in SEQ ID NO: 1, into which an amino acid sequence of at least one modification selected from the group consisting of the following [1´] to [7´] is introduced, and has higher polyethylene terephthalate (PET) decomposition activity than the above mutant protein (B).

[0028] [1´] Modification of substituting the amino acid residue corresponding to the 103rd amino acid residue of SEQ ID NO: 1 with a lysine residue

[0029] [2´] Modification of substituting the amino acid residue corresponding to the 76th amino acid residue of SEQ ID NO: 1 with a cysteine residue

[0030] [3´] Modification of substituting the amino acid residue corresponding to the 144th amino acid residue of SEQ ID NO: 1 with a cysteine residue

[0031] [4´] Modification of substituting the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 with a glycine residue

[0032] [5´] Modification of substituting the amino acid residue corresponding to the 222nd amino acid residue of SEQ ID NO: 1 with a methionine residue

[0033] [6´] Modification of substituting the amino acid residue corresponding to the 73rd amino acid residue of SEQ ID NO: 1 with a glutamine residue

[0034] [7´] Modification of substituting the amino acid residue corresponding to the 203rd amino acid residue of SEQ ID NO: 1 with a threonine residue

[0035] <3> A protein, which is composed of an amino acid sequence of a homologous protein (C) having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1, into which an amino acid sequence of at least one modification selected from the group consisting of the following [1´´] to [7´´] is introduced, and has higher polyethylene terephthalate (PET) decomposition activity than the above homologous protein (C).

[0036] [1´´]Modification of substituting the amino acid residue corresponding to the 103rd amino acid residue of SEQ ID NO: 1 with a lysine residue

[0037] [2´´]Modification of substituting the amino acid residue corresponding to the 76th amino acid residue of SEQ ID NO: 1 with a cysteine residue

[0038] [3´´]Modification of substituting the amino acid residue corresponding to the 144th amino acid residue of SEQ ID NO: 1 with a cysteine residue

[0039] [4´´]Modification of substituting the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 with a glycine residue

[0040] [5´´]Modification of substituting the amino acid residue corresponding to the 222nd amino acid residue of SEQ ID NO: 1 with a methionine residue

[0041] [6´´]Modification of substituting the amino acid residue corresponding to the 73rd amino acid residue of SEQ ID NO: 1 with a glutamine residue

[0042] [7´´]Modification of substituting the amino acid residue corresponding to the 203rd amino acid residue of SEQ ID NO: 1 with a threonine residue

[0043] <4>A DNA encoding the protein according to any one of <1> to <3>.

[0044] <5>A recombinant DNA containing the DNA according to <4>.

[0045] <6>A transformant obtained by transforming a host cell with the recombinant DNA according to <5>.

[0046] <7>A method for decomposing PET, which uses the protein according to any one of <1> to <3> to decompose PET.

[0047] <8>The method for decomposing PET according to <7>, which comprises the following step: decomposing PET in a reaction solution containing the protein according to any one of <1> to <3>, PET, magnesium chloride and sodium carbonate.

[0048] <9>The method for decomposing PET according to <8>, wherein the concentration of magnesium chloride in the above reaction solution is 0.1 mM to 5 mM.

[0049] <10>A method for producing at least one of terephthalic acid (TPA) and mono - hydroxyethyl terephthalate (MHET), which comprises a step of using the protein according to any one of <1> to <3> to decompose PET.

[0050] <11> The method for producing at least one of TPA and MHET according to <10> includes the following steps: decomposing PET in a reaction solution containing the protein according to any one of <1> to <3>, PET, magnesium chloride, and sodium carbonate.

[0051] <12> The method for producing at least one of TPA and MHET according to <11>, wherein the concentration of magnesium chloride in the above reaction solution is 0.1 mM to 5 mM.

[0052] Advantages of the Invention

[0053] The protein of one embodiment of the present invention has higher PET decomposition activity compared to a protein without the substitution by containing amino acid residue substitution at specific positions. Detailed Description of the Invention

[0054] The present invention will be described in detail below, but these show an example of a desired embodiment and are not limited to these contents.

[0055] "~" in the numerical range means a range including the numerical values before and after it. For example, "0 mass% to 100 mass%" means a range of 0 mass% or more and 100 mass% or less.

[0056] 1. Protein with PET Decomposition Activity

[0057] The protein of one embodiment of the present invention is composed of an amino acid sequence obtained by introducing at least one modified amino acid sequence selected from the group consisting of the following [1] to [7] into the amino acid sequence shown in SEQ ID NO: 1.

[0058] [1] Modification of substituting the amino acid residue at position 103 with a lysine residue

[0059] [2] Modification of substituting the amino acid residue at position 76 with a cysteine residue

[0060] [3] Modification of substituting the amino acid residue at position 144 with a cysteine residue

[0061] [4] Modification of substituting the amino acid residue at position 134 with a glycine residue

[0062] [5] Modification of substituting the amino acid residue at position 222 with a methionine residue

[0063] [6] Modification of substituting the amino acid residue at position 73 with a glutamine residue

[0064] [7] Modification of substituting the amino acid residue at position 203 with a threonine residue

[0065] The protein consisting of the amino acid sequence shown in SEQ ID NO: 1 is the PET2 enzyme described in Non-Patent Document 1.

[0066] By setting the protein to be composed of an amino acid sequence in which at least one modification selected from the group consisting of the above [1] to [7] is introduced into the amino acid sequence shown in SEQ ID NO: 1, at least one of the heat resistance and the binding ability to the substrate is improved. Therefore, it is considered that the PET decomposition activity is higher than that of the protein (A) having the amino acid sequence shown in SEQ ID NO: 1.

[0067] As a non-limiting specific example of the protein according to one embodiment of the present invention, for example, a protein composed of an amino acid sequence in which any one of the following (A1) to (A7) is introduced into the amino acid sequence shown in SEQ ID NO: 1 can be cited.

[0068] (A1) The above [1]

[0069] (A2) The above [1] and [2]

[0070] (A3) The above [1] and [3]

[0071] (A4) The above [1], [2] and [3]

[0072] (A5) The above [1], [2], [3] and [4]

[0073] (A6) The above [1], [2], [3], [4] and [5]

[0074] (A7) The above [1], [2], [3], [4], [5] and [6]

[0075] (A8) The above [1], [2], [3], [4], [5], [6] and [7]

[0076] In addition, as a non-limiting specific example of the protein according to one embodiment of the present invention, a protein composed of an amino acid sequence in which at least the modification of [1] is introduced into the amino acid sequence shown in SEQ ID NO: 1, and optionally at least one modification of [2] to [7] is introduced can be cited.

[0077] As another non-limiting specific example of the protein according to one embodiment of the present invention, a protein composed of an amino acid sequence in which at least the modifications of [1] and [2] are introduced into the amino acid sequence shown in SEQ ID NO: 1, and optionally at least one modification of [3] to [7] is introduced can be cited.

[0078] As another non-limiting specific embodiment of the protein according to one aspect of the present invention, there may be mentioned a protein composed of an amino acid sequence in which at least the modifications of [1] and [3] are introduced into the amino acid sequence shown in SEQ ID NO: 1, and optionally at least one modification of [2] and [4] to [7] is further introduced.

[0079] As another non-limiting specific embodiment of the protein according to one aspect of the present invention, there may be mentioned a protein composed of an amino acid sequence in which at least the modifications of [1], [2], and [3] are introduced into the amino acid sequence shown in SEQ ID NO: 1, and optionally at least one modification of [4] to [7] is further introduced.

[0080] As another non-limiting specific embodiment of the protein according to one aspect of the present invention, there may be mentioned a protein composed of an amino acid sequence in which at least the modifications of [1], [2], [3], and [4] are introduced into the amino acid sequence shown in SEQ ID NO: 1, and optionally at least one modification of [5] to [7] is further introduced.

[0081] As another non-limiting specific embodiment of the protein according to one aspect of the present invention, there may be mentioned a protein composed of an amino acid sequence in which at least the modifications of [1], [2], [3], [4], and [5] are introduced into the amino acid sequence shown in SEQ ID NO: 1, and optionally at least one modification of [6] and [7] is further introduced.

[0082] As another non-limiting specific embodiment of the protein according to one aspect of the present invention, there may be mentioned a protein composed of an amino acid sequence in which at least the modifications of [1], [2], [3], [4], [5], and [6] are introduced into the amino acid sequence shown in SEQ ID NO: 1, and optionally the modification of [7] is further introduced.

