Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/44—Polycarboxylic acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/62—Carboxylic acid esters
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y301/00—Hydrolases acting on ester bonds (3.1)
  • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
  • Y02W30/00—Technologies for solid waste management
  • Y02W30/50—Reuse, recycling or recovery technologies
  • Y02W30/62—Plastics recycling; Rubber recycling

Definitions

• the present invention relates to a protein having polyethylene terephthalate decomposition activity, DNA encoding the protein, a transformant obtained by transforming a host cell with the DNA, a method for decomposing polyethylene terephthalate, and a method for producing at least one of terephthalic acid and monohydroxyethyl terephthalate.
• PET Polyethylene terephthalate
• mechanical recycling cannot fully remove impurities, so there is a problem that the uses of PET after decomposition are limited.
• chemical recycling makes it easy to remove impurities because PET can be decomposed and repolymerized on a monomer basis, and it is also attracting attention as a sustainable recycling technology.
• PET decomposition method using enzymes can be said to be an environmentally friendly recycling method because the reaction proceeds under mild conditions and no organic solvents are used.
• PETase derived from Ideonella sakaiensis (Patent Document 1) and Cutinase derived from Thermobifida fusca (Patent Document 2) have been discovered as PET decomposition enzymes.
• LC-Cutinase derived from dead leaf compost metagenome has high decomposition activity and heat resistance (Patent Document 3), and it has been reported that enzyme modification can decompose 97% or more of PET within 24 hours (Non-Patent Document 1).
• Non-Patent Document 2 In addition, in a report on searching for PET decomposition enzymes from metagenomes, PET2, which has a relatively high heat resistance among enzymes, was found, and it is known that it can decompose PET, albeit in small amounts (Non-Patent Document 2). In addition, there are reports that modification of PET2 improves decomposition activity and heat resistance (Non-Patent Document 3).
• Patent Documents 1 and 2 have been researched and developed but have not yet been put to practical use. Furthermore, the inventors' investigations have shown that all of them have low decomposition activity and low heat resistance. Furthermore, the inventors' investigations have shown that even when the enzymes described in Patent Document 3 and Non-Patent Documents 1 to 3 are used, it takes a long time to decompose PET through an enzymatic reaction, so there is a need for the development of a PET-degrading enzyme with higher activity for industrial applications.
• the present invention aims to provide a protein with high PET decomposition activity.
• a protein having an amino acid sequence in which at least one modification has been introduced in which an amino acid residue at a specific position in the amino acid sequence represented by SEQ ID NO:1 is replaced with a specific amino acid residue, has a higher PET degradation activity than a protein having the amino acid sequence represented by SEQ ID NO:1, and thus completed the present invention.
• the present invention is as follows. ⁇ 1> A protein consisting of the amino acid sequence represented by SEQ ID NO: 1, wherein at least one modification selected from the group consisting of the following [1] to [7] has been introduced: [1] A modification in which the 103rd amino acid residue is replaced with a lysine residue; [2] A modification in which the 76th amino acid residue is replaced with a cysteine residue; [3] A modification in which the 144th amino acid residue is replaced with a cysteine residue; [4] A modification in which the 134th amino acid residue is replaced with a glycine residue; [5] A modification in which the 222nd amino acid residue is replaced with a methionine residue; [6] A modification in which the 73rd amino acid residue is replaced with a glutamine residue; [7] A modification in which the 203rd amino acid residue is replaced with a threonine residue.
• PET polyethylene terephthalate
• ⁇ 4> A DNA encoding the protein according to any one of ⁇ 1> to ⁇ 3>.
• ⁇ 5> A recombinant DNA comprising the DNA according to ⁇ 4>.
• ⁇ 6> A transformant obtained by transforming a host cell with the recombinant DNA according to ⁇ 5>.
• ⁇ 7> A method for decomposing PET, using the protein according to any one of ⁇ 1> to ⁇ 3>.
• ⁇ 8> A method for decomposing PET according to ⁇ 7>, comprising decomposing PET in a reaction solution containing the protein according to any one of ⁇ 1> to ⁇ 3>, PET, magnesium chloride, and sodium carbonate.
• ⁇ 9> The method for decomposing PET according to ⁇ 8>, wherein the concentration of magnesium chloride in the reaction solution is 0.1 mM to 5 mM.
• TPA terephthalic acid
• MHET monohydroxyethyl terephthalate
• ⁇ 11> A method for producing at least one of TPA and MHET according to ⁇ 10>, comprising decomposing PET in a reaction solution containing the protein according to any one of ⁇ 1> to ⁇ 3>, PET, magnesium chloride and sodium carbonate.
• the protein according to one embodiment of the present invention contains amino acid residue substitutions at specific sites, and thus has higher PET degradation activity than a protein that does not have those substitutions.
• the present invention will be described in detail below, but these are merely examples of preferred embodiments and the present invention is not limited to these contents.
• the numerical range “to” includes the numerical values before and after it. For example, "0% by mass to 100% by mass” means a range from 0% by mass or more to 100% by mass or less.
• the protein of one embodiment of the present invention has an amino acid sequence represented by SEQ ID NO: 1, in which at least one modification selected from the group consisting of the following [1] to [7] has been introduced: [1] A modification in which the 103rd amino acid residue is replaced with a lysine residue; [2] A modification in which the 76th amino acid residue is replaced with a cysteine residue; [3] A modification in which the 144th amino acid residue is replaced with a cysteine residue; [4] A modification in which the 134th amino acid residue is replaced with a glycine residue; [5] A modification in which the 222nd amino acid residue is replaced with a methionine residue; [6] A modification in which the 73rd amino acid residue is replaced with a glutamine residue; [7] A modification in which the 203rd amino acid residue is replaced with a threonine residue.
