Novel PET degrading enzymes and uses thereof

Abstract

The invention relates to a polypeptide with PET degrading activity and application thereof.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to a modified polypeptide having PET-degrading activity and uses thereof. BACKGROUND

[0002] More than 400 million tons of plastic are newly generated every year, and as environmental problems regarding waste plastics arise, efforts are being made to reduce the production of plastics by establishing regulations on disposable products and using plastic alternatives. However, the use of plastics is actually increasing every year. PET accounts for less than 10% of all plastics, about 360 million tons are newly produced every year, and is considered to be the shortest life cycle plastic because it is mainly used for disposable products. The recycling of waste plastics includes mechanical recycling, pyrolysis, chemical recycling, etc., and various methods have been commercialized or are in the final research stage of commercialization. Various technologies can be a solution to the problem of waste plastics, but none of the existing methods can be considered perfect because there are problems such as quality degradation caused by downcycling, carbon neutralization, resource depletion, effects on seawater and freshwater eutrophication, etc.

[0003] In order to solve the environmental problems caused by waste plastics such as microplastics, greenhouse gas emissions, and resource depletion, a series of research findings were published that degradation of representative plastic PET was achieved by using biotechnology using enzymes (U.S. Patent No. 10851355). SUMMARY

[0004] [TECHNICAL PROBLEM] The present disclosure relates to a new polypeptide having polyethylene terephthalate (PET)-degrading activity, a method of degrading polyesters such as PET, etc. using the same, and a method of synthesizing polyesters such as PET, etc. by recovering mono(2-hydroxymethyl) terephthalate (MHET), terephthalic acid (TPA), and / or ethylene glycol (EG) obtained by degradation, using the same.

[0005] [TECHNICAL SOLUTION] An object of the present disclosure is to provide a modified polypeptide having PET-degrading activity.

[0006] Another object of the present disclosure is to provide a composition comprising the modified polypeptide.

[0007] Another object of the present disclosure is to provide a polynucleotide encoding the modified polypeptide.

[0008] Another object of the present disclosure is to provide a host cell comprising the modified polypeptide; a polynucleotide encoding the polypeptide; a nucleic acid construct comprising the polynucleotide; and / or a vector comprising the polynucleotide or the nucleic acid construct.

[0009] Another object of the present disclosure is to provide a method of preparing a modified polypeptide having PET-degrading activity.

[0010] Another object of the present disclosure is to provide a method of degrading polyester, the method comprising treating the polyester with the modified polypeptide; a host cell expressing the polypeptide; and / or a composition comprising the modified polypeptide.

[0011] Another object of the present disclosure is to provide a method of producing mono(2-hydroxymethyl) terephthalate (MHET), terephthalic acid (TPA), and / or ethylene glycol (EG), the method comprising contacting a polyester with the modified polypeptide; a host cell expressing the polypeptide; and / or a composition comprising the polypeptide.

[0012] Another object of the present disclosure is to provide a method of producing polyester, the method comprising a step of synthesizing polyester using the MHET, TPA, and / or EG produced by the above method.

[0013] Another object of the present disclosure is to provide use of the modified polypeptide, a host cell expressing the modified polypeptide, and / or a composition comprising the modified polypeptide in degrading PET.

[0014] Another object of the present disclosure is to provide use of the modified polypeptide, a host cell expressing the polypeptide, or a composition comprising the modified polypeptide for reaction with a polyester to produce mono(2-hydroxymethyl) terephthalate (MHET), terephthalic acid (TPA), and / or ethylene glycol (EG).

[0015] [Advantages] By using the polypeptide having PET-degrading activity of the present disclosure to enzymatically degrade polyester bonds, polyester such as PET and the like is degraded in an environmentally friendly manner, while producing degradation products such as mono(2-hydroxymethyl) terephthalate (MHET), terephthalic acid (TPA), and / or ethylene glycol (EG) having high commercial value, which can be recycled in the production of polyester such as PET and the like. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Results of investigating PET-degrading activity and Tm values by deriving 10 PETase candidates are shown; and Figure 2 Results of detecting PET-degrading activity of CaPETase are shown. DETAILED DESCRIPTION

[0017] One aspect of the present disclosure provides a modified polypeptide having PET-degrading activity.

[0018] In one embodiment, i) the modified polypeptide is a polypeptide having at least 70% and less than 100% sequence identity to SEQ ID NO: 1; and / or ii) the modified polypeptide is a polypeptide encoded by a polynucleotide having at least 70% and less than 100% sequence identity to the coding sequence of the mature polypeptide of SEQ ID NO: 1; and / or iii) the modified polypeptide is a polypeptide encoded by a polynucleotide that hybridizes under low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with (a) the coding sequence of the mature polypeptide of SEQ ID NO: 1, (b) the cDNA thereof, or (c) the full-length complement of (a) or (b); and / or iv) the modified polypeptide is a functional fragment of the polypeptide of i) to iii) that has PET-degrading activity; and the modified polypeptide comprises any one of the following modifications: an amino acid deletion, an amino acid insertion, a different amino acid substitution at any one or more of positions 129, 198, and 196, and / or combinations thereof; wherein the position numbers are positions corresponding to the positions of the polypeptide of SEQ ID NO: 1.

[0019] In any of the specific embodiments, prior to the modification, the amino acid at position 129 can be valine (V); the amino acid at position 198 can be arginine (R); and / or the amino acid at position 196 can be glycine (G).

[0020] In any of the specific embodiments, the modified polypeptide can comprise a substitution at any one or more of the following positions: 129; 198; 196; 129 and 196; 129 and 198; 196 and 198; or 129, 196, and 198; wherein the position numbers are positions corresponding to the positions of the polypeptide of SEQ ID NO: 1.

[0021] In any of the specific embodiments, the modified polypeptide can comprise any one or more of the following substitutions: the amino acid corresponding to position 129 is substituted with threonine or serine; the amino acid corresponding to position 198 is substituted with lysine or aspartic acid; and the amino acid corresponding to position 196 is substituted with threonine, alanine, isoleucine, valine, or serine; wherein the position number is a position corresponding to a position of the polypeptide of SEQ ID NO: 1.

[0022] In any of the specific embodiments, the modified polypeptide can comprise any one or more modifications selected from the following modifications: a valine at position 129 is modified to threonine or serine; a glycine at position 196 is modified to threonine, alanine, isoleucine, leucine, valine, or serine; an arginine at position 198 is modified to lysine or aspartic acid; a valine at position 129 and a glycine at position 196 are modified to threonine; a valine at position 129 is modified to threonine and an arginine at position 198 is modified to lysine; a glycine at position 196 is modified to threonine and an arginine at position 198 is modified to lysine; a valine at position 129 is modified to threonine, a glycine at position 196 is modified to threonine, and an arginine at position 198 is modified to lysine; wherein the position number is a position corresponding to a position of the polypeptide of SEQ ID NO: 1.

[0023] Another aspect of the present disclosure provides a composition comprising the modified polypeptide having PET-degrading activity.

[0024] In any of the specific embodiments, the composition can be a composition for degrading PET.

[0025] Another aspect of the present disclosure provides a polynucleotide encoding the modified polypeptide.

[0026] Another aspect of the present disclosure provides a nucleic acid construct comprising the polynucleotide.

[0027] Another aspect of the present disclosure provides a vector comprising the polynucleotide or the nucleic acid construct.

[0028] Another aspect of the present disclosure provides a host cell comprising the modified polypeptide, the polynucleotide, the nucleic acid construct, and / or the vector.

[0029] Another aspect of the present disclosure provides a method of preparing the modified polypeptide, the method comprising the steps of culturing a host cell; and recovering the modified polypeptide expressed in the culturing step.

[0030] Another aspect of the present disclosure provides a method of degrading a polyester, the method comprising treating the polyester with the modified polypeptide; a host cell expressing the polypeptide; and / or a composition comprising the modified polypeptide.

[0031] In any of the specific embodiments, the polyester can be PET.

[0032] Another aspect of the present disclosure provides a method of producing mono(2-hydroxymethyl) terephthalate (MHET), terephthalic acid (TPA), and / or ethylene glycol (EG), the method comprising contacting a polyester with the modified polypeptide; a host cell expressing the polypeptide; and / or a composition comprising the polypeptide.

[0033] In any of the specific embodiments, the polyester can be PET.

[0034] In any of the specific embodiments, the method can further comprise the step of recovering the MHET, TPA, and / or EG produced by the above method.

[0035] Another aspect of the present disclosure provides a method of producing a polyester, the method comprising the step of synthesizing a polyester using the produced MHET, TPA, and / or EG.

[0036] In any of the specific embodiments, the polyester can be PET.

[0037] Another aspect of the present disclosure provides the use of the modified polypeptide, a host cell expressing the modified polypeptide, and / or a composition comprising the modified polypeptide in degrading PET.

[0038] Another aspect of the present disclosure provides the use of the modified polypeptide or a composition comprising the modified polypeptide for reacting with a polyester to produce mono(2-hydroxymethyl) terephthalate (MHET), terephthalic acid (TPA), and / or ethylene glycol (EG).

[0039] Modes for carrying out the present invention The present disclosure will be described in detail below. At the same time, each description and embodiment disclosed in the present disclosure can also be applied to other descriptions and embodiments. That is, all combinations of various elements disclosed in the present disclosure fall within the scope of the present disclosure. In addition, the scope of the present disclosure is not limited by the specific descriptions described below.

[0040] In addition, those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents for the specific embodiments of the disclosure described herein. In addition, such equivalents are intended to be within the scope of the present disclosure.