[0083] As another non-limiting specific embodiment of the protein according to one aspect of the present invention, there may be mentioned a protein composed of an amino acid sequence in which the modifications of [1], [2], [3], [4], [5], [6], and [7] are introduced into the amino acid sequence shown in SEQ ID NO: 1.

[0084] The protein according to one aspect of the present invention is composed of an amino acid sequence in which at least one modification selected from the group consisting of deletion, substitution, insertion, and addition of 1 to 20 amino acid residues is introduced into the amino acid sequence of a modified mutant protein (B) and at least one modification selected from the following [1´] to [7´] is introduced, and has higher PET decomposition activity than the above mutant protein (B).

[0085] [1´] Modification of substituting the amino acid residue corresponding to the 103rd amino acid residue of SEQ ID NO: 1 with a lysine residue

[0086] [2´]Modification of replacing the amino acid residue corresponding to the 76th amino acid residue of SEQ ID NO: 1 with a cysteine residue

[0087] [3´]Modification of replacing the amino acid residue corresponding to the 144th amino acid residue of SEQ ID NO: 1 with a cysteine residue

[0088] [4´]Modification of replacing the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 with a glycine residue

[0089] [5´]Modification of replacing the amino acid residue corresponding to the 222nd amino acid residue of SEQ ID NO: 1 with a methionine residue

[0090] [6´]Modification of replacing the amino acid residue corresponding to the 73rd amino acid residue of SEQ ID NO: 1 with a glutamine residue

[0091] [7´]Modification of replacing the amino acid residue corresponding to the 203rd amino acid residue of SEQ ID NO: 1 with a threonine residue

[0092] In this specification, a mutant protein refers to a protein obtained by artificially deleting or replacing amino acid residues in a base protein, or inserting or adding amino acid residues into the protein.

[0093] In the above mutant protein, the modification of the amino acid consisting of at least one of deletion, replacement, insertion, and addition may mean that at any position in the amino acid sequence shown in SEQ ID NO: 1, 1 to 20 amino acids have been modified by at least one of deletion, replacement, insertion, and addition. The number of amino acids modified by at least one of deletion, replacement, insertion, and addition is 1 to 20, preferably 1 to 10, more preferably 1 to 8, and most preferably 1 to 5.

[0094] The amino acids modified by at least one of deletion, replacement, insertion, and addition can be either natural or non-natural. 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, L-cysteine, etc.

[0095] Examples of amino acids that can be mutually replaced are shown below. Amino acids included in the same group can be mutually replaced.

[0096] Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutyric acid, methionine, O-methylserine, tert-butylglycine, tert-butylalanine, cyclohexylalanine

[0097] Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminooctanedioic acid

[0098] Group C: asparagine, glutamine

[0099] Group D: lysine, arginine, ornithine, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid

[0100] Group E: proline, 3-hydroxyproline, 4-hydroxyproline

[0101] Group F: serine, threonine, homoserine

[0102] Group G: phenylalanine, tyrosine

[0103] As a non-limiting specific example of the protein as one embodiment of the present invention, for example, a protein composed of an amino acid sequence in which any one of the following (B1) to (B8) is introduced into the amino acid sequence of a modified mutant protein (B) composed of at least one of deletion, substitution, insertion, and addition of 1 to 20 amino acid residues with respect to the amino acid sequence shown in SEQ ID NO: 1 can be cited.

[0104] (B1) The above [1´]

[0105] (B2) The above [1´] and [2´]

[0106] (B3) The above [1´] and [3´]

[0107] (B4) The above [1´], [2´] and [3´]

[0108] (B5) The above [1´], [2´], [3´] and [4´]

[0109] (B6) The above [1´], [2´], [3´], [4´] and [5´]

[0110] (B7) The above [1´], [2´], [3´], [4´], [5´] and [6´]

[0111] (B8) The above [1´], [2´], [3´], [4´], [5´], [6´] and [7´]

[0112] Further, as a non-limiting specific example of the protein according to one embodiment of the present invention, there may be mentioned a protein composed of an amino acid sequence in which at least the modification of [1´] is introduced into the amino acid sequence of the above-mentioned mutant protein (B), and optionally at least one modification of [2´] to [7´] is further introduced.

[0113] As another non-limiting specific example of the protein according to one embodiment of the present invention, there may be mentioned a protein composed of an amino acid sequence in which at least the modifications of [1´] and [2´] are introduced into the amino acid sequence of the above-mentioned mutant protein (B), and optionally at least one modification of [3´] to [7´] is further introduced.

[0114] As another non-limiting specific example of the protein according to one embodiment of the present invention, there may be mentioned a protein composed of an amino acid sequence in which at least the modifications of [1´] and [3´] are introduced into the amino acid sequence of the above-mentioned mutant protein (B), and optionally at least one modification of [2´] and [4´] to [7´] is further introduced.

[0115] As another non-limiting specific example of the protein according to one embodiment of the present invention, there may be mentioned a protein composed of an amino acid sequence in which at least the modifications of [1´], [2´] and [3´] are introduced into the amino acid sequence of the above-mentioned mutant protein (B), and optionally at least one modification of [4´] to [7´] is further introduced.

[0116] As another non-limiting specific example of the protein according to one embodiment of the present invention, there may be mentioned a protein composed of an amino acid sequence in which at least the modifications of [1´], [2´], [3´] and [4´] are introduced into the amino acid sequence of the above-mentioned mutant protein (B), and optionally at least one modification of [5´] to [7´] is further introduced.

[0117] As another non-limiting specific example of the protein according to one embodiment of the present invention, there may be mentioned a protein composed of an amino acid sequence in which at least the modifications of [1´], [2´], [3´], [4´] and [5´] are introduced into the amino acid sequence of the above-mentioned mutant protein (B), and optionally at least one modification of [6´] and [7´] is further introduced.

[0118] As another non-limiting specific example of the protein according to one embodiment of the present invention, there may be mentioned a protein composed of an amino acid sequence in which at least the modifications of [1´], [2´], [3´], [4´], [5´] and [6´] are introduced into the amino acid sequence of the above-mentioned mutant protein (B), and optionally the modification of [7´] is further introduced.

[0119] As another non-limiting specific embodiment of the protein according to one aspect of the present invention, there may be mentioned a protein consisting of a modified amino acid sequence in which [1´], [2´], [3´], [4´], [5´], [6´] and [7´] are introduced into the amino acid sequence of the mutant protein (B).

[0120] The protein according to one aspect of the present invention is composed of a modified amino acid sequence in which at least one modification selected from the group consisting of the following [1´´] to [7´´] is introduced into the amino acid sequence of a homologous protein (C) having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1, and has higher polyethylene terephthalate (PET) degradation activity than the above homologous protein (C).

[0121] [1´´] A modification in which the amino acid residue corresponding to the 103rd amino acid residue of SEQ ID NO: 1 is replaced with a lysine residue

[0122] [2´´] A modification in which the amino acid residue corresponding to the 76th amino acid residue of SEQ ID NO: 1 is replaced with a cysteine residue

[0123] [3´´] A modification in which the amino acid residue corresponding to the 144th amino acid residue of SEQ ID NO: 1 is replaced with a cysteine residue

[0124] [4´´] A modification in which the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 is replaced with a glycine residue

[0125] [5´´] A modification in which the amino acid residue corresponding to the 222nd amino acid residue of SEQ ID NO: 1 is replaced with a methionine residue

[0126] [6´´] A modification in which the amino acid residue corresponding to the 73rd amino acid residue of SEQ ID NO: 1 is replaced with a glutamine residue

[0127] [7´´] A modification in which the amino acid residue corresponding to the 203rd amino acid residue of SEQ ID NO: 1 is replaced with a threonine residue

[0128] In the present specification, a homologous protein means a protein having a structure and function similar to the underlying protein.

[0129] As a homologous protein, there may be mentioned, for example, an amino acid sequence having 80% or more, preferably 90% or more, particularly preferably 95% or more identity with the amino acid sequence of the protein of interest.

[0130] The identity between amino acid sequences and base sequences can be determined using the algorithms BLAST [Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] and FASTA [Methods Enzymol., 183, 63 (1990)] developed by Karlin and Altschul. Programs called BLASTN and BLASTX [J. Mol. Biol., 215, 403 (1990)] were developed based on this algorithm BLAST. When analyzing base sequences using BLASTN based on BLAST, the parameters are set, for example, as Score = 100 and wordlength = 12. Additionally, when analyzing amino acid sequences using BLASTX based on BLAST, the parameters are set, for example, as score = 50 and wordlength = 3. When using the BLAST and Gapped BLAST programs, the default parameters of each program are used. The specific methods of these analysis methods are well-known.