• the protein consisting of the amino acid sequence represented by SEQ ID NO:1 is the PET2 enzyme described in Non-Patent Document 1.
• at least one modification selected from the group consisting of [1] to [7] into the amino acid sequence represented by SEQ ID NO: 1 to form a protein at least one of heat resistance and substrate binding ability is improved, and it is believed that the protein will exhibit higher PET decomposition activity than protein (A) having the amino acid sequence represented by SEQ ID NO: 1.
• Non-limiting specific examples of the protein of one aspect of the present invention include a protein having an amino acid sequence represented by SEQ ID NO:1 into which any of the following (A1) to (A7) has been introduced: (A1) The above [1] (A2) The above [1] and [2] (A3) The above [1] and [3] (A4) The above [1], [2] and [3] (A5) The above [1], [2], [3] and [4] (A6) The above [1], [2], [3], [4] and [5] (A7) The above [1], [2], [3], [4], [5] and [6] (A8) The above [1], [2], [3], [4], [5], [6] and [7]
• a non-limiting specific example of the protein of one aspect of the present invention includes a protein consisting of an amino acid sequence represented by SEQ ID NO:1 into which at least the modification [1] has been introduced, and further, optionally, at least one of the modifications [2] to [7] has been introduced.
• Other non-limiting specific embodiments of the protein of one aspect of the present invention include a protein consisting of an amino acid sequence represented by SEQ ID NO:1 into which at least the modifications [1] and [2] have been introduced, and further, optionally, at least one of the modifications [3] to [7] has been introduced.
• non-limiting specific embodiments of the protein of one aspect of the present invention include a protein consisting of an amino acid sequence represented by SEQ ID NO:1 into which at least the modifications [1] and [3] have been introduced, and further, optionally, at least one of the modifications [2] and [4] to [7] has been introduced.
• Other non-limiting specific embodiments of the protein of one aspect of the present invention include a protein consisting of an amino acid sequence represented by SEQ ID NO:1 into which at least the modifications [1], [2], and [3] have been introduced, and further, optionally, at least one of the modifications [4] to [7] has been introduced.
• non-limiting specific embodiments of the protein of one aspect of the present invention include a protein consisting of an amino acid sequence represented by SEQ ID NO:1 into which at least the modifications [1], [2], [3], and [4] have been introduced, and further, optionally, at least one of the modifications [5] to [7] has been introduced.
• Other non-limiting specific embodiments of the protein of one aspect of the present invention include a protein consisting of an amino acid sequence represented by SEQ ID NO: 1 into which at least the modifications [1], [2], [3], [4], and [5] have been introduced, and further, optionally, at least one of the modifications [6] and [7] has been introduced.
• non-limiting specific embodiments of the protein of one aspect of the present invention include a protein consisting of an amino acid sequence represented by SEQ ID NO: 1 into which at least the modifications [1], [2], [3], [4], [5] and [6] have been introduced, and further, optionally, the modification [7] has been introduced.
• Other non-limiting specific embodiments of the protein of one aspect of the present invention include a protein consisting of an amino acid sequence represented by SEQ ID NO:1 into which modifications [1], [2], [3], [4], [5], [6], and [7] have been introduced.
• One embodiment of the protein of the present invention comprises an amino acid sequence in which at least one modification selected from the group consisting of [1'] to [7'] below has been introduced into the amino acid sequence of mutant protein (B), which has at least one modification consisting of deletion, substitution, insertion, and addition of 1 to 20 amino acid residues relative to the amino acid sequence represented by SEQ ID NO: 1, and has a higher PET degradation activity than the above-mentioned mutant protein (B).
• [1'] An alteration in which the amino acid residue corresponding to the 103rd amino acid residue of SEQ ID NO:1 is replaced with a lysine residue.
• a mutant protein refers to a protein obtained by artificially deleting or substituting amino acid residues in a base protein, or by inserting or adding amino acid residues into the base protein.
• the amino acid modification consisting of at least one of deletion, substitution, insertion, and addition may mean that 1 to 20 amino acids may be modified by at least one of deletion, substitution, insertion, and addition at any position in the amino acid sequence represented by SEQ ID NO:1.
• the number of amino acids modified by at least one of deletion, substitution, insertion, and addition is 1 to 20, preferably 1 to 10, more preferably 1 to 8, and most preferably 1 to 5.
• the amino acids that are modified by at least one of deletion, substitution, insertion, and addition may be natural or non-natural.
• Natural amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and L-cysteine.
• amino acids in the same group can be substituted for each other.
• Group A leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine
• Group B aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid
• Group C asparagine, glutamine
• D lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid
• Group E proline, 3-hydroxyproline, 4-hydroxyproline
• Group F serine, threonine, homoserine
• Group G phenylalanine, tyros
• a non-limiting specific example of the protein of one aspect of the present invention is a protein having an amino acid sequence in which any of the following (B1) to (B8) has been introduced into the amino acid sequence of mutant protein (B), which has at least one modification consisting of deletion, substitution, insertion, and addition of 1 to 20 amino acid residues relative to the amino acid sequence represented by SEQ ID NO: 1: (B1) The above [1'] (B2) The above [1'] and [2'] (B3) The above [1'] and [3'] (B4) The above [1'], [2'] and [3'] (B5) The above [1'], [2'], [3'] and [4'] (B6) The above [1'], [2'], [3'], [4'] and [5'] (B7) The above [1'], [2'], [3'], [4'], [5'] and [6'] (B8) The above [1'], [2'], [3'], [4'], [5'] and [6
• a non-limiting specific embodiment of a protein according to one aspect of the present invention includes a protein having an amino acid sequence in which at least modification [1'] has been introduced into the amino acid sequence of the above-mentioned mutant protein (B), and further, optionally, at least one modification [2'] to [7'] has been introduced.
