Novel PET-degrading enzyme and its use

Abstract

This application relates to a polypeptide having PET-degrading activity and its use.

Description

[Technical Field]

[0001] This application relates to a mutant polypeptide having PET-degrading activity and its use. [Background technology]

[0002] More than 400 million tons of plastic are newly produced every year, and as environmental issues related to plastic waste gain attention, efforts are being made to reduce production through regulations on single-use products and the use of plastic substitutes, but in reality, production is increasing every year. PET, which accounts for less than 10% of all plastics, is newly produced at a rate of approximately 360 million tons per year and is mainly used in single-use products, making it the plastic with the shortest lifespan. Recycling of plastic waste includes mechanical recycling, pyrolysis, and chemical recycling, and each method is either commercialized or in the final stages of research for commercialization. While each technology offers a solution to the plastic waste problem, no existing method is perfect due to the impacts of downcycling, such as quality degradation, carbon neutrality, resource depletion, and eutrophication of seawater and freshwater.

[0003] To address environmental problems caused by waste plastics, such as microplastics, greenhouse gas emissions, and resource depletion, a series of research findings have been published on the decomposition of PET, one of the most common plastics, using enzymatic biological technology (Patent Document 1). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] U.S. Patent No. 10851355 [Non-patent literature]

[0005] [Non-Patent Document 1] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444 [Non-licensed document 2] Rice et al., 2000, Trends Genet. 16: 276-277 [Non-licensed document 3] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453

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[0006] This application relates to a novel polypeptide having PET (polyethylene terephthalate) decomposition activity, a method for decomposing polyesters such as PET using the polypeptide, and a method for synthesizing polyesters such as PET by recycling mono(2-hydroxymethyl) terephthalate (MHET), terephthalic acid (TPA), and / or ethylene glycol (EG) obtained by the decomposition. [Means for solving the problem]

[0007] This application aims to provide a mutant polypeptide having PET degradation activity.

[0008] Furthermore, this application aims to provide a composition containing the aforementioned mutant polypeptide.

[0009] Furthermore, this application aims to provide a polynucleotide encoding the aforementioned mutant polypeptide.

[0010] Furthermore, this application aims to provide a host cell comprising the mutant polypeptide, a polynucleotide encoding it, a nucleic acid construct comprising the polynucleotide, and / or a vector comprising the nucleotide or the nucleic acid construct.

[0011] Furthermore, this application aims to provide a method for producing mutant polypeptides having PET-degrading activity.

[0012] Furthermore, this application aims to provide a method for degrading polyester, comprising the step of treating the polyester with the mutant polypeptide, a host cell expressing the polypeptide, and / or a composition containing the mutant polypeptide.

[0013] Furthermore, this application aims to provide a method for producing mono(2-hydroxymethyl) terephthalate (MHET), terephthalic acid (TPA), and / or ethylene glycol (EG), comprising the step of contacting a polyester with the mutant polypeptide, a host cell expressing the polypeptide, and / or a composition containing the polypeptide.

[0014] Furthermore, this application aims to provide a method for producing polyester, comprising the step of synthesizing polyester using MHET, TPA and / or EG produced by the above method.

[0015] Furthermore, this application aims to provide the use of the mutant polypeptide, a host cell expressing the mutant polypeptide, and / or a composition containing the mutant polypeptide for PET degradation.

[0016] Furthermore, this application aims to provide the use of the mutant polypeptide, a host cell expressing the polypeptide, or a composition containing the mutant polypeptide for use in the reaction with polyester in the production of mono(2-hydroxymethyl) terephthalate (MHET), terephthalic acid (TPA), and / or ethylene glycol (EG). [Effects of the Invention]

[0017] By enzymatically degrading polyester bonds using the polypeptide having PET-degrading activity of this application, polyesters such as PET can be degraded in an environmentally friendly manner, and mono(2-hydroxymethyl) terephthalate (MHET), terephthalic acid (TPA), and / or ethylene glycol (EG), which are degradation products with high commercial value, can be produced and recycled for the manufacture of polyesters such as PET. [Brief explanation of the drawing]

[0018] [Figure 1]This figure shows the results of determining 10 candidate PETases and examining their PET degradation activity and Tm values. [Figure 2] This figure shows the results of confirming the PET degradation activity of CaPETase. [Modes for carrying out the invention]

[0019] One aspect of this application is a mutant polypeptide having PET-degrading activity.

[0020] As a specific example, the mutant polypeptide is i) a polypeptide having 70% or more and less than 100% sequence identity with SEQ ID NO: 1, and / or ii) a polypeptide encoded by a polynucleotide having 70% or more and less than 100% sequence identity with the sequence encoding the mature polypeptide of SEQ ID NO: 1, and / or iii) the mutant polypeptide is (a) the mature polypeptide coding sequence of SEQ ID NO: 1, (b) its cDNA, or (c) the full-length complementary sequence of (a) or (b). A polypeptide encoded by a polynucleotide that hybridizes with a complement) under low stringency, moderate stringency, upper-medium stringency, high stringency, or very high stringency conditions, and / or iv) the mutant polypeptide is a functional fragment of the polypeptide i) to iii) having PET degradation activity, and the mutant polypeptide comprises any modification selected from deletion, insertion, substitution, and / or combination thereof at at least one amino acid at positions 129, 198 and 196.

[0021] Here, the above position number corresponds to the position of polypeptide sequence number 1.

[0022] In any of the above-mentioned specific examples, the 129th amino acid before modification may be valine (V), the 198th amino acid may be arginine (R), and / or the 196th amino acid may be glycine (G).

[0023] In any of the above-described examples, the mutant polypeptide may include a substitution at at least one position selected from the group consisting of the 129th, 198th, 196th, 129th and 196th, 129th and 198th, 196th and 198th, and 129th, 196th and 198th.

[0024] Here, the above position number corresponds to the position of polypeptide sequence number 1.

[0025] In any of the above-mentioned specific examples, the mutant polypeptide may include at least one substitution selected from the group consisting of a substitution of the amino acid corresponding to the 129th position with threonine or serine, a substitution of the amino acid corresponding to the 198th position with lysine or aspartic acid, and a substitution of the amino acid corresponding to the 196th position with threonine, alanine, isoleucine, valine, or serine.

[0026] Here, the above position number corresponds to the position of polypeptide sequence number 1.

[0027] In any of the above-mentioned specific examples, the mutant polypeptide may include at least one modification selected from the group consisting of: modification of valine at position 129 to threonine or serine; modification of glycine at position 196 to threonine, alanine, isoleucine, leucine, valine or serine; modification of arginine at position 198 to lysine or aspartic acid; modification of valine at position 129 and glycine at position 196 to threonine; modification of valine at position 129 and arginine at position 198 to lysine; modification of glycine at position 196 to threonine and arginine at position 198 to lysine; modification of valine at position 129 to threonine, glycine at position 196 to threonine and arginine at position 198 to lysine.

[0028] Here, the above position number corresponds to the position of polypeptide sequence number 1.

[0029] Another aspect of this application is a composition comprising a mutant polypeptide having the PET-degrading activity.

[0030] In any of the above-mentioned specific examples, the composition may be a PET decomposition composition.

[0031] Another aspect of this application is a polynucleotide encoding the mutant polypeptide.

[0032] Another aspect of this application is a nucleic acid construct comprising the polynucleotide.

[0033] Another aspect of this application is a vector comprising the polynucleotide or the nucleic acid construct.

[0034] A further aspect of this application is a host cell comprising the mutant polypeptide, the polynucleotide, the nucleic acid construct and / or the vector.

[0035] A further aspect of this application is a method for producing a mutant polypeptide, comprising the steps of culturing the host cells and recovering the mutant polypeptide expressed in the culturing step.

[0036] A further aspect of this application is a method for degrading polyester, comprising the step of treating the polyester with the mutant polypeptide, a host cell expressing the polypeptide, and / or a composition comprising the mutant polypeptide.

[0037] In any of the above-mentioned specific examples, the polyester may be PET.

[0038] A further aspect of this application is a method for producing mono(2-hydroxymethyl) terephthalate (MHET), terephthalic acid (TPA), and / or ethylene glycol (EG), comprising the step of contacting a mutant polypeptide, a host cell expressing the polypeptide, and / or a composition comprising the polypeptide with a polyester.

[0039] In any of the above-mentioned specific examples, the polyester may be PET.

[0040] In any of the above-mentioned specific examples, the step of recovering the MHET, TPA, and / or EG produced by the above method may be further included.

[0041] Another aspect of this application is a method for producing polyester, comprising the step of synthesizing polyester using MHET, TPA and / or EG manufactured as described above.

[0042] In any of the above-mentioned specific examples, the polyester may be PET.

[0043] A further aspect of this application is the use of the mutant polypeptide, a host cell expressing the mutant polypeptide, and / or a composition comprising the mutant polypeptide for PET degradation.

[0044] A further aspect of this application is the use of the mutant polypeptide or a composition containing the mutant polypeptide for use in reaction with polyester in the production of mono(2-hydroxymethyl) terephthalate (MHET), terephthalic acid (TPA), and / or ethylene glycol (EG).

[0045] [Modes for carrying out the invention] The following describes the specific details for carrying out the invention. Note that each description and embodiment disclosed in this application applies to other descriptions and embodiments. That is, any combination of the various elements disclosed in this application is included. Furthermore, this application is not limited to the following specific descriptions.

[0046] Furthermore, any person with ordinary skill in the art would be able to recognize and confirm many equivalents of the specific embodiments of this application described herein using only ordinary experiments. Moreover, these equivalents are also intended to be included in this application.