[0041] As used in the specification and the appended claims of the present disclosure, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Singular terms will include the plural and plural terms will include the singular, unless they are clearly indicated to not do so. As used in the specification and the appended claims of the present disclosure, the use of “or” can be used to mean “and / or” unless stated otherwise.

[0042] As used herein, the term “about” can precede a recited numerical value. The term “about” as used herein, includes not only the recited precise number but also a range of numbers close or approximate to that number. Whether a number is close or approximate to a particular recited number can be determined in view of the context in which the number is presented. In one example, the term “about” can refer to a range of -10% to +10% of an index value. In another example, the term “about” can refer to a range of -5% to +5% of a given numerical value, but is not limited thereto.

[0043] As used herein, descriptions such as the terms “first, second, third, …”, “i), ii), iii), …” or “(a), (b), (c), (d), …” are used to distinguish like elements, and these terms do not imply that the elements are continuous or sequential. For example, when the terms are used to refer to steps of a method, use, or assay, there can be no time interval between the steps, or they can be performed simultaneously, or they can be performed several seconds, minutes, hours, days, or months apart.

[0044] As used herein, the term “consisting essentially of’ can mean that, when the features of the subject matter claimed herein are not substantially affected by the presence of the unspecified components, the unspecified components can be present.

[0045] As used herein, the term “consisting of’ refers to a total ratio of 100% of the specified components. The components or features recited after the term “consisting of’ can be necessary or mandatory. In some embodiments, any other components or non-essential components can be excluded in addition to the components or features recited after the term “consisting of’.

[0046] As used herein, the term “comprising” means the presence of the features, steps, or components recited after the term and does not exclude the presence or addition of one or more features, steps, or components. The components or features recited herein after the term “comprising” can be necessary or mandatory. However, in some embodiments, the term can also include any other or non-essential components or features.

[0047] As used herein, in some embodiments, the term "comprising" can be modified to mean "consisting essentially of or "consisting of.

[0048] With respect to the amino acid sequences in the present disclosure, although it is described as a polypeptide "comprising" the amino acid sequence described by a particular sequence number, a polypeptide "consisting of" the amino acid sequence described by a particular sequence number, or a polypeptide or protein "having" the amino acid sequence described by a particular sequence number, it is apparent that any protein having an amino acid sequence in which part of the sequence is deleted, modified, substituted, conservatively substituted, or added can be used in the present disclosure, as long as it has the same or corresponding activity as a polypeptide consisting of the amino acid sequence of the corresponding sequence number. For example, it can be a sequence addition at the N-terminus and / or C-terminus of the amino acid sequence, a naturally occurring mutation, a silent mutation or a conservative substitution thereof, but is not limited thereto.

[0049] As used herein, the term "protein" or "polypeptide" refers to a polymer or oligomer of contiguous amino acid residues. In the present disclosure, "polypeptide", "protein", and "peptide" can be used interchangeably with "amino acid sequence".

[0050] In some cases, an amino acid sequence exhibiting activity can be referred to as an "enzyme". In the present disclosure, the amino acid sequence is described in the direction from N-terminus to C-terminus, unless otherwise specified.

[0051] As used herein, the term "recombinant" with respect to a cell, or a nucleic acid, polypeptide, or vector, means that the cell, nucleic acid, polypeptide, or vector has been modified by the introduction of a heterologous nucleic acid or polypeptide or alteration of a native nucleic acid or polypeptide, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell can express genes that are not found in cells of the same species in nature, or express genes that are found in nature but expressed at a different level, or express genes that are not expressed in nature. In some embodiments, a recombinant cell can express a gene that is not found in cells of the same species in nature.

[0052] As used herein, the term "isolated" refers to a substance or a substance that does not exist in a naturally occurring environment. It includes a substance (sequence, enzyme, or nucleic acid) that has been at least substantially free of naturally associated and found in nature, for example, at least one other component having the sequence, enzyme, or nucleic acid.

[0053] For example, the isolated sequence, enzyme, or nucleic acid provided herein can be provided in a form substantially free of one or more contaminants.

[0054] Examples of isolated substances include: i) any non-naturally occurring substance; ii) any substance in which one or more or all of the naturally occurring components associated with the substance have been removed (e.g., an enzyme, variant, nucleic acid, protein, peptide, or cofactor); iii) any substance that has been artificially modified from a substance found in nature; or iv) any substance that has been modified to alter the amount of the substance relative to other components associated with the substance in nature (e.g., increasing the copy number of a gene encoding a particular substance; modifying a promoter associated with a gene encoding a particular substance to be a high-activity promoter, etc.), but are not limited thereto.

[0055] As used herein, the term “wild-type” refers to a naturally occurring state without artificial modification. When the term “wild-type” is used to refer to a polypeptide, it refers to a naturally occurring polypeptide without artificial mutations (substitutions, insertions, deletions, etc.) at one or more amino acid positions. Similarly, when the term “wild-type” is used to refer to a polynucleotide, it means without artificial modifications (substitutions, insertions, deletions) in one or more nucleotides. However, a polynucleotide encoding a wild-type polypeptide is not limited to a naturally occurring polynucleotide, including a sequence encoding any wild-type polypeptide.

[0056] As used herein, a parent sequence or backbone refers to a reference sequence into which modifications are introduced to become a modified polypeptide. That is, a parent sequence can serve as a starting sequence into which modifications, such as substitutions, additions, and / or deletions, can be introduced. A parent sequence can be naturally occurring or wild-type, or a variant in which one or more substitutions, insertions, or deletions have occurred from the natural or wild-type, or can be an artificially synthesized sequence. When the parent sequence is an amino acid sequence that exhibits activity, i.e., an amino acid sequence of an enzyme, it can be referred to as a parent enzyme.

[0057] As used herein, the term “reference sequence” refers to a sequence used to determine the position of an amino acid within any amino acid sequence. Any amino acid sequence can be aligned with a reference sequence to determine the position of an amino acid in the any amino acid sequence that corresponds to a particular position of the reference sequence.

[0058] With regard to an amino acid or nucleic acid sequence in the present disclosure, the term “fragment” refers to a portion of a parent sequence. For example, it can be a polypeptide in the form of one or more amino acids removed from the C-terminus or N-terminus of a parent sequence.

[0059] As used herein, the term “fragment” of an enzyme can refer to a “functional fragment.” A “functional fragment” can also be referred to as an active fragment, and refers to a polypeptide that is a part of a parent enzyme and has the enzymatic activity of the parent enzyme. For example, a functional fragment of an enzyme can comprise a catalytic site of the enzyme.

[0060] A fragment of an enzyme can comprise a portion of the full length of the parent enzyme. For example, a fragment of an enzyme can comprise at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or more, or about less than 100% of the amino acids of the full length of the parent enzyme, but is not so limited.

[0061] As used herein, the term "modification" means a change or alteration. This can be from the natural state. For example, an enzyme can be altered in such a way that the enzyme is different from a parent sequence or a reference sequence.

[0062] In the present disclosure, a modified enzyme can be an enzyme that does not exist in nature, i.e., a non-naturally occurring enzyme.

[0063] As used herein, the term "modified" refers to a change from its naturally occurring form, for example. The modified enzymes of the present disclosure include non-naturally occurring enzymes or naturally occurring variants. For example, the modified enzymes of the present disclosure are modified enzymes that have not been found in nature. For example, the modified enzymes of the present disclosure can be enzymes that do not occur spontaneously, but are not so limited.

[0064] As used herein, the term "modification" when used in reference to an amino acid / nucleic acid sequence can include substitution of an amino acid / nucleic acid residue of a parent sequence with a different amino acid / nucleic acid residue at one or more positions in the amino acid sequence, deletion of an amino acid / nucleic acid residue (or series of amino acid / nucleic acid residues) at one or more positions of the parent sequence, insertion of an amino acid / nucleic acid residue (or series of amino acid / nucleic acid residues) at one or more positions of the parent sequence, or truncation of the N-terminal and / or C-terminal amino acid sequence or 5' and / or 3' nucleic acid sequence, and any combination thereof.

[0065] As used herein, the term "variant" or "modified polypeptide" of an enzyme refers to a protein having one or more amino acids that are different from a parent enzyme by conservative substitution and / or other modification. "Variant" or "modified polypeptide" can be used interchangeably. The variant or modified polypeptide can be non-naturally occurring, but is not so limited.

[0066] A variant differs from the sequence of a parent enzyme by one or more modifications (e.g., amino acid substitutions, deletions, and / or insertions).

[0067] Such variants can generally be identified by modifying one or more amino acids of a parent enzyme and evaluating the properties of the modified protein. In other words, the ability of a variant can be enhanced, unchanged, or decreased compared to the ability of the parent enzyme.

[0068] In addition, some variants can include modified polypeptides in which one or more regions, such as an N-terminal leader sequence or a transmembrane domain, have been removed.

[0069] Other variants can include variants in which a portion has been removed from the N-terminus and / or C-terminus of the mature protein.

[0070] The term "variant" or "modified polypeptide" can be used interchangeably with terms such as modification, modified protein, mutant, mutein, divergent, variant, and the like, and is not limited thereto, as long as the term is used to indicate a mutation.

[0071] The variant can also include deletion or addition of an amino acid that has minimal effect on the properties and secondary structure of the polypeptide. For example, the polypeptide can be linked to an N-terminal signal (or leader) sequence of the protein, which is involved in protein translocation in a co-translational or post-translational manner. In addition, the polypeptide can also be linked to another sequence or linker to identify, purify, or synthesize the polypeptide.

[0072] As used herein, the term "conservative substitution" refers to the substitution of an amino acid with another amino acid having similar structure and / or chemical properties. Such amino acid substitution can typically occur based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues.