• Other non-limiting specific embodiments of the protein of one aspect of the present invention include a protein having an amino acid sequence in which at least modifications [1'] and [2'] have been introduced into the amino acid sequence of the above-mentioned mutant protein (B), and further, optionally, at least one modification [3'] to [7'] has been introduced.
• non-limiting specific embodiments of the protein of one aspect of the present invention include a protein having an amino acid sequence in which at least modifications [1'] and [3'] have been introduced into the amino acid sequence of the above-mentioned mutant protein (B), and further, optionally, at least one of modifications [2'] and [4'] to [7'] has been introduced.
• Other non-limiting specific embodiments of the protein of one aspect of the present invention include a protein having an amino acid sequence in which at least modifications [1'], [2'], and [3'] have been introduced into the amino acid sequence of the above-mentioned mutant protein (B), and further, optionally, at least one modification [4'] to [7'] has been introduced.
• non-limiting specific embodiments of the protein of one aspect of the present invention include a protein having an amino acid sequence in which at least modifications [1'], [2'], [3'] and [4'] have been introduced into the amino acid sequence of the above-mentioned mutant protein (B), and further, optionally, at least one modification [5'] to [7'] has been introduced.
• Other non-limiting specific embodiments of the protein of one aspect of the present invention include a protein having an amino acid sequence in which at least modifications [1'], [2'], [3'], [4'] and [5'] have been introduced into the amino acid sequence of the above-mentioned mutant protein (B), and further, optionally, at least one modification [6'] and [7'] has been introduced.
• non-limiting specific embodiments of the protein of one aspect of the present invention include a protein having an amino acid sequence in which at least modifications [1'], [2'], [3'], [4'], [5'] and [6'] have been introduced into the amino acid sequence of the above-mentioned mutant protein (B), and further, optionally, a modification [7'] has been introduced.
• Other non-limiting specific embodiments of the protein of one aspect of the present invention include a protein having an amino acid sequence in which modifications [1'], [2'], [3'], [4'], [5'], [6'] and [7'] have been introduced into the amino acid sequence of the above-mentioned mutant protein (B).
• a protein according to one embodiment of the present invention comprises an amino acid sequence in which at least one modification selected from the group consisting of [1′′] to [7′′] below has been introduced into the amino acid sequence of a homologous protein (C) having 80% or more identity to the amino acid sequence represented by SEQ ID NO:1, and has a higher polyethylene terephthalate (PET) decomposition activity than the homologous protein (C).
• C homologous protein
• PET polyethylene terephthalate
• homologous protein refers to a protein that is similar in structure and function to the original protein.
• homologous proteins include amino acid sequences that have an identity of 80% or more, preferably 90% or more, and particularly preferably 95% or more, to the amino acid sequence of the target protein.
• the alignment of the amino acid sequence represented by SEQ ID NO:1 with the amino acid sequence of a homologous protein can be created using the well-known alignment program ClustalW [Nucelic Acids Research 22, 4673, (1994)].
• ClustalW is available from http://www.ebi.ac.uk/clustalw/ (European Bioinformatics Institute).
• ClustalW is available from http://www.ebi.ac.uk/clustalw/ (European Bioinformatics Institute).
• Non-limiting specific examples of the protein of one aspect of the present invention include a protein having an amino acid sequence in which any of the following (C1) to (C8) has been introduced into the amino acid sequence of a homologous protein (C) having 80% or more identity with the amino acid sequence represented by SEQ ID NO:1: (C1) The above [1 ''] (C2) The above [1′′] and [2′′] (C3) The above [1′′] and [3′′] (C4) The above [1′′], [2′′] and [3′′] (C5) The above [1′′], [2′′], [3′′] and [4′′] (C6) The above [1′′], [2′′], [3′′], [4′′] and [5′′] (C7) The above [1′′], [2′′], [3′′], [4′′], [5′′] and [6′′] (C8) The above [1′′], [2′′], [3′′], [4′′], [5′′], [6′′] and [7′′]
• a non-limiting specific embodiment of the protein of one aspect of the present invention includes a protein having an amino acid sequence in which at least the modification [1′′] has been introduced into the amino acid sequence of the above-mentioned homologous protein (C), and further, optionally, at least one of the modifications [2′′] to [7′′] has been introduced.
• Other non-limiting specific embodiments of the protein of one aspect of the present invention include a protein having an amino acid sequence in which at least the modifications [1′′] and [2′′] have been introduced into the amino acid sequence of the above-mentioned homologous protein (C), and further, optionally, at least one of the modifications [3′′] to [7′′] has been introduced.
• non-limiting specific embodiments of the protein of one aspect of the present invention include a protein having an amino acid sequence in which at least the modifications [1′′] and [3′′] have been introduced into the amino acid sequence of the above-mentioned homologous protein (C), and further, optionally, at least one of the modifications [2′′] and [4′′] to [7′′] has been introduced.
• Other non-limiting specific embodiments of the protein of one aspect of the present invention include a protein having an amino acid sequence in which at least the modifications [1′′], [2′′], and [3′′] have been introduced into the amino acid sequence of the above-mentioned homologous protein (C), and further, optionally, at least one of the modifications [4′′] to [7′′] has been introduced.
• non-limiting specific embodiments of the protein of one aspect of the present invention include a protein having an amino acid sequence in which at least the modifications [1′′], [2′′], [3′′] and [4′′] have been introduced into the amino acid sequence of the above-mentioned homologous protein (C), and further, optionally, at least one of the modifications [5′′] to [7′′] has been introduced.