[0047] As used in the specification and claims of this application, singular articles ("a," "an," and "the") include plural subjects unless otherwise specified. Unless otherwise specified, singular terms include plural subjects, and plural terms include singular subjects. Unless otherwise specified, in the specification and claims of this application, "or" is used to include "and / or."

[0048] In this application, "about" is used before a specific number. In this application, "about" includes not only the exact number that follows the term "about," but also a range that is approximately that number or close to it. Considering the context in which the number is used, it is possible to determine whether it is close to or approximately that specific number. For example, "about" indicates a range of -10% to +10% of a given number. Another example is that "about" indicates a range of -5% to +5% of a given number. However, it is not limited to these examples.

[0049] In this application, terms such as "first, second, third," "i), ii), iii)," and "(a), (b), (c), (d)," are used to distinguish similar configurations and do not imply that they are continuous or performed in order. For example, when the above terms are used in relation to steps of a method, use, or analysis, there may be no time interval between those steps, they may be performed simultaneously, or they may be performed with intervals of a few seconds, a few minutes, a few hours, a few days, or a few months.

[0050] In this application, "consisting essentially of" means that the presence of the unspecified component is not substantially affected by the presence of the unspecified component in the manner in which the features of the subject matter claimed by this application are claimed.

[0051] In this application, "consisting of" means that the proportion of a particular component totals 100%. The component or feature referred to as "consisting of" is either essential or mandatory. In a specific example, any other optional or non-essential component is excluded from the component or feature referred to as "consisting of".

[0052] In this application, “comprising” means that the features, steps, or components referred to by the above terms are present, and does not exclude the presence or addition of one or more features, steps, or components. While the components or features referred to as “comprising” in this application are essential or mandatory, in a specific example, other optional or non-essential components or features may be further included.

[0053] In this application, "including" is used in one specific example to mean "essentially consisting of" or "consisting of".

[0054] In this application, even if a polypeptide is described as "containing" an amino acid sequence represented by a specific sequence number, "consisting of" an amino acid sequence represented by a specific sequence number, or "having" an amino acid sequence represented by a specific sequence number, it goes without saying that proteins having amino acid sequences in which some sequences are deleted, modified, substituted, conserved substituted, or added may be used in this application as long as they have the same or equivalent activity as the polypeptide consisting of the amino acid sequence of said sequence number. For example, this includes, but is not limited to, proteins having sequences added to the N-terminus and / or C-terminus that do not alter the function of the protein, naturally occurring mutations, silent mutations, or conserved substitutions.

[0055] In this application, "protein" or "polypeptide" means a polymer or oligomer of continuous amino acid residues. In this application, "polypeptide," "protein," and "peptide" are used interchangeably with "amino acid sequence."

[0056] In some cases, an active amino acid sequence is called an "enzyme." Unless otherwise specified, amino acid sequences in this application are described in the direction from the N-terminus to the C-terminus.

[0057] With respect to cells, nucleic acids, polypeptides, or vectors, “recombinant” in this application means that cells, nucleic acids, polypeptides, or vectors are modified by the introduction of heterologous nucleic acids or polypeptides, or by alteration of native nucleic acids or polypeptides, or that cells are derived from such modified cells. For example, recombinant cells express genes that are not found in the cell’s native (non-recombinant) form, or they express genes that are not expressed at all, or they express native genes that are abnormally expressed.

[0058] In this application, “isolated” means a substance that exists in an environment that does not occur in nature, or a form that does not exist in nature. This includes substances that naturally associate in nature and are similar to those found in nature, such as sequences, enzymes, or nucleic acids, from which at least one other component has said substance (sequence, enzyme, or nucleic acid) that is at least substantially isolated.

[0059] For example, the isolated sequences, enzymes, or nucleic acids provided in this application are provided in a form that is substantially free of at least one contaminant.

[0060] Examples of separated substances include, but are not limited to, i) any non-naturally occurring substance, ii) any substance from which one, more or all naturally occurring components associated in nature have been removed (e.g., enzymes, mutants, nucleic acids, proteins, peptides, or cofactors), iii) any substance found in nature that has been artificially modified, or iv) a substance that has been modified so that its amount is different compared to other naturally associated components (e.g., an increase in the copy number of a gene encoding a particular substance, or modification of a promoter naturally linked to a gene encoding a particular substance to a more active promoter).

[0061] In this application, "wild-type" means a naturally occurring polynucleotide without artificial modification. When "wild-type" is used in reference to a polypeptide, it means a naturally occurring polypeptide that does not have artificial mutations (substitutions, insertions, deletions, etc.) at one or more amino acid positions. Similarly, when "wild-type" is used in reference to a polynucleotide, it means one that does not have artificial modifications (substitutions, insertions, deletions) at one or more nucleotides. However, polynucleotides encoding wild-type polypeptides are not limited to natural polynucleotides, but also include sequences encoding any wild-type polypeptide.

[0062] In this application, the term "parent sequence" or "backbone" refers to a reference sequence that, when modified, becomes a mutant polypeptide. That is, the parent sequence is the starting sequence, which is the target of mutations such as substitutions, insertions, and / or deletions. The parent sequence may be naturally occurring or wild-type, a variant in which one or more substitutions, insertions, or deletions have occurred in the natural or wild-type sequence, or an artificially synthesized sequence. If the parent sequence is an active amino acid sequence, i.e., an enzyme amino acid sequence, it is called the parent enzyme.

[0063] In this application, "reference sequence" refers to a sequence used to determine the position of an amino acid in any given amino acid sequence. By aligning any amino acid sequence with the reference sequence, it is possible to determine the position of an amino acid in the given amino acid sequence that corresponds to a specific position in the reference sequence.

[0064] In this application, with respect to amino acid or nucleic acid sequences, "fragment" means a part of the parent sequence. For example, it may be a polypeptide in which one or more amino acids have been removed from the C or N terminus of the parent sequence.

[0065] In this application, the "fragment" of an enzyme may also be a "functional fragment." A "functional fragment," also called an active fragment, refers to a polypeptide that is part of the parent enzyme and possesses the enzymatic activity of the parent enzyme. For example, an enzyme's functional fragment may include the enzyme's catalytic site.

[0066] An enzyme fragment contains a portion of the full length of the parent enzyme. For example, it may contain, but is not limited to, at least approximately 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or less than 100% of the amino acids of the parent enzyme's full length.

[0067] In this application, "modifying" means changing or altering. This may be a change from something that occurs naturally. For example, an enzyme can be altered in such a way that it is changed from its parent sequence or reference sequence.

[0068] The modified enzyme in this application may be an enzyme that does not exist naturally, i.e., a non-naturally occurring enzyme.

[0069] In this application, "modified" means, for example, that it has been altered from its naturally occurring form. Modified enzymes in this application include non-naturally occurring enzymes and naturally occurring variants. For example, modified enzymes in this application are modified enzymes not found in nature. For example, modified enzymes in this application do not occur spontaneously, but are not limited to that.

[0070] When the term "modification" in this application is used in relation to an amino acid / nucleic acid sequence, it includes substitution of an amino acid / nucleic acid residue of the parent sequence with a different amino acid / nucleic acid residue at at least one site of the amino acid sequence; deletion of an amino acid / nucleic acid residue (or a series of amino acid / nucleic acid residues) of the parent sequence at at least one site; insertion of an amino acid / nucleic acid residue (or a series of amino acid / nucleic acid residues) of the parent sequence at at least one site; truncation of the N-terminal and / or C-terminal amino acid sequence or the 5' and / or 3' nucleic acid sequence; and any combination thereof.

[0071] In this application, an enzyme "variant" or "modified polypeptide" means a protein that differs from the parent enzyme by at least one amino acid through conservative substitution and / or modification. The terms "variant" and "modified polypeptide" are used interchangeably. The aforementioned variant or modified polypeptide is non-naturally occurring, but is not limited to that.

[0072] The aforementioned mutant differs from the sequence of the parent enzyme by at least one modification, such as amino acid substitution, deletion, and / or insertion.

[0073] Such mutants can generally be identified by modifying at least one amino acid in the parent enzyme and evaluating the properties of the modified protein. That is, the performance of the mutant will be improved, unchanged, or decreased compared to the parent enzyme.

[0074] Furthermore, some variant polypeptides include mutant polypeptides in which at least one part, such as the N-terminal leader sequence or the transmembrane domain, has been removed.

[0075] Other variants include those in which a portion of the mature protein's N and / or C-terminus has been removed.

[0076] The terms "mutant" or "mutant polypeptide" are often used interchangeably with terms such as mutant, modified, mutated protein, and mutation (in English, these include modification, modified protein, mutant, mutein, divergent, and variant), but any term that means mutation is acceptable.

[0077] The variants may include the deletion or addition of amino acids that have minimal effect on the polypeptide's properties and secondary structure. For example, the polypeptide may be bound to the N-terminal signal (or leader) sequence of a protein involved in protein transfer, either co-translationally or post-translationally. Alternatively, the polypeptide may be bound to other sequences or linkers to enable the polypeptide to be identified, purified, or synthesized.

[0078] In this application, "conservative substitution" means that one amino acid is replaced by another amino acid having similar structural and / or chemical properties. Such amino acid substitutions can generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues.

[0079] Throughout this specification, the usual one- and three-letter codes for naturally occurring amino acids are used. Furthermore, amino acids referred to by abbreviations in this application are described according to IUPAC-IUB nomenclature. Alanine Ala,A Arginine Arg,R Asparagine Asn,N Aspartic acid (Asp,D) Cysteine ​​(Cys,C) Glutamic acid Glu,E Glutamine Gln,Q Glycine (Gly,G) Histidine His,H Isoleucine (Ile,I) Leucine Leu,L Lysine, K Methionine Met,M Phenylalanine Phe,F Proline Pro,P Serine Ser,S Threonine Thr,T Tryptophan Trp,W Tyrosine Tyr,Y Valin Val,V

[0080] On the other hand, any amino acid is denoted as Xaa or X.

