Novel pet-degrading enzyme and use thereof

Abstract

The present application relates to a novel PET-degrading enzyme and a use thereof.

Description

Novel PET-degrading enzyme and its uses

[0001] The present application relates to a polypeptide having PET degradation activity and the use thereof.

[0002] Over 400 million tons of new plastic are produced annually. As environmental issues regarding waste plastics gain prominence, efforts are being made to reduce production through regulations on single-use items and the use of plastic substitutes; however, in reality, production continues to increase each year. PET, which accounts for less than 10% of total plastics, sees approximately 360 million tons of new production annually. As it is primarily used in single-use products, it is considered to have the shortest lifespan of any plastic. Recycling methods for waste plastics include mechanical recycling, pyrolysis, and chemical recycling, with each method currently entering the commercialization phase or the final research stage for commercialization. While each technology can serve as a solution to the waste plastic issue, no existing method can be considered perfect due to impacts such as quality degradation caused by downcycling, carbon neutrality, resource depletion, and eutrophication of seawater and freshwater.

[0003] A series of research results are being published regarding the decomposition of PET, a representative type of plastic, using enzymatic biological technology to solve environmental problems caused by waste plastics, such as microplastics, greenhouse gas emissions, and resource depletion.

[0004] [Prior Art Literature]

[0005] (Patent Document 1) EP 3909947 A2

[0006] The present application relates to a polypeptide having PET degradation activity and the use thereof.

[0007]

[0008] One objective of the present application is to provide a variant polypeptide having PET degradation activity.

[0009] Another objective of the present application is to provide a composition comprising the polypeptide.

[0010] Another objective of the present application is to provide a polynucleotide encoding the polypeptide.

[0011] Another object of the present application is to provide a host cell comprising the polypeptide; a polynucleotide encoding the same; a nucleic acid structure comprising the polynucleotide; and / or a vector comprising the nucleotide or the nucleic acid structure.

[0012] Another objective of the present application is to provide a method for producing a variant polypeptide having PET degradation activity.

[0013] Another object of the present application is to provide a method for degrading polyester, comprising: a polypeptide having PET degradation activity; a host cell expressing said polypeptide; and / or treating a polyester with a composition comprising said polypeptide.

[0014] Another object of the present application is to provide a method for producing bis-2-hydroxyethyl terephthalate (BHET), (mono(2-hydroxymethyl) terephthalate (MHET), terephthalic acid (TPA), and / or ethylene glycol (EG), comprising: a polypeptide having PET degradation activity; a host cell expressing said polypeptide; and / or contacting a polyester with a composition comprising said polypeptide.

[0015] Another object of the present application is to provide a method for producing a polyester, comprising the step of synthesizing a polyester using BHET, MHET, TPA, and / or EG prepared by the above method.

[0016] Another object of the present application is to provide a polypeptide having PET degradation activity, a host cell expressing said polypeptide, and / or a PET degradation use of a composition comprising said polypeptide.

[0017] Another object of the present application is to provide a polypeptide having PET degradation activity for reaction with a polyester in the production of bis-2-hydroxyethyl terephthalate (BHET), (mono(2-hydroxymethyl) terephthalate (MHET), terephthalic acid (TPA), and / or ethylene glycol (EG), a host cell expressing said polypeptide, or a composition comprising said polypeptide.

[0018]

[0019] The polypeptide having PET degradation activity of the present application can be usefully employed in various industrial fields.

[0020]

[0021] Figure 1 shows the PET degradation activity of MpPETase, LCC, and IsPETase at 30℃, 40℃, and 50℃.

[0022]

[0023] One aspect of the present application is a variant polypeptide having PET degradation activity.

[0024] In one specific example, the variant polypeptide is i) a polypeptide having sequence identity of 70% or more and less than 100% with SEQ ID NO. 1; and / or

[0025] ii) The above variant polypeptide is a polypeptide encoded by a polynucleotide having 70% or more and less than 100% sequence identity with the sequence encoding the mature polypeptide of SEQ ID NO. 1; and / or

[0026] iii) The above variant polypeptide is a polypeptide encoded by (a) the mature polypeptide coding sequence of SEQ ID NO. 1, (b) its cDNA, or (c) a polynucleotide hybridizing with the full-length complement of (a) or (b) under low, medium, medium-high, high, or very high strictness conditions; and / or

[0027] iv) The above variant polypeptide is a functional fragment of polypeptides i) to iii) having PET degradation activity; and

[0028] The above variant polypeptide comprises any one of the modifications selected below:

[0029] Deletion at one or more amino acid positions among 179, 201, 203, 231, 49, 61, 241, 286, 198, 229, 215, 147, 228, 251, 46, 67, 221, 197, and 90, insertion of an amino acid, substitution with another amino acid, formation of a disulfide bond, and combinations thereof;

[0030] Here, the above position number is a position corresponding to the position of the polypeptide of sequence number 1.

[0031] As an embodiment of any one of the above embodiments, the amino acid at position 179 before modification is aspartic acid (D); the amino acid at position 201 is arginine (R); the amino acid at position 203 is threonine (T); the amino acid at position 231 is alanine (A); the amino acid at position 49 is leucine (L); the amino acid at position 61 is serine (S); the amino acid at position 241 is alanine (A); the amino acid at position 286 is serine (S); the amino acid at position 198 is serine (S); the amino acid at position 229 is glycine (G); the amino acid at position 215 is glycine (G); the amino acid at position 147 is serine (S); the amino acid at position 228 is threonine (T); the amino acid at position 251 is alanine (A); the amino acid at position 46 is glutamic acid (E); the amino acid at position 67 is alanine (A); the amino acid at position 221 is serine (S); the amino acid at position 197 is threonine (T); and / or amino acid 90 may be proline (P).

[0032] As an embodiment of any one of the above embodiments, the variant polypeptide may comprise one or more of the following substitutions:

[0033] The amino acid corresponding to position 179 is substituted with cysteine, lysine, glutamic acid, or serine;

[0034] The amino acid corresponding to position 201 is substituted with cysteine, lysine, glutamic acid, or serine;

[0035] The amino acid corresponding to position 203 is substituted with cysteine ​​or alanine;

[0036] The amino acid corresponding to position 231 is substituted with cysteine;

[0037] The amino acid corresponding to position 49 is substituted with cysteine, alanine, glycine, valine, isoleucine, or serine;

[0038] The amino acid corresponding to position 61 is substituted with cysteine, alanine, glycine, valine, or isoleucine;

[0039] The amino acid corresponding to position 241 is substituted with cysteine ​​or serine;

[0040] The amino acid corresponding to position 286 is substituted with cysteine ​​or alanine;

[0041] The amino acid corresponding to position 198 is substituted with cysteine, alanine, threonine, valine, isoleucine, or glycine;

[0042] The amino acid corresponding to position 229 is substituted with cysteine, alanine, threonine, valine, isoleucine, or serine;

[0043] The amino acid corresponding to position 215 is substituted with threonine, arginine, serine, glutamine, or isoleucine;

[0044] The amino acid corresponding to position 147 is substituted with threonine, proline, alanine, or glycine;

[0045] The amino acid corresponding to position 228 is substituted with glutamine, histidine, glutamic acid, serine, or asparagine;

[0046] The amino acid corresponding to position 251 is substituted with aspartic acid, histidine, glutamic acid, serine, asparagine, or glycine;

[0047] The amino acid corresponding to position 46 is substituted with glutamine, arginine, histidine, asparagine, or alanine;

[0048] The amino acid corresponding to position 67 is substituted with glutamine, arginine, lysine, asparagine, valine, isoleucine, or tyrosine;

[0049] The amino acid corresponding to position 221 is substituted with glutamine, arginine, asparagine, or valine;

[0050] The amino acid corresponding to position 197 is substituted with lysine, arginine, glutamic acid, serine, asparagine, leucine, isoleucine, or methionine; and

[0051] The amino acid corresponding to position 90 is substituted with alanine or glycine;

[0052] Here, the above position number is a position corresponding to the position of the polypeptide of sequence number 1.

[0053]

[0054] As an embodiment of any one of the above embodiments, the variant polypeptide is

[0055] Amino acids 179 and 201 are substituted with serine, glutamic acid, cysteine, or lysine;

[0056] Amino acids 203 and 231 are substituted with cysteine;

[0057] Amino acids 49 and 61 are substituted with alanine, cysteine, glycine, valine, or isoleucine;

[0058] Amino acids 241 and 285 are substituted with cysteine; and

[0059] Amino acids 198 and 229 are substituted with cysteine, alanine, valine, isoleucine, or threonine;

[0060] It includes one or more substitutions selected from among, and the position number may be a position corresponding to the position of the polypeptide of sequence number 1.

[0061]

[0062] As an embodiment of any one of the above embodiments, the variant polypeptide is

[0063] Amino acid 179 is replaced with cysteine; amino acid 201 is replaced with cysteine; amino acid 203 is replaced with cysteine; amino acid 231 is replaced with cysteine; amino acid 215 is replaced with threonine; amino acid 147 is replaced with threonine; amino acid 228 is replaced with glutamine; amino acid 251 is replaced with aspartic acid; amino acid 49 is replaced with cysteine; amino acid 61 is replaced with cysteine; amino acid 46 is replaced with glutamine; amino acid 67 is replaced with glutamine; amino acid 241 is replaced with cysteine; amino acid 286 is replaced with cysteine; amino acid 197 is replaced with lysine; amino acid 90 is replaced with alanine; amino acid 221 is replaced with glutamine; amino acid 198 is replaced with cysteine; and amino acid 229 is substituted with cysteine, and

[0064] The above position number may be a position corresponding to the position of the polypeptide of sequence number 1.

[0065]

[0066] As an embodiment of any one of the above embodiments, the variant polypeptide may form a disulfide bond between pairs of cysteine ​​selected from the following:

[0067] Cysteine ​​179 and cysteine ​​201;

[0068] Cysteine ​​203 and cysteine ​​231;

[0069] Cysteine ​​No. 49 and Cysteine ​​No. 61;

[0070] Cysteine ​​241 and cysteine ​​286; and / or

[0071] Cysteine ​​198 and cysteine ​​229;

[0072] Here, the above position number is a position corresponding to the position of the polypeptide of sequence number 1.

[0073]

[0074] As an embodiment of any one of the above embodiments, the variant polypeptide may have one or more modified characteristics of any one of i) to vii) below compared to the polypeptide composed of the amino acid sequence of SEQ ID NO. 1:

[0075] i) Increase or decrease in enzyme activity;

[0076] ii) Increase or decrease in specific activity;

[0077] iii) Increase or decrease in pH stability;

[0078] iv) Increase or decrease in storage stability;

[0079] v) Increase or decrease in acid resistance;

[0080] vi) increase or decrease in heat resistance or thermal stability; and

[0081] vii) Change in substrate specificity.

[0082]

[0083] Another aspect of the present application is a composition comprising a variant polypeptide having the above-mentioned PET degradation activity.

[0084] As an example of any one of the above embodiments, the composition may be a composition for PET degradation.

[0085] Another aspect of the present application is a polynucleotide encoding the variant polypeptide.

[0086] Another aspect of the present application is a nucleic acid structure comprising the above-mentioned polynucleotide.

[0087] Another aspect of the present application is a vector comprising the polynucleotide or the nucleic acid structure.

[0088] Another aspect of the present application is a host cell comprising the variant polypeptide, the polynucleotide, the nucleic acid structure, and / or the vector.

[0089]

[0090] Another aspect of the present application is a method for producing a variant polypeptide, comprising the steps of: culturing the host cell; and recovering the variant polypeptide expressed in the culturing step.

[0091]

[0092] Another aspect of the present application is a method for degrading a polyester, comprising treating the polyester with the variant polypeptide, or SEQ ID NO. 1 or a polypeptide having at least 70% sequence identity therewith; a host cell expressing the polypeptide; and / or a composition comprising the polypeptide.

[0093] As an example of any one of the above embodiments, the polyester may be PET.

[0094] As an embodiment of any one of the above embodiments, the method for degrading the polyester may include a step of performing a glycolysis reaction by a polypeptide having PET degradation activity.

[0095] As an embodiment of any one of the above embodiments, the method for degrading the polyester may include a step in which the polyester is converted to BHET in the presence of a polypeptide having PET degradation activity and ethylene glycol.

[0096]

[0097] Another aspect of the present application is a method for producing BHET, MHET, TPA and / or EG, comprising: the variant polypeptide, or SEQ ID NO. 1 or a polypeptide having at least 70% sequence identity with it; a host cell expressing the polypeptide; and / or contacting a polyester with a composition comprising the polypeptide.

[0098] As an example of any one of the above embodiments, the polyester may be PET.

[0099] As an embodiment of any one of the above embodiments, the method may further include a step of recovering BHET, MHET, TPA, and / or EG produced by the above method.

[0100]

[0101] Another aspect of the present application is a method for producing polyester, comprising the step of synthesizing polyester using the BHET, MHET, TPA, and / or EG prepared above.

[0102] As an example of any one of the above embodiments, the polyester may be PET.

[0103]

[0104] Another aspect of the present application is a host cell overexpressing the variant polypeptide, or SEQ ID NO. 1 or a polypeptide having 70% or more sequence identity therewith. Another aspect of the present application is the PET degradation use of the variant polypeptide, or SEQ ID NO. 1 or a polypeptide having 70% or more sequence identity therewith; a host cell expressing the polypeptide; and / or a composition comprising the polypeptide.

[0105] Another aspect of the present application is the use of said variant polypeptide, or a polypeptide having at least 70% sequence identity with SEQ ID NO. 1, for reaction with a polyester in the production of BHET, MHET, TPA, and / or EG; a host cell expressing said polypeptide; and / or a composition comprising said polypeptide.

[0106]

[0107] The specific details for implementing the invention are described as follows. Meanwhile, each description and embodiment disclosed in this application may be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application should not be considered limited by the specific descriptions provided below.

[0108] In addition, a person skilled in the art can recognize or identify a number of equivalents to the specific embodiments of the present application described herein by using only ordinary experiments. In addition, such equivalents are intended to be included in the present application.

[0109]

[0110] As used in the specification and appended claims of this application, singular articles (“a,” “an,” and “the”) include plural references unless the context clearly indicates otherwise. Unless the context indicates otherwise, singular terms include plural forms and plural terms include singular forms. In the specification and appended claims of this application, unless otherwise noted, the use of “or” may be used to mean “and / or”.