[0073] Throughout the specification of the present disclosure, the conventional one-letter and three-letter codes for naturally occurring amino acids are used. In addition, the amino acids mentioned herein as abbreviations are described according to the following IUPAC-IUB nomenclature rules: Alanine Ala, A Arginine Arg, R Asparagine Asn, N Aspartic Acid Asp, D Cysteine Cys, C Glutamic Acid Glu, E Glutamine Gin, Q Glycine Gly, G Histidine His, H Isoleucine Ile, I Leucine Leu, L Lysine Lys, K Methionine Met, M Phenylalanine Phe, F Proline Pro, P Serine Ser, S Threonine Thr, T Tryptophan Trp, W Tyrosine Tyr, Y Valine Val, V Meanwhile, any amino acid can be described as Xaa or X.

[0074] In addition, the three-letter code is generally not only applicable to naturally occurring amino acids, but also to other amino acids such as Aib (2-amino isobutyric acid), Sar (N-methyl glycine), α-methyl glutamic acid, etc.

[0075] Amino acids can generally be classified based on similarities in their polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties. Thus, amino acid substitutions can generally occur based on similarities in their polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties.

[0076] For example, among amino acids with charged side chains (charged amino acids), positively charged (basic) amino acids include arginine, lysine, and histidine, and negatively charged (acidic) amino acids include glutamic acid and aspartic acid; among amino acids with uncharged side chains (uncharged amino acids), 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.

[0077] As used herein, the term "gene" refers to a polynucleotide that encodes a polypeptide and includes regions upstream and downstream of the coding region. In some embodiments, a gene can have sequences (introns) inserted between individual coding regions (exons).

[0078] As used herein, the terms "homology" or "identity" refer to the relatedness of two given amino acid sequences or nucleotide sequences and can be expressed in percentage. The terms homology and identity can generally be used interchangeably with each other.

[0079] The sequence homology or identity of a conservative polynucleotide or polypeptide can be determined by standard alignment algorithms and can use the default gap penalties established by the program used. Essentially, homologous or identical sequences can generally hybridize to the full-length sequence or at least about 50%, about 60%, about 70%, about 80%, or about 90% of the full-length under moderately or highly stringent conditions. Obviously, hybridization also encompasses the hybridization of a polynucleotide to a polynucleotide containing universal or codons that take into account the codon degeneracy.

[0080] Whether any two polynucleotide or polypeptide sequences are substantially identical, similar, or identical can be determined, e.g., using known computer algorithms, such as the "FASTA" program using default parameters of Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, it can be determined by the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later), using the default parameters of the program (GCG program package (Devereux, J. et al., Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO et al.] (1988) SIAM J Applied Math 48: 1073). For example, the BLAST or ClustalW of the National Center for Biotechnology Information can be used to determine the homology, similarity, or identity, but are not limited thereto.

[0081] Homology, similarity or identity of polynucleotides or polypeptides can be determined, for example, by comparing sequence information using, for example, the GAP computer program, as disclosed in Needleman et al. (1970), J Mol Biol. 48:443, as disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the GAP program defines homology, similarity or identity as the number of similar aligned 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 can include: (1) a unitary matrix (including a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14: 6745, as disclosed in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979) (or the EDNAFULL substitution matrix (EMBOSS version of NCBI NUC4.4)); (2) a penalty of 3.0 for each gap, and an extra penalty of 0.10 for each symbol in each gap (or a gap open penalty of 10, and a gap extension penalty of 0.5); and (3) no penalty for end gaps.

[0082] Furthermore, whether any two polynucleotide or polypeptide sequences have homology, similarity or identity to each other can be determined by comparing sequence information by performing Southern hybridization experiments under defined stringent conditions, and defined suitable hybridization conditions are within the skill in the art and can be determined by methods well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York), but are not limited thereto.

[0083] As used herein, the term "mature polypeptide" refers to a form of the polypeptide without the signal sequence or propeptide sequence. The mature protein / polypeptide / peptide can be a functional form of the protein / polypeptide / peptide. The mature polypeptide can be the final form after translation, or after post-translational modification. Examples of post-translational modifications include, but are not limited to, N-terminal or C-terminal modifications, glycosylation, phosphorylation, leader sequence removal, and the like.

[0084] As used herein, the term "nucleic acid construct" refers to a single- or double- stranded nucleic acid molecule, comprising one or more control sequences, and which is either artificial or man-made, or engineered to contain specific sequences not found in nature or isolated from nature.

[0085] As used herein, the term "expression" encompasses any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0086] As used herein, the term "expression vector" refers to a linear or circular nucleic acid molecule, comprising a coding sequence and operably linked control sequences for its expression.

[0087] As used herein, the term "operably linked" refers to the configuration of control sequences in which they perform their intended function when linked to a coding sequence. Thus, the term "operably linked" includes attachment of a regulatory region (such as a promoter, terminator, signal sequence, or enhancer) to a target (gene or polypeptide) in a manner that allows for expression, secretion, or function of the target in accordance with the known or intended activity of the regulatory region.

[0088] As used herein, the term "cDNA" refers to a DNA sequence that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. A cDNA sequence lacks intervening sequences found in the corresponding genomic DNA sequence. The initial primary RNA transcript is the precursor to mRNA, which emerges as mature, spliced mRNA after a series of steps including splicing.

[0089] As used herein, the term "control sequence" refers to polynucleotide sequences necessary for expression of a coding sequence. Each control sequence can be native with the coding sequence or foreign (derived from a different gene). Examples of control sequences can include a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, operator sequence, ribosome binding domain, and sequences that control transcription and translation termination. A minimal control sequence can include a promoter and sequences that terminate transcription and translation.

[0090] To describe the variants provided in the present disclosure, the following nomenclature is used.

[0091] In the present disclosure, reference to a particular position in an amino acid sequence can include reference to an amino acid present at or substituted at that position. Reference to an amino acid at a particular position can be described in a variety of ways. For example, "position 3" can be described as "3", "amino acid 3", "3rd amino acid". Additionally, for example, when the amino acid at position 3 is serine (S), this can be described as "S3" or "Ser3".

[0092] An amino acid substitution can be represented by describing the amino acid before substitution, the position, and the amino acid to be substituted in order. Amino acids can be expressed using conventional one-letter and three-letter codes. For example, when the amino acid alanine at position 8 of a particular sequence is substituted with valine, this can be described as "A8V" or "Ala8Val".

[0093] An arbitrary amino acid at a particular position can be referred to as "X". For example, X6 refers to an arbitrary amino acid at position 6. Further, when the amino acid to be substituted is represented as X, it means that the amino acid is substituted with an amino acid different from the amino acid present before substitution. For example, "V6X" indicates that V at position 6 is substituted with an arbitrary amino acid other than V.

[0094] Different substitutions can be expressed by describing several types of amino acids at the same time using symbols such as or ",". For example, when the amino acid (D) at position 12 is substituted with S or K, this can be described as D12S,K.

[0095] Multiple mutations can be described using "+" or " / ". For example, a description such as "G2A+M8V" means that the amino acid glycine at position 2 is substituted with alanine and the amino acid methionine at position 8 is substituted with valine, respectively. For example, L180C / A202C / R242C / S291C means that the amino acid leucine at position 180 is substituted with cysteine, the amino acid alanine at position 202 is substituted with cysteine, the amino acid arginine at position 242 is substituted with cysteine, and the amino acid serine at position 291 is substituted with cysteine.

[0096] As used herein, the term "corresponding to" refers to an amino acid residue at the position described in a protein or polypeptide, or an amino acid residue similar, identical, or homologous to the residue described in a protein or polypeptide. Identifying the amino acid at the corresponding position can be determining a particular amino acid in the sequence of a particular sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related protein or a reference protein.

[0097] In the present disclosure, SEQ ID NO: 1 can be used as a reference sequence to determine the position of an amino acid in any amino acid sequence.

[0098] In other words, SEQ ID NO: 1 disclosed herein can be used to determine the corresponding amino acid residues in any polypeptide having PET-degrading activity. The residues of a particular amino acid sequence are numbered based on the numbering of SEQ ID NO: 1, unless otherwise specified in the disclosure.

[0099] For example, any amino acid sequence is aligned with SEQ ID NO: 1, based on which alignment, each amino acid residue of the amino acid sequence can be numbered with reference to the amino acid residue number position corresponding to the amino acid residues of SEQ ID NO: 1. For example, sequence alignment algorithms as described herein can identify the position of an amino acid or the position of a modification such as a substitution, insertion, or deletion compared to a query sequence (also referred to as a “reference sequence”).

[0100] In such alignment, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needleman program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), Trends Genet. 16: 276-277), etc. can be used, but are not limited thereto.

[0101] Further, the corresponding amino acid residues in another PETase can be identified by multiple sequence alignment. Examples of multiple sequence alignment known in the art include programs such as MUSCLE (multiple sequence comparison by log-Expectation; version 3.5 or higher; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT (version 6.857 or higher; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518; Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009, Methods in Molecular Biology 537: 39-64; Katoh and Toh, 2010, Bioinformatics 26: 1899-1900), and EMBOSS EMMA employing ClustalW (version 1.83 or higher; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), and the like, and can be used with their respective default parameters, but are not limited thereto.