[0081] Furthermore, generally accepted three-letter codes are used not only for naturally occurring amino acids, but also for other amino acids such as Aib (2-Aminoisobutyric acid), Sar (N-methylglycine), and α-methylglutamic acid.

[0082] Amino acids are generally classified based on the similarities of their residues in terms of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature. Therefore, amino acid substitutions can generally occur based on the similarities of their residues in terms of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature.

[0083] For example, among amino acids having electrically charged side chains, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; among amino acids having uncharged side chains, nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; polar or hydrophilic amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine; and among the nonpolar amino acids, aromatic amino acids include phenylalanine, tryptophan, and tyrosine.

[0084] In this application, "gene" means a polynucleotide that codes for a polypeptide and a polynucleotide that includes the regions before and after the coding region. In one specific example, the gene may have sequences (introns) inserted between each coding region (exon).

[0085] In this application, "homology" or "identity" refers to the degree to which two given amino acid sequences or base sequences are related, and is expressed as a percentage. Homology and identity are often used interchangeably.

[0086] The sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard sequencing algorithms, which may also be used in conjunction with a default gap penalty established by the program used. Substantively, homologous or identical sequences generally hybridize with at least 50%, 60%, 70%, 80%, or 90% of the entire sequence or its total length under moderate to high stringent conditions. Hybridization also includes hybridization with polynucleotides that have common codons or codons considering codon degeneracy.

[0087] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined, for example, using default parameters as described in Non-Patent Document 1 and known computer algorithms such as the "FASTA" program. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 3), as performed in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) (version 5.0.0 or later) (including the GCG program package (Non-Patent Document 4), BLASTP, BLASTN, and FASTA (Non-Patent Documents 5, 6, and 7)). For example, homology, similarity, or identity can be determined using BLAST or Clustal W from the National Center for Biotechnology Information, but these are not the only options.

[0088] The homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as Non-Patent Document 3, as disclosed in Non-Patent Document 8, for example. In summary, the GAP program is defined as the number of similar sequence symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program include (1) a binary comparison matrix (with a value of 1 for identity and 0 for non-identity) and a weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in Non-Patent Document 9, as in Non-Patent Document 10; (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap open penalty of 10 and a gap extended penalty of 0.5); and (3) no penalty for terminal gaps.

[0089] Furthermore, whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be confirmed by comparing the sequences in a Southern hybridization experiment under defined stringent conditions. The defined appropriate hybridization conditions are within the scope of the art and are determined by methods well known to those skilled in the art (e.g., Non-Patent Documents 11 and 12), but are not limited to these.

[0090] In this application, "mature polypeptide" refers to a polypeptide in which there is no signal sequence or propeptide sequence. A mature protein / polypeptide / peptide may also be a functional form of a protein / polypeptide / peptide. A mature polypeptide may also be the final form after translation or post-translational modification. Examples of post-translational modification include, but are not limited to, alteration of the N or C terminus, glycosylation, phosphorylation, and removal of the leader sequence.

[0091] In this application, "nucleic acid construct" means a single-stranded or double-stranded nucleic acid molecule that includes at least one regulatory sequence and is either artificially synthesized, manipulated to include a specific sequence in a manner not found in nature, or isolated from nature.

[0092] In this application, "expression" includes, but is not limited to, any steps involved in the production of polypeptides, such as transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0093] In this application, "expression vector" means a linear or cyclic nucleic acid molecule comprising a coding sequence and a regulatory sequence operably linked for its expression.

[0094] In this application, “operably linked” means a configuration in which a regulatory sequence is positioned appropriately so that the regulatory sequence indicates the expression of a coding sequence. Thus, “operably linked” includes a regulatory region of a well-known or desired functional domain, such as a promoter, terminator, signal sequence, or enhancer region, that is attached to or linked to a target (gene or polypeptide) so that the expression, secretion, or function of that target can be regulated according to its well-known or desired activity.

[0095] In this application, "cDNA" means a DNA sequence produced by reverse transcription from a mature, spliced ​​mRNA molecule obtained from a eukaryotic or prokaryotic cell. The cDNA sequence does not contain the intron sequences present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor of mRNA that appears as a mature, spliced ​​mRNA after being processed through a series of steps including splicing.

[0096] In this application, "regulatory sequence" refers to a polynucleotide sequence necessary for the expression of a coding sequence. Each regulatory sequence may be a native (same origin as the coding sequence) or a foreign (derived from another gene) sequence. Examples of regulatory sequences include leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, operator sequences, sequences encoding ribosome binding sites, and sequences that regulate transcription and translation termination. The smallest unit of a regulatory sequence includes a promoter and transcription and translation termination sequences.

[0097] The following nomenclature is used to describe the variants provided in this application.

[0098] In this application, indicating a specific position in the amino acid sequence indicates both the amino acid present at that position and the amino acid being substituted. The amino acid at a specific position can be described in various ways. For example, "position 003" can be described as "position 3," "amino acid number 3," or "the third amino acid." Also, for example, if the amino acid at position 3 is serine (S), it can be described as "S3" or "Ser3."

[0099] Amino acid substitutions are indicated by listing the amino acid before substitution, its position, and the amino acid being substituted, in that order. The amino acids are represented using standard one- and three-letter codes. For example, if alanine, the amino acid at position 8 of a particular sequence, is substituted with valine, it would be written as "A8V" or "Ala8Val".

[0100] At a specific position, any amino acid is indicated by "X". For example, X6 indicates any amino acid at the 6th position. Also, when an amino acid to be substituted is denoted as X, it means that it is being replaced with a different amino acid than the one that was present before the substitution. For example, "V6X" indicates that V at the 6th position is being replaced with any amino acid other than V.

[0101] By using symbols such as "," to list several amino acids simultaneously, different alterations can be indicated. For example, if the amino acid at the 12th position (D) is replaced with S or K, it is written as D12S,K.

[0102] Multiple mutations are indicated using "+" or " / ". For example, "G2A+M8V" means that glycine, the amino acid at the second position, is replaced by alanine, and methionine, the amino acid at the eighth position, is replaced by valine. As another example, L180C / A202C / R242C / S291C means that leucine, the amino acid at the 180th position, is replaced by cysteine, alanine, the amino acid at the 202nd position, is replaced by cysteine, arginine, the amino acid at the 242nd position, is replaced by cysteine, and serine, the amino acid at the 291st position, is replaced by cysteine.

[0103] In this application, "corresponding to" means an amino acid residue at a position listed in a protein or polypeptide, or an amino acid residue that is similar, identical, or equivalent to a residue listed in a protein or polypeptide. Identifying the amino acid at the corresponding position will determine the specific amino acid in the sequence referencing the particular sequence. In this application, "corresponding region" generally means a similar or corresponding position in the related protein or reference protein.

[0104] In this application, Sequence ID 1 may be used as a reference sequence to determine the position of an amino acid in any amino acid sequence.

[0105] In other words, Sequence ID No. 1 disclosed in this application is used to determine the corresponding amino acid residue in any polypeptide having PET degradation activity, and unless otherwise specified, the residues of a specific amino acid sequence are numbered based on Sequence ID No. 1.

[0106] For example, by aligning any amino acid sequence with Sequence ID No. 1, each amino acid residue in the sequence can be numbered based on the number and position of amino acid residues corresponding to the amino acid residues in Sequence ID No. 1. For example, the sequence alignment algorithm in this application can be used to identify the positions of amino acids, or the positions where modifications such as substitutions, insertions, or deletions occur, by comparing it with a query sequence (also called a "reference sequence").

[0107] Such alignments can be performed using, but are not limited to, the Needleman-Wunsch algorithm (Non-Patent Document 3) or the Needle program from the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2).

[0108] Furthermore, multiple sequence alignment allows for the identification of corresponding amino acid residues in other PETases. Examples of multiple sequence alignment programs known in the art include MUSCLE (multiple sequence comparison by log-expectation; version 3.5 or later; Non-Patent Document 13), MAFFT (version 6.857 or later; Non-Patent Document 14; Non-Patent Document 15; Non-Patent Document 16; Non-Patent Document 17; Non-Patent Document 18), and EMBOSS EMMA using Clustal W (version 1.83 or later; Non-Patent Document 19). The default parameters of each of the above programs can be used, but are not limited to these.

[0109] In addition, if enzymes isolated from the mature polypeptide of Sequence ID No. 1 cannot be found to be related by conventional sequence-based comparisons, other pairwise sequence comparison algorithms are used (Non-Patent Literature 20). In sequence-based searches, high sensitivity can be obtained by search programs that use probabilistic representations of polypeptide families (profiles) for searching databases. For example, the PSI-BLAST program can calculate profiles and detect distant homologs through an iterative database search process (Non-Patent Literature 21). Much greater sensitivity can be obtained if the polypeptide family or superfamily has at least one representation in the protein structure database. Programs such as GenTHREADER (Non-Patent Literature 22; Non-Patent Literature 23) use information from various sources such as PSI-BLAST, secondary structure predictions, structural alignment profiles, and solvation potentials as input to a neural network that predicts the structural folding of the query sequence. Similarly, the method in Non-Patent Literature 24 is used to align the sequence of an unknown structure with a superfamily model present in the SCOP database. These alignments are sequentially used to generate homology models for polypeptides, and these models are evaluated for accuracy using various tools developed for that purpose.