[0111]

[0112] In this application, the term "about" may be placed before a specific numerical value. As used in this application, the term "about" includes not only the exact number specified after the term, but also a range that is approximately that number or close to that number. Whether the number is close to or nearly that specific number may be determined by considering the context in which the number is presented. For example, the term "about" may refer to a range of -10% to +10% of a numerical value. For another example, the term "about" may refer to a range of -5% to +5% of a given numerical value. However, it is not limited thereto.

[0113]

[0114] In this application, terms such as “first, second, third…”, “i), ii), iii)…”, or “(a), (b), (c), (d)…” are used to distinguish similar configurations and do not imply that they are performed sequentially or in order. For example, when the terms are used in relation to steps of a method, use, or analysis, there may be no time interval between these steps, they may be performed simultaneously, or they may be performed with intervals of seconds, minutes, hours, days, or months.

[0115]

[0116] In this application, the term "consisting essentially of" means that said unspecified component may be present in such a case that the features of the subject matter claimed in this application are not substantially affected by the presence of said unspecified component.

[0117] In this application, the term “consisting of” means that the proportion of a specific component(s) is 100% of the total. The component or feature following the term “consisting of” may be essential or mandatory. In some embodiments, any other component or non-essential component may be excluded in addition to the component or feature following the term “consisting of”.

[0118] In this application, the term "comprising" means the presence of the features, steps, or components described below the above term, and does not exclude the presence or addition of one or more features, steps, or components. In this application, the components or features described below "comprising" may be essential or mandatory, but some embodiments may further include other optional or non-essential components or features.

[0119] In this application, the term "comprising" may be modified to refer to "essentially made of" or "made of" in some embodiments.

[0120] In relation to amino acid sequences in this application, even if a polypeptide "comprising" the amino acid sequence described by a specific sequence number, a polypeptide "consisting" of the amino acid sequence described by a specific sequence number, or a polypeptide or protein "having" the amino acid sequence described by a specific sequence number, it is obvious that a protein having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added may also be used in this application, provided that it has the same or corresponding activity as the polypeptide consisting of the amino acid sequence of said sequence number. For example, this may include, but is not limited to, the addition of a sequence that does not alter the function of the protein at the N-terminus and / or C-terminus of the said amino acid sequence, a naturally occurring mutation, a silent mutation thereof, or a conservative substitution.

[0121]

[0122] In this application, the terms "protein" or "polypeptide" refer to a polymer or oligomer of a sequence of amino acid residues. In this application, "polypeptide," "protein," and "peptide" may be used interchangeably with "amino acid sequence."

[0123] In some cases, an amino acid sequence exhibiting activity may be referred to as an "enzyme." In this application, amino acid sequences are described in an N-terminal → C-terminal orientation unless otherwise indicated.

[0124]

[0125] With respect to cells, nucleic acids, polypeptides, or vectors, the term “recombinant” in this application means that a cell, nucleic acid, polypeptide, or vector has been modified by the introduction of a heterologous nucleic acid or polypeptide or by a modification of a natural nucleic acid or polypeptide, or that a cell is derived from a cell so modified. Thus, for example, a recombinant cell may express a gene not found in the natural (non-recombinant) form of the cell, or may express a natural gene that is expressed, not expressed at all, or otherwise abnormally expressed.

[0126]

[0127] In this application, the term “isolated” refers to a substance that exists in an environment that does not occur naturally or is of a form that does not exist naturally. This includes at least one other component having a substance that is naturally associated in nature and found in nature, such as a sequence, enzyme, or nucleic acid, from which said substance (sequence, enzyme, or nucleic acid) is at least substantially free.

[0128] For example, the isolated sequence, enzyme, or nucleic acid provided in this application may be provided in a form substantially free of one or more contaminants.

[0129] Examples of isolated substances may include, but are not limited to: i) any non-naturally occurring substance; ii) any substance from which one, more, or all naturally occurring components associated in nature have been removed (e.g., enzymes, variants, nucleic acids, proteins, peptides, or cofactors); iii) any substance found in nature that has been artificially modified; or iv) a substance modified to alter the amount of that substance relative to other naturally associated components (e.g., increasing the copy number of a gene encoding a specific substance; modifying a promoter naturally associated with a gene encoding a specific substance into a highly active promoter).

[0130]

[0131] In this application, the term "wild type" means naturally occurring and not having artificial modifications. When the term "wild type" is used in relation to a polypeptide, it means a naturally occurring polypeptide that does not have artificial modifications (substitutions, insertions, deletions, etc.) at one or more amino acid positions. Similarly, when the term "wild type" is used in relation to a polynucleotide, it means that it does not have artificial modifications (substitutions, insertions, deletions) of one or more nucleotides. However, polynucleotides encoding wild type polypeptides are not limited to naturally occurring polynucleotides and include sequences encoding any wild type polypeptide.

[0132]

[0133] In this application, the term "parent sequence" or "backbone" refers to a reference sequence into which a variant polypeptide is formed by introducing modifications. That is, the parent sequence may serve as a starting sequence to which modifications such as substitutions, insertions, and / or deletions are introduced. The parent sequence may be a naturally occurring or wild type, a variant in which one or more substitutions, insertions, or deletions have occurred in the naturally occurring or wild type, or an artificially synthesized sequence. If the parent sequence is an amino acid sequence that exhibits activity, i.e., an amino acid sequence of an enzyme, it may be referred to as a parent enzyme.

[0134]

[0135] In this application, the term "reference sequence" refers to a sequence used to determine the position of an amino acid within any amino acid sequence. By aligning any amino acid sequence with the reference sequence, the position of an amino acid corresponding to a specific position of the reference sequence within any amino acid sequence can be determined.

[0136]

[0137] In this application, with respect to amino acid or nucleic acid sequences, the term "fragment" means a part of a parent sequence. For example, it may be a polypeptide in which one or more amino acids from the parent sequence have been removed at the C or N terminus.

[0138] In this application, the “fragment” of an enzyme may refer to a “functional fragment.” The “functional fragment” may also be referred to as an active fragment and means a polypeptide that is part of a parent enzyme and possesses the enzymatic activity of the parent enzyme. For example, the functional fragment of an enzyme may include a catalytic site of the enzyme.

[0139] The enzyme fragment may contain a portion of the full length of the parent enzyme. For example, it may contain at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more and less than 100% of the full length of the parent enzyme, but is not limited thereto.

[0140]

[0141] In this application, "modifying" means changing or altering. This may be a change from a naturally occurring one. For example, an enzyme may be modified in such a way that the enzyme is altered from a parent sequence or a reference sequence.

[0142] In the present application, the modified enzyme may be an enzyme that does not exist in nature itself, that is, a non-naturally occurring enzyme.

[0143] In this application, the term “modified” means, for example, that it has been altered from its naturally occurring form. The modified enzymes of this application include enzymes that do not occur naturally or naturally occurring variants. For example, the modified enzymes of this application are modified enzymes not found in nature. For example, the modified enzymes of this application may not occur spontaneously, but are not limited thereto.

[0144] Where the term “modification” is used in relation to an amino acid / nucleic acid sequence in this application, it may include substitution of an amino acid / nucleic acid residue of the parent sequence for a different amino acid / nucleic acid residue at one or more sites in the amino acid sequence, deletion of an amino acid / nucleic acid residue (or a series of amino acid / nucleic acid residues) of the parent sequence at one or more sites, insertion of an amino acid / nucleic acid residue (or a series of amino acid / nucleic acid residues) of the parent sequence at one or more sites, truncation of an N-terminal and / or C-terminal amino acid sequence or a 5’ and / or 3’ nucleic acid sequence, and any combination thereof.

[0145]

[0146] In this application, an enzyme “variant” or “modified polypeptide” refers to a protein that differs from the parent enzyme in that one or more amino acids undergo conservative substitution and / or modification. “Variant” or “modified polypeptide” may be used interchangeably. The variant or modified polypeptide may be non-naturally occurring, but is not limited thereto.

[0147] The above variant differs from the sequence of the parent enzyme by one or more modifications, e.g., amino acid substitution, deletion, and / or insertion.

[0148] These variants generally modify one or more amino acids in the parent enzyme and can be identified by evaluating the characteristics of the modified protein. That is, the ability of the variant may be increased, unchanged, or decreased compared to the parent enzyme.

[0149] In addition, some variants may include variant polypeptides in which one or more parts, such as an N-terminal leader sequence or a transmembrane domain, have been removed.

[0150] Other variants may include variants in which a portion of the N- and / or C-terminus of the mature protein has been removed.

[0151] The terms "variant" or "variant polypeptide" may be used interchangeably with terms such as variant, modification, mutated protein, and mutation (in English, modification, modified protein, mutant, mutein, divergent, variant, etc.), and are not limited to these as long as they are used with the meaning of being mutated.

[0152] Variants may include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the polypeptide may be conjugated with a signal (or leader) sequence at the N-terminus of a protein involved in the co-translational or post-translational transfer of the protein. Additionally, the polypeptide may be conjugated with another sequence or linker to enable identification, purification, or synthesis of the polypeptide.

[0153]

[0154] In this application, the term "conservative substitution" means substituting one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitution may generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues.

[0155]

[0156] Throughout this application specification, standard one- and three-character codes for naturally occurring amino acids are used. Additionally, amino acids referred to by abbreviations in this application are described according to the IUPAC-IUB nomenclature.

[0157]

[0158] Alanine Ala, A Arginine Arg, R

[0159] Asparagine Asn, N Aspartic acid Asp, D

[0160] Cysteine ​​(Cys, C) and Glutamic acid (Glu, E)

[0161] Glutamine Gln, Q Glycine Gly, G

[0162] Histidine His, H Isoleucine Ile, I

[0163] Leu, L Lysine Lys, K

[0164] Methionine Met, M Phenylalanine Phe, F

[0165] Proline Pro, P Serine Ser, S

[0166] Threonine Thr, T Tryptophan Trp, W

[0167] Tyrosine Tyr, Y Valine Val, V

[0168]

[0169] Meanwhile, any amino acid can be written as Xaa or X.

[0170] In addition, 3-character codes that are generally accepted for other amino acids, such as Aib (2-Aminoisobutyric acid), Sar (N-methylglycine), and alpha-methyl-glutamic acid, may be used as well as naturally occurring amino acids.

[0171]

[0172] Amino acids can generally be classified based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Accordingly, amino acid substitutions can generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues.

[0173] For example, among amino acids having electrically charged side chains, 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 having uncharged side chains, nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline, polar or hydrophilic amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine, and among the nonpolar amino acids, aromatic amino acids include phenylalanine, tryptophan, and tyrosine.

[0174]

[0175] In this application, the term "gene" means a polynucleotide coding for a polypeptide and a polynucleotide comprising regions before and after the coding region. In some embodiments, the gene may have a sequence (intron) inserted between each coding region (exon).

[0176]

[0177] In this application, the terms “homology” or “identity” refer to the degree of relationship between two given amino acid sequences or base sequences and may be expressed as a percentage. The terms homology and identity may often be used interchangeably.

[0178] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard arrangement algorithms, and a default gap penalty established by the program used may be utilized. Substantially, homologous or identical sequences can generally be hybridized under moderate or high stringent conditions along at least about 50%, 60%, 70%, 80%, or 90% of the entire sequence or its total length. It is evident that hybridization also involves polynucleotides containing common codons or codons that account for codon degeneracy.

[0179] Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined using a known computer algorithm, such as the “FASTA” program, using default parameters as in, for example, Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as performed 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) (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.,] (Including Academic Press, San Diego, 1994, and [CARILLO ET al.](1988) SIAM J Applied Math 48: 1073). For example, homology, similarity, or identity can be determined using BLAST from the National Biotechnology Information Database Center or ClustalW, but is not limited thereto.

[0180] The homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as that described in, for example, Smith and Waterman, Adv. Appl. Math (1981) 2:482, or Needleman et al. (1970), J Mol Biol. 48:443. In summary, a GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly arranged symbols (i.e., nucleotides or amino acids). The default parameters for a GAP program are (1) a unitary matrix (containing values ​​of 1 for identity and 0 for non-identity) and, as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979), or Gribskov et al. (1986) Nucl. Acids Res. 14: A weighted comparison matrix of 6745 (or an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0181] In addition, whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined by comparing the sequences by Southern hybridization experiments under defined strict conditions, and the defined appropriate hybridization conditions may be determined by methods within the scope of the art and well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York), but are not limited thereto.

[0182]

[0183] In this application, the term "mature polypeptide" refers to a polypeptide in a form that lacks a signal sequence or a propeptide sequence. A mature protein / polypeptide / peptide may be a functional form of a protein / polypeptide / peptide. A mature polypeptide may be a final form that has undergone post-translation or post-translation modification. Examples of post-translation modification include, but are not limited to, N- or C-terminal modification, glycosylation, phosphorylation, and removal of a leader sequence.

[0184]

[0185] In this application, the term "nucleic acid construct" refers to a single or double-stranded nucleic acid molecule comprising one or more regulatory sequences, which is artificially synthesized, manipulated to include a specific sequence in a manner not found in nature, or isolated from nature.

[0186]

[0187] In this application, the term "expression" includes, but is not limited to, any step involved in the generation of a polypeptide, e.g., transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0188] In this application, the term "expression vector" means a linear or circular nucleic acid molecule comprising a coding sequence and a regulatory sequence operably linked for the expression thereof.

[0189]

[0190] In this application, the term "operably linked" refers to a configuration in which a regulatory sequence is positioned at an appropriate location so that the regulatory sequence directs the expression of a coding sequence. Accordingly, "operably linked" includes a regulatory region of a functional domain having known or desired activity, such as a promoter, terminator, signal sequence, or enhancer region, being attached to or linked to a target (gene or polypeptide) so as to regulate the expression, secretion, or function of the target according to said known or desired activity.

[0191]

[0192] In this application, the term "cDNA" refers to a DNA sequence that can be prepared by reverse transcription from a mature, spliced ​​mRNA molecule obtainable from a eukaryotic or prokaryotic cell. The cDNA sequence does not contain intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor of mRNA before being processed through a series of steps including splicing to appear as mature, spliced ​​mRNA.