[0131] The alignment of the amino acid sequence shown in SEQ ID NO: 1 with the amino acid sequences of homologous proteins can be created using the well-known alignment program ClustalW [Nucelic Acids Research 22, 4673, (1994)]. ClustalW can be obtained from http: / / www.ebi.ac.uk / clustalw / (European Bioinformatics Institute). Regarding the parameters when creating an alignment using ClustalW, for example, the default values are used.

[0132] As a non-limiting specific example of the protein as one aspect of the present invention, for example, a protein composed of an amino acid sequence in which any one of the following (C1) to (C8) is introduced into the amino acid sequence of a homologous protein (C) having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1 can be cited.

[0133] (C1) The above [1´´]

[0134] (C2) The above [1´´] and [2´´]

[0135] (C3) The above [1´´] and [3´´]

[0136] (C4) The above [1´´], [2´´] and [3´´]

[0137] (C5) The above [1´´], [2´´], [3´´] and [4´´]

[0138] (C6) The above [1´´], [2´´], [3´´], [4´´] and [5´´]

[0139] (C7) The above [1´´], [2´´], [3´´], [4´´], [5´´] and [6´´]

[0140] (C8) The above [1´´], [2´´], [3´´], [4´´], [5´´], [6´´] and [7´´]

[0141] In addition, as a non-limiting specific embodiment of the protein according to one aspect of the present invention, there may be mentioned a protein composed of an amino acid sequence in which at least the modification of [1´´] has been introduced into the amino acid sequence of the above-mentioned homologous protein (C), and optionally at least one modification of [2´´] to [7´´] has been introduced.

[0142] As another non-limiting specific embodiment of the protein according to one aspect of the present invention, there may be mentioned a protein composed of an amino acid sequence in which at least the modifications of [1´´] and [2´´] have been introduced into the amino acid sequence of the above-mentioned homologous protein (C), and optionally at least one modification of [3´´] to [7´´] has been introduced.

[0143] As another non-limiting specific embodiment of the protein according to one aspect of the present invention, there may be mentioned a protein composed of an amino acid sequence in which at least the modifications of [1´´] and [3´´] have been introduced into the amino acid sequence of the above-mentioned homologous protein (C), and optionally at least one modification of [2´´] and [4´´] to [7´´] has been introduced.

[0144] As another non-limiting specific embodiment of the protein according to one aspect of the present invention, there may be mentioned a protein composed of an amino acid sequence in which at least the modifications of [1´´], [2´´] and [3´´] have been introduced into the amino acid sequence of the above-mentioned homologous protein (C), and optionally at least one modification of [4´´] to [7´´] has been introduced.

[0145] As another non-limiting specific embodiment of the protein according to one aspect of the present invention, there may be mentioned a protein composed of an amino acid sequence in which at least the modifications of [1´´], [2´´], [3´´] and [4´´] have been introduced into the amino acid sequence of the above-mentioned homologous protein (C), and optionally at least one modification of [5´´] to [7´´] has been introduced.

[0146] As another non-limiting specific embodiment of the protein according to one aspect of the present invention, there may be mentioned a protein composed of an amino acid sequence in which at least the modifications of [1´´], [2´´], [3´´], [4´´] and [5´´] have been introduced into the amino acid sequence of the above-mentioned homologous protein (C), and optionally at least one modification of [6´´] and [7´´] has been introduced.

[0147] As another non-limiting specific embodiment of the protein according to one aspect of the present invention, there may be mentioned a protein composed of an amino acid sequence in which modifications of [1´´], [2´´], [3´´], [4´´], [5´´] and [6´´] are introduced into the amino acid sequence of the above-mentioned homologous protein (C), and optionally a modification of [7´´] is also introduced.

[0148] As another non-limiting specific embodiment of the protein according to one aspect of the present invention, there may be mentioned a protein composed of an amino acid sequence in which modifications of [1´´], [2´´], [3´´], [4´´], [5´´], [6´´] and [7´´] are introduced into the amino acid sequence of the above-mentioned homologous protein (C).

[0149] The protein according to one aspect of the present invention can decompose the main chain of PET. Therefore, the protein according to one aspect of the present invention can decompose bis(2-hydroxyethyl) terephthalate (hereinafter also referred to as "BHET"), which is an intermediate product of PET decomposition, into mono(2-hydroxyethyl) terephthalate (hereinafter also referred to as "MHET"). Furthermore, it can decompose MHET into terephthalic acid (hereinafter also referred to as "TPA") and ethylene glycol (hereinafter also referred to as "EG"). That is, the protein according to one aspect of the present invention can hydrolyze PET or BHET, which is a partial structure of PET, as a substrate to generate MHET, and further generate TPA and EG.

[0150] The PET decomposition activity of the protein can be confirmed as follows: For example, a reaction solution containing the protein and PET film fragments is prepared, an enzyme reaction is carried out, and the amounts of TPA and MHET generated after the reaction is completed are measured. If the total amount of TPA and MHET generated from the reaction solution containing the protein according to one aspect of the present invention and PET film fragments is greater than the total amount of TPA and MHET generated from the reaction solution containing the protein of the comparative object and PET film fragments, it can be said that the protein according to one aspect of the present invention has a higher PET decomposition activity than the protein of the comparative object.

[0151] The amounts of TPA and MHET generated can be measured using high performance liquid chromatography (hereinafter also referred to as "HPLC").

[0152] The protein according to one aspect of the present invention is also referred to as the mutant PET2 of the present embodiment.

[0153] 2. DNA

[0154] As the DNA according to one aspect of the present invention, there may be mentioned DNA encoding the protein according to one aspect of the present invention described in the above 1.

[0155] 3. Transformant

[0156] As a transformant obtained by DNA transformation of a protein as a mode of the present invention, a transformant obtained by transforming a host cell with a recombinant DNA containing the DNA of 2. above by a known method can be mentioned. As the host cell, any of prokaryotes, yeasts, animal cells, insect cells, plant cells, etc. can be used, and prokaryotes such as bacteria are preferably mentioned, and microorganisms belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc. are more preferably mentioned.

[0157] 4. Preparation of DNA of a mode of the present invention

[0158] As the DNA of a mode of the present invention, the DNA (a) encoding a protein composed of an amino acid sequence in which at least one modified amino acid sequence selected from the group consisting of the above [1] to [7] is introduced into the amino acid sequence shown in SEQ ID NO: 1 can be obtained by the following method.

[0159] The above DNA (a) can be obtained by using the DNA encoding the protein composed of the amino acid sequence shown in SEQ ID NO: 1 and using, for example, the site-directed mutagenesis methods described in Molecular Cloning 3rd Edition and Current Protocols in Molecular Biology, etc. to replace the base sequence of the part encoding the amino acid residue before replacement with the base sequence encoding the amino acid residue after replacement at at least one modification position selected from the group consisting of the above [1] to [7] described in 1. above.

[0160] In addition, as the DNA of a mode of the present invention, the DNA (b) encoding a protein composed of an amino acid sequence in which at least one modified amino acid sequence selected from the group consisting of the above [1´] to [7´] is introduced into the amino acid sequence of a modified mutant protein (B) composed of at least one of deletion, substitution, insertion, and addition of 1 to 20 amino acid residues relative to the amino acid sequence shown in SEQ ID NO: 1 and having higher PET-decomposing activity than the above mutant protein (B) can be obtained by the following method.

[0161] The above DNA (b) can be obtained by using the DNA encoding the modified mutant protein (B) composed of at least one of deletion, substitution, insertion, and addition of 1 to 20 amino acid residues in the amino acid sequence shown in SEQ ID NO: 1, aligning the amino acid sequence shown in SEQ ID NO: 1 and the above mutant protein (B) by the method described in 1. above, and by the site-directed mutagenesis method, replacing the base sequence of the part encoding the amino acid residue before replacement with the base sequence encoding the amino acid residue after replacement at at least one modification position selected from the group consisting of the above [1´] to [7´] described in 1. above in the amino acid sequence of the above mutant protein (B).