[0110] For proteins with known structures, multiple tools and resources are used to search for and create structural alignments. For example, the SCOP superfamily of proteins is structurally aligned, and this alignment is accessible and downloadable. Two or more protein structures can be aligned using various algorithms such as distance alignment matrices (Non-Patent Literature 25) and Combinatorial Extension (CE) (Non-Patent Literature 26). Implementations of these algorithms may be further used to query structural databases containing the target structure in order to find possible structural homologs (Non-Patent Literature 27).

[0111] The methods described above are just examples and are not the only ones that can be used.

[0112] The following provides a more detailed explanation of a specific example of this application.

[0113] In this application, "polypeptide having PET degrading activity" and "PET-degrading enzyme (PETase)" refer to polypeptides having depolymerization activity of polyethylene terephthalate (PET), and also include polypeptides having depolymerization activity of low polymers obtained by depolymerizing PET, such as bis(2-hydroxyethyl) terephthalate (BHET). "Depolymerization" means the process in which a polymer or at least one polymer of the plastic substance is depolymerized into even smaller molecules such as monomers and oligomers.

[0114] In this application, PET degradation activity can be measured and evaluated using methods known in the art, including those described in the embodiments of this application. For example, it can be evaluated by measuring the amounts of BHET, MHET, TPA, and EG produced.

[0115] In this application, "parent PET-degrading enzyme" means a PET-degrading enzyme that is modified to produce the variant or mutant polypeptide of this application. Specifically, the parent PET-degrading enzyme, parent enzyme, or parent sequence may be a naturally occurring polypeptide or a wild-type polypeptide, a mature polypeptide thereof, a variant thereof, or a polypeptide containing a functional fragment thereof, and may be any polypeptide that has PET-degrading activity and serves as the parent of a variant.

[0116] The parent PET-degrading enzyme provided in this application is, but is not limited to, the polypeptide of SEQ ID NO: 1. Furthermore, any polypeptide having PET-degrading activity may be included as the parent PET-degrading enzyme, even if it has approximately 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with the polypeptide of SEQ ID NO: 1. Any polypeptide having the same or equivalent activity as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is included as the parent PET-degrading enzyme.

[0117] The parent PETase of the mutant provided in this application may be derived from a microorganism of the genus Cryptosporangium. Specifically, it may be derived from Cryptosporangium aurantiacum.

[0118] On the other hand, the aforementioned microorganisms are examples of microorganisms from which the parent PETase provided in this application originates, and also include microorganisms that are taxonomically homologous to them, regardless of their name.

[0119] The aforementioned microorganisms are distributed from well-known microorganism depositaries such as ATCC, DSMZ, CBS, NRRL, KCTC, and KCCM.

[0120] In this application, sequences "derived from" a specific microorganism include not only sequences that are naturally generated or can be generated from that microorganism, but also sequences encoded by genes generated and isolated from microorganisms containing that gene.

[0121] For example, PETase derived from Cryptosporangium sp. includes not only enzymes with PETase activity that are naturally produced from Cryptosporangium sp., but also those produced from Cryptosporangium sp. sources, and those produced from other host cells by genetic modification known in the art (e.g., transformation to the sequence encoding the enzyme).

[0122] Furthermore, this application found that the polypeptide of SEQ ID NO: 1 has PET-degrading activity, and created mutant polypeptides with altered or enhanced properties by introducing mutations to the parent sequence of SEQ ID NO: 1. The description of the parent PET-degrading enzyme of the mutants provided in this application applies to polypeptides having the same or equivalent activity as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0123] In this application, the "mutant polypeptide having PET-degrading activity" may be a mutant of the parent PET-degrading enzyme.

[0124] In this application, "PET-degrading enzyme variant" or "PET-degrading enzyme variant" means a protein in which at least one amino acid differs from the amino acid sequence of the parent PETase and has PET-degrading activity.

[0125] The aforementioned "mutant polypeptides with PET-degrading activity," "PETase variants," and "PET-degrading enzyme variants" are used interchangeably.

[0126] The mutants provided in this application may have PETase activity and may also include at least one amino acid modification in the parent PETase sequence. The modification may be an amino acid deletion, an amino acid insertion, an amino acid substitution, and / or a combination thereof, and specifically, it may be an amino acid substitution, and a hydrogen bond may be formed by the amino acid substitution.

[0127] Furthermore, the variant may be i) a polypeptide having 70% or more and less than 100% sequence identity with SEQ ID NO: 1, and / or ii) a polypeptide encoded by a polynucleotide having 70% or more and less than 100% sequence identity with the sequence encoding the mature polypeptide of SEQ ID NO: 1, and / or iii) a polypeptide encoded by a polynucleotide that hybridizes with (a) the mature polypeptide coding sequence of SEQ ID NO: 1, (b) its cDNA, or (c) a full-length complementary sequence of (a) or (b) under low stringency, moderate stringency, upper-medium stringency, high stringency, or very high stringency conditions, and / or iv) a functional fragment of the polypeptide of i), ii), or iii) having PET degradation activity.

[0128] Specifically, the mutants provided in this application may have PET degradation activity and also include at least one amino acid modification in the parent PETase sequence, and may have at least one altered function or characteristic compared to the parent PETase.

[0129] As a specific example, the mutant provided in this application may have PETase activity, include at least one amino acid modification of the parent PETase sequence, have at least one altered function or characteristic compared to the parent PETase, and have at least one conservative substitution.

[0130] The mutants provided in this application may be mutants of the parent PETase and may be polypeptides having PET degradation activity.

[0131] As a specific example, the variant provided in this application may include a modification at at least one position selected from the amino acids corresponding to positions 129, 198, and 196 of Sequence ID No. 1. Specifically, the modification may be an amino acid deletion, an amino acid insertion, and / or a substitution with another amino acid, and more specifically, it may be an amino acid substitution.

[0132] In this application, the position number corresponds to the position of the polypeptide in Sequence ID No. 1, and the meaning of "corresponding" is as described above.

[0133] In any of the examples described above, the variant of this application may include an amino acid modification corresponding to at least one of V129, R198, and G196 of SEQ ID NO: 1.

[0134] In any of the examples described above, the 129th amino acid of Sequence ID No. 1 before modification of this application may be valine (V), the 198th amino acid may be arginine (R), and / or the 196th amino acid may be glycine (G).

[0135] In any of the examples described above, the variant of this application may include a substitution of the amino acid corresponding to the 129th position of SEQ ID NO: 1 with G, A, L, I, M, F, W, P, S, T, C, Y, N, Q, D, E, K, R, or H, and more specifically, may include a substitution of T or S.

[0136] In any of the examples described above, the variant of this application may include a substitution of the amino acid corresponding to position 198 of Sequence ID No. 1 with G, A, V, L, I, M, F, W, P, S, T, C, Y, N, Q, D, E, K, or H, and more specifically, may include a substitution with K or D.

[0137] In any of the examples described above, the variant of this application may include a substitution of A, V, L, I, M, F, W, P, S, T, C, Y, N, Q, D, E, K, R, or H of the amino acid corresponding to the 196th position of SEQ ID NO: 1, and more specifically, it may include a substitution of T, A, I, L, V, or S.

[0138] In any of the examples described above, the variant of this application may include at least one substitution selected from the group consisting of V129T,S, R198K,D, and G196T,A,L,I,V,S.

[0139] In any of the examples described above, the variant of this application may include at least one substitution selected from the group consisting of a substitution of the amino acid corresponding to the 129th position with threonine or serine, a substitution of the amino acid corresponding to the 198th position with lysine or aspartic acid, and a substitution of the amino acid corresponding to the 196th position with threonine, alanine, isoleucine, valine, or serine.

[0140] In any of the examples described above, the variant of this application may include a substitution of the amino acid corresponding to the 129th position of the amino acid sequence of SEQ ID NO: 1 with threonine, a substitution of the amino acid corresponding to the 198th position with lysine, and a substitution of the amino acid corresponding to the 196th position with threonine.

[0141] In any of the examples described above, the variants of this application include all possible combinations of modifications as described above.

[0142] In any of the examples described above, the variant of this application may have about 60% or more sequence identity with the parent PET-degrading enzyme, its mature polypeptide, or its functional fragment, for example, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, and less than 100%.

[0143] In any of the examples described above, the variant of this application may have approximately 60% or more sequence identity with SEQ ID NO: 1, for example, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more, and less than 100%.

[0144] In any of the examples described above, the variant of this application may be a polypeptide encoded by a polynucleotide having approximately 60% or more sequence identity with the sequence encoding the mature polypeptide of SEQ ID NO: 1, for example, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more, and less than 100%.

[0145] In any of the above-mentioned examples, the variant of this application may have approximately 60% or more sequence identity with the functional fragment of Sequence ID No. 1, for example, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more, and less than 100%.

[0146] The variants provided in this application may have at least one modification in any characteristic or attribute of the selected or detected polypeptide compared to other parent PET-degrading enzymes, such as wild-type PET-degrading enzymes, parent PET-degrading enzymes, or other PET-degrading enzyme variants.

[0147] The aforementioned properties or attributes include oxidative stability, substrate specificity, catalytic activity, thermal stability, alkaline stability, pH activity profile, resistance to proteolysis, Km, and k. cat , k cat This includes, but is not limited to, the ability to: / Km ratio, protein folding, immune response induction, ligand binding ability, receptor binding ability, secretion ability, cell surface presentation ability, oligomer formation ability, signaling ability, cell proliferation promotion ability, cell proliferation inhibition ability, apoptosis induction ability, modification by phosphorylation or glycosylation, and / or disease treatment ability.

[0148] Specifically, the mutants provided in this application may have at least one of the following modified activities compared to the parent sequence. i) Increase or decrease in enzyme activity ii) Increase or decrease in specific activity iii) Improvement or decrease in pH stability iv) Improvement or decrease in storage stability v) Improvement or decrease in acid resistance vi) Improvement or decrease in heat resistance vii) Modification of substrate specificity

[0149] However, this is not the only example.