[0193]

[0194] In this application, the term "regulatory sequence" refers to a polynucleotide sequence required for the expression of a coding sequence. Each regulatory sequence may be of the same origin (of the same origin) or foreign (derived from a different gene) form with respect to the coding sequence. Examples of the regulatory sequences include a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, an operator sequence, a sequence coding for a ribosome binding site, and a sequence regulating transcription and translation termination. The minimum unit of the regulatory sequence may include a promoter, a transcription and translation termination sequence.

[0195]

[0196] To describe the variants provided in this application, the following nomenclature is used.

[0197] In the present application, designating a specific position of an amino acid sequence may include designating an amino acid present at or substituted at that position. The designation of an amino acid at a specific position may be described in various ways. For example, "position 003" may be described as "position 3," "3rd amino acid," or "3rd amino acid." Additionally, for example, if the amino acid at the 3rd position is serine (S), it may be described as "S3" or "Ser3."

[0198] Amino acid substitutions can be expressed by listing the amino acid before substitution, the position, and the substituted amino acid in that order. The amino acids can be expressed using standard 1-character and 3-character codes. For example, if alanine, the amino acid at position 8 of a specific sequence, is substituted with valine, it can be written as "A8V" or "Ala8Val".

[0199] Any amino acid at a specific position can be designated as "X". For example, X6 refers to any amino acid at position 6. Additionally, when a substituted amino acid is denoted as X, it signifies that the amino acid is replaced by one different from the amino acid present prior to the substitution. For example, "V6X" indicates that V at position 6 is replaced by any amino acid other than V.

[0200] Different alterations can be expressed by simultaneously listing different types of amino acids using symbols such as ",". For example, an amino acid (D) at position 12 that is substituted with S or K can be written as D12S,K.

[0201] Multiple variations can be indicated using " / ". For example, D179C / R201C means that aspartic acid, the amino acid at position 179, is substituted with cysteine, and arginine, the amino acid at position 201, is substituted with cysteine.

[0202] In this application, the term “corresponding to” refers to an amino acid residue at a listed position in a protein or polypeptide, or an amino acid residue that is similar, identical, or homologous to a listed residue in a protein or polypeptide. Identifying the amino acid at the corresponding position may involve determining a specific amino acid of a sequence that references a specific sequence. As used in this application, “corresponding region” generally refers to a similar or corresponding position in a related protein or reference protein.

[0203] In the present application, Sequence No. 1 may be used as a reference sequence to determine the position of an amino acid within any amino acid sequence.

[0204] That is, SEQ ID NO. 1 disclosed in this application may be used to determine corresponding amino acid residues in polypeptides having any PET degradation activity, and unless otherwise indicated in this application, residues of a specific amino acid sequence are numbered based on SEQ ID NO. 1.

[0205] For example, any amino acid sequence can be aligned with sequence number 1, and based on this, each amino acid residue of the said amino acid sequence can be numbered by referring to the numerical position of the amino acid residue corresponding to the amino acid residue of sequence number 1. For example, a sequence alignment algorithm such as that described in the present application can identify the position of an amino acid, or the position where modifications such as substitution, insertion, or deletion occur, by comparing with a query sequence (also referred to as a "reference sequence").

[0206] For such alignment, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), Trends Genet. 16: 276-277) can be used, but is not limited thereto.

[0207] In addition, corresponding amino acid residues in different PETases can be identified through multiple sequence alignment. Examples of multiple sequence alignment programs known in the art include 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 using ClustalW. (1.83 or higher; Thompson et al., 1994, Nucleic Acids Research 22 : 4673-4680) etc., and the basic parameters of each of the above programs may be used, but are not limited thereto.

[0208] In addition, if the relationship between enzymes branched from the mature polypeptide of SEQ NO. 1 cannot be detected by conventional sequence-based comparison, other pairwise sequence comparison algorithms may be used (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615). Higher sensitivity than in sequence-based search can be achieved by using a search program that utilizes probabilistic representations of polypeptide families (profiles) for searching databases. For example, the PSI-BLAST program can generate profiles through an iterative database search process and detect remote homologs with low relationality (Atschul et al., 1997, Nucleic Acids Res. 25: 3389-3402). Much greater sensitivity can be achieved if a family or superfamily for a polypeptide has one or more representations 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 various sources, such as PSI-BLAST, secondary structure prediction, structural alignment profiles, and solvation potentials, as input to neural networks that predict structural folding for query sequences. Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919 can be used to align sequences of unknown structure with superfamily models existing in the SCOP database. These alignments can, in turn, be used to create homology models for polypeptides, and these models can be evaluated for accuracy using various tools developed for that purpose.

[0209] For proteins of known structures, several tools and resources can be utilized to search for and generate structural alignments. For example, the SCOP superfamily of proteins is structurally aligned, and this alignment is accessible and downloadable. Two or more protein structures can be aligned using various algorithms, such as distance alignment matrix (Holm and Sander, 1998, Proteins 33: 88-96) or CE (Combinational Extension) (Shindyalov and Bourne, 1998, Protein Engineering 11: 739-747). Implementations of these algorithms can be further utilized to query structural databases containing the target structure to discover possible structural homologs (Holm and Park, 2000, Bioinformatics 16: 566-567).

[0210] The above methods are examples and are not limited thereto.

[0211]

[0212] The following is a more detailed description of the specific embodiments of the present application.

[0213]

[0214] In this application, "polypeptide having PET degradation activity" and "PET degradation enzyme (PETase)" are polypeptides having depolymerization activity of polyethylene terephthalate (PET), and may also include polypeptides having depolymerization activity of a low polymer obtained by depolymerizing PET, for example, bis(2-hydroxyethyl) terephthalate (BHET). The term "depolymerization" means a process in which a polymer or at least one polymer of the plastic material is depolymerized into smaller molecules, such as monomers and / or oligomers.

[0215] In this application, PET degradation activity can be measured and evaluated by using methods known in the art, including embodiments described in this application. For example, it can be evaluated by measuring the amount of BHET, MHET, or TPA produced.

[0216]

[0217] In this application, "mother PET degrading enzyme" refers to a PET degrading enzyme that is modified to produce the variant or variant polypeptide of this application. Specifically, the mother PET degrading enzyme, the mother enzyme, or the mother sequence may be a naturally occurring polypeptide or a wild-type polypeptide, may be a mature polypeptide thereof, or may include a variant or functional fragment thereof, but is not limited thereto as long as it is a polypeptide that possesses PET degrading enzyme activity and can serve as the parent of a variant.

[0218] The parent PET degrading enzyme provided in this application may be the polypeptide of SEQ ID NO. 1, though not limited thereto. Additionally, as long as it has PET degradation activity, it may be a polypeptide having about 60%, 70%, 75%, 77%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with the polypeptide of SEQ ID NO. 1, and may be included without limitation in the range of the parent PET degrading enzyme if it has the same or corresponding activity as the polypeptide consisting of the amino acid sequence of SEQ ID NO. 1.

[0219] The parent PETase of the variant provided in this application may be derived from a microorganism of the genus Micromonospora. Specifically, it may be derived from Micromonospora pattaloongensis.

[0220] Meanwhile, the aforementioned microorganism is an example of a microorganism from which the parent PETase provided in this application may be derived, and includes microorganisms derived from taxonomically homologous microorganisms regardless of the name of the microorganism.

[0221] The aforementioned microorganisms can be obtained from known microorganism deposit institutions such as ATCC, DSMZ, CBS, NRRL, KCTC, and KCCM.

[0222] In this application, the sequence "derived from" a specific microorganism is not limited to those naturally produced or produceable in that microorganism, but also includes sequences encoded by genes produced and isolated from microorganisms containing that gene.

[0223] For example, PETase derived from Micromonospora sp. includes not only enzymes having PETase activity naturally produced in Micromonospora sp., but also those produced from Micromonospora sp. sources, and those produced in other host cells through genetic modification known in the art (e.g., transformation with a sequence encoding said enzyme).

[0224] Furthermore, the present application newly identifies that the polypeptide of SEQ ID NO. 1 possesses PET degradation activity, and prepares variant polypeptides with modified or enhanced properties by introducing a mutation to SEQ ID NO. 1 as the parent sequence. Accordingly, one aspect of the present application provides a use of a polypeptide having the same or corresponding activity as the polypeptide composed of the amino acid sequence of SEQ ID NO. 1 as a PET degradation enzyme. The description of the parent PET degradation enzyme of the variant provided in the present application may be applied to the polypeptide having the same or corresponding activity as the polypeptide composed of the amino acid sequence of SEQ ID NO. 1.

[0225] In the present application, "variant polypeptide having PET degradation activity" may be a variant of the parent PET degradation enzyme.

[0226] In this application, the terms "variant of PET degrading enzyme" or "PET degrading enzyme variant" refer to a protein having PET degradation activity in which one or more amino acids differ from the amino acid sequence of parent PETase.

[0227] The above "variant polypeptide having PET degradation activity," "variant of PETase," and "variant of PET degradation enzyme" may be used interchangeably.

[0228] The variant provided in this application may have PETase activity and may include one or more modifications of amino acids in the parent PETase sequence. Such modifications may be the deletion, insertion, substitution with another amino acid, formation of a disulfide bond, and / or a combination thereof. Specifically, it may be the substitution of amino acids and / or the formation of a disulfide bond, and more specifically, a combination thereof.

[0229] Additionally, the variant is i) a polypeptide having sequence identity of 70% or more and less than 100% with SEQ ID NO. 1; and / or ii) the variant is a polypeptide encoded by a polynucleotide having sequence identity of 70% or more and less than 100% with a sequence encoding the mature polypeptide of SEQ ID NO. 1; and / or iii) the variant is a polypeptide encoded by a polynucleotide that hybridizes with (a) the mature polypeptide coding sequence of SEQ ID NO. 1, (b) its cDNA, or (c) the full-length complement of (a) or (b) under low, medium, medium, high, or very high strictness conditions; and / or iv) the variant may be a functional fragment of the polypeptide i), ii), or ii) having PET degradation activity.

[0230] Specifically, the variant provided in this application may have PET degradation activity and may have one or more modified functions or characteristics compared to the parent PETase by including a modification of one or more amino acids in the parent PETase sequence.

[0231] In one embodiment, the variant provided in this application may have PETase activity, have one or more altered functions or characteristics relative to the parent PETase including modification of one or more amino acids in the parent PETase sequence, and may have one or more conservative substitutions.

[0232]

[0233] The variant provided in this application may be a variant of the parent PETase and may be a polypeptide having PET degradation activity.

[0234] In one specific example, the variant provided in the present application may include a modification at one or more selected positions among the amino acids corresponding to positions 179, 201, 203, 231, 49, 61, 241, 286, 198, 229, 215, 147, 228, 251, 46, 67, 221, 197, and 90 of SEQ ID NO. 1.

[0235] In this application, the position number is a position corresponding to the position of the polypeptide of sequence number 1, and "corresponding" is as described above.

[0236] As an embodiment of any one of the aforementioned embodiments, the variant provided in the present application may include a modification of an amino acid corresponding to one or more of D179, R201, T203, A231, L49, S61, A241, S286, S198, G229, G215, S147, T228, A251, E46, A67, S221, T197, and P90 of SEQ ID NO. 1.

[0237] In any one of the embodiments described above, the amino acid at position 179 before modification provided in this application is aspartic acid (D); amino acid at position 201 is arginine (R); amino acid at position 203 is threonine (T); amino acid at position 231 is alanine (A); amino acid at position 49 is leucine (L); amino acid at position 61 is serine (S); amino acid at position 241 is alanine (A); amino acid at position 286 is serine (S); amino acid at position 198 is serine (S); amino acid at position 229 is glycine (G); amino acid at position 215 is glycine (G); amino acid at position 147 is serine (S); amino acid at position 228 is threonine (T); amino acid at position 251 is alanine (A); amino acid at position 46 is glutamic acid (E); amino acid at position 67 is alanine (A); amino acid at position 221 is serine (S); amino acid at position 197 is threonine (T); and / or amino acid 90 may be proline (P).

[0238] In any one of the aforementioned embodiments, the variant provided in the present application may include a substitution of G, A, V, L, I, M, F, W, P, S, T, C, Y, N, Q, E, K, R, or H of the amino acid corresponding to position 179 of SEQ ID NO. 1, and specifically may include a substitution of C, K, E, or S.

[0239] As an embodiment of any one of the aforementioned embodiments, the variant provided in the present application may include a substitution of G, A, V, L, I, M, F, W, P, S, T, C, Y, N, Q, D, E, K, or H of the amino acid corresponding to position 201 of SEQ ID NO. 1, and specifically may include a substitution of C, K, E, or S.

[0240] As any one of the aforementioned embodiments, the variant provided in the present application may include a substitution of G, A, V, L, I, M, F, W, P, S, C, Y, N, Q, D, E, K, R, or H of the amino acid corresponding to position 203 of SEQ ID NO. 1, and specifically may include a substitution of C or A.

[0241] As an embodiment of any one of the aforementioned embodiments, the variant provided in the present application may include a substitution of G, V, L, I, M, F, W, P, S, T, C, Y, N, Q, D, E, K, R, or H of the amino acid corresponding to position 231 of SEQ ID NO. 1, and specifically may include a substitution of C.

[0242] As any one of the embodiments described above, the variant provided in the present application may include a substitution of G, A, V, I, M, F, W, P, S, T, C, Y, N, Q, D, E, K, R, or H of the amino acid corresponding to position 49 of SEQ ID NO. 1, and specifically may include a substitution of C, A, G, V, I, S.

[0243] As an embodiment of any one of the aforementioned embodiments, the variant provided in the present application may include a substitution of G, A, V, L, I, M, F, W, P, T, C, Y, N, Q, D, E, K, R, or H of the amino acid corresponding to position 61 of SEQ ID NO. 1, and specifically may include a substitution of C, A, G, V, I.

[0244] As any one of the aforementioned embodiments, the variant provided in the present application may include a substitution of G, V, L, I, M, F, W, P, S, T, C, Y, N, Q, D, E, K, R, or H of the amino acid corresponding to position 241 of SEQ ID NO. 1, and specifically may include a substitution of C or S.