[0102] Furthermore, where enzymes derived from the mature polypeptide of SEQ ID NO: 1 are unable to detect relationships among them through traditional sequence-based comparisons, other pairwise sequence comparison algorithms can be used (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615). Greater sensitivity in sequence-based searching can be attained using search programs that utilize profiles (profiles) of a family of polypeptides to search a database. For example, the PSI BLAST program generates profiles through a iterative database search process and is capable of detecting remote homologs (Atschul et al., 1997, Nucleic Acids Res. 25: 3389-3402). Still greater sensitivity can be achieved if a family, or superfamily, of polypeptides has one or more representatives in a protein structure database. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffin and Jones, 2003, Bioinformatics 19: 874-881) utilize information from a variety of sources, such as PSI BLAST, secondary structure prediction, structure alignment profiles, and solvation potentials, as input to neural networks that predict the structural fold for a query sequence. Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919 can be used to align an unstructured sequence to a family model in the SCOP database. These alignments can be used to generate homology models for a sequence and the accuracy of the models can be assessed using a variety of tools developed for this purpose.

[0103] For proteins of known structure, several tools and resources are available to retrieve and generate structure alignments. For example, the SCOP superfamily for a protein has been subjected to structural alignment, and these alignments are accessible and downloadable. Two or more protein structures can be aligned using a variety of algorithms, such as the distance alignment matrix (Holm and Sander, 1998, Proteins 33: 88-96) or CE (combinatorial extension) (Shindyalov and Bourne, 1998, Protein Engineering 11 : 739-747). Implementation of these algorithms can also be used to query a structural database with a structure of interest in order to discover possible structural homologs (Holm and Park, 2000, Bioinformatics 16: 566-567).

[0104] The above-described method is exemplary and is not limited thereto.

[0105] Hereinafter, specific embodiments of the present disclosure will be described in more detail.

[0106] In the present disclosure, a "polypeptide having PET-degrading activity" or "PETase" is a polypeptide having depolymerization activity for polyethylene terephthalate (PET), and can also include a polypeptide having depolymerization activity for oligomers obtained by PET depolymerization, for example, bis(2-hydroxyethyl) terephthalate (BHET). The term "depolymerization" refers to a process in which a polymer or at least one polymer of a plastic material is depolymerized into smaller molecules, such as monomers and / or oligomers.

[0107] In the present disclosure, PET-degrading activity can be measured and evaluated by including the embodiments described herein using methods known in the art, for example, can be evaluated by measuring the production of BHET, MHET, TPA, or EG.

[0108] As used herein, the term "parent PETase" refers to a PETase that is modified in order to produce a variant or modified polypeptide of the present disclosure. Specifically, the parent PETase, parent enzyme, or parent sequence can be a naturally occurring polypeptide or wild-type polypeptide, can be a mature polypeptide thereof, and can include a variant or functional fragment thereof, but is not limited thereto, as long as the polypeptide has PETase activity and can be a parent of a variant.

[0109] The parent PETase provided in the present disclosure can be a polypeptide of SEQ ID NO: 1, but is not limited thereto. In addition, it can be a polypeptide having about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with the polypeptide of SEQ ID NO: 1, as long as it has PET-degrading activity, and any polypeptide can be included in the scope of the parent PETase, as long as it has the same or corresponding activity as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0110] The parent PETase of the variant provided herein can be derived from a Cryptosporangium sp. microorganism, specifically, Cryptosporangium aurantiacum.

[0111] Meanwhile, the above-described microorganism is an exemplary microorganism from which the parent PETase provided herein can be derived, and includes a microorganism derived from a microorganism homologous to the microorganism in taxonomy, regardless of the name of the microorganism.

[0112] The above-described microorganism can be obtained from a known microorganism preservation agency such as ATCC, DSMZ, CBS, NRRL, KCTC, KCCM.

[0113] As used herein, a sequence "derived from" a particular microorganism is not limited to a sequence naturally produced in or producible in the microorganism, but also includes a sequence encoded by a gene produced and isolated from the microorganism comprising the gene.

[0114] For example, a PETase derived from the genus Lecythospora can include not only an enzyme having PETase activity naturally produced in the genus Lecythospora, but also those produced in a source derived from the genus Lecythospora, and those produced in other host cells by genetic modification known in the art (e.g., transformation into a sequence encoding the enzyme).

[0115] Further, in the present disclosure, it is newly investigated that the polypeptide of SEQ ID NO: 1 has PET degradation activity, and a modified polypeptide having an altered or enhanced property is prepared by introducing a mutation into the parent sequence of SEQ ID NO: 1. The description of the parent PETase of the variant provided in the present disclosure can be applied to a polypeptide having the same or corresponding activity as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0116] As used herein, a "modified polypeptide having PET degradation activity" can be a variant of a parent PETase.

[0117] As used herein, the term "variant of a PETase" or "PETase variant" refers to a protein having PET degradation activity in which one or more amino acids are different from the amino acid sequence of a parent PETase.

[0118] A "modified polypeptide having PET degradation activity", "variant of a PETase", and "PETase variant" can be used interchangeably.

[0119] The variant provided in the present disclosure can comprise one or more amino acid modifications in the sequence of a parent PETase, while having PETase activity. The modification can be an amino acid deletion, an amino acid insertion, a substitution with another amino acid, and / or a combination thereof, particularly an amino acid substitution, whereby a hydrogen bond can be formed.

[0120] In addition, the variant can be i) a polypeptide having at least 70% and less than 100% sequence identity to SEQ ID NO: 1; and / or ii) the variant can be a polypeptide encoded by a polynucleotide having at least 70% and less than 100% sequence identity to the coding sequence of the mature polypeptide of SEQ ID NO: 1; and / or iii) the variant can be a polypeptide encoded by a polynucleotide that hybridizes under low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with (a) the coding sequence of the mature polypeptide of SEQ ID NO: 1, (b) the cDNA thereof, or (c) the full-length complement of (a) or (b); and / or iv) the variant can be a functional fragment of the polypeptide of i), ii), or iii) having PET-degrading activity.

[0121] In particular, the variants provided by the present disclosure comprise a modification of one or more amino acids in a parent PETase sequence, while having PET-degrading activity, thereby having one or more altered functions or properties compared to the parent PETase.

[0122] In one particular embodiment, the variants provided by the present disclosure comprise a modification of one or more amino acids in a parent PETase sequence, while having PET-degrading activity, thereby having one or more altered functions or properties compared to the parent PETase, and having one or more conservative substitutions.

[0123] The variants provided by the present disclosure are variants of a parent PETase, and can be a polypeptide having PET-degrading activity.

[0124] In one particular embodiment, the variants provided by the present disclosure can comprise a modification corresponding to one or more of positions 129, 198, and 196 of SEQ ID NO: 1. In particular, the modification can be an amino acid deletion, an amino acid insertion, and / or a substitution with another amino acid, in particular an amino acid substitution.

[0125] In the present disclosure, position numbering is the position corresponding to the position of the polypeptide of SEQ ID NO: 1, and the term “corresponding” is as described above.

[0126] In any of the foregoing particular embodiments, the variants provided by the present disclosure can comprise an amino acid modification corresponding to one or more of V129, R198, and G196 of SEQ ID NO: 1.

[0127] In any of the foregoing embodiments, the amino acid at position 129 of SEQ ID NO: 1 provided by the present disclosure prior to modification can be valine (V); the amino acid at position 198 can be arginine (R); and / or the amino acid at position 196 can be glycine (G).

[0128] In any of the foregoing embodiments, the variant provided by the present disclosure can comprise substitution of the amino acid corresponding to position 129 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, particularly with T or S.

[0129] In any of the foregoing embodiments, the variant provided by the present disclosure can comprise substitution of the amino acid corresponding to position 198 of SEQ ID NO: 1 with G, A, V, L, I, M, F, W, P, S, T, C, Y, N, Q, D, E, K, or H, particularly with K or D.

[0130] In any of the foregoing embodiments, the variant provided by the present disclosure can comprise substitution of the amino acid corresponding to position 196 of SEQ ID NO: 1 with A, V, L, I, M, F, W, P, S, T, C, Y, N, Q, D, E, K, R, or H, particularly with T, A, I, L, V, or S.

[0131] In any of the foregoing embodiments, the variant provided by the present disclosure can comprise any one or more of the following substitutions: V129T,S; R198K,D; and G196T,A,L,I,V,S.

[0132] In any of the foregoing embodiments, the variant provided by the present disclosure can comprise any one or more of the following substitutions: the amino acid corresponding to position 129 is substituted with threonine or serine; the amino acid corresponding to position 198 is substituted with lysine or aspartic acid; and the amino acid corresponding to position 196 is substituted with threonine, alanine, isoleucine, valine, or serine.

[0133] In any of the foregoing embodiments, the variant provided by the present disclosure can comprise substitution of the amino acid at position 129 of the amino acid sequence of SEQ ID NO: 1 with threonine, substitution of the amino acid at position 198 of the amino acid sequence of SEQ ID NO: 1 with lysine, and substitution of the amino acid at position 196 of the amino acid sequence of SEQ ID NO: 1 with threonine.

[0134] In any of the foregoing embodiments, the variants provided by the present disclosure comprise all possible combinations of the modifications described above.

[0135] In any of the foregoing embodiments, the variants provided by the present disclosure can have about 60% or more, e.g., 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% sequence identity to the parent PETase, mature polypeptide thereof, or functional fragment thereof.

[0136] In any of the foregoing embodiments, the variants provided by the present disclosure can have about 60% or more, e.g., 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% sequence identity to SEQ ID NO: 1.

[0137] In any of the foregoing embodiments, the variants provided by the present disclosure can be polypeptides encoded by a polynucleotide having about 60% or more, e.g., 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% sequence identity to the coding sequence of the mature polypeptide of SEQ ID NO: 1.

[0138] In any of the foregoing embodiments, the variants provided by the present disclosure can have about 60% or more, e.g., 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% sequence identity to a functional fragment of SEQ ID NO: 1.

[0139] In the variant polypeptides provided by the present disclosure, one or more arbitrary selectable or detectable properties or attributes of the polypeptide can be altered as compared to other parent PETases (e.g., wild-type PETase, parent PETase, other PETase variants, etc.).