[0150] As another example, the PET-degrading enzyme provided in this application may have at least one of the following modified activities compared to the PET-degrading enzyme derived from Ideonella sakaiensis or Thermobifida fusca. i) Increase or decrease in enzyme activity ii) Increase or decrease in specific activity iii) Improvement or decrease in pH stability iv) Improvement or decrease in storage stability v) Improvement or decrease in acid resistance vi) Improvement or decrease in heat resistance vii) Modification of substrate specificity

[0151] However, this is not the only example.

[0152] As yet another example, the PET-degrading enzyme provided in this application may have at least one of the following modified activities compared to the PET-degrading enzyme derived from Ideonella sakaiensis or the PET-degrading enzyme derived from Thermobifida fusca. i) Improvement of enzyme activity ii) Improvement of specific activity iii) Improved pH stability iv) Improvement of storage stability v) Improved acid resistance vi) Improved heat resistance vii) Modification of substrate specificity

[0153] However, this is not the only example.

[0154] In this application, "enzymatic activity" means at least one catalytic activity. Specifically, mainly k cat The enzyme conversion efficiency is expressed in / Km, but it is not limited to this expression.

[0155] k cat This refers to the catalytic constant, also known as the turnover number, which is the rate at which a single enzyme converts a substrate into a product per unit time when the enzyme is completely saturated with the substrate. Km is the substrate concentration at which the reaction rate is half of its maximum value (Vmax).

[0156] An example of a method for expressing enzyme activity is specific activity (umol of converted substrate × mg). -1 ×min -1 ), volumetric activity(umol of converted substrate × mL -1 ×min -1 ) are some examples.

[0157] However, enzyme activity is not limited to the above and can be defined and evaluated based on the information disclosed in Non-Patent Documents 28, 29, 30, 31, 32, 33, etc.

[0158] As a specific example, the mutants provided in this application may have enzyme activity that is approximately 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% or more improved compared to the parent enzyme.

[0159] As another specific example, the mutants provided in this application may have enzyme activity reduced by about 99%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, or about 20% or less compared to the parent enzyme.

[0160] In this application, "specific activity" refers to enzyme activity per unit weight of protein, expressed as units / mg. Protein quantification can be performed using methods such as SDS-PAGE or Bradford assay.

[0161] Enzyme stability refers to the preservation of enzyme activity or its ability to be maintained throughout the reaction time. To measure this change in stability, the level at which enzyme activity is lost or the overall enzyme stability can be expressed by measuring and comparing the enzyme activity at 0 hours (time zero) (100%) and after a predetermined time (x%) under the conditions under which the enzyme activity was initially determined.

[0162] Factors that affect enzyme activity include, for example, pH, heat, and the presence of other substances (e.g., oxidizing agents, chelating agents).

[0163] In this application, "pH stability" refers to the ability of a protein to function within a specific pH range. As a specific example, the mutant provided in this application is active in the range of approximately pH 4.0 to approximately pH 12.0, but is not limited to this range.

[0164] If a protein maintains its function within a specific pH range, it is defined as having "pH stability," and depending on the pH range, it is also defined as having "acid resistance," "alkali resistance," etc.

[0165] In this application, "thermal stability" refers to the ability of a protein to function within a specific temperature range. For example, the mutants provided in this application are active in the range of approximately 20°C to 70°C, specifically in the range of approximately 25°C to 65°C, but are not limited to this range.

[0166] In this application, "thermal tolerance" means the ability of a protein to function after being exposed to a specific temperature, such as high heat or extremely low temperature. For example, a thermally tolerant protein may not function at the temperature to which it was exposed, but may function again when returned to an optimal temperature environment.

[0167] Improved stability includes maintaining high enzyme activity compared to other enzymes, such as the wild-type enzyme, the parent enzyme, and / or other variants, and increasing the range of pH, temperature, and / or time over which the protein maintains its function.

[0168] Reduced stability includes lower enzyme activity maintenance compared to other enzymes, such as the wild-type enzyme, the parent enzyme, and / or other variants, and a decrease in the range of pH, temperature, and / or time over which the protein maintains its function.

[0169] In this application, "substrate specificity" refers to the ability of an enzyme to distinguish between a substrate and molecules that compete with it. Substrate specificity can be determined by measuring the activity of the enzyme against different substrates. As one specific example, the change in substrate specificity may be such that the specificity to the substrate that produces the target product is improved. As another specific example, the change in substrate specificity may be such that the specificity to the substrate that produces the target product is decreased.

[0170] The "polynucleotide" encoding the variant of this application may include the coding sequence of the variant described above. The polynucleotide can be modified in various ways in the coding region, either by codon degeneracy or by considering the preferred codons in the organism that intends to express the polypeptide, as long as the amino acid sequence of the polypeptide does not change.

[0171] Furthermore, the polynucleotide of this application may be any sequence that encodes a variant of this application by hybridizing under stringent conditions with a probe prepared from a known gene sequence, for example, a complementary sequence to all or part of the base sequence.

[0172] The aforementioned "stringent conditions" refer to conditions that enable specific hybridization between polynucleotides. Such conditions are specifically described in the literature (e.g., Non-Patent Documents 11 and 12).

[0173] For example, this could involve hybridizing polynucleotides with high homology or identity, specifically 40% or more, more specifically 90% or more, more specifically 95% or more, 96% or more, 97% or more, 98% or more, and even more specifically 99% or more homology or identity, while not hybridizing polynucleotides with lower homology or identity. Alternatively, it could involve washing once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions of a typical Southern hybridization: 60°C, 1×SSC, 0.1%SDS, specifically 60°C, 0.1×SSC, 0.1%SDS, or more specifically 68°C, 0.1×SSC, 0.1%SDS.

[0174] Hybridization requires that the two nucleic acids have complementary sequences, even if mismatches between bases are possible depending on the stringency of the hybridization. "Complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of this application may include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary to the entire sequence.

[0175] Specifically, polynucleotides having homology or identity can be detected using hybridization conditions in which the hybridization step is performed at a Tm value of 55°C and the conditions described above. The Tm value may be 60°C, 63°C, or 65°C, but is not limited to these, and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0176] The appropriate stringency for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides, and these variables are known in the art (see Non-Patent Document 34).

[0177] For example, "high stringency" occurs approximately 5-10°C below the probe's Tm, "moderate stringency" occurs approximately 10-20°C below the probe's Tm, and "low stringency" occurs approximately 20-25°C below Tm, but these are not the only possibilities.

[0178] For example, "low stringency conditions" may involve prehybridization and hybridization of probes with a length of at least 100 nucleotides using 5×SSPE, 0.3% SDS, 200 micrograms / ml of sheared and denatured salmon sperm DNA, and 25% formamide at 42°C for 12-24 hours, following the Southern blotting standard procedure. The aforementioned carrier material is then washed two to three times with 2×SSC and 0.1-0.2% SDS at 50°C for 15 minutes each.

[0179] For example, "medium stringency conditions" may involve prehybridization and hybridization of probes with a length of at least 100 nucleotides using 5×SSPE, 0.3% SDS, 200 micrograms / ml of sheared and denatured salmon sperm DNA, and 35% formamide at 42°C for 12-24 hours, following the Southern blotting standard procedure. The aforementioned carrier material is then washed two to three times with 2×SSC and 0.1-0.2% SDS at 55°C for 15 minutes each. For example, a "medium-high stringency condition" may involve prehybridization and hybridization of probes with a length of at least 100 nucleotides using 5×SSPE, 0.3% SDS, 200 micrograms / ml of sheared and denatured salmon sperm DNA, and 35% formamide at 42°C for 12-24 hours, following the Southern blotting standard procedure. The aforementioned carrier material is then washed 2-3 times each with 1-2×SSC and 0.1-0.2% SDS at 60°C for 15 minutes each.

[0180] For example, "high stringency conditions" may refer to prehybridization and hybridization performed at 42°C for 12-24 hours on probes of at least 100 nucleotide lengths using 5×SSPE, 0.3% SDS, 200 micrograms / ml of sheared and denatured salmon sperm DNA, and 35% formamide, following the Southern blotting standard procedure. The aforementioned carrier material is then washed two to three times at 65°C for 15 minutes each with 2×SSC and 0.1-0.2% SDS.

[0181] The “nucleic acid construct” provided in this application comprises a polynucleotide encoding a variant provided in this application, which is operably linked to at least one regulatory sequence that exhibits expression of the coding sequence in a suitable host cell under conditions suitable for the regulatory sequence.

[0182] Polynucleotides are manipulated in various ways to enable the expression of mutants. Depending on the expression vector, it may be preferable, or even necessary, to manipulate the polynucleotides before inserting them into the vector. Such manipulations are performed using methods known in the art.

[0183] In this application, “vector” means a DNA product comprising a polynucleotide sequence encoding a mutant operably ligated to a suitable regulatory region (or regulatory sequence) so as to enable the expression of the mutant in a suitable host. The regulatory region includes a promoter for initiating transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable mRNA-ribosome binding site, and sequences for regulating the termination of transcription and translation. When transformed into a suitable host cell, the vector can replicate and function independently of the host genome and is integrated into the genome itself.

[0184] The vectors used in this application are not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant state. For example, as phage vectors or cosmid vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc., can be used, and as plasmid vectors, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, pET series, etc., can be used. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc., can be used.