• non-limiting specific embodiments of the protein of one aspect of the present invention include a protein having an amino acid sequence in which at least the modifications [1′′], [2′′], [3′′], [4′′] and [5′′] have been introduced into the amino acid sequence of the above-mentioned homologous protein (C), and further, optionally, at least one of the modifications [6′′] and [7′′] has been introduced.
• non-limiting specific embodiments of the protein of one aspect of the present invention include a protein having an amino acid sequence in which at least the modifications [1′′], [2′′], [3′′], [4′′], [5′′] and [6′′] have been introduced into the amino acid sequence of the above-mentioned homologous protein (C), and further, optionally, the modification [7′′] has been introduced.
• Other non-limiting specific embodiments of the protein of one aspect of the present invention include a protein having an amino acid sequence in which modifications [1′′], [2′′], [3′′], [4′′], [5′′], [6′′] and [7′′] have been introduced into the amino acid sequence of the above-mentioned homologous protein (C).
• the protein of one embodiment of the present invention can degrade the main chain of PET.
• the protein of one embodiment of the present invention can degrade bis(2-hydroxyethyl)terephthalate (hereinafter also referred to as "BHET”), an intermediate product of PET degradation, into monohydroxyethyl terephthalate (hereinafter also referred to as "MHET”), and can further degrade MHET into terephthalic acid (hereinafter also referred to as "TPA”) and ethylene glycol (hereinafter also referred to as "EG”).
• BHET bis(2-hydroxyethyl)terephthalate
• MHET monohydroxyethyl terephthalate
• TPA terephthalic acid
• EG ethylene glycol
• the protein of one embodiment of the present invention can hydrolyze PET or BHET, a partial structure of PET, as a substrate to produce MHET, and further produce TPA and EG.
• the PET decomposition activity of a protein can be confirmed, for example, by preparing a reaction solution containing the protein and a PET film fragment, carrying out an enzymatic reaction, and measuring the amounts of TPA and MHET produced after the reaction is completed. If the total amount of TPA and MHET produced from a reaction solution containing the protein of one embodiment of the present invention and a PET film fragment is greater than the total amount of TPA and MHET produced from a reaction solution containing a comparative protein and a PET film fragment, it can be said that the protein of one embodiment of the present invention has a higher PET decomposition activity than the comparative protein.
• the amounts of TPA and MHET produced can be measured by high performance liquid chromatography (hereinafter also referred to as "HPLC").
• the protein of one aspect of the present invention is also referred to as the mutant PET2 of this embodiment.
• DNA An example of the DNA according to one embodiment of the present invention is a DNA encoding the protein according to one embodiment of the present invention described in 1 above.
• Transformant An example of a transformant transformed with a DNA encoding a protein according to one embodiment of the present invention is a transformant obtained by transforming a host cell by a known method using a recombinant DNA containing the DNA described in 2.
• the host cell may be any of prokaryotes, yeast, animal cells, insect cells, plant cells, etc., but is preferably a prokaryote such as a bacterium, and more preferably a microorganism belonging to the genera Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc.
• DNA (a) which is a DNA of one embodiment of the present invention, encoding a protein consisting of an amino acid sequence having at least one modification selected from the group consisting of [1] to [7] above in the amino acid sequence represented by SEQ ID NO:1, can be obtained by the following method.
• the DNA (a) can be obtained by using a DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO:1, for example, by site-directed mutagenesis described in Molecular Cloning, 3rd Edition and Current Protocols in Molecular Biology, and by substituting a nucleotide sequence encoding an amino acid residue before substitution at least one modification site selected from the group consisting of [1] to [7] described in 1. above, with a nucleotide sequence encoding an amino acid residue after substitution.
• DNA (b), which is one embodiment of the present invention has an amino acid sequence in which at least one modification selected from the group consisting of [1'] to [7'] has been introduced into the amino acid sequence of mutant protein (B), which has at least one modification consisting of deletion, substitution, insertion, and addition of 1 to 20 amino acid residues relative to the amino acid sequence represented by SEQ ID NO: 1, and encodes a protein having higher PET degradation activity than the mutant protein (B), and can be obtained by the following method.
• the DNA (b) can be obtained by using a DNA encoding a mutant protein (B) having at least one of the modifications consisting of deletion, substitution, insertion, and addition of 1 to 20 amino acid residues in the amino acid sequence represented by SEQ ID NO: 1, aligning the amino acid sequence represented by SEQ ID NO: 1 with the mutant protein (B) by the method described in 1. above, and replacing the base sequence of a portion encoding the amino acid residue before substitution with a base sequence encoding the amino acid residue after substitution at at least one modification site selected from the group consisting of [1'] to [7'] described in 1. above in the amino acid sequence of the mutant protein (B) by site-directed mutagenesis.
• the above DNA (b) can also be obtained by using a DNA encoding a protein consisting of an amino acid sequence in which at least one modification selected from the group consisting of [1] to [7] has been introduced into the amino acid sequence represented by SEQ ID NO: 1, and introducing mutations into the base sequence of a portion encoding the amino acid residues such that 1 to 20 amino acid residues other than the site of the modification are modified by at least one of deletion, substitution, insertion, and addition.
• the above DNA (b) can also be obtained by using a DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO:1 and introducing a mutation into the base sequence of a portion encoding the modification site so that at least one modification selected from the group consisting of the above [1'] to [7'] and at least one modification consisting of deletion, substitution, insertion, and addition of 1 to 20 amino acid residues are made at a site other than the modification site.
• a primer designed so as to introduce the target modification can be used.
• DNA (c) which is one embodiment of the DNA of the present invention, has an amino acid sequence in which at least one modification selected from the group consisting of [1′′] to [7′′] has been introduced into the amino acid sequence of a homologous protein (C) having 80% or more identity with the amino acid sequence represented by SEQ ID NO:1, and encodes a protein having higher PET degradation activity than the homologous protein (C), and can be obtained by the following method.