[0162] The above DNA (b) can also be obtained by the following method: Using DNA encoding a protein composed of an amino acid sequence in which at least one modified amino acid selected from the group consisting of the above [1] to [7] is introduced into the amino acid sequence shown in SEQ ID NO: 1, introducing a mutation into the base sequence of the part encoding the amino acid residue in such a way that at least one of deletion, substitution, insertion, and addition of 1 to 20 amino acid residues at positions other than the modified site is performed.

[0163] The above DNA (b) can also be obtained by the following method: Using DNA encoding a protein composed of the amino acid sequence shown in SEQ ID NO: 1, introducing a mutation into the base sequence of the part encoding the modified position in such a way that at least one of the modifications selected from the group consisting of the above [1´] to [7´] and at least one of deletion, substitution, insertion, and addition of 1 to 20 amino acid residues at positions other than the modified position are performed.

[0164] In the above obtaining method, primers designed in such a way that the desired modification can be introduced can be used.

[0165] In addition, DNA (c) encoding a protein composed of an amino acid sequence in which at least one modified amino acid selected from the group consisting of the above [1´´] to [7´´] is introduced into a homologous protein (C) having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1 and having higher PET-degrading activity than the above homologous protein (C), which is a DNA of one embodiment of the present invention, can be obtained by the following method.

[0166] Regarding the above DNA (c), DNA encoding a homologous protein (C) having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1 can be used, and the amino acid sequence shown in SEQ ID NO: 1 and the above homologous protein (C) are aligned by the method described in the above 1., and the base sequence of the part encoding the amino acid residue before substitution is replaced with the base sequence encoding the amino acid residue after substitution at at least one of the modified positions selected from the group consisting of the above [1´´] to [7´´] described in the above 1. in the amino acid sequence of the above homologous protein (C) by site-directed mutagenesis.

[0167] The above DNA (c) can be obtained by using DNA encoding a protein consisting of an amino acid sequence modified by introducing at least one modification selected from the group consisting of the above [1] to [7] into the amino acid sequence shown in SEQ ID NO: 1, and introducing a modification into the base sequence of the portion other than the position encoding the modification site. At this time, the modification site is designed such that the amino acid sequence other than at least one modification site selected from the group consisting of the above [1] to [7] has 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1.

[0168] In addition, the DNA of one aspect of the present invention can be obtained by using DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, introducing at least one modification selected from the group consisting of the above [1´´] to [7´´], and introducing a modification into the base sequence of the portion other than the position encoding the modification site. At this time, the modification site is designed such that the amino acid sequence other than at least one modification site selected from the group consisting of the above [1´´] to [7´´] has 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1.

[0169] In the above obtaining method, primers designed in such a manner as to be able to introduce the desired modification can be used.

[0170] 5. Production of a transformant of one aspect of the present invention

[0171] Examples of the transformant of one aspect of the present invention include a transformant obtained by introducing a recombinant DNA containing the DNA of one aspect of the present invention into a host cell, and a transformant obtained by introducing a recombinant DNA obtained by integrating the DNA of one aspect of the present invention into a vector DNA into a host cell.

[0172] Examples of the vector for integrating the DNA of the present invention include pET28a (manufactured by Sigma-Aldrich), pBluescriptII KS(+)(manufactured by Stratagene), pDIRECT [Nucleic Acids Res., 18, 6069(1990)], pCR-Script Amp SK(+)(manufactured by Stratagene), pT7Blue (manufactured by Novogene), pCRII (manufactured by Invitrogen), pCR-TRAP (manufactured by GeneHunter), pQE-60 (manufactured by Qiagen), and the like.

[0173] Examples of the host cell include microorganisms belonging to the genus Escherichia. Examples of microorganisms belonging to the genus Escherichia include Escherichia coli SHuffle T7 Express Competent, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli DH5α, Escherichia coli MC1000, Escherichia coli ATCC 12435, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No. 49, Escherichia coli W3110, Escherichia coli NY49, Escherichia coli MP347, Escherichia coli NM522, Escherichia coli BL21, Escherichia coli ME8415, and the like.

[0174] As a method for introducing the recombinant DNA, any method can be used as long as it is a method for introducing DNA into the above host cell. Examples include the method using calcium ions [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)], the protoplast method (Japanese Patent Laid-Open No. 63-248394), the electroporation method [Nucleic Acids Res., 16, 6127 (1988)], and the like.

[0175] 6. Method for producing the protein according to one embodiment of the present invention

[0176] (1) Production of the transformant for producing the protein according to one embodiment of the present invention

[0177] Based on the DNA according to one embodiment of the present invention, a DNA fragment of an appropriate length containing a portion encoding the protein according to one embodiment of the present invention is prepared as needed. In addition, by substituting the base sequence of the portion encoding the protein with codons that are most suitable for expression in the host, a transformant with improved production efficiency of the protein can be obtained.

[0178] By introducing the recombinant DNA into a host cell suitable for the expression vector, a transformant for producing the protein according to one embodiment of the present invention can be obtained.

[0179] As the host cell, any cell capable of expressing the target gene can be used, such as bacteria, yeast, animal cells, insect cells, plant cells, and the like.

[0180] As the expression vector, an expression vector that can replicate autonomously in the above host cell or can be integrated into the chromosome and contains a promoter at a position where the DNA of the present invention can be transcribed can be used.

[0181] When using prokaryotes such as bacteria as host cells, the recombinant DNA having the DNA of the present invention is preferably a recombinant DNA capable of autonomous replication in prokaryotes and composed of a promoter, a ribosome binding sequence, the DNA of the present invention, and a transcription termination sequence, and may contain a gene for controlling the promoter.

[0182] As expression vectors, pET28a (manufactured by Sigma-Aldrich), pColdI (manufactured by Takara Bio Inc.), pCDF-1b, pRSF-1b (both manufactured by Novogene), pMAL-c2x (manufactured by New England Biolabs), pGEX-4T-1 (manufactured by GE Healthcare Life Sciences), pTrcHis (manufactured by Invitrogen), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-30 (manufactured by Qiagen), pQE-60 (manufactured by Qiagen), pET-3 (manufactured by Novogene), pKYP10 (Japanese Patent Laid-Open 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 coli JM109 / pTrS30 (FERM BP-5407)], pTrS32 [prepared from Escherichia coli 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 Laid-Open No. 63-233798), pGHA2 [prepared from Escherichia coli IGHA2 (FERM B-400), Japanese Patent Laid-Open No. 60-221091], pGKA2 [prepared from Escherichia coli IGKA2 (FERM BP-6798), Japanese Patent Laid-Open No. 60-221091], pTerm2 (U.S. Patent No. 4686191, U.S. Patent No. 4939094, U.S. Patent No. 5160735), pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol., 172, 2392 (1990)], pCG1 (Japanese Patent Laid-Open No. 57-134500), pCG2 (Japanese Patent Laid-Open No. 58-35197), pCG4 (Japanese Patent Laid-Open No. 57-183799), pCG11 (Japanese Patent Laid-Open No. 57-134500), pCG116, pCE54, pCB101 (all see Japanese Patent Laid-Open No. 58-105999), pRI109 (International Publication No. 00 / 044886), pCE51, pCE52, pCE53 [all see Molecular and General Genetics, 196, 175 (1984)] and the like.

[0183] As the promoter, any promoter can be used as long as it functions in host cells such as Escherichia coli. Examples include promoters derived from Escherichia coli, phages, etc., such as trp promoter (Ptrp), lac promoter (Plac), PL promoter, PR promoter, PSE promoter, SPO1 promoter, SPO2 promoter, penP promoter, etc. In addition, artificially designed and modified promoters such as the promoter formed by two Ptrps in series (Ptrp × 2), tac promoter, lacT7 promoter, let I promoter, etc. can also be used.

[0184] Preferably, a plasmid in which the distance between the Shine-Dalgarno sequence as the ribosome binding sequence and the start codon is adjusted to an appropriate distance (for example, 6 to 18 bases) is used. In the expression of the DNA of one embodiment of the present invention, a transcription termination sequence is not necessarily required, but it is preferable to arrange a transcription termination sequence immediately downstream of the structural gene in the recombinant DNA.