[0185] For example, a chromosome vector can be used to insert a polynucleotide encoding the mutant provided in this application into a chromosome. The insertion of the polynucleotide into the chromosome can be carried out by any method known in the art, such as homologous recombination, but is not limited thereto. A selection marker may further be included to confirm whether or not the polynucleotide has been inserted into the chromosome. The selection marker is for selecting cells transformed by the vector, i.e., to confirm whether or not the target nucleic acid molecule has been inserted, and markers that confer selectable phenotypes such as drug resistance, nutritional requirements, resistance to cytotoxic agents, and expression of surface polypeptides are used. In an environment treated with a selective agent, only cells expressing the selection marker will survive or exhibit different phenotypes, so transformed cells can be selected.

[0186] The “host cell” in this application may be any cell that expresses the variant of this application.

[0187] The host cells of this application may include the aforementioned mutants, polynucleotides encoding the mutants, nucleic acid constructs and / or vectors containing the same.

[0188] The nucleic acid construct or vector may be maintained as an extrachromosomal vector that is incorporated into a chromosome or self-replicates, as described above.

[0189] The host cells of this application include any offspring of the parent cell that are not identical to the parent cell due to mutations occurring during replication.

[0190] The host cell may be any cell useful for the recombination generation of the mutant, such as a prokaryotic cell or a eukaryotic cell.

[0191] The prokaryotic host cell may be any Gram-positive or Gram-negative bacterium.

[0192] Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, Corynebacterium, and Streptomyces.

[0193] Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia, Flavobacterium, Fusobacterium, Helicobacter, Iliobacter, Neisseria, Pseudomonas, Salmonella, Vibrio (e.g., Vibrio natriegens) and Ureaplasma.

[0194] As a specific example, the bacterial host cells mentioned above are Bacillus host cells, specifically including, but not limited to, Bacillus alkarophilus, Bacillus amyloricephasiens, Bacillus brevis, Bacillus circulans, Bacillus clauzi, Bacillus coagulans, Bacillus filums, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megatherium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

[0195] As a specific example, the bacterial host cells mentioned above are Streptococcus genus host cells, specifically including, but not limited to, cells of Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and the Streptococcus equi subspecies Zooepidemicus.

[0196] As a specific example, the bacterial host cells mentioned above are Streptomyces genus host cells, and specifically include, but are not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces cericara, Streptomyces griseus, and Streptomyces lividans cells.

[0197] As a specific example, the bacterial host cells mentioned above are Corynebacterium host cells, including Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, and Corynebacterium striatum. This includes, but is not limited to, *Corynebacterium striatum*, *Corynebacterium ammoniagenes*, *Corynebacterium pollutisoli*, *Corynebacterium imitans*, *Corynebacterium testudinoris*, or *Corynebacterium flavescens*.

[0198] As a specific example, the bacterial host cell is an Escherichia genus host cell, specifically Escherichia coli (E. coli), but is not limited to these.

[0199] The host cell may be a eukaryote, such as a mammal, insect, plant, or fungal cell.

[0200] The host cell may be a fungal cell. In this application, “fungus” includes not only Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, but also Oomycota and all imperfect fungi.

[0201] The fungal host cell may be a yeast cell. The term "yeast" in this application includes ascosporogenous yeasts (Endomycetales), basidiosporogenous yeasts, and yeasts belonging to the imperfect fungi (Blastomycetes). However, these classifications are subject to change, and the classifications are defined as described in Non-Patent Document 35.

[0202] Yeast host cells include Candida, Hansenula, Kluyveromyces, Pichia, Komagataella, Saccharomyces, Schizosaccharomyces, or Yarrowia cells, such as Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, and Saccharomyces cruiberi. These may be cells of *Saccharomyces kluyveri*, *Saccharomyces norbensis*, *Saccharomyces oviformis*, *Komagataella phaffii*, or *Yarrowia lipolytica*.

[0203] The fungal host cell may also be a filamentous fungal cell. "Filamentous fungi" include all filamentous forms of the phylum Mycomycota and subphylum Oomycetes (as defined in the above-mentioned literature (Hawksworth et al., 1995)). Filamentous fungi are generally characterized by hyphae walls composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphae elongation, and carbon catabolism is obligate aerobic. In contrast, vegetative growth by yeast, such as Saccharomyces cerevisiae, is by the germination of unicellular thallus, and carbon catabolism is fermentative.

[0204] The host cells of filamentous fungi include Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Fumicola, Magnaporthe, Mucor, Myserioftra, and Neocaridix. The cells may be callimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cells.

[0205] For example, filamentous fungal host cells include Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidurans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, and Ceriporiopsis librosa. Chrysosporium rivulosa), Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum Fusarium zonatum), Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium clockwellensFusarium crookwellense), Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulfureum Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Miseriophtera thermophylla, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris Trichoderma terrestris), Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum (TrichodermaThese may be Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cells, but are not limited to these.

[0206] The “composition” of this application may include a mutant polypeptide having PET degradation activity, or a host cell expressing the mutant polypeptide.

[0207] The mutant polypeptide having the PET degradation activity, or the host cells expressing the mutant polypeptide, are subject to the descriptions provided in this application regarding the mutant polypeptide and the host cells expressing it.

[0208] The composition of this application may also be used to convert polyester into a final product.

[0209] "Polyester" refers to a polymer whose main chain contains an ester functional group. For example, polyethylene terephthalate is a semi-aromatic copolymer composed of two monomers: terephthalic acid and ethylene glycol.

[0210] The polyester may be selected from polyethylene terephthalate (PET), polyethylene terephthalate glycol (PETG), polyethylene coisosorbide terephthalate (PEIT), polytrimethylene terephthalate (PTT), polybutylene adipate terephthalate (PBAT), polycyclohexylene dimethylene terephthalate (PCT), and polybutylene terephthalate (PBT). Specifically, the polyester may be PET.

[0211] The polypeptide of this application or a composition containing the same may be used to depolymerize PET into bis(2-hydroxyethyl) terephthalate (BHET) or to decompose PET into mono(2-hydroxyethyl) terephthalate (MHET) and terephthalic acid (TPA).

[0212] The polypeptide of this application or a composition containing the same may be used for the decomposition of polymers (e.g., low polymers) derived from PET depolymerization. For example, it may be used to decompose BHET into MHET and TPA.

[0213] The composition of this application may further include other components in addition to the PET-degrading mutant provided herein. Those skilled in the art can appropriately select additional components to be added to the composition of this application.

[0214] As a specific example, the composition of this application may further include any components suitable for use in converting PET into a final product.

[0215] As a specific example, the composition of this application may further include any components suitable for use in PET decomposition.

[0216] Examples of substances that may be added include, but are not limited to, stabilizers, surfactants, builders, chelating agents, dispersants, enzymes, enzyme stabilizers, catalysts, activators, carriers, mixtures, lubricants, disintegrants, excipients, solubilizers, suspending agents, dyes, fragrances, buffers, preservatives, analgesics, isotonic agents, diluents, and lubricants.

[0217] As a specific example, the composition provided in this application may further contain, in addition to the variant provided in this application, naturally occurring or non-naturally occurring substances.

[0218] As a specific example, the composition provided in this application may further contain additional enzymes in addition to the variants provided in this application.

[0219] The method for producing the mutant according to this application may include the steps of culturing host cells and recovering the mutant expressed in the culturing step.

[0220] In this application, "cultivation" means growing the host cells under appropriately controlled environmental conditions. The cultivation process in this application is carried out using suitable culture media and conditions known in the art. Such a cultivation process can be easily adjusted and used by those skilled in the art depending on the selected bacterial strain. Specifically, the cultivation is batch, continuous, and fed-batch culture, but is not limited to these.

[0221] In this application, "culture medium" refers to a substance mixed mainly with nutrients necessary for culturing the host cells, supplying nutrients and growth factors, including water, which are essential for survival and growth. Specifically, the culture medium and other culture conditions used for culturing the host cells in this application may be any that are normally used for culturing host cells, and the host cells in this application can be cultured in a normal culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, under aerobic conditions, with the temperature, pH, etc., adjusted.

[0222] In this application, the carbon source can be carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn maceration liquid can be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) can be used, and any other carbon source in an appropriate amount may be used. These carbon sources can be used individually or in combination of two or more, but are not limited to these uses.

[0223] As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate can be used, as well as organic nitrogen sources such as amino acids like glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn maceration liquid, casein hydrolysates, fish or their decomposition products, defatted soybean cake or its decomposition products. These nitrogen sources can be used individually or in combination of two or more, but are not limited to these uses.

[0224] As the phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or equivalent sodium-containing salts can be used. As inorganic compounds, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc., can be used, and in addition, amino acids, vitamins, and / or suitable precursors can be used. These components or precursors can be added to the culture medium in batches or continuously, but are not limited to these.

[0225] Furthermore, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture medium in a suitable manner during the culture of the host cells. In addition, during culture, the formation of bubbles can be suppressed using an antifoaming agent such as fatty acid polyglycol ester. Furthermore, in order to maintain an aerobic state in the culture medium, oxygen or oxygen-containing gas may be injected into the culture medium, and in order to maintain an anaerobic and microaerobic state, it is not necessary to inject gas, but nitrogen, hydrogen, or carbon dioxide gas may be injected, but the invention is not limited to these.

[0226] The culture medium temperature is 20°C to 50°C, specifically 25°C to 40°C, but is not limited to this range. The incubation period continues until the desired amount of useful substance is produced, specifically 24 hours to 196 hours, but is not limited to this range.

[0227] As a specific example, the mutants expressed in the culture step can be recovered using methods well known in the art to which this application belongs. For example, the mutants can be recovered from the nutrient medium by conventional procedures including, but are not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, and precipitation.

[0228] The aforementioned recovery method may involve collecting mutants using a suitable method known in the art, depending on the host cell culture method of this application, such as batch, continuous, or fed-batch culture. For example, various chromatography methods such as centrifugation, filtration, crystallization, treatment with protein precipitants (salting-out method), extraction, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, and combinations thereof can be used, and mutants can be recovered from the culture medium or host cells using a suitable method known in the art.