• the DNA (c) can be prepared by aligning the amino acid sequence of SEQ ID NO: 1 with the homologous protein (C) by the method described in 1 above using a DNA encoding the homologous protein (C) having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1, and by site-directed mutagenesis, replacing a nucleotide sequence of a portion encoding an amino acid residue before substitution with a nucleotide sequence encoding an amino acid residue after substitution at at least one modification site selected from the group consisting of [1′′] to [7′′] described in 1 above in the amino acid sequence of the homologous protein (C).
• the DNA (c) can be obtained by introducing an alteration into the base sequence of a portion encoding a site other than the alteration site, using a DNA encoding a protein consisting of an amino acid sequence in which at least one alteration selected from the group consisting of [1] to [7] has been introduced into the amino acid sequence represented by SEQ ID NO: 1.
• the alteration site is designed so that the amino acid sequence other than the at least one alteration site selected from the group consisting of [1] to [7] has 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1.
• the DNA of one embodiment of the present invention can be obtained by using a DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO:1 and introducing at least one modification selected from the group consisting of [1′′] to [7′′] above and modifications into the base sequence of a portion encoding the other parts than the modification.
• the modification parts are designed so that the amino acid sequence other than the at least one modification part selected from the group consisting of [1′′] to [7′′] above has 80% or more identity with the amino acid sequence represented by SEQ ID NO:1.
• a primer designed so as to introduce the target modification can be used.
• Examples of the transformant according to one embodiment of the present invention include a transformant obtained by introducing a recombinant DNA containing the DNA according to one embodiment of the present invention into a host cell, and a transformant obtained by introducing a recombinant DNA obtained by incorporating the DNA according to one embodiment of the present invention into a vector DNA into a host cell.
• vectors into which the DNA of the present invention can be incorporated include pET28a (Sigma-Aldrich), pBluescriptII KS(+) (Stratagene), pDIRECT [Nucleic Acids Res., 18, 6069 (1990)], pCR-Script Amp SK(+) (Stratagene), pT7Blue (Novagen), pCR II (Invitrogen), pCR-TRAP (Gene Hunter), and pQE-60 (Qiagen).
• Host cells include microorganisms belonging to the genus Escherichia.
• microorganisms belonging to the genus Escherichia include, for example, Escherichia coli SHuffle T7 Express Competent, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli DH5 ⁇ , Escherichia coli MC1000, Escherichia coli ATCC 12435, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, and Escherichia coli No.
• Escherichia coli W3110 Escherichia coli NY49, Escherichia coli MP347, Escherichia coli NM522, Escherichia coli BL21, Escherichia coli ME8415, etc.
• Any method for introducing recombinant DNA into the host cells can be used, such as the calcium ion method [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)], the protoplast method (JP Patent Publication 63-248394), and the electroporation method [Nucleic Acids Res., 16, 6127 (1988)].
• Method for Producing a Protein of an Embodiment of the Present Invention (1) Production of a Transformant Producing a Protein of an Embodiment of the Present Invention Based on the DNA of an embodiment of the present invention, a DNA fragment of an appropriate length containing a portion encoding the protein of an embodiment of the present invention is prepared as necessary. In addition, by substituting bases in the base sequence of the portion encoding the protein so that it has optimal codons for expression in the host, a transformant with improved production efficiency of the protein can be obtained.
• the recombinant DNA can be introduced into a host cell compatible with the expression vector to obtain a transformant that produces the protein of one embodiment of the present invention.
• a host cell any cell capable of expressing the target gene can be used, including bacteria, yeast, animal cells, insect cells, plant cells, and the like.
• the expression vector used is one which is capable of autonomous replication in the above-mentioned host cells or can be integrated into a chromosome and contains a promoter at a position where the DNA of the present invention can be transcribed.
• the recombinant DNA having the DNA of the present invention is preferably capable of autonomously replicating in the prokaryote and is composed of a promoter, a ribosome binding sequence, the DNA of the present invention, and a transcription termination sequence, and may also contain a gene that controls the promoter.
• Expression vectors include pET28a (Sigma-Aldrich), pColdI (Takara Bio), pCDF-1b, pRSF-1b (both Novagen), pMAL-c2x (New England Biolabs), pGEX-4T-1 (GE Healthcare Biosciences), pTrcHis (Invitrogen), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-30 (Qiagen), and pQE-60 ( Qiagen), pET-3 (Novagen), pKYP10 (JP Patent Publication 58-110600), pKYP200 [Agric. Biol.
• the promoter may be any promoter that functions in a host cell such as E. coli.
• Examples include promoters derived from E. coli or phages, such as the trp promoter (Ptrp), lac promoter (Plac), PL promoter, PR promoter, and PSE promoter, as well as the SPO1 promoter, SPO2 promoter, and penP promoter.
• artificially designed and modified promoters such as a promoter with two Ptrp promoters in series (Ptrp x 2), the tac promoter, the lacT7 promoter, and the let I promoter can also be used.
• a transcription termination sequence is not necessarily required for the expression of the DNA of one embodiment of the present invention, but in recombinant DNA, it is preferable to place a transcription termination sequence immediately downstream of the structural gene.
• Prokaryotes include microorganisms belonging to the genera Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, and Pseudomonas, such as Escherichia coli SHuffle T7 Expr.
• Escherichia coli XL1-Blue Escherichia coli XL2-Blue
• Escherichia coli DH1 Escherichia coli DH5 ⁇
• Escherichia coli NM522 Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.
• the microorganism belongs to the genus Escherichia or Corynebacterium, more preferably Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli DH5 ⁇ , Escherichia coli MC1000, Escherichia coli MM294, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.