[0185] Examples of prokaryotes include microorganisms belonging to the genera Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc. Examples include Escherichia coli SHuffle T7 Express Competent, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli DH5α, Escherichia coli NM522, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No. 49, Escherichia coli W3110, Escherichia coli NY49, Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillus amyloliquefaciens, Brevibacterium immariophilum ATCC14068, Brevibacterium saccharolyticum ATCC14066, Corynebacterium ammoniagenes, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum ATCC13869, Corynebacterium acetoacidophilum ATCC13870, Microbacterium ammoniaphilum ATCC15354, Pseudomonas sp. D-0110, etc.Preferably, microorganisms belonging to the genus Escherichia or Corynebacterium are exemplified, and more preferably Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli DH5α, Escherichia coli MC1000, Escherichia coli MM294, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No. 49, Escherichia coli W3110, Escherichia coli NY49, Escherichia coli GI698, Corynebacterium ammoniagenes ATCC6872, Corynebacterium ammoniagenes ATCC21170, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC21171, etc. are exemplified.

[0186] As a method for introducing the recombinant vector, any method can be used as long as it is a method for introducing DNA into the above host cells. Examples include the method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], the protoplast method (Japanese Patent Laid-Open No. 57-186492, Japanese Patent Laid-Open No. 57-18649), the electroporation method [e.g., the methods described in Journal of Bacteriology, 175, 4096 (1993), Appl. Microbiol. Biotechnol., 52, 541 (1999)], Gene, 17, 107 (1982), and Molecular & General Genetics, 168, 111 (1979), etc.

[0187] When using a yeast strain as the host cell, as the expression vector, for example, YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, pHS15, etc. can be used.

[0188] As the promoter, any promoter that functions in the yeast strain can be used. Examples include promoters such as the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock polypeptide promoter, MFα1 promoter, CUP 1 promoter, etc.

[0189] Examples of the host cell include yeast strains belonging to the genera Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Schwanniomyces, Pichia, Candida, etc. Specifically, examples include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichia pastoris, Candida utilis, etc.

[0190] As a method for introducing recombinant DNA, any method can be used as long as it is a method for introducing DNA into yeast. Examples include electroporation (Methods Enzymol., 194, 182 (1990)), the protoplast method (Proc. Natl. Acad. Sci., USA, 81, 4889 (1984)), the lithium acetate method (J. Bacteriol., 153, 163 (1983)), etc.

[0191] (2) Method for producing the protein of one embodiment of the present invention

[0192] The transformant obtained by the method described in the above (1) is cultured in a medium, and the protein of one embodiment of the present invention is generated and accumulated in the culture, and then collected from the culture, whereby the protein can be produced.

[0193] The host of the above transformant used for producing the protein of one embodiment of the present invention can be any one of prokaryotes, yeast, animal cells, insect cells, plant cells, etc. Examples of microorganisms belonging to prokaryotes such as bacteria are preferred, and microorganisms belonging to the genera Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas are more preferred.

[0194] When expressed using yeast, animal cells, insect cells or plant cells, a protein added with sugar or sugar chains can be obtained.

[0195] The method for culturing the above transformant in a medium can be carried out according to the conventional methods for culturing the host.

[0196] As a medium for culturing transformants obtained using prokaryotes such as Escherichia coli or eukaryotes such as yeast as hosts, either a natural medium or a synthetic medium can be used as long as it contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the organism and enables efficient culturing of the transformants.

[0197] As the carbon source, any carbon source that can be assimilated by the organism can be used, such as carbohydrates like glucose, fructose, sucrose, molasses containing these, starch or starch hydrolyzates; organic acids such as acetic acid and propionic acid; and alcohols such as ethanol and propanol.

[0198] As the nitrogen source, ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented bacterial cells and their digests, etc. can be used.

[0199] As the inorganic salts, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, etc. can be used.

[0200] Culturing is usually carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culturing temperature is preferably 15 - 40°C, and the culturing time is usually 5 hours to 7 days. During culturing, the pH is maintained at 3.0 - 11. The pH is adjusted using inorganic or organic acid or base solutions, urea, calcium carbonate, ammonia, etc.

[0201] In addition, during culturing, antibiotics such as kanamycin, ampicillin, and tetracycline can be added to the medium as needed.

[0202] When culturing a microorganism transformed with an expression vector using an inducible promoter as the promoter, an inducer can be added to the medium as needed. For example, when culturing a microorganism transformed with an expression vector using the lac promoter, isopropyl-β-D-thiogalactopyranoside, etc. can be added to the medium, and when culturing a microorganism transformed with an expression vector using the trp promoter, indoleacrylic acid, etc. can be added to the medium.

[0203] As a method for producing a protein according to one aspect of the present invention, there are a method of producing it inside the host cell, a method of secreting it outside the host cell, or a method of producing it on the outer membrane of the host cell, and depending on the selected method, the structure of the produced protein can be changed.

[0204] When producing the protein of one embodiment of the present invention inside a host cell or on the outer membrane of a host cell, the protein can be actively secreted outside the host cell by following the methods of Paulson et al. [J. Biol. Chem., 264, 17619 (1989)], Luo et al. [Proc. Natl. Acad. Sci., USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)] or the methods described in Japanese Patent Laid-Open No. 05-336963, International Publication No. 94 / 23021, etc.

[0205] That is, by using genetic recombination methods to produce the protein of one embodiment of the present invention with a signal peptide added in front of the protein containing the active site of the protein, the protein can be actively secreted outside the host cell.

[0206] In addition, based on the method described in Japanese Patent Laid-Open No. 2-227075, the yield can also be increased by using a gene amplification system using a dihydrofolate reductase gene or the like.

[0207] To isolate and purify the protein produced by the transformant of the present invention, conventional methods for isolating and purifying enzymes can be used.

[0208] For example, when the protein of one embodiment of the present invention is expressed in a soluble state inside the cell, after the cultivation is completed, the cells are recovered by centrifugation, suspended in an aqueous buffer, and then the cells are disrupted using an ultrasonic crusher, a French press, a MANTON-GAULIN homogenizer, a Dyno mill, etc. to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, conventional methods for isolating and purifying enzymes, namely, solvent extraction, salting-out using ammonium sulfate or the like, desalting, precipitation using an organic solvent, anion exchange chromatography using resins such as diethylaminoethyl (DEAE)-agarose, DIAION (registered trademark) HPA-75 (manufactured by Mitsubishi Chemical Corporation), cation exchange chromatography using resins such as S-Sepharose FF (manufactured by Amersham Biosciences), hydrophobic chromatography using resins such as butyl agarose and phenyl agarose, gel filtration using a molecular sieve, affinity chromatography, chromatofocusing, electrophoresis methods such as isoelectric focusing electrophoresis, etc., can be used alone or in combination to obtain a purified preparation.

[0209] In addition, when expressing the protein by forming an insoluble body in the cell, after collecting the cells, they are disrupted and centrifuged in the same manner, and thus the insoluble body of the protein is recovered in the form of a precipitate fraction. The recovered insoluble body of the protein is solubilized with a protein denaturant. The solubilized solution is diluted or dialyzed to reduce the concentration of the protein denaturant in the solubilized solution, whereby the protein restores its normal three-dimensional structure. After this operation, a purified preparation of the protein can be obtained by the same isolation and purification method as described above.

[0210] When the protein of one embodiment of the present invention or a derivative thereof such as a protein having a sugar chain added thereto is secreted outside the cell, the protein or the derivative of the protein can be recovered from the culture supernatant. That is, the culture supernatant can be obtained by treating the culture using the same methods such as centrifugation as described above, and a purified preparation can be obtained from the culture supernatant by using the same isolation and purification method as described above.

[0211] Examples of the protein thus obtained include the protein of one embodiment of the present invention.

[0212] In addition, the protein of one embodiment of the present invention can also be produced in the form of a fusion protein with another protein and purified by affinity chromatography using a substance having an affinity for the fused protein. For example, based on the methods described by Luo et al. [Proc. Natl. Acad. Sci., USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)], Japanese Patent Laid-Open No. 5-336963, and International Publication No. 94 / 23021, the protein of one embodiment of the present invention can be produced in the form of a fusion protein with protein A and purified by affinity chromatography using immunoglobulin G.

[0213] In addition, the protein of one embodiment of the present invention can also be produced in the form of a fusion protein with a Flag peptide and purified by affinity chromatography using an anti-Flag antibody [Proc. Natl. Acad. Sci., USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)], or the protein of one embodiment of the present invention can be produced in the form of a fusion protein with polyhistidine and purified by affinity chromatography using a metal coordination resin having a high affinity for polyhistidine. In addition, it can also be purified by affinity chromatography using an antibody against the protein itself.

[0214] The protein of one embodiment of the present invention can be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethoxycarbonyl method) and the tBoc method (tert-butyloxycarbonyl method) based on the amino acid sequence information of the protein obtained above. In addition, chemical synthesis can also be performed using peptide synthesizers of Advanced ChemTech, PerkinElmer, Pharmacia, Protein Technology Instrument, Synthecell-Vega, Applied Biosystems, Shimadzu Corporation, etc.