[0229] As another example, the mutants expressed by the host cells during the culture step do not need to be recovered. In this example, the host cells expressing the mutants themselves can be used as the source of the mutants.

[0230] This application may also relate to a method for producing mono(2-hydroxymethyl) terephthalate (MHET), terephthalic acid (TPA), and / or ethylene glycol (EG), comprising the step of contacting a polyester with the aforementioned mutant polypeptide, a host cell expressing the polypeptide, or a composition containing the same.

[0231] Specifically, the details regarding polyester are as described above.

[0232] In this application, "contact" of a mutant polypeptide, a host cell expressing the polypeptide, or a composition containing them with a polyester means, but is not limited to, decomposing the polyester. Decomposition is also used in depolymerization.

[0233] The time required to decompose polyester varies depending on various process parameters (i.e., temperature, pH, additional chemicals, etc.), as well as the polyester-containing article itself (i.e., the attributes, origin, composition, shape, etc. of the plastic product), the form and amount of the variant polypeptide used. Process parameters can be readily applied using techniques known in the art to suit polyester-containing articles.

[0234] For example, the decomposition step may be carried out at 20°C to 90°C, preferably 40°C to 80°C, and more preferably 50°C to 70°C. More specifically, the temperature may be maintained below the inactivation temperature, which corresponds to the temperature at which the mutant polypeptide is inactivated, and / or below the temperature at which the host cell no longer synthesizes the mutant polypeptide.

[0235] For example, the decomposition process may be carried out at a pH of 5 to 11, but it is preferable to carry it out at a pH of 6 to 9.

[0236] As a specific example of this application, an article containing polyester may be pre-treated before contact with the mutant polypeptide of this application, thereby increasing the surface area of ​​contact with the mutant polypeptide by physically or chemically modifying its structure.

[0237] Specifically, monomers and / or oligomers that may be generated by the decomposition by contact may be recovered sequentially or continuously.

[0238] Specifically, the recovered monomers and / or oligomers may be further purified using a variety of suitable purification methods to produce a repolymerizable form. Purification methods more preferably include, but are not limited to, stripping, separation with aqueous solution, steam selective condensation, filtration and concentration of the culture medium after the bioprocess, separation, distillation, vacuum evaporation, extraction, electrodialysis, adsorption, ion exchange, precipitation, crystallization, concentration and acid addition dehydration, precipitation, nanofiltration, acid catalytic treatment, semi-continuous mode distillation or continuous distillation, solvent extraction, evaporation concentration, evaporation crystallization, liquid / liquid extraction, hydrogenation, azeotropic distillation process, column chromatography, simple vacuum distillation, and microfiltration.

[0239] The final products obtained using the composition of this application, namely MHET, TPA, and / or EG, may be recycled for polyester polymerization.

[0240] Specifically, the final products obtained as described above, MHET, TPA, and / or EG, may be recycled to synthesize polyesters as repolymerizable monomers and / or oligomers. Specifically, polyesters of the same properties may be repolymerized, or they may be mixed with other monomers and / or oligomers to synthesize, for example, new copolymers.

[0241] The polyester may be selected from polyethylene terephthalate (PET), polyethylene terephthalate glycol (PETG), polyethylene coisosorbide terephthalate (PEIT), polytrimethylene terephthalate (PTT), polybutylene adipate terephthalate (PBAT), polycyclohexylene dimethylene terephthalate (PCT), and polybutylene terephthalate (PBT). Specifically, the polyester may be PET.

[0242] Methods for synthesizing polyester using MHET, TPA, and / or EG are known in the art. [Examples]

[0243] The present application will be described in more detail below with reference to examples and experimental cases. However, these examples and experimental cases are merely illustrative of the present application and the present application is not limited to these examples and experimental cases. [Examples]

[0244] Discovery of a novel CaPETase In addition to the well-known PET hydrolase (IsPETase) from Ideonella sakaiensis 201-F6, sequence homology analysis was performed using the NCBI database to identify 10 candidate PETases. To investigate these 10 candidate PETases, we first attempted to produce them in signal peptide cleavage form, successfully producing 9 candidate PETases. Next, using PET bottle powder (B-PET) as a substrate, we monitored the amount of PET hydrolysis products released from MHET and TPA to measure the PET hydrolysis activity of the 9 candidate PETases. Surprisingly, while most candidate PETases released very small amounts of PET hydrolysis products, SHM40309.1 (PC2 in Figure 1) released a significantly larger amount of PET hydrolysis products compared to the other enzymes. Furthermore, to investigate the isothermia of these enzymes, the melting temperatures (Tm) of the 9 candidate PETases were measured. These candidate PETases exhibited a variety of Tm values ​​ranging from 38.6°C to 70.5°C. Surprisingly, SHM40309.1, which exhibited extremely high PET hydrolysis activity compared to other enzymes, also showed high temperature stability with a Tm value of 66.8°C. SHM40309.1 also had the highest soluble expression level compared to other enzymes (Figure 1). These results indicate that SHM40309.1 possesses excellent properties for efficient PET degradation, including enzyme activity, isothermal stability, and protein expression level. Therefore, SHM40309.1 (PETase of Cryptosporangium aurantiacum, CaPETase) was selected as the novel PETase in this application. [Examples]

[0245] Production of CaPETase and well-known PET degradation proteins Expression and purification were carried out under the following conditions. A gene optimized for codons in E. coli was synthesized and amplified by polymerase chain reaction (PCR). The nucleotide sequence corresponding to the signal peptide was removed from the synthetic DNA. Then, the PCR product was subcloned (Nco I and Xho I) into pET22b(+) (Novagen) without a signal peptide, which was constructed independently. The generated expression vector pET22b(+):CaPETase was used to transform the E. coli BL21(DE3)-T1R strain. The E. coli strain was cultured in a flask containing 1 L of lysogeny broth medium containing 200 mg / L of ampicillin at 37 °C until the optical density at 600 nm reached 0.6.

[0246] Protein expression was induced by the addition of 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and the culture medium was further cultured at 18 °C for 16 hours. Next, the cells were collected by centrifugation at 4 °C and 4000 × g for 20 minutes.

[0247] The cell pellet was resuspended in buffer A (50 mM Na2HPO4-HCl, pH 7.0) and then disrupted by sonication. Cell debris was removed by centrifugation at 13,500×g for 25 minutes, and the supernatant was applied to a Ni-NTA agarose column (Qiagen). It was washed with buffer A containing 30 mM imidazole, and then the bound protein was eluted with 300 mM imidazole in buffer A. Finally, trace amounts of contaminants were removed by size-exclusion chromatography using a Superdex 200 prepgrade column (320 ml, GE Healthcare) equilibrated with buffer A. All purification steps were performed at 4°C. The protein purity was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified protein was concentrated with 50 mM Na2HPO4-HCl (pH 7.0) and 100 mM NaCl. Similarly, well-known PET hydrolases LCC (GenBank: AEV21261.1), IsPETase (GenBank: GAP38373.1), and TfCut2 (Uniprot Accession E5BBQ3) were prepared and used as a comparison group. The primers used for cloning are shown in Table 1.

[0248]

Table 1

Example

[0249] Analysis of PET-degrading activity of CaPETase and well-known PET-degrading proteins To compare the PET hydrolysis activity of CaPETase with that of the three PET hydrolases (LCC, IsPETase, and TfCut2) prepared in Example 2, 15 mg of B-PET was prepared and immersed in 1 mL of 50 mM Glycine-NaOH (pH 9.0) buffer with 500 nM enzyme. B-PET (PET sample derived from a PET bottle) was obtained by the following process: A transparent PET bottle was crushed in a crusher, and then the crushed PET was melted in a high-temperature oven at 270°C. The molten PET was immediately impregnated in water at 4°C and cured. The cured PET was subjected to a cryogenic grinding process, and then PET powder of 300 μm or less was obtained using a steel mesh. The reaction mixture was reacted at 30°C and 40°C for 12 hours. Next, the reaction mixture was analyzed by HPLC. After this reaction, CaPETase completely converted the PET powder into MHET and TPA. The products were analyzed by HPLC to evaluate the PET degradation activity.

[0250] In reactions at 30°C, CaPETase exhibited extremely high PET degradation activity compared to LCC and TfCut2, and showed activity equivalent to or even higher than IsPETase, which is known to have the highest activity at ambient temperature among PET degradation proteins reported to date. In particular, CaPETase showed 1.6 times higher activity than IsPETase in B-PET, a highly crystalline PET sample. The difference in PET hydrolysis activity between these two PET hydrolases was much larger than that of other PET hydrolases reacted at 40°C, with CaPETase showing 7.5 times higher activity than IsPETase in B-PET. These results indicate that CaPETase has higher temperature controllability and PET hydrolysis activity than IsPETase (Figure 2). [Examples]

[0251] Structural analysis of CaPETase To provide a structural basis for the high PET hydrolysis performance of CaPETase, the crystal structure of CaPETase was determined at a resolution of 1.36 Å. Crystallization was carried out at 20 °C using the sitting-drop vapor diffusion method (Non-Patent Document 36) with crystallization screening kits: Index and PEG / Ion (Hampton Research) and Wizard I and II (Rigaku). The experiment was conducted with 1.0 μl of the protein solution and 1.0 μl of the reservoir solution, and then equilibrated with 50 μl of the reservoir solution. The above-mentioned crystals were transferred to a cryoprotectant solution containing 25% (v / v) glycerol, extracted with a loop larger than the crystals, and immersed in liquid nitrogen for rapid freezing. For the collection of analysis data of the protein crystals, data were collected at 100 K by Beamline 7A of the Pohang Accelerator Laboratory (Pohang, Korea). The data were indexed, integrated, and scaled using the HKL2000 software suite. As a result, the CaPETase crystals belonged to the space group P21212 and had unit cell parameters of a = 82.31 Å, b = 82.45 Å, c = 87.39 Å, α = β = γ = 90° (Non-Patent Document 37). Assuming 1 molecule of CaPETase per asymmetric unit, the Matthews coefficient was 2.68 Å 3 / Da, which corresponds to a solvent content of 52.25%. To elucidate the structural characteristics of the protein crystals, the structure of cutinase 1 derived from Termobifida cellulosilytica (PDB code 5LUI) was used as a search model, and the structure of CaPETase was elucidated by the molecular replacement method using MOLREP of the CCP4 version. Model building was performed using the WinCoot program, and refinement was performed using REFMAC5. The above-mentioned statistical data are shown in Table 2. The refined model of CaPETase is deposited in the Protein Data Bank with the PDB code 7YM9.