• Any method for introducing a recombinant vector can be used so long as it is a method for introducing DNA into the host cells, such as the method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], the protoplast method (JP Patent Publication Nos. 57-186492 and 57-18649), the electroporation method [e.g., Journal of Bacteriology, 175, 4096 (1993); Appl. Microbiol. Biotechnol., 52, 541 (1999)], and the methods described in Gene, 17, 107 (1982) and Molecular & General Genetics, 168, 111 (1979).
• YEp13 ATCC37115
• YEp24 ATCC37051
• YCp50 ATCC37419
• pHS19 pHS15, etc.
• Any promoter may be used as long as it functions in a yeast strain, and examples of such promoters include the PHO5 promoter, the PGK promoter, the GAP promoter, the ADH promoter, the gal 1 promoter, the gal 10 promoter, the heat shock polypeptide promoter, the MF ⁇ 1 promoter, and the CUP 1 promoter.
• Host cells include yeast strains belonging to the genera Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Siwanniomyces, Pichia, Candida, etc., and more specifically, examples thereof include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichia pastoris, Candida utilis, etc.
• Any method for introducing recombinant DNA into yeast can be used, such as electroporation (Methods Enzymol., 194, 182 (1990)), the spheroplast method (Proc. Natl. Acad. Sci., USA, 81, 4889 (1984)), and the lithium acetate method (J. Bacteriol., 153, 163 (1983)).
• the host for the transformant for producing the protein of one embodiment of the present invention may be any of prokaryotes, yeast, animal cells, insect cells, plant cells, etc., but is preferably a prokaryote such as a bacterium, and more preferably a microorganism belonging to the genera Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc.
• the above transformant can be cultured in a medium according to a conventional method used for culturing a host.
• a medium for culturing a transformant obtained using a prokaryote such as Escherichia coli or a eukaryote such as yeast as a host either a natural medium or a synthetic medium may be used as long as it contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the organism and allows efficient cultivation of the transformant.
• the carbon source may be any that can be assimilated by the organism, and examples of the carbon source that can be used include carbohydrates such as glucose, fructose, sucrose, molasses containing these, starch or starch hydrolysates, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol.
• carbohydrates such as glucose, fructose, sucrose, molasses containing these, starch or starch hydrolysates, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol.
• nitrogen sources examples include ammonia, ammonium salts of inorganic or organic acids such as 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 hydrolysate, soybean meal and soybean meal hydrolysate, various fermentation bacteria, and digested products thereof.
• ammonia ammonium salts of inorganic or organic acids such as 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 hydrolysate, soybean meal and soybean meal hydrolysate, various fermentation bacteria, and digested products thereof.
• Inorganic salts that can be used include potassium dihydrogen phosphate, potassium dihydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, etc.
• Cultivation is usually carried out under aerobic conditions such as shaking culture or deep aeration agitation culture.
• the culture temperature is preferably 15 to 40°C, and the culture time is usually 5 hours to 7 days.
• the pH during cultivation is maintained at 3.0 to 11.
• the pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.
• antibiotics such as kanamycin, ampicillin, tetracycline, etc. may be added to the medium, if necessary.
• an inducer may be added to the medium as necessary.
• isopropyl- ⁇ -D-thiogalactopyranoside or the like may be added to the medium when culturing a microorganism transformed with an expression vector using a lac promoter, and indoleacrylic acid or the like may be added to the medium when culturing a microorganism transformed with an expression vector using a trp promoter.
• the protein can be produced within a host cell, secreted outside the host cell, or produced on the outer membrane of the host cell.
• the structure of the protein produced can be altered depending on the method selected.
• a protein according to one embodiment of the present invention When a protein according to one embodiment of the present invention is produced inside a host cell or on the outer membrane of the host cell, the protein can be actively secreted outside the host cell by applying, mutatis mutandis, the method of Paulson et al. [J. Biol. Chem., 264, 17619 (1989)], the method of Rowe et al. [Proc. Natl. Acad. Sci., USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)], or the methods described in Japanese Patent Application Laid-Open No. 05-336963 and International Publication No. 94/23021, etc.
• the protein can be actively secreted outside the host cell. Furthermore, the production amount can be increased by utilizing a gene amplification system using a dihydrofolate reductase gene or the like, according to the method described in Japanese Patent Application Laid-Open No. 2-227075.
• a purified preparation can be obtained by using, alone or in combination, a conventional enzyme isolation and purification method, i.e., solvent extraction, salting out with ammonium sulfate or the like, desalting, precipitation with an organic solvent, anion exchange chromatography using resins such as diethylaminoethyl (DEAE)-Sepharose, 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 Sepharose and phenyl Sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, electrophoresis such as isoelectric focusing, and the like.
• a conventional enzyme isolation and purification method i.e., solvent extraction, salting out with ammonium sulfate or the like, desalting
• the cells are similarly recovered, disrupted, and centrifuged to recover the insoluble protein as a precipitate fraction.
• the recovered insoluble 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, thereby returning the protein to its normal three-dimensional structure.
• a purified sample of the protein can be obtained by the same isolation and purification method as above.
• the protein or the derivative can be recovered from the culture supernatant. That is, the culture can be treated by a method such as centrifugation similar to that described above to obtain a culture supernatant, and a purified specimen can be obtained from the culture supernatant by the same isolation and purification method as described above.
• the protein of one embodiment of the present invention may be produced as a fusion protein with another protein, and purified by affinity chromatography using a substance having affinity for the fusion protein.
• the protein of one embodiment of the present invention may be produced as a fusion protein with protein A, and purified by affinity chromatography using immunoglobulin G, in accordance with the methods described in Lowe et al. [Proc. Natl. Acad. Sci., USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)], JP-A-5-336963, and WO 94/23021.
