Aspartic acid protease variant

Abstract

The present application relates to: a novel aspartic acid protease variant having increased thermal stability; and a use thereof.

Description

Aspartic protease mutant

[0001] Cross-citation with related application(s)

[0002] This application claims the benefit of priority to Korean Patent Application No. 10-2023-0084460, filed June 29, 2023, the entire contents of which are incorporated herein by reference.

[0003] The present application relates to a mutant polypeptide having aspartic protease activity and its use.

[0004]

[0005] Proteases (or proteases) are enzymes that catalyze the hydrolysis of peptide bonds by acting on proteins or peptides. According to the Hartley classification system, which classifies proteases according to their active structure, they are classified into serine proteases, thiol proteases, aspartic proteases, metalloproteases, and non-metalloproteases. Depending on the optimal pH, they are classified into acid proteases, neutral proteases, and alkaline proteases. Depending on the location of hydrolysis, they are divided into endopeptidases and exopeptidases. In addition to these, there are enzymes that work well on specific proteins, such as collagenase, elastase, keratinase, and gelatinase.

[0006] These various proteases are widely used in the food industry, chemical industry, leather, medical, detergent, paper and photography industries, as digestive agents, analytical reagents, agricultural and marine product processing, cosmetics manufacturing, and environmental purification (US 2022-0174980 A1). In particular, proteases improve the digestibility of poultry by decomposing protein anti-nutrient factors found in some plant-based feed ingredients such as corn / soybean meal feed. Specifically, the effect of alleviating anti-nutrient factors increases feed efficiency and also increases the utilization of dietary protein. Therefore, proteases are used as feed additives that improve poultry productivity by assisting endogenous enzymes secreted by poultry to hydrolyze proteins and thereby increasing the utilization of amino acids contained in feed.

[0007] The stomachs of poultry, including monogastric animals, have a highly acidic pH (pH 2.5–3.5). The forage consumed by poultry (broilers) is broken down and absorbed in the digestive system, which includes the proventriculus, which performs chemical functions, the gizzard, which performs mechanical functions, and the small intestine, which includes the duodenum, jejunum, and ileum. Chickens have two stomachs: the proventriculus and the gizzard. In the gizzard, feed is mixed with gastric acid and digestive juices, while in the gizzard, strong muscles grind the feed and mix it with gastric juices, where actual digestion begins. The activity of the gizzard is stimulated by coarse feed ingredients and grains. Therefore, proteases are needed that not only possess the stability necessary to pass through the acidic conditions of the gizzard (pH 2.5–3.5), but also assist endogenous enzymes in the gizzard to facilitate digestion.

[0008] Acid proteases are protein-degrading enzymes that undergo enzymatic action under acidic pH conditions. They are widely used industrially in baking, cheesemaking, milk coagulation, digestive enzymes, and burn treatments. However, improvements in various aspects, including heat stability and activity, are required to ensure greater industrial economic viability and efficacy. In particular, heat stability at high temperatures (70-90°C) during feed manufacturing processes is essential due to heat-induced enzyme inactivation.

[0009]

[0010] One object of the present application is to provide a mutant polypeptide having aspartic protease activity.

[0011] Another object of the present application is to provide a composition comprising the mutant polypeptide.

[0012] Another object of the present application is to provide a reaction composition comprising the mutant polypeptide or the mutant polypeptide for reaction with a protein.

[0013] Another object of the present application is to provide a method for degrading a protein, comprising the step of treating a substrate with the mutant polypeptide, a host cell expressing the mutant polypeptide, and / or a composition comprising the mutant polypeptide.

[0014] Another object of the present application is to provide a polynucleotide encoding the mutant polypeptide; a nucleic acid construct comprising the polynucleotide; a vector comprising the polynucleotide or the nucleic acid construct; and a host cell comprising at least one of the polynucleotide, the nucleic acid construct, and the vector.

[0015] Another object of the present application is to provide a method for producing the mutant polypeptide.

[0016] Another object of the present application is to provide a protein degradation use of the mutant polypeptide, composition, and host cell.

[0017] Another object of the present application is to provide a use for producing a feed composition, a food composition, or an industrial composition of the mutant polypeptide and the host cell.