[0140] The properties or attributes can include, but are not limited to, oxidative stability, substrate specificity, catalytic activity, thermal stability, alkaline stability, pH activity profile, proteolytic degradation resistance, Km, kcat, kcat / Km ratio, protein folding, ability to induce an immune response, ability to bind a ligand, ability to bind a receptor, ability to be secreted, ability to be displayed on a cell surface, ability to oligomerize, ability to signal, ability to stimulate cell proliferation, ability to inhibit cell proliferation, ability to induce apoptosis, ability to be modified by phosphorylation or glycosylation, and / or ability to treat a disease, etc.

[0141] In particular, the variants provided by the present disclosure can have any one or more of the following altered activities as compared to the parent sequence: i) increased or decreased enzyme activity; ii) increased or decreased specific activity; iii) increased or decreased pH stability; iv) increased or decreased storage stability; v) increased or decreased acid tolerance; vi) increased or decreased thermal tolerance; and vii) altered substrate specificity; but are not limited thereto.

[0142] For another example, the PETases provided by the present disclosure can have any one or more of the following altered activities as compared to a PETase derived from Ideonella sakaiensis or a PETase derived from Thermobifida fusca: i) increased or decreased enzyme activity; ii) increased or decreased specific activity; iii) increased or decreased pH stability; iv) increased or decreased storage stability; v) increased or decreased acid tolerance; vi) increased or decreased thermal tolerance; and vii) altered substrate specificity; but are not limited thereto.

[0143] For another example, the PETases provided by the present disclosure can have any one or more of the following altered activities as compared to a PETase derived from Ideonella sakaiensis or a PETase derived from Thermobifida fusca: i) increased enzyme activity; ii) increased specific activity; iii) increased pH stability; iv) increased storage stability; v) increased acid tolerance; vi) increased heat tolerance; and vii) altered substrate specificity; but not limited thereto.

[0144] As used herein, "enzyme activity" represents at least one catalytic activity. Specifically, it can be the conversion efficiency of an enzyme expressed mainly as kcat / Km, but not limited thereto.

[0145] kcat is the rate constant (catalytic constant) of an enzyme in converting a substrate into a product per unit time, also known as turnover number, when the enzyme is completely saturated with the substrate. cat Km is the substrate concentration at which the reaction rate is half of the maximum (Vmax).

[0146] Examples of the means to express enzyme activity include specific activity (umol converted substrate x mg enzyme x min) or volumetric activity (umol converted substrate x mL enzyme x min), etc. -1 -1 -1 -1

[0147] ​​​​However, the definition of enzyme activity is not limited to the above description and can be defined and evaluated based on the information disclosed in Irwin H. Segel, Enzyme kinetics, John Wiley & Sons, 1979; A. G. Marangoni, Enzyme kinetics, Wiley-Interscience, 2003; A. Fersht, Enzyme structure and mechanisms, John Wiley & Sons, 1981; Structure and Mechanism in Protein Science: A guide to enzyme catalysis and protein folding, Alan Fersht, W. H Freeman, 1999; Fundamentals of Enzyme Kinetics, Athel Cornish-Bowden, Wiley-Blackwell 2012 and Voet et al., “Biochemie” [Biochemistry], 1992, VCH-Verlag, Chapter 13, pages 331-332, etc. regarding enzyme activity.

[0148] In one specific embodiment, the variants provided by the present disclosure can have an enzyme activity that is increased by about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about 200% or more compared to the parent enzyme.

[0149] In another specific embodiment, the variants provided by the present disclosure can have an enzyme activity that is 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.

[0150] As used herein, the term "specific activity" is the enzyme activity per unit weight of protein, which can be expressed in units / mg. The quantification of protein can be performed using, for example, SDS-PAGE or Bradford analysis.

[0151] Enzyme stability refers to the maintenance of enzyme activity during storage or reaction. In order to measure the change in such stability, the initial enzyme activity can be measured and compared at zero time (100%) and after a predetermined time (x %) under defined conditions, and thus, the level of enzyme activity loss or enzyme stability can be expressed.

[0152] Factors that affect enzyme activity include, for example, pH, heat, presence of other substances (e.g., oxidizing agents, chelating agents), etc.

[0153] As used herein, the term "pH stability" refers to the ability of a protein to function within a particular pH range. In one specific embodiment, the variants provided by the present disclosure can have activity at about pH 4.0 to about pH 12.0, but are not limited thereto.

[0154] A protein can be defined as having "pH stability" or "acid tolerance," "alkaline tolerance," etc. according to the pH range when the protein maintains its function within a particular pH range.

[0155] As used herein, the term "thermal stability" refers to the ability of a protein to function within a particular temperature range. In one specific embodiment, the variants provided by the present disclosure can have activity at about 20°C to about 70°C, specifically, at about 25°C to about 65°C, but are not limited thereto.

[0156] As used herein, the term "thermostability" refers to the ability of a protein to function after exposure to a particular temperature (e.g., high heat or low temperature). For example, a protein having thermostability can not function at the temperature to which they are exposed, but can become functional when returned to an optimal temperature environment.

[0157] An increase in stability can include, for example, maintaining high enzyme activity compared to other enzymes (e.g., wild-type enzymes, parent enzymes, and / or other variants); increasing the range of pH, temperature, and / or time, etc. at which a protein maintains its function.

[0158] A decrease in stability can include, for example, maintaining low enzyme activity compared to other enzymes (e.g., wild-type enzymes, parent enzymes, and / or other variants); decreasing the range of pH, temperature, and / or time, etc. at which a protein maintains its function.

[0159] As used herein, the term "substrate specificity" refers to the ability of an enzyme to recognize a substrate and molecules that compete with the substrate. Substrate specificity can be determined by measuring the activity of an enzyme on different substrates. In one specific embodiment, a change in substrate specificity can be a change in the direction of increased specificity for a substrate that is capable of producing a desired product. In another specific embodiment, a change in substrate specificity can be a change in the direction of decreased specificity for a substrate that is capable of producing a desired product.

[0160] A "polynucleotide" encoding a variant of the present disclosure can include a coding sequence of the above-described variant. The polynucleotide can be variously modified in the coding region within the range that does not change the amino acid sequence of the polypeptide, due to codon degeneracy or in consideration of preferred codons in an organism in which the polypeptide is to be expressed.

[0161] Further, the polynucleotide of the present disclosure can include a probe that can be prepared from a known gene sequence, for example, any sequence encoding a variant of the present disclosure by hybridizing under stringent conditions to a sequence complementary to all or a portion of the nucleotide sequence, without limitation.

[0162] "Stringent conditions" refer to conditions that enable specific hybridization between polynucleotides. Such conditions are specifically described in the literature (for example, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

[0163] For example, stringent conditions can include conditions under which polynucleotides having 40% or more, in particular 90% or more, more particularly 95% or more, 96% or more, 97% or more, 98% or more, even more particularly 99% or more of high homology or identity hybridize to each other, while polynucleotides having less homology or identity than the above do not hybridize to each other, or washing conditions of a general Southern hybridization, which is performed once, in particular twice or three times, at a salt concentration and temperature corresponding to 60°C, 1xSSC, 0.1% SDS, specifically 60°C, 0.1xSSC, 0.1% SDS, more specifically 68°C, 0.1xSSC, 0.1% SDS.

[0164] Hybridization requires that the two nucleic acids have complementary sequences, although mismatches between bases are possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize to each other. For example, for DNA, adenine nucleosides are complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotide of the present disclosure can include isolated nucleotide fragments complementary to the entire sequence as well as nucleic acid sequences substantially similar thereto.

[0165] Specifically, polynucleotides having homology or identity can be detected under the above conditions using hybridization conditions including a hybridization step with a Tm value of 55°C. Further, the Tm value can be 60°C, 63°C, or 65°C, but is not limited thereto, and can be appropriately adjusted by those skilled in the art according to their purposes.

[0166] The appropriate stringency for hybridizing polynucleotides depends on the length and complementarity of the polynucleotides, and these variables are well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

[0167] For example, "high stringency" can occur at about 5°C to 10°C below the Tm of the probe; "moderate stringency" can occur at about 10°C to 20°C below the Tm of the probe; and "low stringency" can occur at about 20°C to 25°C below the Tm of the probe, although these are not absolute limits.

[0168] For example, "low stringency conditions" for a probe of at least about 100 nucleotides in length can mean prehybridization and hybridization at 42°C in 5 x SSPE, 0.3% SDS, 200 micrograms / ml of sheared and denatured salmon sperm DNA, and 25% formamide, followed by two or three washes in 2 X SSC, 0.1% to 0.2% SDS at 50°C, for 15 minutes each. The support material can be washed finally in 0.1 X SSC, 0.1% to 0.2% SDS at 50°C, for 15 minutes.

[0169] For example, "moderate stringency conditions" for a probe of at least about 100 nucleotides in length can mean prehybridization and hybridization at 42°C in 5 x SSPE, 0.3% SDS, 200 micrograms / ml of sheared and denatured salmon sperm DNA, and 35% formamide, followed by two or three washes in 2 X SSC, 0.1% to 0.2% SDS at 55°C, for 15 minutes each. For example, "moderate-high stringency conditions" can mean, for a probe of at least about 100 nucleotides in length, prehybridization and hybridization at 42°C in 5 x SSPE, 0.3% SDS, 200 micrograms / ml of sheared and denatured salmon sperm DNA, and 35% formamide, followed by two or three washes in 1 X to 2 X SSC, 0.1% to 0.2% SDS at 60°C, for 15 minutes each. For example, "high stringency conditions" for a probe of at least about 100 nucleotides in length can mean prehybridization and hybridization at 42°C in 5 x SSPE, 0.3% SDS, 200 micrograms / ml of sheared and denatured salmon sperm DNA, and 35% formamide, followed by two or three washes in 2 X SSC, 0.1% to 0.2% SDS at 65°C, for 15 minutes each.