[0245] As any one of the aforementioned embodiments, the variant provided in the present application may include a substitution of G, A, V, L, I, M, F, W, P, T, C, Y, N, Q, D, E, K, R, or H of the amino acid corresponding to position 286 of SEQ ID NO. 1, and specifically may include a substitution of C or A.

[0246] As any one of the embodiments described above, the variant provided in the present application may include a substitution of G, A, V, L, I, M, F, W, P, T, C, Y, N, Q, D, E, K, R, or H of the amino acid corresponding to position 198 of SEQ ID NO. 1, and specifically may include a substitution of C, A, T, V, I, or G.

[0247] In any one of the aforementioned embodiments, the variant provided in the present application may include a substitution of the amino acid corresponding to position 229 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, and specifically may include a substitution with C, A, T, V, I, or S.

[0248] As any one of the embodiments described above, the variant provided in the present application may include a substitution of the amino acid corresponding to position 215 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, and specifically may include a substitution with T, R, S, Q, or I.

[0249] As any one of the embodiments described above, the variant provided in the present application may include a substitution of the amino acid corresponding to position 147 of SEQ ID NO. 1 with G, A, V, L, I, M, F, W, P, T, C, Y, N, Q, D, E, K, R, or H, and specifically may include a substitution with T, P, A, or G.

[0250] As any one of the embodiments described above, the variant provided in the present application may include a substitution of the amino acid corresponding to position 228 of SEQ ID NO. 1 with G, A, V, L, I, M, F, W, P, S, C, Y, N, Q, D, E, K, R, or H, and specifically may include a substitution with Q, H, E, S, N.

[0251] As an embodiment of any one of the aforementioned embodiments, the variant provided in the present application may include a substitution of the amino acid corresponding to position 251 of SEQ ID NO. 1 with G, V, L, I, M, F, W, P, S, T, C, Y, N, Q, D, E, K, R, or H, and specifically may include a substitution with D, H, E, S, N, or G.

[0252] As any one of the embodiments described above, the variant provided in the present application may include a substitution of the amino acid corresponding to position 46 of SEQ ID NO. 1 with G, A, V, L, I, M, F, W, P, S, T, C, Y, N, Q, D, K, R, or H, and specifically may include a substitution with Q, R, H, N, or A.

[0253] As any one of the embodiments described above, the variant provided in the present application may include a substitution of the amino acid corresponding to position 67 of SEQ ID NO. 1 with G, V, L, I, M, F, W, P, S, T, C, Y, N, Q, D, E, K, R, or H, and specifically may include a substitution with Q, R, K, N, V, I, Y.

[0254] As any one of the embodiments described above, the variant provided in the present application may include a substitution of the amino acid corresponding to position 221 of SEQ ID NO. 1 with G, A, V, L, I, M, F, W, P, T, C, Y, N, Q, D, E, K, R, or H, and specifically may include a substitution with Q, R, N, or V.

[0255] As any one of the embodiments described above, the variant provided in the present application may include a substitution of the amino acid corresponding to position 197 of SEQ ID NO. 1 with G, A, V, L, I, M, F, W, P, S, C, Y, N, Q, D, E, K, R, or H, and specifically may include a substitution with K, R, E, S, N, L, I, M.

[0256] In any one of the aforementioned embodiments, the variant provided in the present application may include a substitution of G, A, V, L, I, M, F, W, S, T, C, Y, N, Q, D, E, K, R, or H of the amino acid corresponding to position 90 of SEQ ID NO. 1, and specifically may include a substitution of A or G.

[0257]

[0258] As an embodiment of any one of the aforementioned embodiments, the variant provided in this application may include one or more of the following substitutions:

[0259] The amino acid corresponding to position 179 is substituted with cysteine, lysine, glutamic acid, or serine;

[0260] The amino acid corresponding to position 201 is substituted with cysteine, lysine glutamic acid, or serine;

[0261] The amino acid corresponding to position 203 is substituted with cysteine ​​or alanine;

[0262] The amino acid corresponding to position 231 is substituted with cysteine;

[0263] The amino acid corresponding to position 49 is substituted with cysteine, alanine, glycine, valine, isoleucine, or serine;

[0264] The amino acid corresponding to position 61 is substituted with cysteine, alanine, glycine, valine, or isoleucine;

[0265] The amino acid corresponding to position 241 is substituted with cysteine ​​or serine;

[0266] The amino acid corresponding to position 286 is substituted with cysteine ​​or alanine;

[0267] The amino acid corresponding to position 198 is substituted with cysteine, alanine, threonine, valine, isoleucine, or glycine;

[0268] The amino acid corresponding to position 198 is substituted with cysteine, alanine, threonine, valine, isoleucine, or glycine;

[0269] The amino acid corresponding to position 229 is substituted with cysteine, alanine, threonine, valine, isoleucine, or serine;

[0270] The amino acid corresponding to position 215 is substituted with threonine, arginine, serine, glutamine, or isoleucine;

[0271] The amino acid corresponding to position 147 is substituted with threonine, proline, alanine, or glycine;

[0272] The amino acid corresponding to position 228 is substituted with glutamine, histidine, glutamic acid, serine, or asparagine;

[0273] The amino acid corresponding to position 251 is substituted with aspartic acid, histidine, glutamic acid, serine, asparagine, or glycine;

[0274] The amino acid corresponding to position 46 is substituted with glutamine, arginine, histidine, asparagine, or alanine;

[0275] The amino acid corresponding to position 67 is substituted with glutamine, arginine, lysine, asparagine, valine, isoleucine, or tyrosine;

[0276] The amino acid corresponding to position 221 is substituted with glutamine, arginine, asparagine, or valine;

[0277] The amino acid corresponding to position 197 is substituted with lysine, arginine, glutamic acid, asparagine, serine, leucine, isoleucine, or methionine; and

[0278] The amino acid corresponding to position 90 is substituted with alanine or glycine;

[0279] Here, the position number is a position corresponding to the position of the polypeptide of sequence number 1.

[0280]

[0281] As an embodiment of any one of the aforementioned embodiments, the variant provided in this application may include one or more of the following substitutions:

[0282] D179C, K, E, S;

[0283] R201C, K, E, S;

[0284] T203C, A;

[0285] A231C;

[0286] L49C, A, G, V, I, S;

[0287] S61C, A, G, V, I;

[0288] A241C, S;

[0289] S286C, A;

[0290] S198C, A, T, V, I, G;

[0291] G229C, A, T, V, I, S;

[0292] G215T, R, S, Q, I;

[0293] S147T, P, A, G;

[0294] T228Q, H, E, S, N;

[0295] A251D, H, E, S, N, G;

[0296] E46Q, R, H, N, A;

[0297] A67Q, R, K, N, V, I, Y;

[0298] S221Q, R, N, V;

[0299] T197K, R, E, S, N, L, I, M;

[0300] P90A, G;

[0301] Here, the position number is a position corresponding to the position of the polypeptide of sequence number 1.

[0302]

[0303] As an embodiment of any one of the aforementioned embodiments, the variant provided in this application may include the substitution of amino acids 179 and 201 of SEQ ID NO. 1 with cysteine, and may form a disulfide bridge (disulfide bond; disulfide bond) between the substituted amino acids.

[0304] In any one of the aforementioned embodiments, the variant provided in this application may include the substitution of amino acids 203 and 231 of SEQ ID NO. 1 with cysteine, and may form a disulfide bridge between the substituted amino acids.

[0305] In any one of the aforementioned embodiments, the variant provided in this application may include the substitution of amino acids 49 and 61 of SEQ ID NO. 1 with cysteine, and may form a disulfide bridge between the substituted amino acids.

[0306] In any one of the aforementioned embodiments, the variant provided in this application may include the substitution of amino acids 241 and 286 of SEQ ID NO. 1 with cysteine, and may form a disulfide bridge between the substituted amino acids.

[0307] In any one of the aforementioned embodiments, the variant provided in this application may comprise the substitution of amino acids 198 and 229 of SEQ ID NO. 1 with cysteine, and may form a disulfide bridge between the substituted amino acids.

[0308]

[0309] As an embodiment of any one of the aforementioned embodiments, the variant provided in this application may include any one of the following substitutions:

[0310] D179C / R201C;

[0311] D179K / R201K;

[0312] D179E / R201E;

[0313] D179S / R201S;

[0314] T203C / A231C;

[0315] T203A;

[0316] L49C / S61C;

[0317] L49A / S61A;

[0318] L49G / S61G;

[0319] L49S;

[0320] L49A / S61A;

[0321] S198G;

[0322] L49V / S61V;

[0323] L49I / S61I;

[0324] A241C / S286C;

[0325] S286A;

[0326] A241S;

[0327] S198C / G229C;

[0328] S198A / G229A;

[0329] G229S;

[0330] S198T / G229T;

[0331] S198V / G229V;

[0332] S198I / G229I;

[0333] G215T, R, S, Q, I;

[0334] S147T, P, A, G;

[0335] T228Q, H, E, S, N;

[0336] A251D, H, E, S, N, G;

[0337] E46Q, R, H, N, A;

[0338] A67Q, R, K, N, V, I, Y;

[0339] S221Q, R, N, V;

[0340] T197K, R, E, S, N, L, I, M;

[0341] P90A, G;

[0342] D179C / R201C / T203C / A231C / G215T / S147T / T228Q / A251D / L49C / S61C / E46Q / A67Q / A241C / S286C / T197K / P90A / S221Q / S198C / G229C;

[0343] Here, the position number is a position corresponding to the position of the polypeptide of sequence number 1.

[0344]

[0345] In one specific example,

[0346] A variant containing the G215T substitution of SEQ ID NO. 1 is SEQ ID NO. 3,

[0347] A variant containing the S147T substitution of SEQ ID NO. 1 is SEQ ID NO. 4,

[0348] A variant containing the T228Q substitution of SEQ ID NO. 1 is SEQ ID NO. 5,

[0349] A variant containing the A251D substitution of SEQ ID NO. 1 is SEQ ID NO. 6,

[0350] Variants containing the E46Q substitution of SEQ ID NO. 1 are SEQ ID NO. 7,

[0351] Variants containing the A67Q substitution of SEQ ID NO. 1 are SEQ ID NO. 8,

[0352] A variant containing the S221Q substitution of SEQ ID NO. 1 is SEQ ID NO. 9,

[0353] Variants containing the T197K substitution of SEQ ID NO. 1 are SEQ ID NO. 10,

[0354] Variants containing the P90A substitution of SEQ ID NO. 1 are SEQ ID NO. 11,

[0355] A variant containing the D179C / R201C substitution of SEQ ID NO. 1 is SEQ ID NO. 12,

[0356] Variants containing the T203C / A231C substitution of SEQ ID NO. 1 are SEQ ID NO. 13,

[0357] A variant containing the L49C / S61C substitution of SEQ ID NO. 1 is SEQ ID NO. 14,

[0358] A variant containing the A241C / S286C substitution of SEQ ID NO. 1 is SEQ ID NO. 15,

[0359] A variant containing the S198C / G229C substitution of SEQ ID NO. 1 is SEQ ID NO. 16,

[0360] Variants containing the D179C / R201C / T203C / A231C / G215T / S147T / T228Q / A251D / L49C / S61C / E46Q / A67Q / A241C / S286C / T197K / P90A / S221Q / S198C / G229C substitution of SEQ ID NO. 17,

[0361] Variants containing the G215R substitution of SEQ ID NO. 1 are SEQ ID NO. 18,

[0362] A variant containing the G215S substitution of SEQ ID NO. 1 is SEQ ID NO. 19,

[0363] A variant containing the G215Q substitution of SEQ ID NO. 1 is SEQ ID NO. 20,

[0364] Variants containing the G215I substitution of SEQ ID NO. 1 are SEQ ID NO. 21,

[0365] A variant containing the S147P substitution of SEQ ID NO. 1 is SEQ ID NO. 22,

[0366] A variant containing the S147A substitution of SEQ ID NO. 1 is SEQ ID NO. 23,

[0367] A variant containing the S147G substitution of SEQ ID NO. 1 is SEQ ID NO. 24,

[0368] A variant containing the T228H substitution of SEQ ID NO. 1 is SEQ ID NO. 25,

[0369] A variant containing the T228E substitution of SEQ ID NO. 1 is SEQ ID NO. 26,

[0370] A variant containing the T228S substitution of SEQ ID NO. 1 is SEQ ID NO. 27,

[0371] A variant containing the T228N substitution of SEQ ID NO. 1 is SEQ ID NO. 28,

[0372] A variant containing the A251H substitution of SEQ ID NO. 1 is SEQ ID NO. 29,

[0373] A variant containing the A251E substitution of SEQ ID NO. 1 is SEQ ID NO. 30,

[0374] Variants containing the A251S substitution of SEQ ID NO. 1 are SEQ ID NO. 31,

[0375] A variant containing the A251N substitution of SEQ ID NO. 1 is SEQ ID NO. 32,

[0376] A variant containing the A251G substitution of SEQ ID NO. 1 is SEQ ID NO. 33,

[0377] A variant containing the E46R substitution of SEQ ID NO. 1 is SEQ ID NO. 34,

[0378] A variant containing the E46H substitution of SEQ ID NO. 1 is SEQ ID NO. 35,

[0379] A variant containing the E46N substitution of SEQ ID NO. 1 is SEQ ID NO. 36,

[0380] A variant containing the E46A substitution of SEQ ID NO. 1 is SEQ ID NO. 37,

[0381] Variants containing the A67R substitution of SEQ ID NO. 1 are SEQ ID NO. 38,

[0382] A variant containing the A67K substitution of SEQ ID NO. 1 is SEQ ID NO. 39,

[0383] A variant containing the A67N substitution of SEQ ID NO. 1 is SEQ ID NO. 40,

[0384] A variant containing the A67V substitution of SEQ ID NO. 1 is SEQ ID NO. 41,

[0385] A variant containing the A67I substitution of SEQ ID NO. 1 is SEQ ID NO. 42,

[0386] A variant containing the A67Y substitution of SEQ ID NO. 1 is SEQ ID NO. 43,

[0387] A variant containing the S221R substitution of SEQ ID NO. 1 is SEQ ID NO. 44,

[0388] A variant containing the S221N substitution of SEQ ID NO. 1 is SEQ ID NO. 45,

[0389] A variant containing the S221V substitution of SEQ ID NO. 1 is SEQ ID NO. 46,