[0215] 7. Methods for decomposing PET

[0216] The method for decomposing PET according to one embodiment of the present invention may include a method using the protein according to one embodiment of the present invention. The method for decomposing PET according to one embodiment of the present invention preferably includes the step of decomposing PET in a reaction solution containing the protein according to one embodiment of the present invention, PET, magnesium chloride, and sodium carbonate, but is not limited thereto.

[0217] When decomposing PET, the concentration of the protein of one embodiment of the present invention in the reaction solution is preferably 1 to 100 μg / mL, more preferably 5 to 20 μg / mL, from the viewpoint of PET decomposition efficiency, but is not limited to this concentration and can be appropriately set depending on the amount of PET to be decomposed, etc.

[0218] When decomposing PET, the concentration of magnesium chloride in the reaction solution is preferably 0.05 to 10 mM, more preferably 0.1 mM to 5 mM. Magnesium chloride contributes to the stabilization of the protein in 1. above. When it is 0.05 mM or more, it has the advantage of enhancing the heat resistance of the protein, and when it is 10 mM or less, it has the advantage of not easily reducing the binding ability of the protein to the substrate.

[0219] From the viewpoint of the heat resistance of the protein in 1. above or the PET decomposition activity, the reaction temperature is preferably 40 to 70°C, more preferably 55 to 65°C.

[0220] From the viewpoint of the PET decomposition activity of the protein in 1. above, the pH during the reaction is preferably 6 to 11, more preferably 8.5 to 9.5.

[0221] The reaction time can be appropriately set depending on the amount of PET to be decomposed, etc., and is preferably 12 to 48 hours. When the treatment is performed for a long time, the protein described in 1. above may be added periodically.

[0222] When the protein of one embodiment of the present invention is used to decompose PET, the form of the PET to be decomposed is not limited, and examples thereof include fiber, granular, flake, pellet, film, block, and bottle. In addition, a mixture of these may also be used.

[0223] The protein according to one embodiment of the present invention can process waste such as PET bottles. The protein according to one embodiment of the present invention can decompose PET and use the decomposition products for recycling. Furthermore, by using the protein according to one embodiment of the present invention, surface modification of PET processed products such as PET films, surface modification of PET fibers, cleaning of clothing using PET fibers, cleaning of PET resins for recycling, etc. can also be performed.

[0224] 8. Method for manufacturing at least one of TPA and MHET

[0225] As a method for manufacturing at least one of TPA and MHET according to one embodiment of the present invention, a method including a step of decomposing PET using the protein according to one embodiment of the present invention can be cited. The method for manufacturing at least one of TPA and MHET according to one embodiment of the present invention preferably includes a step of decomposing PET in a reaction solution containing the protein according to one embodiment of the present invention, PET, magnesium chloride, and sodium carbonate, but is not limited thereto.

[0226] The protein according to one embodiment of the present invention can decompose the main chain of PET. Therefore, the protein according to one embodiment of the present invention can decompose BHET, an intermediate product of PET decomposition, into MHET, and further decompose MHET into TPA and EG. That is, the protein according to one embodiment of the present invention can hydrolyze using BHET, which is PET or a partial structure of PET, as a substrate to generate MHET, and further generate TPA and EG.

[0227] Regarding the concentration of the protein according to one embodiment of the present invention in the reaction solution, the concentration of magnesium chloride in the reaction solution when decomposing PET, the reaction temperature, the pH during the reaction, the reaction time, and the form of PET to be decomposed, they are as described in the above 7.

[0228] [Analysis Example]

[0229] In the examples, the analysis and quantification of TPA and MHET were performed through the steps shown below.

[0230] The solution after the enzyme reaction was centrifuged to recover the supernatant. The TPA and MHET contained in the supernatant were analyzed using HPLC (manufactured by Shimadzu Corporation).

[0231] [Analysis Conditions]

[0232] Column: YMC-Triart C18 / S-5μm / 12nm 150×4.6 mm (manufactured by YMC Co., Ltd.)

[0233] Column temperature: 25°C

[0234] Mobile phase: 0.1% formic acid, 20% acetonitrile (v / v)

[0235] Flow rate: 0.8 mL / min

[0236] Detection wavelength: 260 nm

[0237] Example

[0238] The following examples specifically illustrate the present invention, but the present invention is not limited by the following examples as long as it does not exceed its gist.

[0239] [Example 1] Preparation of mutant PET2 expression strain

[0240] (1) Preparation of wild-type PET2 expression plasmid

[0241] Using the DNA (SEQ ID NO: 2) composed of the base sequence of the gene encoding the amino acid sequence (SEQ ID NO: 1) of wild-type PET2 described in Non-Patent Document 2 as a template, and using the DNA composed of the base sequences shown in SEQ ID NOs: 5 and 6 as a primer set, PCR was performed to obtain a DNA fragment of PET2. The DNA shown in SEQ ID NO: 2 was prepared by artificial synthesis. The DNA fragment of wild-type PET2 obtained above and the expression vector pET28a (manufactured by Sigma-Aldrich) were ligated using an In-Fusion HD cloning kit (manufactured by Takara Bio Inc.), thereby obtaining the wild-type PET2 expression plasmid pET28a-PET2.

[0242] (2) Preparation of mutant PET2 expression plasmid of the present embodiment - 1

[0243] Using the plasmid pET28a-PET2 obtained in (1) above as a template, and using the DNA composed of the base sequences shown in SEQ ID NOs: 7 and 8 as a primer set, PCR was performed to prepare the expression plasmid pET28a-L103K of the mutant PET2 of the present embodiment in which the 103rd L-leucine residue in the amino acid sequence of PET2 shown in SEQ ID NO: 1 was replaced with an L-lysine residue.

[0244] Then, using pET28a-L103K obtained above as a template, and using the DNA composed of the base sequences shown in SEQ ID NOs: 9 and 10 as a primer set, PCR was performed to prepare the expression plasmid pET28a-L103K / G76C of the mutant PET2 of the present embodiment in which the 103rd L-leucine residue in the amino acid sequence of PET2 was replaced with an L-lysine residue and the 76th L-glycine residue was replaced with an L-cysteine residue.

[0245] Similarly, using the plasmid obtained through the previous operations as a template, PCR was performed using DNA composed of the base sequences corresponding to the respective sequence numbers shown in Table 1 as a primer set, thereby successively producing expression plasmids of the mutant PET2 of the present embodiment in which mutations were added to the amino acid sequence of PET2. The respective mutation points added and the sequence numbers of the corresponding primer sets are shown in Table 1.

[0246] [Table 1]

[0247]

[0248] (3) Preparation of the mutant PET2 expression plasmid of the present embodiment - 2

[0249] Using the mutant PET2 expression plasmid (pET28a-L103K / G76C / A144C / Q134G / A222M) of the present embodiment in which all the mutations shown in Table 1 were added as a template, DNA composed of the base sequences shown in sequence numbers 5 and 6 was used as a primer set, and error-prone PCR was performed using a Diversify PCR Random Mutagenesis Kit (manufactured by Takara Bio Inc.). As a result, a DNA fragment of the mutant PET2 of the present embodiment with a newly added mutation point L73Q (substituting the 73rd L-leucine residue with an L-glutamine residue) was obtained.

[0250] The obtained DNA fragment and the expression vector pET28a were ligated using an In-Fusion HD Cloning Kit, thereby producing an expression plasmid of the mutant PET2 of the present embodiment in which mutations of L103K, G76C, A144C, Q134G, A222M, and L73Q were added to the amino acid sequence of PET2.

[0251] Then, using the plasmid (pET28a-L103K / G76C / A144C / Q134G / A222M / L73Q) obtained above as a template, DNA composed of the base sequences shown in sequence numbers 5 and 6 was used as a primer set, and error-prone PCR was performed in the same manner as above to obtain a DNA fragment of the mutant PET2 of the present embodiment with a newly added mutation point I203T (substituting the 203rd L-isoleucine residue with an L-threonine residue). The obtained DNA fragment and the expression vector pET28a were ligated, thereby producing an expression plasmid of the mutant PET2 of the present embodiment in which mutations of L103K, G76C, A144C, Q134G, A222M, L73Q, and I203T were added to the amino acid sequence of PET2.