[0170] The "nucleic acid construct" provided in the present disclosure comprises a polynucleotide encoding the variant provided in the present disclosure, which is operably linked to one or more regulatory sequences that direct the expression of the coding sequence in a suitable host cell under conditions suitable for the regulatory sequences.

[0171] The polynucleotide can be manipulated in various ways to allow expression of the variant. Depending on the expression vector, it can be necessary or desirable to manipulate the polynucleotide prior to insertion into the vector. Such manipulation can be carried out using methods known in the art.

[0172] The "vector" provided in the present disclosure refers to a DNA construct comprising a nucleotide sequence of a polynucleotide encoding the variant, which is operably linked to a suitable expression regulatory region (expression regulatory sequence), so that the variant of the present disclosure can be expressed in a suitable host cell. The expression regulatory region can comprise a promoter capable of initiating transcription, any operator sequence that controls transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence that regulates transcription and translation termination. Once transformed into a suitable host cell, the vector can replicate independently of the host genome or function, or can be integrated into the genome thereof.

[0173] The vector that can be used in the present disclosure is not particularly limited, and any vector known in the art can be used. Examples of the vector that is generally used can include natural or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A, etc. can be used as a bacteriophage vector or a cosmid vector; those based on pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET, etc. can be used as a plasmid vector. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector, etc. can be used.

[0174] For example, the polynucleotide encoding the variant provided in the present disclosure can be inserted into a chromosome through a vector for intracellular chromosomal insertion. 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. The vector can further comprise a selection marker to confirm insertion into the chromosome. The selection marker is used to select cells transformed by the vector, i.e., to confirm whether the target nucleic acid molecule has been inserted, and a marker that provides a selectable phenotype (such as drug resistance, auxotrophy, cytotoxic agent resistance, or surface polypeptide expression) can be used. Only cells expressing the selection marker can survive in an environment treated with a selection agent or show a different phenotype, so transformed cells can be selected.

[0175] A "host cell" of the present disclosure can include any host cell without limitation so long as it is capable of expressing a variant of the present disclosure.

[0176] A host cell of the present disclosure can comprise a variant described above, a polynucleotide encoding the variant, a nucleic acid construct and / or a vector comprising the polynucleotide.

[0177] The nucleic acid construct or vector can be integrated into a chromosome as described previously, or can be maintained as a self-replicating extrachromosomal vector.

[0178] A host cell of the present disclosure includes any progeny of a parent cell that is different from the parent cell due to mutations that occur during replication.

[0179] A host cell can be any cell that can be used for recombinant production of a variant, such as a prokaryotic cell or a eukaryotic cell.

[0180] A prokaryotic host cell can be any Gram-positive or Gram-negative bacteria.

[0181] Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, Corynebacterium, and Streptomyces.

[0182] Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, Vibrio (e.g., Vibrio natriegens), and Ureaplasma.

[0183] In a specific embodiment, the bacterial host cell can be a host cell belonging to the genus Bacillus, specifically including Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells, but is not limited thereto.

[0184] In a specific embodiment, the bacterial host cell can be a host cell belonging to the genus Streptococcus, specifically including Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells, but is not limited thereto.

[0185] In a specific embodiment, the bacterial host cell can be a host cell belonging to the genus Streptomyces, specifically including Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells, but is not limited thereto.

[0186] In one embodiment, the bacterial host cell can be a host cell belonging to the genus Corynebacterium, specifically including Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens cells, but not limited thereto.

[0187] In one embodiment, the bacterial host cell can be a host cell belonging to the genus Escherichia, and can be Escherichia coli (E. coli), but not limited thereto.

[0188] The host cell can be a eukaryotic cell, such as a mammalian, insect, plant, or fungal cell.

[0189] The host cell can be a fungal cell. As used herein, "fungal" can include Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as Oomycota and all Fungi imperfecti.

[0190] The fungal host cell can be a yeast cell. As used herein, "yeast" includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the fungus-like group (Blastomycetes). However, this classification can change and can be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0191] The yeast host cell can be a Candida, Hansenula, Kluyveromyces, Pichia, Komagataella, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, for example, a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, Komagataella phaffii, or Yarrowia lipolytica cell.

[0192] The fungal host cell can be a filamentous fungus cell. "Filamentous fungi" include all of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth of yeasts such as Saccharomyces cerevisiae is by budding of single celled thalli and carbon catabolism can be fermentative.

[0193] The filamentous fungal host cell can be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

[0194] For example, the filamentous fungal host cell can be an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium verticilliodes, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliopthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Promyces clavus, Rhizomucor miehei, Schizophyllum commune, Trichoderma harzianum, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cells, but are not limited thereto.

[0195] The "compositions" of the present disclosure can comprise the modified polypeptide having PET-degrading activity or the host cell expressing the modified polypeptide.

[0196] The description of the modified polypeptide and the host cell expressing the same provided in the present disclosure can be applied to the modified polypeptide having PET-degrading activity or the host cell expressing the modified polypeptide.

[0197] The compositions of the present disclosure can be used to convert a polyester into a final product.

[0198] The term "polyester" refers to a polymer comprising ester functional groups in the backbone of its structure. For example, polyethylene terephthalate is a semi-aromatic copolymer composed of two monomers, terephthalic acid and ethylene glycol.

[0199] The polyester can be selected from the group consisting of polyethylene terephthalate (PET), polyethylene terephthalate glycol (PETG), polyethylene co-isosorbide-terephthalate (PEIT), polytrimethylene terephthalate (PTT), polybutylene adipate terephthalate (PBAT), poly(cyclohexane dimethylene terephthalate) (PCT), and polybutylene terephthalate (PBT). In particular, the polyester can be PET.

[0200] The polypeptides of the present disclosure, or compositions comprising the same, can be used to depolymerize PET into bis(2-hydroxyethyl) terephthalate (BHET), or to decompose PET into mono(2-hydroxymethyl) terephthalate (MHET) and terephthalic acid (TPA).

[0201] The polypeptides of the present disclosure, or compositions comprising the same, can be used to degrade polymers (e.g., oligomers) resulting from the depolymerization of PET. For example, it can be used to degrade BHET into MHET and TPA.

[0202] In addition to the variants provided by the present disclosure having PET-degrading activity, the compositions of the present disclosure can also comprise other components. The components added to the compositions of the present disclosure can be appropriately selected by one skilled in the art.

[0203] In one embodiment, the compositions of the present disclosure can also comprise any components suitable for use in converting PET into a final product.

[0204] In one embodiment, the compositions of the present disclosure can also comprise any components suitable for use in PET degradation.

[0205] Examples of materials that can be added include stabilizers, surfactants, builders, chelating agents, dispersants, enzymes, enzyme stabilizers, catalysts, activators, carriers, binders, lubricants, disintegrants, excipients, solubilizers, suspending agents, coloring agents, flavoring agents, buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, diluents, lubricants, preservatives, etc., but are not limited thereto.

[0206] In one embodiment, the compositions provided by the present disclosure can comprise naturally occurring materials or non-naturally occurring materials in addition to the variants provided by the present disclosure.

[0207] In one specific embodiment, the compositions provided in this disclosure may contain additional enzymes in addition to the variants provided in this disclosure.

[0208] Methods for preparing variants of this disclosure may include culturing host cells; and recovering the variants expressed in the culturing step.

[0209] As used herein, the term "culture" refers to the growth of host cells under appropriately controlled environmental conditions. The culture process of this disclosure can be performed in suitable culture media and culture conditions known in the art. Such a culture process can be readily adapted for use by those skilled in the art according to the strain to be selected. Specifically, the culture can be a batch culture, a continuous culture, or a fed-batch culture, but is not limited thereto.

[0210] As used herein, the term "culture medium" refers to a mixture of substances containing nutrients required for culturing host cells as its main components, providing nutrients and growth factors, as well as water necessary for survival and growth. Specifically, the culture medium and other culture conditions used to culture the host cells of this disclosure can be any culture medium commonly used for culturing host cells without any particular limitation. However, the host cells of this disclosure can be cultured under aerobic conditions in a general culture medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids and / or vitamins, while adjusting temperature, pH, etc.

[0211] In this disclosure, carbon sources may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. Furthermore, carbon sources may include natural organic nutrients such as starch hydrolysate, molasses, molasses, rice bran, cassava, cane molasses, and corn steep liquor. Specifically, carbohydrates such as glucose and aseptically pretreated molasses (i.e., molasses converted to reducing sugars) may be used. In addition, various other carbon sources may be used without limitation in appropriate amounts. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0212] Nitrogen sources can include inorganic nitrogen sources, such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; amino acids, such as glutamic acid, methionine, and glutamine; and organic nitrogen sources, such as peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn steep liquor, casein hydrolysate, fish or its decomposition products, and defatted soybean meal or its decomposition products. These nitrogen sources can be used alone or in combination of two or more of them, but are not limited thereto.

[0213] Phosphorus sources may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or their corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc. Additionally, amino acids, vitamins, and / or suitable precursors may be included. These components or precursors may be added to the culture medium in batches or continuously, but these phosphorus sources are not limited to these.

[0214] Furthermore, during host cell culture, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid in an appropriate manner. Additionally, antifoaming agents such as polyethylene glycol fatty acids can be used during culture to prevent bubble formation. Moreover, oxygen or oxygen-containing gases can be injected into the culture medium to maintain aerobic conditions, or no gas can be injected, or nitrogen, hydrogen, or carbon dioxide can be injected to maintain anaerobic or micro-aerobic conditions, but these are not limited to these methods.