[0390] A variant containing the T197R substitution of SEQ ID NO. 1 is SEQ ID NO. 47,

[0391] A variant containing the T197E substitution of SEQ ID NO. 1 is SEQ ID NO. 48,

[0392] A variant containing the T197S substitution of SEQ ID NO. 1 is SEQ ID NO. 49,

[0393] A variant containing the T197N substitution of SEQ ID NO. 1 is SEQ ID NO. 50,

[0394] Variants containing the T197L substitution of SEQ ID NO. 1 are SEQ ID NO. 51,

[0395] A variant containing the T197I substitution of SEQ ID NO. 1 is SEQ ID NO. 52,

[0396] A variant containing the T197M substitution of SEQ ID NO. 1 is SEQ ID NO. 53,

[0397] A variant containing the P90G substitution of SEQ ID NO. 1 is SEQ ID NO. 54,

[0398] A variant containing the D179K / R201K substitution of SEQ ID NO. 1 is SEQ ID NO. 55,

[0399] A variant containing the D179E / R201E substitution of SEQ ID NO. 1 is SEQ ID NO. 56,

[0400] A variant containing the D179S / R201S substitution of SEQ ID NO. 1 is SEQ ID NO. 57,

[0401] A variant containing the T203A substitution of SEQ ID NO. 1 is SEQ ID NO. 58,

[0402] A variant containing the L49A / S61A substitution of SEQ ID NO. 1 is SEQ ID NO. 59,

[0403] A variant containing the L49G / S61G substitution of SEQ ID NO. 1 is SEQ ID NO. 60,

[0404] A variant containing the L49S substitution of SEQ ID NO. 1 is SEQ ID NO. 61,

[0405] A variant containing the L49V / S61V substitution of SEQ ID NO. 1 is SEQ ID NO. 62,

[0406] A variant containing the L49I / S61I substitution of SEQ ID NO. 1 is SEQ ID NO. 63,

[0407] A variant containing the S286A substitution of SEQ ID NO. 1 is SEQ ID NO. 64,

[0408] A variant containing the A241S substitution of SEQ ID NO. 1 is SEQ ID NO. 65,

[0409] A variant containing the S198A / G229A substitution of SEQ ID NO. 1 is SEQ ID NO. 66,

[0410] A variant containing the S198G substitution of SEQ ID NO. 1 is SEQ ID NO. 67,

[0411] A variant containing the G229S substitution of SEQ ID NO. 1 is SEQ ID NO. 68,

[0412] A variant containing the S198T / G229T substitution of SEQ ID NO. 1 is SEQ ID NO. 69,

[0413] A variant containing the S198V / G229V substitution of SEQ ID NO. 1 is SEQ ID NO. 70,

[0414] A variant containing the S198I / G229I substitution of SEQ ID NO. 1 may be represented by SEQ ID NO. 71.

[0415]

[0416] In one specific example, the variants provided in this application include all possible combinations of the aforementioned variants.

[0417] In one embodiment, the variant provided in this application may have sequence identity of at least about 60%, e.g., at least 65%, at least 70%, at least 75%, at least 77%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% and less than 100% with the parent PET degrading enzyme; its mature polypeptide or its functional fragment.

[0418] In one specific example, the variant provided in this application may have sequence identity with SEQ ID NO. 1 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 77%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% and less than 100%.

[0419] In one specific example, the variant provided in this application may be a polypeptide encoded by a polynucleotide having sequence identity of about 60% or more, e.g., 65% or more, 70% or more, 75% or more, 77% 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% with respect to the sequence encoding the mature polypeptide of SEQ ID NO. 1.

[0420] In one specific example, the variant provided in this application may have sequence identity with the functional fragment of SEQ ID NO. 1 of about 60% or more, e.g., 65% or more, 70% or more, 75% or more, 77% 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%.

[0421]

[0422] The variant provided in this application may have one or more characteristics or properties of a polypeptide that can be selected or detected modified compared to other parent PET degrading enzymes, e.g., wild-type PET degrading enzyme, parent PET degrading enzyme, other PET degrading enzyme variants, etc.

[0423] The above characteristics or properties include oxidation stability, substrate specificity, catalytic activity, thermal stability, alkali stability, pH activity profile, resistance to proteolysis, Km, k cat , k cat / Km ratio, protein folding, induction of immune response, ability to bind to ligands, ability to bind to receptors, ability to secrete, ability to display on the surface of cells, ability to form oligomers, ability to send signals, ability to promote cell proliferation, ability to inhibit cell proliferation, ability to induce apoptosis, ability to be modified by phosphorylation or glycosylation, and / or ability to treat diseases are included, but not limited to.

[0424]

[0425] Specifically, the variant provided in this application may have one or more of the following modified activities compared to the parent sequence:

[0426] i) Increase or decrease in enzyme activity;

[0427] ii) Increase or decrease in specific activity;

[0428] iii) Increase or decrease in pH stability;

[0429] iv) Increase or decrease in storage stability;

[0430] v) Increase or decrease in acid resistance;

[0431] vi) increase or decrease in heat resistance or thermal stability; and

[0432] vii) Change in substrate specificity;

[0433] However, it is not limited to this.

[0434]

[0435]

[0436] As another example, the PET degrading enzyme provided in this application may have one or more modified activities compared to LCC (GenBank: AEV21261.1) and IsPETase (GenBank: GAP38373.1):

[0437] i) Increase in enzyme activity;

[0438] ii) Increase in specific activity;

[0439] iii) Increased pH stability;

[0440] iv) Increased storage stability;

[0441] v) Increased acid resistance;

[0442] vi) Increase in heat resistance; and

[0443] vii) Change in substrate specificity;

[0444] However, it is not limited to this.

[0445]

[0446] In the present application, "enzymatic activity" represents at least one catalytic activity. Specifically, k cat It may be the enzyme conversion efficiency, mainly expressed in / Km, but is not limited thereto.

[0447] k cat τ represents the catalytic constant representing the rate at which a single enzyme converts a substrate into a product per unit time when the enzyme is completely saturated with the substrate, and is also referred to as the turnover number. Km is the substrate concentration at which the reaction rate is half of the maximum value (Vmax).

[0448] As an example of a method to express enzyme activity, specific activity (umol of converted substrate x mg) -1 x min -1 ) or volumetric activity (umol of converted substrate x mL -1 x min-1 There are ) etc.

[0449] However, defining enzyme activity is not limited to the aforementioned content and can be defined and evaluated based on known information such as Irwin H. Segel, Enzyme kinetics, John Wiley & Sons, 1979; AG 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, WH 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 with respect to enzymatic activity.

[0450]

[0451] In one specific example, the variant provided in this application may have increased enzyme activity of 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.

[0452] In another specific example, the variant provided in this application may have reduced enzyme activity of 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.

[0453]

[0454] In this application, the term "specific activity" refers to the enzyme activity per unit weight of protein and can be expressed in unit / mg. Protein quantification can be performed, for example, using SDS-PAGE or the Bradford assay.

[0455]

[0456] Enzyme stability refers to the preservation of enzyme activity during storage or reaction time. To measure changes in this stability, the level of enzyme activity loss or enzyme stability can be expressed by measuring the initial enzyme activity under defined conditions at time zero (100%) and after a certain period of time (x%) and comparing them.

[0457]

[0458] Factors that affect enzyme activity include, for example, pH, heat, and the presence of other substances (e.g., oxidizing agents, chelating agents).

[0459]

[0460] In this application, the term “pH stability” means the ability of a protein to function within a specific pH range. In one embodiment, the variant provided in this application may be active at about pH 4.0 to about pH 12.0, but is not limited thereto.

[0461] If a protein maintains its function within a specific pH range, it can be defined as having "pH stability," and depending on the pH range, it can be defined as having "acid resistance," "alkali resistance," etc.

[0462]

[0463] In this application, the term "thermal stability" refers to the ability of a protein to function within a specific temperature range. In one embodiment, the variant provided in this application may have activity in a range of about 20°C to about 90°C, and specifically may have activity in a range of about 25°C to about 75°C, but is not limited thereto.

[0464] In this application, the term "thermal tolerance" refers to the ability of a protein to function after being exposed to a specific temperature, for example, high heat or cryogenic temperature. For example, a thermally toleranced protein may not function at the exposed temperature, but may regain function when returned to an optimal temperature environment.

[0465]

[0466] Increased stability includes maintaining high enzyme activity compared to other enzymes, e.g., wild-type enzymes, parent enzymes, and / or other variants; and an increased range of pH, temperature, and / or time over which the protein maintains its function.

[0467] Reduced stability includes maintaining lower enzyme activity compared to other enzymes, e.g., wild-type enzymes, parent enzymes, and / or other variants; and a reduced range of pH, temperature, and / or time, etc., over which the protein maintains its function.

[0468]

[0469] In this application, the term "substrate specificity" refers to the ability of an enzyme to identify a substrate and molecules competing with the substrate. Substrate specificity can be determined by measuring the enzyme's activity toward different substrates. In one embodiment, the change in substrate specificity may be a change in the direction of increasing specificity toward a substrate capable of producing a desired product. In another embodiment, the change in substrate specificity may be a change in the direction of decreasing specificity toward a substrate capable of producing a desired product.

[0470]

[0471] The "polynucleotide" encoding the variant of the present application may include the coding sequence of the aforementioned variant. Various modifications to the coding region of the polynucleotide may be made within a range that does not alter the amino acid sequence of the polypeptide, due to codon degeneracy or considering the codons preferred by the organism intended to express the polypeptide.

[0472] In addition, the polynucleotide of the present application may include, without limitation, any sequence encoding a variant of the present application, provided that it is hybridized under strict conditions with a probe that can be prepared from a known gene sequence, for example, a sequence complementary to all or part of the base sequence.

[0473] The above "stringent condition" refers to a condition that enables specific hybridization between polynucleotides. Such conditions are specifically described in the literature (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

[0474] For example, conditions may be listed in which polynucleotides with high homology or identity are hybridized with each other with 40% or more, specifically 90% or more, more specifically 95% or more, 96% or more, 97% or more, 98% or more, and even more specifically 99% or more homology or identity, and polynucleotides with lower homology or identity are not hybridized with each other, or conditions in which the polynucleotides are washed once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions of conventional southern hybridization, which are 60°C, 1 χSSC, 0.1% SDS, specifically 60°C, 0.1 χSSC, 0.1% SDS, more specifically 68°C, 0.1 χSSC, 0.1% SDS.

[0475] Hybridization requires that two nucleic acids have complementary sequences, even though a mismatch between bases may be possible depending on the degree of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, regarding DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Accordingly, the polynucleotides of this application may also include isolated nucleic acid fragments that are complementary to the entire sequence, as well as substantially similar nucleic acid sequences.

[0476] Specifically, polynucleotides having homology or identity can be detected using hybridization conditions including a hybridization step at a Tm value of 55°C and using the conditions described above. Additionally, the Tm value may be 60°C, 63°C, or 65°C, but is not limited thereto and can be appropriately adjusted by a person skilled in the art according to the purpose.

[0477] The appropriate strictness for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides, and the variables are well known in the field (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

[0478] For example, “high severity” occurs about 5 to 10°C below the probe’s Tm; “medium severity” occurs about 10 to 20°C below the probe’s Tm; and “low severity” may occur about 20 to 25°C below the Tm, but is not limited thereto.

[0479] For example, a “low stringency condition” may be prehybridization and hybridization at 42°C for 12–24 hours for a probe of at least 100 nucleotides in 5X SSPE, 0.3% SDS, shear and denatured salmon sperm DNA 200 micrograms / ml and 25% formamide according to Southern blotting standard procedures. The carrier material may finally be washed 2 to 3 times for 15 minutes each using 2 X SSC and 0.1 to 0.2% SDS at 50°C.

[0480] For example, a "medium stringency condition" may be prehybridization and hybridization at 42°C for 12-24 hours for a probe of at least 100 nucleotides in 5X SSPE, 0.3% SDS, shear and denatured salmon sperm DNA 200 micrograms / ml and 35% formamide according to Southern blotting standard procedures. The carrier material may finally be washed 2 to 3 times for 15 minutes each using 2 X SSC and 0.1 to 0.2% SDS at 55°C. For example, a "medium-high stringency condition" may be prehybridization and hybridization at 42°C for 12-24 hours for a probe of at least 100 nucleotides in 5X SSPE, 0.3% SDS, shear and denatured salmon sperm DNA 200 micrograms / ml and 35% formamide according to Southern blotting standard procedures. The carrier material may finally be washed 2 to 3 times for 15 minutes each using 1 to 2 X SSC and 0.1 to 0.2% SDS at 60°C.

[0481] For example, a "high stringency condition" may be prehybridization and hybridization at 42°C for 12 to 24 hours for a probe of at least 100 nucleotides in 5 X SSPE, 0.3% SDS, shear and denatured salmon sperm DNA 200 micrograms / ml and 35% formamide according to Southern blotting standard procedures. The carrier material may finally be washed 2 to 3 times for 15 minutes each using 2 X SSC and 0.1 to 0.2% SDS at 65°C.

[0482] The “nucleic acid structure” provided in this application comprises a polynucleotide encoding a variant provided in this application, which is operably linked to one or more regulatory sequences that direct the expression of a coding sequence in a suitable host cell under conditions suitable for the regulatory sequence.

[0483] Polynucleotides can be manipulated in various ways to enable the expression of variants. Depending on the expression vector, it may be desirable or necessary to manipulate the polynucleotide before inserting it into the vector. Such manipulation may be performed using methods known in the art.

[0484]

[0485] The “vector” provided in this application means a DNA product containing a sequence of nucleotides of a polynucleotide encoding said variant, which is operably linked to a suitable expression control region (or expression control sequence) so as to enable the expression of said variant in a suitable host. The expression control region may include a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence coding for a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. After being transformed into a suitable host cell, the vector may replicate or function independently of the host genome and may be incorporated into the genome itself.

[0486] The vectors that can be used in this application are not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage vectors or cosmid vectors, and pBR-based, pUC-based, pBluescriptII-based, pGEM-based, pTZ-based, pCL-based, and pET-based vectors may be used as plasmid vectors. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors may be used.