[0252] (4) Preparation of the comparative mutant PET2 expression plasmid

[0253] Using the plasmid pET28a-PET2 obtained in (1) above as a template, and using DNA composed of the base sequences shown in SEQ ID NOs: 17 and 18 as a primer set, PCR was performed to thereby prepare an expression plasmid pET28a-W254F of a comparative mutant PET2 in which the L-tryptophan residue at position 254 in the amino acid sequence of PET2 shown in SEQ ID NO: 1 was replaced with an L-phenylalanine residue.

[0254] In addition, using the plasmid pET28a-PET2 obtained in (1) above as a template, and using DNA composed of the base sequences shown in SEQ ID NOs: 19 and 20 as a primer set, PCR was performed to thereby prepare an expression plasmid pET28a-N257D of a comparative mutant PET2 in which the L-asparagine residue at position 257 in the amino acid sequence of PET2 shown in SEQ ID NO: 1 was replaced with an L-aspartic acid residue.

[0255] In addition, using the plasmid pET28a-PET2 obtained in (1) above as a template, and using DNA composed of the base sequences shown in SEQ ID NOs: 21 and 22 as a primer set, PCR was performed to thereby prepare an expression plasmid pET28a-L103D of a comparative mutant PET2 in which the L-leucine residue at position 103 in the amino acid sequence of PET2 shown in SEQ ID NO: 1 was replaced with an L-aspartic acid residue.

[0256] [Table 2]

[0257]

[0258] (5) Preparation of transformants having various PET2 expression plasmids

[0259] Each of the plasmids prepared in (1) to (4) was separately transformed into SHuffle T7 Express Competent E. coli (manufactured by New England BioLabs) to obtain a total of 12 transformants (PET2 strain, L103K strain, L103K / G76C strain, L103K / A144C strain, L103K / G76C / A144C strain, L103K / G76C / A144C / Q134G strain, L103K / G76C / A144C / Q134G / A222M strain, L103K / G76C / A144C / Q134G / A222M / L73Q strain, L103K / G76C / A144C / Q134G / A222M / L73Q / I203T strain, W254F strain, N257D strain, and L103D strain).

[0260] [Example 2] Evaluation of each mutation point by measuring the PET degradation activity of various PET2 purified enzymes

[0261] (1) Obtaining various PET2 purification enzymes

[0262] A total of 12 transformants obtained in Example 1(5) were cultured on an LB plate at 30 °C for 24 hours, inoculated into a large test tube containing 5 mL of LB medium supplemented with 100 mg / L kanamycin, and cultured with shaking at 30 °C for 20 hours. Thereafter, 0.6 mL of the obtained culture solution was transferred to a flask containing 30 mL of LB medium supplemented with 100 mg / L kanamycin, and cultured with shaking at 37 °C. After the OD600 of the cells reached the range of 0.4 - 0.6, isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 0.5 mM, and then cultured with shaking for another 20 hours. After the culture was completed, the culture solution was centrifuged to remove the supernatant, and the cells were recovered.

[0263] From the obtained cells, a crude protein extract was obtained by a conventional method, and various PET2 enzyme solutions were recovered from this extract using TALON metal affinity resin (manufactured by Takara Bio Inc.). The enzyme solution was diluted with 100 mM sodium carbonate buffer (pH 9.2) and concentrated and desalted using Amicon Ultra-4 (manufactured by Merck Millipore) to obtain various PET2 purification enzymes.

[0264] (2) Measurement of PET decomposition activity

[0265] The PET decomposition activities of various PET2 purification enzymes were measured by the following enzyme reaction, and the effects brought about by introducing each mutation point were compared.

[0266] A reaction solution of 0.5 mL containing 5 μg of various PET2 purification enzymes, 100 mM sodium carbonate buffer (pH 9.2), 1 mM or 10 mM magnesium chloride, and 0.01 g of PET film fragments (A-PET manufactured by Minelon Kasei Kogyo Co., Ltd.) was prepared, and an enzyme reaction was carried out at 60 °C for 24 hours. After the reaction was completed, TPA and MHET generated by PET decomposition were analyzed by HPLC. The mutation points of various PET2 purification enzymes, the concentration (mM) of magnesium chloride (MgCl2) in the reaction solution, the concentrations (mM) of TPA and MHET generated by the enzyme reaction of various PET2 purification enzymes, and the total value (mM) of these are shown in Table 3.

[0267] [Table 3]

[0268]

[0269] As shown in Table 3, compared with the purified enzyme of wild-type PET2 and the purified enzyme of the comparative mutant PET2, for the purified enzyme of any mutant PET2 of the present embodiment, the concentration amounts of TPA and MHET generated by the enzymatic reaction and the total value thereof (hereinafter referred to as the decomposition amount) are all increased, indicating that the PET decomposition activity is improved by introducing the mutation points. In particular, for the purified enzyme into which the mutation point L73Q is introduced, compared with the purified enzyme into which this mutation is not introduced, the decomposition amount is increased by 6.7 mM, indicating that this mutation point is very helpful for improving the PET decomposition activity. Similarly, for the purified enzyme into which the mutation point Q134G is introduced, compared with the purified enzyme into which this mutation is not introduced, the decomposition amount is increased by 5.7 mM, indicating that this mutation point is very helpful for improving the PET decomposition activity. In addition, regarding the mutation point A144C, by introducing it in combination with the mutation point G76C, the decomposition amount is increased by 2.5 mM, indicating that the combination of the mutation points A144C and G76C is very helpful for improving the PET decomposition activity.

[0270] Furthermore, compared with the case where the magnesium chloride concentration in the reaction solution is 10 mM, the decomposition amount is more in the case of 1 mM, indicating that it is a concentration suitable for decomposing PET using the mutant PET2 of the present embodiment.

[0271] [Example 3] Comparison of PET decomposition activities brought by the reported mutant PET2 and the mutant PET2 of the present embodiment

[0272] (1) Preparation of the expression plasmid of the reported mutant PET2

[0273] As the reported mutant PET2, using the DNA (SEQ ID NO: 4) composed of the base sequence of the gene encoding the mutant PET2 (SEQ ID NO: 3) described in Non-Patent Document 3 (hereinafter referred to as PET2s) as a template, and using the DNA composed of the base sequences shown in SEQ ID NOs: 23 and 24 as a primer set, PCR was performed to obtain the DNA fragment of PET2s. The DNA shown in SEQ ID NO: 4 was prepared by artificial synthesis.

[0274] The DNA fragment of PET2s obtained above and the expression vector pET28a were ligated using the In-Fusion HD Cloning Kit, thereby obtaining the expression plasmid pET28a-PET2s of PET2s.

[0275] (2) Preparation of the microorganism having the PET2s expression plasmid

[0276] The plasmid prepared in (1) was transformed into SHuffle T7 Express Competent E. Coli to obtain the PET2s strain.

[0277] (3) Obtaining of the PET2s purified enzyme

[0278] The transformants obtained in (2) were cultured on an LB plate at 30 °C for 24 hours, inoculated into a large test tube containing 5 mL of LB medium supplemented with 100 mg / L kanamycin, and cultured with shaking at 30 °C for 20 hours. Then, 0.6 mL of the obtained culture solution was inoculated into a flask containing 30 mL of LB medium supplemented with 100 mg / L kanamycin, and cultured with shaking at 37 °C. After the OD600 of the cells reached the range of 0.4 - 0.6, IPTG was added at a final concentration of 0.5 mM, and further cultured with shaking for 20 hours. After the culture was completed, the culture solution was centrifuged to remove the supernatant, and the cells were recovered.

[0279] From the obtained cells, a crude protein extract was obtained by a conventional method, and the PET2s enzyme solution was recovered from this extract using TALON (registered trademark) metal affinity resin (manufactured by Takara Bio Inc.). The enzyme solution was diluted with 100 mM sodium carbonate buffer (pH 9.2), and concentrated and desalted using Amicon (registered trademark) Ultra- (manufactured by Merck Millipore) to obtain purified PET2s enzyme.

[0280] (4) Evaluation of PET degradation activity

[0281] The PET degradation activities of the purified PET2s enzyme and the four mutant PET2 purified enzymes of this embodiment obtained in Example 1 were measured by the following enzyme reaction.