[0215] The temperature of the culture medium can be from 20°C to 50°C, specifically from 25°C to 40°C, but is not limited thereto. The culture can continue until the desired amount of useful material is obtained, and can specifically be carried out from 24 hours to 196 hours, but is not limited thereto.

[0216] In one specific embodiment, variants expressed during the culture step can be recovered using methods known in the art to which this disclosure pertains. For example, variants can be recovered from nutrient media via conventional procedures, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

[0217] The recovery method involves collecting variants using the methods for culturing host cells disclosed herein, for example, using suitable methods known in the art according to batch culture, continuous culture, or fed-batch culture methods. For example, methods such as centrifugation, filtration, treatment with a protein crystallizing precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc.), HPLC, and combinations thereof can be used, and variants can be recovered from the culture medium or host cells using suitable methods known in the art.

[0218] In another specific embodiment, the variant expressed by the host cell during the culture step may not be recovered. In this embodiment, the host cell expressing the variant may be used directly as the source of the variant.

[0219] This disclosure may include methods for producing mono(2-hydroxymethyl) terephthalate (MHET), terephthalic acid (TPA), and / or ethylene glycol (EG), said methods comprising contacting the polyester with the modified polypeptide or a host cell expressing said polypeptide or a composition comprising thereto.

[0220] Specifically, the polyester may include the foregoing description.

[0221] In this disclosure, polyesters can be degraded by, but are not limited to, contacting a modified polypeptide or a host cell expressing the polypeptide or a composition containing the polypeptide. Degradation may also be referred to as depolymerization.

[0222] The time required for polyester degradation can vary depending on the polyester-containing product itself (i.e., the properties and source of the plastic product, its composition, shape, etc.), the type and amount of the modifying peptides used, and various process parameters (i.e., temperature, pH, additives, etc.). These process parameters can be readily applied to polyester-containing products using techniques known in the art.

[0223] For example, the degradation process can be carried out at 20°C to 90°C, preferably at 40°C to 80°C, and more preferably at 50°C to 70°C. More specifically, the temperature can be maintained below the inactivation temperature (which corresponds to the temperature at which the modified peptide becomes inactive) and / or below the temperature at which the host cell no longer synthesizes the modified peptide.

[0224] For example, the degradation process can be carried out at pH 5 to pH 11, preferably pH 6 to pH 9.

[0225] In this disclosure, in specific embodiments, polyester-containing articles may be pretreated before contact with the modified peptides of this disclosure to physically or chemically alter their structure, thereby increasing the contact area with the modified peptides.

[0226] Specifically, monomers and / or oligomers generated from contact degradation can be recovered sequentially or continuously.

[0227] Specifically, the recovered monomers and / or oligomers can be further purified using all suitable purification methods and can be produced in a repolymerizable form. More preferably, purification may include, but is not limited to, stripping processes, separation via aqueous solutions, selective steam condensation, filtration and concentration of post-bioprocess culture media, separation, distillation, vacuum evaporation, extraction, electrodialysis, adsorption, ion exchange, precipitation, crystallization, concentration and acid addition dehydration and precipitation, nanofiltration, acid catalyst treatment, semi-continuous or continuous distillation, solvent extraction, evaporative concentration, evaporative crystallization, liquid / liquid extraction, hydrogenation, azeotropic distillation processes, adsorption, column chromatography, simple vacuum distillation and microfiltration.

[0228] The final products MHET, TPA, and / or EG obtained by using the compositions disclosed herein can be recycled in the polymerization of polyester.

[0229] Specifically, the resulting final products (MHET, TPA, and / or EG) can be recycled as repolymerizable monomers and / or oligomers to synthesize polyesters. Specifically, polyesters with the same properties can be repolymerized and blended with another monomer and / or oligomer to synthesize, for example, new copolymers.

[0230] The polyester can be selected from polyethylene terephthalate (PET), polyethylene terephthalate-1,4-cyclohexanediol (PETG), polyethylene terephthalate-isosorbide copolyester (PEIT), polyethylene terephthalate (PTT), polybutylene terephthalate (PBAT), polycyclohexanediol terephthalate (PCT), and polybutylene terephthalate (PBT). Specifically, the polyester can be PET.

[0231] Methods for synthesizing polyesters using MHET, TPA, and / or EG are known in the art.

[0232] The present disclosure will be described in more detail below with reference to embodiments and experimental examples. However, these embodiments and experimental examples are for illustrative purposes only, and the scope of the present disclosure is not intended to be limited by these embodiments and experimental examples.

[0233] Example 1: Finding a new CaPETase To identify novel PETases beyond the well-known PET hydrolase (IsPETase) of *Sakaiella ossaka* 201-F6, sequence homology analysis using the NCBI database yielded 10 PETase candidates. To investigate these 10 candidates, an initial attempt was made to generate them via signal peptide cleavage, successfully producing 9 candidates. Then, using PET bottle powder (B-PET) as a substrate, the PET hydrolytic activity of the 9 candidates was measured by monitoring the amount of PET hydrolysates (MHET and TPA) released. Surprisingly, most PETase candidates showed very low levels of PET hydrolysates compared to other enzymes, while SHM40309.1 (…)… Figure 1 PC2 in the study showed a significant amount of PET hydrolysates. Furthermore, the melting temperatures (Tm) of nine PETase candidates were measured to investigate the temperature stability of these enzymes, which exhibited a variety of Tm values ​​ranging from 38.6 °C to 70.5 °C. Surprisingly, SHM40309.1, exhibiting extremely high PET hydrolytic activity compared to other enzymes, also showed high-temperature stability with a Tm value of 66.8 °C, and SHM40309.1 showed the highest soluble expression level compared to other enzymes (PC2). Figure 1 ).

[0234] These results indicate that SHM40309.1 possesses excellent properties for the efficient degradation of PET, such as enzyme activity, temperature stability, and protein expression levels. Therefore, SHM40309.1 (the PETase of Cryptosporidium orange, CaPETase) has been selected as a novel PETase in this disclosure.

[0235] Example 2: Preparation of CaPETase and known PET-degrading proteins Expression and purification were performed under the following conditions. A gene optimized for *E. coli* codons was synthesized and amplified by polymerase chain reaction (PCR). The nucleotide sequence corresponding to the signal peptide was removed from the synthesized DNA. The PCR products were then subcloned (NcoI and XhoI) into pET22b(+) (Novagen), which lacks its own signal peptide. The resulting expression vector pET22b(+):CaPETase was used to transform *E. coli* strain BL21(DE3)-T1R. The *E. coli* strain was cultured at 37°C in 1 L of lysogenic broth supplemented with 200 mg / L ampicillin until the optical density at 600 nm reached 0.6.

[0236] Protein expression was induced by adding 0.1 mM isopropyl β-D-1-thiogalactoside (IPTG), and the culture medium was then incubated at 18°C ​​for 16 hours. Cells were then harvested by centrifugation at 4000 × g for 20 minutes at 4°C.

[0237] The cell pellet was resuspended in buffer A (50 mM Na₂HPO₄-HCl, pH 7.0) and sonicated. Cell debris was removed by centrifugation at 13500 × g for 25 min, and the supernatant was applied to a Ni-NTA agarose column (Qiagen). After washing with buffer A containing 30 mM imidazole, bound proteins were eluted with 300 mM imidazole in buffer A. Finally, trace 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. Protein purity was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified protein was concentrated in 50 mM Na₂HPO₄-HCl (pH 7.0) and 100 mM NaCl. In the same manner, the well-known PET hydrolases LCC (GenBank: AEV21261.1), IsPETase (GenBank: GAP38373.1), and TfCut2 (Uniprot accession number E5BBQ3) were prepared and used as the control group. Primers used for cloning are listed in Table 1 below. Example 3: Analysis of the PET degradation activity of CaPETase and known PET-degrading proteins To compare the PET hydrolytic activity of CaPETase with 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 along with 500 nM enzyme. B-PET (a PET sample derived from a PET bottle) was obtained through the following processing: A clear PET bottle was pulverized using a grinder, and the pulverized PET was then melted in a high-temperature furnace at 270°C. The molten PET was immediately immersed in water at 4°C to solidify. The resulting quenched PET was then subjected to a cryogenic grinding process, and PET powder of 300 μm or smaller was obtained through a stencil. The reaction mixture was reacted at 30°C and 40°C for 12 hours. The reaction mixture was then analyzed using HPLC. After the reaction, CaPETase completely converted the PET powder into MHET and TPA, and the products were analyzed by HPLC to evaluate the PET degradation activity.

[0238] In the reaction at 30°C, CaPETase exhibited extremely high PET degradation activity compared to LCC and TfCut2, similar to or even higher than IsPETase, the highest known room-temperature activity among PET degradation proteins reported to date. Specifically, for the highly crystalline PET sample B-PET, CaPETase showed 1.6 times higher activity than IsPETase. The difference in PET hydrolytic activity between these two PET hydrolases was significantly greater than that of other PET hydrolases reacting at 40°C; for B-PET, CaPETase's activity was 7.5 times higher than IsPETase. These results suggest that CaPETase may offer greater temperature controllability and PET hydrolytic activity than IsPETase. Figure 2 ).