[0487] For example, a polynucleotide encoding the variant provided in this application can be inserted into a chromosome using a vector for intracellular chromosome insertion. The insertion of said polynucleotide into the chromosome may be achieved by any method known in the art, for example, homologous recombination, but is not limited thereto. A selection marker may be additionally included to confirm whether the chromosome insertion has occurred. The selection marker is intended to select cells transformed with the vector, that is, to confirm whether the target nucleic acid molecule has been inserted, and markers conferring selectable phenotypes such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of surface polypeptides may be used. Since only cells expressing the selection marker survive or exhibit other phenotypes in an environment treated with a selective agent, the transformed cells can be selected.

[0488] The "host cell" of the present application may be included without limitation as long as it is capable of expressing the polypeptide having the PET degradation activity of the present application.

[0489] The host cell of the present application may include the aforementioned variant, a polynucleotide encoding the variant, a nucleic acid structure containing the same, and / or a vector.

[0490] In one specific example, the host cell of the present application may overexpress a polypeptide having PET degradation activity, and said polypeptide having PET degradation activity may be the aforementioned variant and / or its parent PET degradation enzyme.

[0491] The above nucleic acid structure or vector may be incorporated into a chromosome as previously described, or maintained as an extrachromosomal vector that self-replicates.

[0492] The host cell of the present application includes any offspring of a parent cell that is not identical to the parent cell due to mutations occurring during replication.

[0493] The host cell can be any cell useful for generating the recombination of variants, for example, a prokaryotic or eukaryotic cell.

[0494] The prokaryotic host cell can be any Gram-positive or Gram-negative bacterium.

[0495] Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, Micromonospora, Corynebacterium, and Streptomyces.

[0496] Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia, Flavobacterium, Fusobacterium, Helicobacter, Illiobacter, Neisseria, Pseudomonas, Salmonella, Vibrio (e.g., Vibrio natriegens) and Ureaplasma.

[0497] In one specific example, the bacterial host cell may be a host cell of the genus Bacillus, specifically including, but not limited to, 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.

[0498] In one specific example, the bacterial host cell may be a host cell of the genus Streptococcus, specifically including but not limited to cells of Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and the subspecies Zooepidemicus of Streptococcus equi.

[0499] In one specific example, the bacterial host cell may be a host cell of the genus Streptomyces, specifically including but not limited to cells of Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicol, Streptomyces griseus, and Streptomyces lividans.

[0500] In one specific example, the bacterial host cell may be a host cell of the genus Corynebacterium, and Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes It may be, but is not limited to, ammoniagenes), Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens.

[0501] In one specific example, the bacterial host cell may be an Escherichia genus host cell and may be Escherichia coli (E. coli), but is not limited thereto.

[0502] The host cell can be a eukaryote, such as a mammal, insect, plant, or fungal cell.

[0503] The host cell may be a fungal cell. In this application, "fungus" includes Ascomycota, Basidiomycota, Tongomycota, and Zygomycota, as well as Oomycota and all imperfect fungi.

[0504] The fungal host cell may be a yeast cell. The term "yeast" in this application includes ascosporogenous yeasts (Endomycetales), basidiosporogenous yeasts, and yeasts belonging to Fungi imperfecti (Blastomycetes). However, this classification may change and may be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0505] Yeast host cells are Candida, Hansenula, Kluyveromyces, Pichia, Komagatella, Saccharomyces, Schizosaccharomyces, or Yarrowia cells, for example, Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norvensis It may be cells of Saccharomyces orbensis, Saccharomyces oviformis, Komagatella phaffii, or Yarrowia lipolytica.

[0506] Fungal host cells may be filamentous fungal cells. "Filamentous fungi" includes all filamentous forms of the Mycophylum and Oomyceta subphylum (as defined in the aforementioned literature (Hawksworth et al., 1995)). Filamentous fungi generally feature hyphal walls composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation, and carbon catabolism is strictly aerobic. In contrast, vegetative growth by yeasts, e.g., Saccharomyces cerevisiae, is by the germination of a unicellular thallus, and carbon catabolism may be fermentable.

[0507] Filamentous fungal host cells include Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Mycelioptora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, and Pleurotus, It may be a Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

[0508] For example, filamentous fungal host cells include 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, and Ceriporiopsis Subrufa (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 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 Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, *Mucor miehei*, Mycelioptora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum,It may be Trichoderma reesei or Trichoderma viride cells. However, it is not limited to these.

[0509]

[0510] The “composition” of the present application may comprise a polypeptide having PET degradation activity having activity corresponding to a polypeptide consisting of the amino acid sequence of SEQ ID NO. 1; and / or a variant thereof or a host cell expressing said polypeptide.

[0511] For a polypeptide having PET degradation activity having activity corresponding to the polypeptide composed of the amino acid sequence of SEQ ID NO. 1, the description of the parent PET degradation enzyme of the variant provided in this application may be applied. In addition, for a variant of the polypeptide and a host cell expressing the same, the description of the variant polypeptide and the host cell expressing the same provided in this application may be applied.

[0512] The composition of the present application can be used to convert polyester into a final product.

[0513] The term "polyester" refers to a polymer that contains ester functional groups in the main chain of its structure. For example, polyethylene terephthalate is a semi-aromatic copolymer composed of two monomers, terephthalic acid and ethylene glycol.

[0514] The polyester may be selected from polyethylene terephthalate (PET), polyethylene terephthalate glycol (PETG), polyethylene co-isosorbide-terephthalate (PEIT), polytrimethylene terephthalate (PTT), polybutylene adipate terephthalate (PBAT), polycyclohexylenedimethylene terephthalate (PCT), and polybutylene terephthalate (PBT). Specifically, the polyester may be PET.

[0515] The polypeptide of the present application or a composition containing the same may be used to depolymerize PET into bis(2-hydroxyethyl) terephthalate (BHET) and / or decompose PET into mono(2-hydroxyethyl) terephthalate (MHET), terephthalic acid (TPA) and ethylene glycol (EG).

[0516] In one embodiment, the polypeptide of the present application can decompose polyester by a glycolysis reaction.

[0517] For example, the degradation of a polyester may be performed in the presence of the polypeptide of the present application and ethylene glycol (EG), including the step of converting the polyester to BHET.

[0518] For example, in the presence of the polypeptide of the present application and ethylene glycol (EG), the ester group of the polyester and the amino acid residue of the polypeptide react to form an acyl-enzyme intermediate; and the formed intermediate reacts with ethylene glycol in a de-acylation step, wherein the polyester is converted to BHET and the degradation of the polyester can be performed.

[0519]

[0520] The composition of the present application may further include other components in addition to the polypeptide having PET degradation activity provided in the present application. Those skilled in the art may appropriately select the components added to the composition of the present application.

[0521] In one embodiment, the composition of the present application may further include any component suitable for application in converting PET into a final product.

[0522] In one embodiment, the composition of the present application may further include any component suitable for application to PET degradation.

[0523] Examples of substances that may be added include, but are not limited to, stabilizers, surfactants, builders, chelating agents, dispersants, enzymes, enzyme stabilizers, catalysts, activators, carriers, composites, lubricants, disintegrants, excipients, solubilizers, suspending agents, colorants, fragrances, buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, diluents, lubricants, preservatives, etc.

[0524] In one embodiment, the composition provided in this application may further include a naturally occurring or non-naturally occurring substance in addition to the variant provided in this application. In one embodiment, the composition provided in this application may further include water.

[0525] In one embodiment, the composition provided in this application may further include additional enzymes in addition to the variant provided in this application.

[0526]

[0527] A method for producing a variant of the present application may include the step of culturing a host cell; and the step of recovering a variant expressed in the culture step.

[0528] In this application, the term "culture" means growing the host cells under appropriately controlled environmental conditions. The culture process of this application may be carried out according to suitable media and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culture may be batch, continuous, and fed-batch, but is not limited thereto.

[0529] In this application, the term "medium" refers to a substance mixed with nutrients as the main component required to culture the host cell, and supplies nutrients and growth factors, including water, which is indispensable for survival and development. Specifically, the medium and other culture conditions used for culturing the host cell of this application may be any medium used for culturing conventional host cells without any particular limitations; however, the host cell of this application may be cultured under aerobic conditions while controlling the temperature, pH, etc., in a conventional medium containing a suitable carbon source, nitrogen source, phosphorus, inorganic compounds, amino acids, and / or vitamins.

[0530] In the present application, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvate, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. Additionally, natural organic nutrient sources such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane residue, and corn steeping liquid may be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted into reducing sugars) may be used, and other carbon sources in appropriate amounts may be used without limitation. These carbon sources may be used individually or in combination of two or more types, but are not limited thereto.

[0531] The above nitrogen sources may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; and organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquid, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. These nitrogen sources may be used alone or in combination of two or more types, but are not limited thereto.

[0532] The above ingredients may include monopotassium phosphate, dipotassium phosphate, or corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc., and may also include amino acids, vitamins, and / or suitable precursors. These components or precursors may be added to the culture medium in a batch or continuous manner. However, they are not limited thereto.

[0533] In addition, during the culture of the host cells, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc., may be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during culture, an antifoaming agent such as fatty acid polyglycol ester may be used to suppress bubble formation. Furthermore, to maintain an aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, or nitrogen, hydrogen, or carbon dioxide gas may be injected without gas injection to maintain an anaerobic and microaerobic state, but is not limited thereto.

[0534] The temperature of the medium may be 20°C to 50°C, specifically 25°C to 40°C, but is not limited thereto. The culture period may continue until a desired amount of useful substance is obtained, specifically 24 hours to 196 hours, but is not limited thereto.

[0535]

[0536] In one specific example, the variant expressed during the culture step may be recovered using methods known to the art to which the present invention belongs. For example, the variant may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.

[0537] The above recovery method may involve collecting variants using a suitable method known in the art according to the host cell culture method of the present application, such as a batch, continuous, or fed-batch culture method. For example, various chromatographs such as centrifugation, filtration, treatment with a crystallizing protein precipitating agent (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, and combinations thereof may be used, and variants may be recovered from the culture medium or host cells using a suitable method known in the art.

[0538] In another specific example, the variant expressed by the host cell during the culture step may not be recovered. In the above specific example, the host cell itself expressing the variant may be used as a source of the variant.

[0539]

[0540] The present application may include a method for producing bis-2-hydroxyethyl terephthalate (BHET), (mono(2-hydroxymethyl) terephthalate (MHET), terephthalic acid (TPA) and / or ethylene glycol (EG), comprising contacting a polyester with a polypeptide having the PET degradation activity described above, a variant thereof, a host cell expressing said polypeptide, or a composition comprising these.

[0541] Specifically, polyester may include the aforementioned details.

[0542] In this application, "disintegration" of a polyester is used, but is not limited to, "contacting" the polyester with a variant polypeptide, a host cell expressing said polypeptide, or a composition containing said polypeptide. Disintegration may also be used for depolymerization.

[0543] The time required to decompose the polyester may vary depending on various process parameters (i.e., temperature, pH, additional chemicals, etc.), as well as the article containing the polyester itself (i.e., properties and origin of the plastic product, its composition, shape, etc.) and the form and amount of the variant polypeptide used. Techniques known in the art can be readily applied to process parameters suitable for articles containing polyester.

[0544] For example, the degradation process may be carried out at 20°C to 90°C, specifically at 40°C to 80°C, and more specifically at 50°C to 70°C. More specifically, the temperature may be maintained at an inactivation temperature corresponding to the temperature at which the variant polypeptide is inactivated and / or at a temperature below which the host cell no longer synthesizes the variant polypeptide.

[0545] For example, the decomposition process can be carried out at pH 5 to pH 11, and specifically at pH 6 to pH 9.

[0546] In a specific example of the present application, an article containing polyester may be pretreated before contacting the variant polypeptide of the present invention to physically or chemically modify its structure, thereby increasing the contact area with the variant polypeptide.

[0547] Specifically, BHET, MHET, TPA, and / or EG that may be generated from the decomposition by the above contact can be recovered sequentially or continuously.

[0548] Specifically, the recovered BHET, MHET, TPA, and / or EG may be further purified using any suitable purification method and produced in a repolymerizable form. More specifically, purification may include, but is not limited to, a stripping process, separation by aqueous solution, steam selective condensation, filtration and concentration of the medium after the bioprocess, separation, distillation, vacuum evaporation, extraction, electrodialysis, absorption, ion exchange, precipitation, crystallization, concentration and acid addition dehydration and precipitation, nanofiltration, acid catalytic treatment, semi-continuous mode distillation or continuous distillation, solvent extraction, evaporative concentration, evaporative crystallization, liquid / liquid extraction, hydrogenation, azeotropic distillation process, absorption, column chromatography, simple vacuum distillation, and microfiltration.

[0549]

[0550] The final products BHET, MHET, TPA, and / or EG obtained using the composition of the present application can be reused in the polymerization of polyester.

[0551] Specifically, the obtained final products, BHET, MHET, TPA, and / or EG, can be reused to synthesize polyesters as repolymerizable monomers and / or oligomers. Specifically, polyesters of the same properties can be repolymerized and mixed with other monomers and / or oligomers to synthesize, for example, new copolymers.

[0552] The polyester may be selected from polyethylene terephthalate (PET), polyethylene terephthalate glycol (PETG), polyethylene co-isosorbide-terephthalate (PEIT), polytrimethylene terephthalate (PTT), polybutylene adipate terephthalate (PBAT), polycyclohexylenedimethylene terephthalate (PCT), and polybutylene terephthalate (PBT). Specifically, the polyester may be PET.

[0553] Methods for synthesizing polyester using BHET, MHET, TPA, and / or EG are known in the art.

[0554]

[0555] Examples

[0556] The present application will be explained in more detail below through examples and experimental examples. However, these examples and experimental examples are intended to illustrate the present application, and the scope of the present application is not limited to these examples and experimental examples.