• the protein of one embodiment of the present invention can be produced as a fusion protein with Flag peptide and purified by affinity chromatography using anti-Flag antibody [Proc. Natl. Acad. Sci., USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)], or produced as a fusion protein with polyhistidine and purified by affinity chromatography using a metal coordination resin that has high affinity for polyhistidine. Furthermore, it can be purified by affinity chromatography using an antibody against the protein itself.
• the protein of one embodiment of the present invention can be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).
• chemical synthesis can also be performed using peptide synthesizers manufactured by Advanced ChemTech, PerkinElmer, Pharmacia, Protein Technology Instrument, Synthecell-Vega, Applied Biosystems, Shimadzu Corporation, etc.
• Method for Degrading PET An example of a method for degrading PET according to an embodiment of the present invention is a method using the protein according to an embodiment of the present invention.
• the method for degrading PET according to an embodiment of the present invention preferably includes, but is not limited to, degrading PET in a reaction solution containing the protein according to an embodiment of the present invention, PET, magnesium chloride, and sodium carbonate.
• the concentration of the protein of one embodiment of the present invention in the reaction solution when decomposing PET is preferably 1 to 100 ⁇ g/mL, more preferably 5 to 20 ⁇ g/mL, from the viewpoint of the efficiency of PET decomposition, but is not limited to this concentration and can be set appropriately depending on the amount of PET to be decomposed, etc.
• the concentration of magnesium chloride in the reaction solution when decomposing PET is preferably 0.05 to 10 mM, and more preferably 0.1 to 5 mM.
• Magnesium chloride contributes to the stabilization of the protein mentioned in 1 above, and 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 that the binding ability of the protein to the substrate is less likely to decrease.
• the reaction temperature is preferably 40 to 70°C, more preferably 55 to 65°C, from the viewpoint of the heat resistance or PET decomposition activity of the protein described in 1 above.
• the pH during the reaction is preferably 6 to 11, more preferably 8.5 to 9.5, from the viewpoint of the PET decomposition activity of the protein described in 1 above.
• the reaction time can be set appropriately depending on the amount of PET to be decomposed, and is preferably 12 to 48 hours. If the treatment is carried out over a long period of time, the protein described above in 1 may be added periodically.
• PET When decomposing PET using a protein according to one embodiment of the present invention, there is no limitation on the form of the PET to be decomposed, and examples of such forms include fibrous, granular, flake, pellet, film, block, and bottle forms. Mixtures of these can also be used.
• Waste materials such as PET bottles can be treated using the protein of one embodiment of the present invention.
• PET can be decomposed using the protein of one embodiment of the present invention, and the decomposition products can be used for recycling.
• the protein of one embodiment of the present invention can also be used to modify the surface of PET processed products such as PET films, modify the surface of PET fibers, wash clothes made of PET fibers, wash PET resin to be recycled, etc.
• the method for producing at least one of TPA and MHET according to one embodiment of the present invention includes a method comprising decomposing PET using the protein according to one embodiment of the present invention.
• the method for producing at least one of TPA and MHET according to one embodiment of the present invention preferably comprises 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.
• the protein of one embodiment of the present invention can degrade the main chain of PET.
• the protein of one embodiment of the present invention can degrade BHET, an intermediate product of PET degradation, into MHET, and can further degrade MHET into TPA and EG.
• the protein of one embodiment of the present invention can hydrolyze PET or BHET, a partial structure of PET, as a substrate to produce MHET, and further produce TPA and EG.
• the concentration of the protein in the reaction solution according to one embodiment of the present invention, 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 the decomposed PET are as described in 7. above.
• Example 1 Construction of mutant PET2 expression strain (1) Construction of wild-type PET2 expression plasmid Using DNA (SEQ ID NO: 2) consisting of the base sequence of a gene encoding the amino acid sequence (SEQ ID NO: 1) of wild-type PET2 described in Non-Patent Document 2 as a template, PCR was performed using DNA consisting of the base sequences represented by SEQ ID NOs: 5 and 6 as a primer set to obtain a DNA fragment of PET2. The DNA represented by SEQ ID NO: 2 was prepared by artificial synthesis.
• the wild-type PET2 DNA fragment obtained above and the expression vector pET28a (Sigma-Aldrich) were ligated using In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain a wild-type PET2 expression plasmid pET28a-PET2.
• PCR was performed using the pET28a-L103K obtained above as a template and DNA consisting of the base sequences represented by SEQ ID NOs: 9 and 10 as a primer set to produce an expression plasmid pET28a-L103K/G76C for the mutant PET2 of this 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.
• the plasmid obtained in the previous step was used as a template, and PCR was performed using DNAs consisting of base sequences corresponding to each of the sequence numbers shown in Table 1 as a primer set to sequentially prepare expression plasmids for mutant PET2 according to this embodiment in which mutations were added to the amino acid sequence of PET2.
• Table 1 shows each added mutation point and the sequence number of the corresponding primer set.
• mutant PET2 expression plasmid according to this embodiment-2
• mutant PET2 expression plasmid pET28a-L103K/G76C/A144C/Q134G/A222M
• SEQ ID NOs: 5 and 6 DNA consisting of the base sequences represented by SEQ ID NOs: 5 and 6 as a primer set
• error-prone PCR was performed using Diversify PCR Random Mutagenesis Kit (manufactured by Takara Bio Inc.).
• a DNA fragment of mutant PET2 according to this embodiment to which a new mutation point L73Q the 73rd L-leucine residue is replaced with an L-glutamine residue
• the resulting DNA fragment was linked to the expression vector pET28a using the In-Fusion HD Cloning Kit to produce an expression plasmid for the mutant PET2 of this embodiment, which contains the following mutations in the amino acid sequence of PET2: L103K, G76C, A144C, Q134G, A222M, and L73Q.