[0239] Example 4: 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 performed at 20°C using a sitting drop vapor diffusion method with crystal screening kits: Index and PEG / Ion (Hampton Research) and Wizard I and II (Rigaku) ​​(Fowlis, William W., et al., “Experimental and theoretical analysis of the rate of solvent equilibration in the hanging drop method of protein crystal growth.” Journal of Crystal Growth 90.1-3 (1988): 117-129.). The experiment consisted of 1.0 μl of protein solution and 1.0 μl of stock solution, followed by equilibration with 50 μl of stock solution. The crystals were transferred to a cryoprotectant solution containing 25% (v / v) glycerol, extracted using a ring larger than the crystals, immersed in liquid nitrogen, and rapidly frozen. Data for protein crystal analysis were collected at 100 K using Beamline 7A at the Pohang Accelerator Laboratory (Pohang, South Korea). The data was indexed, integrated, and scaled using the HKL2000 software suite. The resulting CaPETase crystal belongs to space group P21212 and has cell parameters a = 82.31 Å, b = 82.45 Å, c = 87.39 Å, α = β = γ = 90° (Aroyo, Mois Ilia. International tables for crystallography. John Wiley and Sons Limited, 2013). For a one-molecule CaPETase based on asymmetric units, the Metathows modulus is 2.68 Å. 3 / Da corresponds to a solvent content of 52.25%. To investigate the structural features of the protein crystal, the structure of cutinase 1 derived from *Thermobifida cellulosilytica* (PDB code 5LUI) was used as a search model. The structure of CaPETase was identified using the molecular substitution method of the CCP4 version of MOLREP. The model was constructed using the WinCoot program and purified using REFMAC5. Statistical data are shown in Table 2. The fine model of CaPETase is stored in the Protein DataBank under the PDB code 7YM9. Example 5: Preparation of CaPETase variants and determination of their thermal stability and PET degradation activity Based on the structure analyzed in Example 4, the aim was to improve the PET degradation activity and thermal stability of the enzyme. Seven types of CaPETase variants with mutations introduced through the structural analysis in Example 4 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.

[0240] M1: CaPETaseV129T M2: CaPETaseG196T M3: CaPETaseR198K M4: CaPETaseV129T / G196T M5: CaPETaseV129T / R198K M6: CaPETaseG196T / R198K M7: CaPETaseV129T / G196T / R198K Thermal stability was assessed by measuring the melting temperature (Tm) of each variant. Melting temperatures were determined using a protein thermoshift dye (Applied Biosystems) via StepOnePlus real-time PCR (Thermo Fisher Scientific). Specifically, 5 μg of CaPETase was mixed with 20 μl of the protein thermoshift dye, and the temperature was increased in 1-degree increments from 20°C to 90°C, while monitoring changes in signals reflecting protein denaturation. Based on the melting curves, the melting temperatures (Tm) of CaPETase WT and seven variants were determined.

[0241] The M1 variant showed a Tm value 2.4 °C higher than that of CaPETaseWT, and its PET degradation activity increased by approximately 1.38 times. The M2 variant showed a Tm value 4.6 °C higher than that of CaPETaseWT, and its PET degradation activity increased by approximately 1.19 times. The M3 variant showed a Tm value 1.9 °C higher than that of CaPETaseWT, and its PET degradation activity increased by approximately 1.32 times (Table 3).

[0242] Next, combinations of the three identified mutations and structurally derived mutations were introduced to prepare variants M4 through M7, and the PET degradation activity and Tm values ​​of the four variant types were examined. The results showed that variant M4 had a Tm value 5.4 °C higher than that of CaPETase WT, and its PET degradation activity increased by approximately 1.32-fold. Variant M5 showed a Tm value 3.5 °C higher than that of CaPETase WT, and its PET degradation activity increased by approximately 1.22-fold. Variant M6 showed a Tm value 6.2 °C higher than that of CaPETase WT, and its PET degradation activity increased by approximately 1.36-fold. Variant M7 showed a Tm value 8.0 °C higher than that of CaPETase WT, and its PET degradation activity increased by approximately 1.35-fold (Table 4). Based on this, it is confirmed that the PET degradation activity and temperature stability of the CaPETase protein are improved when mutations are introduced. Example 6: Effects based on CaPETase mutation sites To examine the changes in protein thermal stability and PET degradation activity when the mutation sites identified in Example 5 were replaced with different amino acids, the following variants were prepared in the same manner as in Example 2, and their PET degradation activity and thermal stability were evaluated in the same manner as in Example 4 and are shown in Table 5.

[0243] [Table 5] The above results confirm that when the three mutation sites of CaPETase are changed to different amino acids, the substitution of some amino acids affects the improvement of protein activity or thermal stability. This means that the tested amino acid positions directly or indirectly affect the protein's activity or thermal stability.

[0244] Based on the foregoing description, those skilled in the art will understand that this disclosure can be implemented in different specific forms without altering its technical spirit or essential features. In this regard, it should be understood that the above embodiments are not restrictive but illustrative in all respects. The scope of this disclosure is defined by the appended claims and not by the description that follows them; therefore, all changes and modifications falling within the boundaries and scope of the claims, or their equivalents, are intended to be covered by the claims.

Claims

1. A modified polypeptide with polyethylene terephthalate (PET) degradation activity, Wherein i) the modified polypeptide has at least 70% and less than 100% sequence identity with SEQ ID NO: 1; and / or ii) The modified polypeptide is a polypeptide encoded by a polynucleotide having at least 70% and less than 100% sequence identity with the coding sequence of the mature polypeptide of SEQ ID NO: 1; and / or iii) The modified polypeptide is a polypeptide encoded by a polynucleotide, which hybridizes under low-strict, medium-strict, medium-high-strict, high-strict, or very high-strict conditions with (a) the coding sequence of the mature polypeptide of SEQ ID NO: 1, (b) its cDNA, or (c) the full-length complementary sequence of (a) or (b); and / or iv) The modified polypeptide is a functional fragment of the polypeptide of i), ii), or iii) that has PET degradation activity; and The modified polypeptide contains any of the following modifications: Amino acid deletions, amino acid insertions, different amino acid substitutions, and / or combinations thereof at any one or more of positions 129, 198, and 196; The position number refers to the position of the polypeptide corresponding to SEQ ID NO:

1.

2. The modified polypeptide according to claim 1, wherein prior to modification with the PET-degrading modified polypeptide, the amino acid at position 129 is valine (V); the amino acid at position 198 is arginine (R); and / or the amino acid at position 196 is glycine (G).

3. The modified polypeptide of claim 1, wherein the modified polypeptide comprises modifications of amino acids selected from the following positions: 129； 198； 196； 129 and 196; 129 and 198; 196 and 198; 129, 196, and 198; The position number refers to the position of the polypeptide corresponding to SEQ ID NO:

1.

4. The modified polypeptide of claim 1, wherein the modified polypeptide comprises one or more substitutions selected from the following: The amino acid corresponding to position 129 is replaced by threonine or serine; The amino acid at position 198 is replaced by either lysine or aspartic acid; and The amino acid corresponding to position 196 is replaced by threonine, alanine, isoleucine, valine, or serine. The position number refers to the position of the polypeptide corresponding to SEQ ID NO:

1.

5. The modified polypeptide of claim 1, wherein the modified polypeptide comprises one or more modifications selected from the following: The valine at position 129 is modified to threonine or serine; The glycine at position 196 is modified to threonine, alanine, isoleucine, leucine, valine, or serine; Arginine at position 198 is modified to lysine or aspartic acid; The valine at position 129 is modified to threonine and the glycine at position 196 is modified to threonine. The valine at position 129 was modified to threonine and the arginine at position 198 was modified to lysine. The glycine at position 196 was modified to threonine and the arginine at position 198 was modified to lysine. Valine at position 129 is modified to threonine, glycine at position 196 is modified to threonine, and arginine at position 198 is modified to lysine. The position number refers to the position of the polypeptide corresponding to SEQ ID NO:

1.

6. A composition comprising a modified polypeptide according to any one of claims 1 to 5.

7. The composition according to claim 6, wherein the composition is used to degrade PET.

8. A polynucleotide encoding a modified polypeptide according to any one of claims 1 to 5.

9. A host cell comprising: a modified polypeptide according to any one of claims 1 to 5; a polynucleotide encoding the modified polypeptide; a nucleic acid construct comprising the polynucleotide; and / or a vector comprising the nucleotide or the nucleic acid construct.

10. A method for preparing a modified polypeptide with PET degradation activity, the method comprising the following steps: Cultivate the host cells according to claim 9; and The modified polypeptide with PET degradation activity expressed during the culture step according to any one of claims 1 to 5 is recovered.

11. A method for degrading polyester, the method comprising: The modified polypeptide according to any one of claims 1 to 5; Host cells expressing the polypeptide; The substrate is treated with a composition containing the modified polypeptide.

12. The method of claim 11, wherein the polyester is PET.

13. A method for producing mono(2-hydroxymethyl) terephthalate (MHET), terephthalic acid (TPA), and / or ethylene glycol (EG), said method comprising: The polyester is reacted with the modified polypeptide according to any one of claims 1 to 5; Host cells expressing the polypeptide; Contact with a composition containing the polypeptide.

14. The method of claim 13, wherein the polyester is PET.

15. The method of claim 13, further comprising the step of recovering the produced MHET, TPA and / or EG.

16. A method for producing polyester, the method comprising the following steps: Synthetic polyesters are produced using MHET, TPA, and / or EG produced by the method according to any one of claims 13 to 15.

17. The method of claim 16, wherein the polyester is PET.

18. The modified polypeptide according to any one of claims 1 to 6; or a host cell expressing the modified polypeptide; or the use of a composition comprising the modified polypeptide in the degradation of PET.

19. Use of the modified polypeptide or composition comprising any one of claims 1 to 6 for reaction with polyester to produce mono(2-hydroxymethyl) terephthalate (MHET), terephthalic acid (TPA), and / or ethylene glycol (EG).

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