[0557]

[0558] Example 1: Discovery of a Novel MpPETase

[0559] To discover novel PETases in addition to the well-known PET hydrolase (IsPETase) of Ideonella sakaiensis 201-F6, sequence homology analysis was performed using the NCBI database to identify 10 PETase candidates. To investigate the selected 10 PETase candidates, attempts were first made to produce these enzymes in a signal peptide-cleavage form, and 9 PETase candidates were successfully produced. Subsequently, the PET hydrolysis activity of the 9 PETase candidates was measured by monitoring the amounts of released PET hydrolysis products, MHET and TPA, using PET bottle powder (PET bottle-derived PET samples; hereinafter referred to as "B-PET") as a substrate. B-PET was obtained through the following processing: Clear PET bottles were crushed using a crusher, and the crushed PET was subsequently melted in a high-temperature oven at 270°C. The molten PET was immediately immersed in water at 4°C to cure. The obtained cured PET underwent a cryogenic grinding process, after which PET powder with a particle size of less than 300 μm was obtained through a steel mesh. The B-PET thus obtained was mixed with each PETase candidate and reacted at 50°C for 24 hours. While most PETase candidates exhibited very small amounts of PET hydrolysis products, SDZ16714.1, a cutinase of Micromonospora pattaloongensis, showed a significant amount of PET hydrolysis products compared to other enzymes. In addition, to investigate the thermal stability of these enzymes, the melting temperature (Tm) of nine PETase candidates was measured, and these PETase candidates showed various Tm values ​​ranging from 38.6°C to 86.2°C. SDZ16714.1, which exhibited extremely high PET hydrolysis activity compared to other enzymes, showed high temperature stability with a Tm value of 70.2°C and a relatively high level of solubility expression. These results suggest that SDZ16714.1. It was shown that it possesses excellent characteristics for efficient PET degradation, such as enzyme activity, thermal stability, and protein expression levels. Accordingly, SDZ16714.1 (PETase of Micromonospora pattaloongensis, hereinafter referred to as "MpPETase"; SEQ ID NO. 1) was selected as a novel PETase in the present invention.

[0560]

[0561] Example 2: Preparation of PET-Degraded Protein

[0562] The expression and purification of MpPETase were performed under the following conditions. A codon-optimizing MpPETase gene (Sequence No. 2) for E. coli was synthesized and amplified via polymerase chain reaction (PCR). The nucleotide sequence corresponding to the signal peptide was removed from the synthesized DNA. Subsequently, the PCR product was subcloned (Nde I and Xho I) into pET22b(+) (Novagen), which lacks its own signal peptide. The E. coli Rosetta gami-B (DE3) strain was transformed with the generated expression vector pET22b(+):MpPETase. The E. coli strain was cultured in a flask containing 1 L of lysogeny broth medium containing 100 mg / L ampicillin at 37°C at an optical density of 0.6 at 600 nm.

[0563] Protein expression was induced by the addition of 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and the culture medium was further cultured at 18°C ​​for 18 hours. Subsequently, cells were harvested by centrifugation at 4000 rpm for 20 minutes at 4°C.

[0564] The cell pellet was resuspended in Buffer A (40 mM Tris-HCl, pH 8.0) and then pulverized by sonication. Cell debris was removed by centrifugation at 13,500 rpm for 30 minutes, and the supernatant was transferred to a Ni-NTA agarose column (Qiagen). After washing with Buffer A containing 30 mM imidazole, the binding protein was eluted with 300 mM imidazole in Buffer A. All purification steps were performed at 4°C. Protein purity was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified protein was concentrated in Buffer A. PET hydrolases known by the same method, LCC (GenBank: AEV21261.1) and IsPETase (GenBank: GAP38373.1), were prepared and used as control groups. The primers used for cloning are listed in Table 1.

[0565]

[0566] Sequence Number Enzyme Primer Sequence 72MpPETase WT FTATATCATATGGCACCCCCAGCAAGTGCAACG73RTATATCTCGAGTACGGGCACGTACTACGAAATTG74IsPETase WT FTATACATATGCGCGGTCCGAATCCGACAGCCGCC75RGCGCCTCGAGGCTGCAATTCGCTGTACGAAAATC76LCC WT FTATATCATATGCAATCCAACCCGTACCAGCGCGG77RTATATCTCGAGCTGGCAGTGGCGGTTGTTCGTC

[0567]

[0568] Example 3: PET degradation activity analysis

[0569] To compare the PET-hydrolysis activity of MpPETase with the two PET hydrolyzing enzymes (LCC and IsPETase) prepared in Example 2, 15 mg of B-PET prepared by the same method as in Example 1 was immersed in 1 mL of 50 mM Glycine-NaOH (pH 9.0) buffer with each 500 nM enzyme. The mixture of each enzyme and B-PET was reacted at 50°C for 24 hours. After the reaction, the PET degradation activity was evaluated by analyzing each product via HPLC.

[0570] MpPETase showed 2.6 times higher activity compared to LCC enzyme and 14.5 times higher activity compared to IsPETase at 50°C. (Fig. 1) These results indicate that MpPETase can have higher PET hydrolysis activity at high temperatures than IsPETase and LCC.

[0571] Example 4: Measurement of thermal stability and PET degradation activity of MpPETase variants

[0572] Fifteen variant MpPETases discovered by utilizing enzyme structures to improve the PET degradation activity and thermal stability of the enzyme 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.

[0573] The M1 variant showed a Tm value of 5.3°C increase compared to MpPETaseWT, and its PET degradation activity increased by approximately 1.04 times. The M2 variant showed a Tm value of 1.2°C increase compared to MpPETaseWT, and its PET degradation activity increased by approximately 1.18 times. The M3 variant showed a Tm value of 1.1°C increase compared to MpPETaseWT, and its PET degradation activity increased by approximately 1.3 times. The M4 variant showed a Tm value of 2.5°C increase compared to MpPETaseWT, and its PET degradation activity increased by approximately 1.11 times. The M5 variant showed a Tm value of 0.9°C increase compared to MpPETaseWT, and its PET degradation activity increased by approximately 1.05 times. The M6 ​​variant showed a Tm value of 1.1°C increase compared to MpPETaseWT, and its PET degradation activity increased by approximately 1.2 times. The M7 variant showed a Tm value increase of 2.1°C compared to MpPETaseWT, and its PET degradation activity increased by approximately 1.08 times. The M8 variant showed a Tm value increase of 0.3°C compared to MpPETaseWT, and its PET degradation activity increased by approximately 1.12 times. The M9 variant showed a Tm value increase of 0.3°C compared to MpPETaseWT, and its PET degradation activity increased by approximately 1.13 times. The M10 variant showed a Tm value increase of 5.6°C compared to MpPETaseWT, and its PET degradation activity increased by approximately 1.22 times. The M11 variant showed a Tm value increase of 6.6°C compared to MpPETaseWT, and its PET degradation activity increased by approximately 1.12 times. The M12 variant showed a Tm value increase of 1.5°C compared to MpPETaseWT, and its PET degradation activity increased by approximately 1.18 times. The M13 variant showed a Tm value of 4.6°C increase compared to MpPETaseWT, and its PET degradation activity increased by approximately 1.14 times. The M14 variant showed a Tm value of 4.3°C increase compared to MpPETaseWT, and its PET degradation activity increased by approximately 1.32 times. The M15 variant showed a Tm value of 22.2°C increase compared to MpPETaseWT, and its PET degradation activity increased by approximately 2.39 times.

[0574]

[0575] Based on this, it was confirmed that introducing mutations into the MpPETase protein increased the protein's temperature stability or enhanced PET degradation activity.

[0576]

[0577] Mutant product concentration (uM)Tm (℃)MpPETaseWT867.470.2M1 (MpPETaseG215T)905.8575.5M2 (MpPETaseS147T)1020.0171.4M3 (MpPETaseT228Q)1129.7871.3M4 (MpPETaseA251D)958.7272.7M5 (MpPETaseE46Q)906.7171.1M6 (MpPETaseA67Q)1038.2171.3M7 (MpPETaseS221Q)935.9672.3M8 (MpPETaseT197K)966.7570.5M9 (MpPETaseP90A)977.3170.5

[0578]

[0579] Variant product concentration (uM)Tm (°C)MpPETaseWT 1199.6870.2M10 (MpPETaseD 179C / R 201C) 1467.8875.8M11 (MpPETaseT 203C / A 231C) 1345.7776.8M12 (MpPETaseL 49C / S 61C) 1412.1371.7M13 (MpPETaseA 241C / S 286C) 1369.2974.8M14 (MpPETaseS 198C / G 229C) 1581.9574.5M15 (MpPETaseD179C / R201C / T203C / A231C / G215T / S147T / T228Q / A251D / L49C / S6 1C / E46Q / A67Q / A241C / S286C / T197K / P90A / S221Q / S198C / G229C)2871.0592.4

[0580]

[0581] Example 5: Effect on MpPETase variant site

[0582] In order to confirm the changes in protein thermal stability and PET degradation activity when the position of the introduced variant identified in Example 4 is substituted with another amino acid, the following variants were prepared in the same manner as in Example 2, and the PET degradation activity and thermal stability were evaluated in the same manner as in Example 4 and are shown in Tables 4 and 5.

[0583]

[0584]

[0585]

[0586]

[0587]

[0588]

[0589]

[0590]

[0591]

[0592]

[0593]

[0594]

[0595]

[0596]

[0597] From the above results, it was confirmed that when 19 mutation sites of MpPETase were changed to other amino acids, substitution with some amino acids affected the improvement of protein activity or thermal stability. This means that the evaluated amino acid sites have a direct or indirect effect on the protein's activity or thermal stability.

[0598]

[0599] Example 6: Measurement of PET depolymerization activity by glycolysis-catalyzed reaction of MpPETase variant

[0600] The resistance of the M15 variant MpPETase with enhanced durability to ethylene glycol (EG) and the PET depolymerization activity via glycolysis-catalyzed reactions were evaluated. The M15 variant MpPETase was prepared in the same manner as in Example 2 to prepare an enzyme-EG mixture containing 5 μL of an enzyme solution at a concentration of 10.2 mg / mL, 45 μL of buffer A, and 950 μL of a 99.5% EG solution. To evaluate resistance to EG, the enzyme-EG reaction mixture was exposed to conditions of 40, 50, 60, and 70 °C for 0, 2, 4, 6, 8, and 10 days. 50 mg of B-PET powder was immersed in each of the treated enzyme-EG reaction mixture solutions. The reaction mixture solutions were reacted at 40 °C for 24 hours. After the above reaction, the products of each reaction solution were analyzed via HPLC to measure the PET depolymerization activity catalytically catalyzed by glycolysis. The concentrations of the major products (TPA, BHET, and MHET) for the enzymatic PET glycolysis depolymerization reaction are presented in Table 6. As a result of the reaction under these conditions, BHET was produced at a rate ranging from a minimum of 88.4% to a maximum of 97.4% of the major reaction products. In particular, a BHET concentration of 1.315 mM was obtained as a result of reacting at 40 °C for 24 hours.

[0601]

[0602] PET glycolysis depolymerization reaction product of M15 variant MpPETase EG exposure temperature (°C) EG exposure time (days) TPA (mM) MHET (mM) BHET (mM) 4000 0.001683 0.033972 1.315544402 0.000481 0.03721 71.326569404 0.000441 0.034033 1.238827406 0.000408 0.0324781.190423408 0.000570.0305771.167745401 00.00 18230.0361551.3024135000.0016830.0339721.3155445020.0003830.0366071.2791455040.0004440.0335521.1998495060.0005650.0363341.2865225080.0004070.0297771.10288450100.0008250. 0315931.135456000.0016830.0339721.3155446020.0009720.0337431.1757346040.0007430.0283151.0214416060.0007710.0244860.8741566080.0003950.0211250.77189760100.0011140.0216810 .7965547000.0016830.0339721.3155447020.0002240.0026810.0725517040.0003080.0015730.0374717060.000390.0010070.0177697080.0002970.0006130.00955270100.0004360.0004480.006773

[0603]

[0604] From the foregoing description, those skilled in the art to which this application pertains will understand that this application may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of this application should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

[0605]

[0606] [Ranking List]