[0252] [Table 2] [Examples]

[0253] Production of CaPETase mutants, and measurement of the thermal stability and PET degradation activity of the mutants. Based on the structure analyzed in Example 4, we attempted to improve the PET degradation activity and thermal stability of the enzyme. Structural analysis was performed in Example 4, and seven mutant CaPETases with introduced mutations were prepared in the same manner as in Example 2, and their PET degradation activity was evaluated in the same manner as in Example 3. M1:CaPETaseV129T M2:CaPETaseG196T M3:CaPETaseR198K M4:CaPETaseV129T / G196T M5:CaPETaseV129T / R198K M6:CaPETaseG196T / R198K M7:CaPETaseV129T / G196T / R198K

[0254] The thermal stability of each mutant was confirmed by measuring its solubility temperature (the temperature at which protein modification occurs, Tm). The solubility temperature was determined by measuring the melting curve using StepOnePlus Real-Time PCR (Thermo Fisher Scientific) with Protein thermal shift dye (Applied Biosystems). Specifically, 5 μg of CaPETase was mixed with 20 μl of protein thermal shift dye, and the temperature was then varied by 1 degree increments from 20°C to 90°C, monitoring the signal change at which protein modification began. Based on the melting curves, the solubility temperature (Tm) of CaPETaseWT and seven mutants was determined.

[0255] The M1 mutant had a Tm value increased by 2.4 °C and a PET degradation activity increased by about 1.38-fold compared to CaPETaseWT. The M2 mutant had a Tm value increased by 4.6 °C and a PET degradation activity increased by about 1.19-fold compared to CaPETaseWT. The M3 mutant had a Tm value increased by 1.9 °C and a PET degradation activity increased by about 1.32-fold compared to CaPETaseWT (Table 3).

[0256] Next, the three mutations confirmed as described above and the mutations derived from the structural analysis were combined and introduced to prepare M4 to M7 mutants. When the PET degradation activities and Tm values of the four mutants were confirmed, the M4 mutant had a Tm value increased by 5.4 °C and a PET degradation activity increased by about 1.32-fold compared to CaPETaseWT. The M5 mutant had a Tm value increased by 3.5 °C and a PET degradation activity increased by about 1.22-fold compared to CaPETaseWT. The M6 mutant had a Tm value increased by 6.2 °C and a PET degradation activity increased by about 1.36-fold compared to CaPETaseWT. The M7 mutant had a Tm value increased by 8.0 °C and a PET degradation activity increased by about 1.35-fold compared to CaPETaseWT (Table 4). From these results, it was confirmed that introducing mutations into the CaPETase protein improved the thermal stability of the protein and also improved the PET degradation activity.

[0257] [Table 3]

[0258] [Table 4]

Example

[0259] Effect according to the position of the CaPETase mutant To confirm the changes in the thermal stability and PET degradation activity of the protein when the positions of the mutations confirmed in Example 5 were substituted with other amino acids, the following mutants were prepared in the same manner as in Example 2, and the PET degradation activity and thermal stability were evaluated in the same manner as in Example 4. The results are shown in Table 5.

[0260] [Table 5] JPEG2026517275000007.jpg43149

[0261] These results confirm that substituting certain amino acids when changing the three mutation sites in CaPETase to other amino acids affects the improvement of protein activity or thermal stability. This means that the evaluated amino acid positions have a direct or indirect impact on protein activity or thermal stability.

[0262] From the above explanation, a person skilled in the art to which this application pertains will understand that this application can be implemented in other specific forms without altering its technical idea or essential features. It should be understood that the above embodiments are merely illustrative and not limiting. This application should be interpreted as including all modified or altered forms derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. A mutant polypeptide having PET (polyethylene terephthalate) degradation activity, i) The mutant polypeptide has 70% or more and less than 100% sequence identity with SEQ ID NO: 1, and / or ii) The mutant polypeptide is a polypeptide encoded by a polynucleotide having 70% or more and less than 100% sequence identity with the sequence encoding the mature polypeptide of SEQ ID NO: 1, and / or iii) The mutant polypeptide is a polypeptide encoded by (a) the mature polypeptide coding sequence of Sequence ID No. 1, (b) its cDNA, or (c) a polynucleotide that hybridizes with the full-length complementary sequence of (a) or (b) under low stringency, moderate stringency, upper-medium stringency, high stringency, or very high stringency conditions, and / or iv) The mutant polypeptide is a functional fragment of the i), ii), or iii) polypeptide having PET degradation activity, and This includes any modification selected from deletion, insertion, substitution, and / or combination thereof at at least one amino acid position 129, 198, and 196, The above position number corresponds to the position of polypeptide in sequence number 1. Mutant polypeptide.

2. The original mutant polypeptide having PET degradation activity has valine (V) as its 129th amino acid, arginine (R) as its 198th amino acid, and / or glycine (G) as its 196th amino acid. The mutant polypeptide according to claim 1.

3. The aforementioned mutant polypeptide is The 129th, The 198th, The 196th, The 129th and 196th, The 129th and 198th, The 196th and 198th, This includes a modification of an amino acid at any position selected from the group consisting of the 129th, 196th, and 198th positions. The above position number corresponds to the position of polypeptide in sequence number 1. The mutant polypeptide according to claim 1.

4. The aforementioned mutant polypeptide is Substitution of the amino acid corresponding to the 129th position with threonine or serine, Substitution of the amino acid corresponding to the 198th position with lysine or aspartic acid, The molecule comprises at least one substitution selected from the group consisting of substitutions of the amino acid corresponding to the 196th position with threonine, alanine, isoleucine, valine, or serine, The above position number corresponds to the position of polypeptide in sequence number 1. The mutant polypeptide according to claim 1.

5. The aforementioned mutant polypeptide is Modification of valine at position 129 to threonine or serine, Modification of glycine at position 196 to threonine, alanine, isoleucine, leucine, valine, or serine, Modification of arginine at position 198 to lysine or aspartic acid, The modification of valine at position 129 to threonine and the modification of glycine at position 196 to threonine, The modification of valine at position 129 to threonine and the modification of arginine at position 198 to lysine, The modification of glycine at position 196 to threonine and the modification of arginine at position 198 to lysine, The invention comprises at least one modification selected from the group consisting of modification of valine at position 129 to threonine, modification of glycine at position 196 to threonine, and modification of arginine at position 198 to lysine, The above position number corresponds to the position of polypeptide in sequence number 1. The mutant polypeptide according to claim 1.

6. A mutant polypeptide comprising any one of claims 1 to 5, composition.

7. The aforementioned composition is for PET decomposition. The composition according to claim 6.

8. Encoding a mutant polypeptide according to any one of claims 1 to 5, Polynucleotide.

9. A mutant polypeptide according to any one of claims 1 to 5, The polynucleotide encoding the mutant polypeptide, Nucleic acid constructs comprising the aforementioned polynucleotides, and / or A vector comprising the nucleotide or nucleic acid construct host cell.

10. The step of culturing host cells according to claim 9, The step includes recovering the mutant polypeptide having PET degradation activity according to any one of claims 1 to 5 that is expressed in the culture step, A method for producing mutant polypeptides having PET degradation activity.

11. A mutant polypeptide according to any one of claims 1 to 5, A host cell expressing the polypeptide, and / or The process includes treating a substrate with a composition containing the aforementioned mutant polypeptide. How to break down polyester.

12. The aforementioned polyester is PET. The disassembly method according to claim 11.

13. A mutant polypeptide according to any one of claims 1 to 5, A host cell expressing the polypeptide, and / or The process includes contacting a composition containing the polypeptide with a polyester. A method for producing mono(2-hydroxymethyl) terephthalate (MHET), terephthalic acid (TPA), and / or ethylene glycol (EG).

14. The aforementioned polyester is PET. A method for producing mono(2-hydroxymethyl) terephthalate (MHET), terephthalic acid (TPA), and / or ethylene glycol (EG) as described in claim 13.

15. The method further includes the step of recovering the manufactured MHET, TPA and / or EG. A method for producing mono(2-hydroxymethyl) terephthalate (MHET), terephthalic acid (TPA), and / or ethylene glycol (EG) as described in claim 13.

16. The step includes synthesizing polyester using MHET, TPA and / or EG produced by the method described in any one of claims 13 to 15, How to produce polyester.

17. The aforementioned polyester is PET. A method for producing polyester according to claim 16.

18. A mutant polypeptide according to any one of claims 1 to 6, A host cell expressing the aforementioned mutant polypeptide, or A composition containing the aforementioned mutant polypeptide, Used for PET decomposition.

19. For use in the reaction with polyester in the production of mono(2-hydroxymethyl) terephthalate (MHET), terephthalic acid (TPA) and / or ethylene glycol (EG), Use of a mutant polypeptide according to any one of claims 1 to 6 or a composition containing the mutant polypeptide.

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