• the obtained DNA fragment was linked to the expression vector pET28a to prepare an expression plasmid of mutant PET2 according to this embodiment with the mutations L103K, G76C, A144C, Q134G, A222M, L73Q, and I203T added to the amino acid sequence of PET2.
• an expression plasmid pET28a-N257D of a comparative mutant PET2 in which the 257th L-asparagine residue in the amino acid sequence of PET2 represented by SEQ ID NO: 1 was replaced with an L-aspartic acid residue was prepared.
• an expression plasmid pET28a-L103D of a comparative mutant PET2 in which the 103rd L-leucine residue in the amino acid sequence of PET2 represented by SEQ ID NO: 1 was replaced with an L-aspartic acid residue was prepared.
• Example 2 Evaluation of each mutation point based on measurement of PET decomposition activity of various PET2 purified enzymes (1) Acquisition of various PET2 purified enzymes 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 containing 100 mg / L of kanamycin, and cultured with shaking at 30 ° C. for 20 hours. Then, 0.6 mL of the resulting culture was transferred to a flask containing 30 mL of LB medium containing 100 mg / L of kanamycin, and cultured with shaking at 37 ° C.
• IPTG isopropyl- ⁇ -D-thiogalactopyranoside
• PET decomposition activity of various PET2 purified enzymes was measured by the following enzyme reaction, and the effects of introducing each mutation were compared.
• a reaction solution 0.5 mL
• the concentration of TPA and MHET produced by the enzyme reaction and the total value of these (hereinafter referred to as the decomposition amount) are increased compared to the purified enzyme of the wild-type PET2 and the purified enzyme of the comparative mutant PET2, and it was shown that the introduction of the mutation improved the PET decomposition activity.
• the purified enzyme into which the mutation L73Q was introduced had a decomposition amount increased by 6.7 mM compared to the purified enzyme without the mutation, indicating that the mutation greatly contributes to the improvement of the PET decomposition activity.
• the purified enzyme into which the mutation Q134G was introduced had a decomposition amount increased by 5.7 mM compared to the purified enzyme without the mutation, indicating that the mutation greatly contributes to the improvement of the PET decomposition activity.
• the decomposition amount of the mutation A144C was increased by 2.5 mM by introducing it in combination with the mutation G76C, indicating that the combination of the mutations A144C and G76C greatly contributes to the improvement of the PET decomposition activity.
• the amount of decomposition was greater when the magnesium chloride concentration in the reaction solution was 1 mM than when it was 10 mM, indicating that this is a suitable concentration for the decomposition of PET by the mutant PET2 according to this embodiment.
• Example 3 Comparison of PET degradation activity between previously reported mutant PET2 and mutant PET2 according to the present embodiment
• DNA SEQ ID NO: 4 consisting of the base sequence of a gene encoding mutant PET2 (SEQ ID NO: 3) (hereinafter referred to as PET2s) described in Non-Patent Document 3 was used as a template, and PCR was performed using DNA consisting of the base sequences represented by SEQ ID NOs: 23 and 24 as a primer set to obtain a DNA fragment of PET2s.
• the DNA represented by SEQ ID NO: 4 was prepared by artificial synthesis.
• the above-obtained DNA fragment of PET2s and the expression vector pET28a were ligated using In-Fusion HD Cloning Kit to obtain the expression plasmid pET28a-PET2s of PET2s.
• the culture solution was centrifuged to remove the supernatant, and the bacterial cells were collected.
• a crude protein extract was obtained from the obtained bacterial cells by a standard method, and a PET2s enzyme solution was recovered from the 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 a purified PET2s enzyme.
• PET decomposition activity of the PET2s purified enzyme and the four mutant PET2 purified enzymes according to this embodiment obtained in Example 1 was measured by the following enzyme reaction.
• a reaction solution of 0.5 mL consisting of 5 ⁇ g of each purified enzyme, 100 mM sodium carbonate buffer (pH 9.2), 10 mM magnesium chloride, and 0.01 g of PET film fragments was prepared, and the enzyme reaction was carried out at 60 ° C. for 24 hours. After the reaction was completed, the TPA and MHET produced by PET decomposition were analyzed by HPLC.
• the mutation points of various PET2 purified enzymes, the concentrations (mM) of TPA and MHET produced by the enzymatic reaction of each purified enzyme, and the total value (mM) of these (decomposition amount) are shown in Table 4.
• the purified enzyme of the mutant PET2 according to the present embodiment having the mutations L103K, G76C, A144C, and Q134G introduced the purified enzyme of the mutant PET2 according to the present embodiment having the mutations L103K, G76C, A144C, Q134G, and A222M introduced
• the protein according to one embodiment of the present invention contains amino acid residue substitutions at specific sites, and thus has higher PET degradation activity than a protein that does not have those substitutions.
• SEQ ID NO: 1 Amino acid sequence of PET2 SEQ ID NO: 2: Nucleotide sequence of PET2 SEQ ID NO: 3: Amino acid sequence of PET2s SEQ ID NO: 4: Nucleotide sequence of PET2s SEQ ID NO: 5: Nucleotide sequence of primer Fw for amplifying PET2 fragment SEQ ID NO: 6: Nucleotide sequence of primer Rv for amplifying PET2 fragment SEQ ID NO: 7: Nucleotide sequence of primer Fw for amplifying L103K fragment SEQ ID NO: 8: Nucleotide sequence of primer Rv for amplifying L103K fragment SEQ ID NO: 9: Nucleotide sequence of primer Fw for amplifying G76C fragment SEQ ID NO: 10: Nucleotide sequence of primer Rv for amplifying G76C fragment SEQ ID NO: 11: Nucleotide sequence of primer Fw for amplifying A144C fragment SEQ ID NO: 12: Nucleotide sequence of primer Rv for

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