[0607] #NameSequence1GenBank: SDZ16714.1MpPETaseWT(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV2MpPETaseWT(Ntsequence)ATGCAGCGACACGAGACCATTCCCCGACCGTCCCATCGCCGCCCCCGGGGGCTGCACCGGCTGACCGCGATCGCGGTGGCCGCGGCCATGGCCCTGACCACGCTCGGAGTGGCCGCGCCGCCCGCGTCGGCCACCGAGCGCGGCCTCGCCCCGACCGCGGCGAACATCACCGGCGACGGCAGCTACGGCGTCGTCTCCGCGACCATCACCGGCGCCAGCGGGTTCGGTGGCGGCGTCGTCTACTACCCGAACGCCACCGAACGGTTCCCGGTCGTCGCCATCTCGCCCGGCTACACCGAACGCTGGTCATCGTTCGCGTGGCTCGGGCGGCGGCTGGCCTCGTGGGGATTCGTGGTCGTCGGCATCGAGACGAACTCGCTCTTCGACCAGCCGAACAGCCGGGGTACCCAGTTGCTGCGCGCGCTGGACTGGGCCAGCTCCTCCGCCCCGGCCGCGGTGCGGGACCGCGTCGACGCCACCCGGCAGGGAGTGTCCGGGCACTCGATGGGCGGTGGCGGAACGCTGTCGGCCATGGACCAGCGCCCCTCCGTGCGCGCCGGCGTGCCGCTGGCCCCGTGGCACACCACCACCTCGTGGCCGAGGGTGACGAACCCGGTCATGATCCTGGGGGGCCAGAACGACGGGATCGCGCCGGTGTCGTCCCACGCGATCCCGATGTACACCGGCGTGGCCTCCGGAGAAAAGGCGTACGTCGAGCTGGCGGGCGCCGGGCACAACTTCCCCAACAGCGCCAACCCGATCGTCTCCAGGGCCGCGGTGTCGTGGTTCAAGCGGTTCCTCGACGACGACACCCGGTTCGCCCCGTTCGCCTGCGACTTCGGCGGCGCTTCGATTTCGCAGTTCCGCAGCACCTGTCCGGTCTGA3MpPETaseM1(AAsequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDTIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV4MpPETaseM2(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASTSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV5MpPETaseM3(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYQGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV6MpPETaseM4(AAsequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSDNPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV7MpPETaseM5(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATQRGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV8MpPETaseM6(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSQTITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV9MpPETaseM7(AAsequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSQHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV10MpPETaseM8(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTKSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV11MpPETaseM9(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFAVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV12MpPETaseM10(AAsequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMCQRPSVRAGVPLAPWHTTTSWPCVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV13MpPETaseM11(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVCNPVMILGGQNDGIAPVSSHAIPMYTGVCSGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV14MpPETaseM12(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGCAPTAANITGDGCYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV15MpPETaseM13(AAsequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELCGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASICQFRSTCPV16MpPETaseM14(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTCWPRVTNPVMILGGQNDGIAPVSSHAIPMYTCVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV17MpPETaseM15(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATQRGCAPTAANITGDGCYGVVSQTITGASGFGGGVVYYPNATERFAVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASTSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMCQRPSVRAGVPLAPWHTTKCWPCVCNPVMILGGQNDTIAPVSQHAIPMYQCVCSGEKAYVELCGAGHNFPNSDNPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASICQFRSTCPV18MpPETaseM16(AAsequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDRIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV19MpPETaseM21(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDSIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV20MpPETaseM23(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDQIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV21MpPETaseM29(AAsequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDIIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV22MpPETaseM41(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASPSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV23MpPETaseM43(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASASAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV24MpPETaseM44(AAsequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASGSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV25MpPETaseM54(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYHGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV26MpPETaseM56(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYEGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV27MpPETaseM57(AAsequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYSGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV28MpPETaseM58(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYNGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV29MpPETaseM72(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSHNPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV30MpPETaseM73(AAsequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSENPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV31MpPETaseM74(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSSNPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV32MpPETaseM76(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSNNPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV33MpPETaseM80(AAsequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSGNPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV34MpPETaseM88(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATRRGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV35MpPETaseM90(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATHRGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV36MpPETaseM94(AAsequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATNRGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV37MpPETaseM98(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATARGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV38MpPETaseM106(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSRTITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV39MpPETaseM107(AAsequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSKTITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV40MpPETaseM113(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSNTITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV41MpPETaseM117(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSVTITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV42MpPETaseM119(AAsequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSITITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV43MpPETaseM123(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSYTITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV44MpPETaseM124(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSRHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV45MpPETaseM130(AAsequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSNHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV46MpPETaseM135(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSVHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV47MpPETaseM142(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTRSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV48MpPETaseM145(AAsequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTESWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV49MpPETaseM146(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTSSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV50MpPETaseM147(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTNSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV51MpPETaseM154(AAsequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTLSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV52MpPETaseM155(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTISWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV53MpPETaseM156(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTMSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV54MpPETaseM170(AAsequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFGVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV55MpPETaseM181(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMKQRPSVRAGVPLAPWHTTTSWPKVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV56MpPETaseM184(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMEQRPSVRAGVPLAPWHTTTSWPEVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV57MpPETaseM185(AAsequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMSQRPSVRAGVPLAPWHTTTSWPSVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV58MpPETaseM196(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVANPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV59MpPETaseM213(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGAAPTAANITGDGAYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV60MpPETaseM214(AAsequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGGAPTAANITGDGGYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV61MpPETaseM219(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGSAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV62MpPETaseM224(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGVAPTAANITGDGVYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV63MpPETaseM226(AAsequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGIAPTAANITGDGIYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV64MpPETaseM230(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASIAQFRSTCPV65MpPETaseM237(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELSGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV66MpPETaseM246(AAsequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTAWPRVTNPVMILGGQNDGIAPVSSHAIPMYTAVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV67MpPETaseM247(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTGWPRVTNPVMILGGQNDGIAPVSSHAIPMYTGVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV68MpPETaseM253(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTSWPRVTNPVMILGGQNDGIAPVSSHAIPMYTSVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV69MpPETaseM254(AAsequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTTWPRVTNPVMILGGQNDGIAPVSSHAIPMYTTVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV70MpPETaseM258(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTVWPRVTNPVMILGGQNDGIAPVSSHAIPMYTVVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV71MpPETaseM260(AA sequence)MQRHETIPRPSHRRPRGLHRLTAIAVAAAMALTTLGVAAPPASATERGLAPTAANITGDGSYGVVSATITGASGFGGGVVYYPNATERFPVVAISPGYTERWSSFAWLGRRLASWGFVVVGIETNSLFDQPNSRGTQLLRALDWASSSAPAAVRDRVDATRQGVSGHSMGGGGTLSAMDQRPSVRAGVPLAPWHTTTIWPRVTNPVMILGGQNDGIAPVSSHAIPMYTIVASGEKAYVELAGAGHNFPNSANPIVSRAAVSWFKRFLDDDTRFAPFACDFGGASISQFRSTCPV72MpPETase WTTATATCATATGGCACCCCCAGCAAGTGCAACG73TATATCTCGAGTACGGGGCACGTACTACGAAATTG74IsPETase WT TATACATATGCGCGGTCCGAATCCGACAGCCGCC75GCGCCTCGAGGCTGCAATTCGCTGTACGAAAATC76LCC WT TATATCATATGCAATCCAACCCGTACCAGCGCGG77TATATCTCGAGCTGGCAGTGGCGGTTGTTCGTC

Claims

1. As a variant polypeptide having PET (polyethylene terephthalate) degradation activity, i) The above variant polypeptide has sequence identity of 70% or more and less than 100% with SEQ ID NO. 1; and / or ii) The above variant polypeptide is a polypeptide encoded by a polynucleotide having 70% or more and less than 100% sequence identity with the sequence encoding the mature polypeptide of SEQ ID NO. 1; and / or iii) The above variant polypeptide is a polypeptide encoded by (a) the mature polypeptide coding sequence of SEQ ID NO. 1, (b) its cDNA, or (c) a polynucleotide hybridizing with the full-length complement of (a) or (b) under low, medium, medium-high, high, or very high strictness conditions; and / or iv) the variant polypeptide is a functional fragment of polypeptide i), ii) or iii) having PET degradation activity; and Variant polypeptide comprising any one of the modifications selected from the following: Deletion at one or more amino acid positions among 179, 201, 203, 231, 49, 61, 241, 286, 198, 229, 215, 147, 228, 251, 46, 67, 221, 197, and 90, insertion of an amino acid, substitution with another amino acid, formation of a disulfide bond, and / or combinations thereof; Here, the above position number is a position corresponding to the position of the polypeptide of sequence number 1.

2. In claim 1, the amino acid at position 179 of the variant polypeptide having PET degradation activity before modification is aspartic acid (D); amino acid at position 201 is arginine (R); amino acid at position 203 is threonine (T); amino acid at position 231 is alanine (A); amino acid at position 49 is leucine (L); amino acid at position 61 is serine (S); amino acid at position 241 is alanine (A); amino acid at position 286 is serine (S); amino acid at position 198 is serine (S); amino acid at position 229 is glycine (G); amino acid at position 215 is glycine (G); amino acid at position 147 is serine (S); amino acid at position 228 is threonine (T); amino acid at position 251 is alanine (A); amino acid at position 46 is glutamic acid (E); amino acid at position 67 is alanine (A); amino acid at position 221 is serine (S); amino acid at position 197 is threonine (T); and / or a variant polypeptide in which amino acid 90 is proline (P).

3. The variant polypeptide of claim 1, wherein the variant polypeptide comprises one or more of the following substitutions: The amino acid corresponding to position 179 is substituted with cysteine, lysine, glutamic acid, or serine; The amino acid corresponding to position 201 is substituted with cysteine, lysine, glutamic acid, or serine; The amino acid corresponding to position 203 is substituted with cysteine ​​or alanine; The amino acid corresponding to position 231 is substituted with cysteine; The amino acid corresponding to position 49 is substituted with cysteine, alanine, glycine, valine, isoleucine, or serine; The amino acid corresponding to position 61 is substituted with cysteine, alanine, glycine, valine, or isoleucine; The amino acid corresponding to position 241 is substituted with cysteine ​​or serine; The amino acid corresponding to position 286 is substituted with cysteine ​​or alanine; The amino acid corresponding to position 198 is substituted with cysteine, alanine, threonine, valine, isoleucine, or glycine; The amino acid corresponding to position 229 is substituted with cysteine, alanine, threonine, valine, isoleucine, or serine; The amino acid corresponding to position 215 is substituted with threonine, arginine, serine, glutamine, or isoleucine; The amino acid corresponding to position 147 is substituted with threonine, proline, alanine, or glycine; The amino acid corresponding to position 228 is substituted with glutamine, histidine, glutamic acid, serine, or asparagine; The amino acid corresponding to position 251 is substituted with aspartic acid, histidine, glutamic acid, serine, asparagine, or glycine; The amino acid corresponding to position 46 is substituted with glutamine, arginine, histidine, asparagine, or alanine; The amino acid corresponding to position 67 is substituted with glutamine, arginine, lysine, asparagine, valine, isoleucine, or tyrosine; The amino acid corresponding to position 221 is substituted with glutamine, arginine, asparagine, or valine; The amino acid corresponding to position 197 is substituted with lysine, arginine, glutamic acid, asparagine, serine, leucine, isoleucine, or methionine; and The amino acid corresponding to position 90 is substituted with alanine or glycine; Here, the above position number is a position corresponding to the position of the polypeptide of sequence number 1.

4. In paragraph 3, the variant polypeptide is Amino acids 179 and 201 are substituted with serine, glutamic acid, cysteine, or lysine; Amino acids 203 and 231 are substituted with cysteine; Amino acids 49 and 61 are substituted with alanine, cysteine, glycine, valine, or isoleucine; Amino acids 241 and 285 are substituted with cysteine; and Amino acids 198 and 229 are substituted with cysteine, alanine, valine, isoleucine, or threonine; Includes one or more substitutions selected from among, The above position number is a variant polypeptide that is a position corresponding to the position of the polypeptide of sequence number 1.

5. In paragraph 3, the variant polypeptide is Amino acid 179 is replaced with cysteine; amino acid 201 is replaced with cysteine; amino acid 203 is replaced with cysteine; amino acid 231 is replaced with cysteine; amino acid 215 is replaced with threonine; amino acid 147 is replaced with threonine; amino acid 228 is replaced with glutamine; amino acid 251 is replaced with aspartic acid; amino acid 49 is replaced with cysteine; amino acid 61 is replaced with cysteine; amino acid 46 is replaced with glutamine; amino acid 67 is replaced with glutamine; amino acid 241 is replaced with cysteine; amino acid 286 is replaced with cysteine; amino acid 197 is replaced with lysine; amino acid 90 is replaced with alanine; amino acid 221 is replaced with glutamine; amino acid 198 is replaced with cysteine; and amino acid 229 is substituted with cysteine, and The above position number is a variant polypeptide that is a position corresponding to the position of the polypeptide of sequence number 1.

6. In paragraph 3, the variant polypeptide is one that forms a disulfide bond between pairs of cysteine ​​selected from the following: Cysteine ​​179 and cysteine ​​201; Cysteine ​​203 and cysteine ​​231; Cysteine ​​No. 49 and Cysteine ​​No. 61; Cysteine ​​241 and cysteine ​​286; and / or Cysteine ​​198 and cysteine ​​229; Here, the above position number is a position corresponding to the position of the polypeptide of sequence number 1.

7. A variant polypeptide according to claim 1, wherein the variant polypeptide has one or more of the following modified characteristics compared to a polypeptide composed of the amino acid sequence of SEQ ID NO. 1: i) Increase or decrease in enzyme activity; ii) Increase or decrease in specific activity; iii) Increase or decrease in pH stability; iv) Increase or decrease in storage stability; v) Increase or decrease in acid resistance; vi) increase or decrease in heat resistance or thermal stability; and vii) Change in substrate specificity.

8. A composition comprising a variant polypeptide of any one of claims 1 to 7.

9. A variant polypeptide of any one of claims 1 to 7, or SEQ ID NO. 1 or a polypeptide having at least 70% sequence identity with respect to it; A host cell expressing the above polypeptide; or Or use for degrading the polyester of a composition containing the above polypeptide.

10. A polynucleotide encoding a variant polypeptide of any one of claims 1 to 7.

11. A variant polypeptide of any one of claims 1 to 7; a polynucleotide encoding said polypeptide; a nucleic acid structure comprising said polynucleotide; and / or a host cell comprising a vector comprising said nucleotide or said nucleic acid structure.

12. A host cell overexpressing a variant polypeptide of any one of claims 1 to 7, or a polypeptide having at least 70% sequence identity with SEQ ID NO.

1.

13. A variant polypeptide of any one of claims 1 to 7; a polynucleotide encoding the variant polypeptide; a nucleic acid structure comprising the polynucleotide; and / or a host cell comprising a vector comprising the nucleotide or the nucleic acid structure; and A method comprising the step of recovering a variant polypeptide having PET degradation activity expressed in the above culture step, Method for preparing a variant polypeptide having PET degradation activity.

14. A variant polypeptide of any one of claims 1 to 7, or SEQ ID NO. 1 or a polypeptide having at least 70% sequence identity with respect to it; A host cell expressing the above polypeptide; and / or A method for degrading polyester comprising treating the polyester with a composition containing the above polypeptide.

15. A method for decomposing polyester, wherein the polyester is PET, in accordance with claim 14.

16. A method for degrading a polyester according to claim 14, wherein the method comprises the step of performing a glycolysis reaction by the polypeptide.

17. A method for degrading a polyester according to claim 14, wherein the method comprises the step of converting the polyester to BHET in the presence of the polypeptide and ethylene glycol.

18. A variant polypeptide of any one of claims 1 to 7, or a polypeptide having at least 70% sequence identity with SEQ ID NO. 1; A host cell expressing the above polypeptide; and / or A method for producing bis-2-hydroxyethyl terephthalate (BHET), (mono(2-hydroxymethyl) terephthalate (MHET), terephthalic acid (TPA), and / or ethylene glycol (EG), comprising contacting a polyester with a composition containing the above polypeptide.

19. A method for producing bis-2-hydroxyethyl terephthalate (BHET), (mono(2-hydroxymethyl) terephthalate (MHET), terephthalic acid (TPA) and / or ethylene glycol (EG), wherein the method further comprises the step of recovering the produced BHET, MHET, TPA and / or EG.

20. A method for producing a polyester, comprising the step of synthesizing a polyester using BHET, MHET, TPA, and / or EG prepared according to claim 19.

21. A method for producing polyester, wherein, in paragraph 20, the polyester is PET.

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