[0282] A reaction solution of 0.5 mL containing 5 μg of each purified enzyme, 100 mM sodium carbonate buffer (pH 9.2), 10 mM magnesium chloride, and 0.01 g of PET membrane fragments was prepared, and an enzyme reaction was carried out at 60 °C for 24 hours. After the reaction was completed, TPA and MHET generated by PET degradation were analyzed by HPLC. The mutation sites of various PET2 purified enzymes, the concentrations (mM) of TPA and MHET generated by the enzyme reactions of each purified enzyme, and the total value (mM) (degradation amount) of these are shown in Table 4.

[0283] [Table 4]

[0284]

[0285] As shown in Table 4, for the purified enzymes of the mutant PET2 of the present embodiment into which the mutations of L103K, G76C, A144C, and Q134G are introduced, the purified enzymes of the mutant PET2 of the present embodiment into which the mutations of L103K, G76C, A144C, Q134G, and A222M are introduced, the purified enzymes of the mutant PET2 of the present embodiment into which the mutations of L103K, G76C, A144C, Q134G, A222M, and L73Q are introduced, and the purified enzymes of the mutant PET2 of the present embodiment into which the mutations of L103K, G76C, A144C, Q134G, A222M, L73Q, and I203T are introduced, the decomposition amounts are all more than those of PET2s, indicating high PET decomposition activity.

[0286] The above has described various embodiments, but it goes without saying that the present invention is not limited to the examples. It is obvious that those skilled in the art can conceive of various modification examples or correction examples within the scope described in the claims, and they of course also belong to the technical scope of the present invention. In addition, the constituent elements in the above embodiments can be arbitrarily combined without departing from the gist of the invention.

[0287] It should be noted that this application is based on the Japanese patent application filed on December 9, 2022 (Japanese Patent Application No. 2022-197409), and its content is incorporated herein by reference.

[0288] Industrial Applicability

[0289] The protein according to one embodiment of the present invention has higher PET decomposition activity compared to a protein without the substitution by containing an amino acid residue substitution at a specific position.

[0290] Sequence Listing Free Text

[0291] Sequence No. 1: Amino acid sequence of PET2

[0292] Sequence No. 2: Base sequence of PET2

[0293] Sequence No. 3: Amino acid sequence of PET2s

[0294] Sequence No. 4: Base sequence of PET2s

[0295] Sequence No. 5: Base sequence of primer Fw for amplifying PET2 fragment

[0296] Sequence No. 6: Base sequence of primer Rv for amplifying PET2 fragment

[0297] Sequence No. 7: Base sequence of primer Fw for amplifying L103K fragment

[0298] Serial number 8: Base sequence of primer Rv for amplifying L103K fragment

[0299] Serial number 9: Base sequence of primer Fw for amplifying G76C fragment

[0300] Serial number 10: Base sequence of primer Rv for amplifying G76C fragment

[0301] Serial number 11: Base sequence of primer Fw for amplifying A144C fragment

[0302] Serial number 12: Base sequence of primer Rv for amplifying A144C fragment

[0303] Serial number 13: Base sequence of primer Fw for amplifying Q134G fragment

[0304] Serial number 14: Base sequence of primer Rv for amplifying Q134G fragment

[0305] Serial number 15: Base sequence of primer Fw for amplifying A222M fragment

[0306] Serial number 16: Base sequence of primer Rv for amplifying A222M fragment

[0307] Serial number 17: Base sequence of primer Fw for amplifying W254F fragment

[0308] Serial number 18: Base sequence of primer Rv for amplifying W254F fragment

[0309] Serial number 19: Base sequence of primer Fw for amplifying N257D fragment

[0310] Serial number 20: Base sequence of primer Rv for amplifying N257D fragment

[0311] Serial number 21: Base sequence of primer Fw for amplifying L103D fragment

[0312] Serial number 22: Base sequence of primer Rv for amplifying L103D fragment

[0313] Serial number 23: Base sequence of primer Fw for amplifying PET2s fragment

[0314] Serial number 24: Base sequence of primer Rv for amplifying PET2s fragment

Claims

1. A protein, which is composed of a modified amino acid sequence into which at least one modification selected from the group consisting of the following [1] to [7] has been introduced in the amino acid sequence shown in SEQ ID NO: 1, [1] A modification in which the amino acid residue at position 103 is replaced with a lysine residue; [2] A modification in which the amino acid residue at position 76 is replaced with a cysteine residue; [3] A modification in which the amino acid residue at position 144 is replaced with a cysteine residue; [4] A modification in which the amino acid residue at position 134 is replaced with a glycine residue; [5] A modification in which the amino acid residue at position 222 is replaced with a methionine residue; [6] A modification in which the amino acid residue at position 73 is replaced with a glutamine residue; [7] A modification in which the amino acid residue at position 203 is replaced with a threonine residue.

2. A protein, which is composed of a modified amino acid sequence into which at least one modification selected from the group consisting of the following [1´] to [7´] has been introduced in the amino acid sequence of a modified mutant protein (B) having at least one of deletion, substitution, insertion, and addition of 1 to 20 amino acid residues relative to the amino acid sequence shown in SEQ ID NO: 1, and has higher polyethylene terephthalate (PET) decomposition activity than the mutant protein (B), [1´] A modification in which the amino acid residue corresponding to the amino acid residue at position 103 of SEQ ID NO: 1 is replaced with a lysine residue; [2´] A modification in which the amino acid residue corresponding to the amino acid residue at position 76 of SEQ ID NO: 1 is replaced with a cysteine residue; [3´] A modification in which the amino acid residue corresponding to the amino acid residue at position 144 of SEQ ID NO: 1 is replaced with a cysteine residue; [4´] A modification in which the amino acid residue corresponding to the amino acid residue at position 134 of SEQ ID NO: 1 is replaced with a glycine residue; [5´] A modification in which the amino acid residue corresponding to the amino acid residue at position 222 of SEQ ID NO: 1 is replaced with a methionine residue; [6´] A modification in which the amino acid residue corresponding to the amino acid residue at position 73 of SEQ ID NO: 1 is replaced with a glutamine residue; [7´] A modification in which the amino acid residue corresponding to the amino acid residue at position 203 of SEQ ID NO: 1 is replaced with a threonine residue.

3. A protein, which is composed of a modified amino acid sequence into which at least one modification selected from the group consisting of the following [1´´] to [7´´] has been introduced in the amino acid sequence of a homologous protein (C) having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1, and has higher polyethylene terephthalate (PET) decomposition activity than the homologous protein (C), [1´´] A modification in which the amino acid residue corresponding to the amino acid residue at position 103 of SEQ ID NO: 1 is replaced with a lysine residue; [2´´] A modification in which the amino acid residue corresponding to the amino acid residue at position 76 of SEQ ID NO: 1 is replaced with a cysteine residue; [3´´] A modification in which the amino acid residue corresponding to the amino acid residue at position 144 of SEQ ID NO: 1 is replaced with a cysteine residue; [4´´]Modification of replacing the amino acid residue corresponding to the 134th amino acid residue of SEQ ID NO: 1 with a glycine residue; [5´´]Modification of replacing the amino acid residue corresponding to the 222nd amino acid residue of SEQ ID NO: 1 with a methionine residue; [6´´]Modification of replacing the amino acid residue corresponding to the 73rd amino acid residue of SEQ ID NO: 1 with a glutamine residue; [7´´]Modification of replacing the amino acid residue corresponding to the 203rd amino acid residue of SEQ ID NO: 1 with a threonine residue.

4. A DNA encoding the protein according to any one of claims 1 to 3.

5. A recombinant DNA containing the DNA according to claim 4.

6. A transformant obtained by transforming a host cell with the recombinant DNA according to claim 5.

7. A method for decomposing PET, which uses the protein according to any one of claims 1 to 3 to decompose PET.

8. The method for decomposing PET according to claim 7, which comprises the following step: decomposing PET in a reaction solution containing the protein according to any one of claims 1 to 3, PET, magnesium chloride and sodium carbonate.

9. The method for decomposing PET according to claim 8, wherein, The concentration of magnesium chloride in the reaction solution is 0.1 mM to 5 mM.

10. A method for producing at least one of terephthalic acid (TPA) and mono - 2 - hydroxyethyl terephthalate (MHET), which comprises the step of decomposing PET using the protein according to any one of claims 1 to 3.

11. The method for producing at least one of TPA and MHET according to claim 10, which comprises the following step: decomposing PET in a reaction solution containing the protein according to any one of claims 1 to 3, PET, magnesium chloride and sodium carbonate.

12. The method for manufacturing at least one of TPA and MHET according to claim 11, wherein, The concentration of magnesium chloride in the reaction solution is 0.1 mM to 5 mM.

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