[0018]

[0019] The specific details for implementing the invention are as follows. Each description and embodiment disclosed in this application may also be applied to other descriptions and embodiments. Furthermore, the scope of this application is not limited by the specific descriptions described below.

[0020] Furthermore, those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments of the present application described herein. Furthermore, such equivalents are intended to be encompassed by this application.

[0021]

[0022] definition

[0023] As used in the specification and appended claims of this application, the singular articles "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Unless the context clearly dictates otherwise, the singular terms include the plural and the plural terms include the singular. In the specification and appended claims of this application, unless otherwise indicated, the use of "or" is intended to include "and / or."

[0024]

[0025] In this application, the term "about" may be used before a specific numerical value. As used herein, the term "about" encompasses not only the exact number described after the term, but also a range that is or is nearly that number. Whether a number is or is nearly the specific number described can be determined based on the context in which it is presented. For example, the term "about" may refer to a range of -10% to +10% of a numerical value. In another example, the term "about" may refer to a range of -5% to +5% of a given numerical value. However, this is not a limitation.

[0026]

[0027] In this application, the terms "first, second, third," "i), ii), iii)…" or "(a), (b), (c), (d)…" are used to distinguish similar configurations, and these terms do not imply that the steps are performed consecutively or in order. For example, when the terms are used in connection with steps of a method, use, or analysis, these steps may be performed simultaneously, without a time interval, or may be performed at intervals of seconds, minutes, hours, days, or months.

[0028]

[0029] In this application, the term "consisting essentially of" means that a non-specific component may be present if the characteristics of the subject matter claimed in this application are not substantially affected by the presence of the non-specific component.

[0030] In this application, the term "consisting of" means that the proportion of a specific component(s) totals 100%. The components or features listed below the term "consisting of" may be essential or mandatory. In some embodiments, other than the components or features listed below "consisting of," other optional or nonessential components may be excluded.

[0031] In this application, the term "comprising" means the presence of a feature, step, or component described below, and does not exclude the presence or addition of one or more features, steps, or components. The components or features described below "comprising" in this application may be essential or mandatory, but in some embodiments, other optional or non-essential components or features may be further included.

[0032] In this application, the term “comprising” may, in some embodiments, be modified to refer to “consisting essentially of” or “consisting of.”

[0033] In the present application, with respect to an amino acid sequence, even if it is described as a polypeptide “comprising” an amino acid sequence set forth in a specific sequence number, a polypeptide “consisting of” an amino acid sequence set forth in a specific sequence number, or a polypeptide or protein “having” an amino acid sequence set forth in a specific sequence number, it is obvious that a protein having an amino acid sequence in which a portion of the sequence is deleted, modified, substituted, conservatively substituted, or added may also be used in the present application, as long as it has the same or corresponding activity as a polypeptide consisting of the amino acid sequence of the corresponding sequence number. For example, it may be a case in which the amino acid sequence has an addition of a sequence that does not alter the function of the protein at the N-terminus and / or C-terminus, a mutation that may occur naturally, a silent mutation thereof, or a conservative substitution, but is not limited thereto.

[0034]

[0035] In this application, the term "protein" or "polypeptide" refers to a polymer or oligomer of consecutive amino acid residues. In this application, "polypeptide," "protein," and "peptide" may be used interchangeably with "amino acid sequence."

[0036] In some cases, an amino acid sequence that exhibits activity may be referred to as an "enzyme." In this application, amino acid sequences are described in N-terminal → C-terminal orientation, unless otherwise indicated.

[0037]

[0038] In relation to a cell, nucleic acid, polypeptide, or vector, the term "recombinant" in this application means that the cell, nucleic acid, polypeptide, or vector has been modified by the introduction of a heterologous nucleic acid or polypeptide or by alteration of a native nucleic acid or polypeptide, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell may express a gene not found in the native (non-recombinant) form of the cell, or may express a native gene that is expressed or not expressed at all, or otherwise abnormally expressed.

[0039]

[0040] As used herein, the term "isolated" refers to a substance that exists in an environment where it does not occur naturally, or in a form that does not occur naturally. This includes the substance (sequence, enzyme, or nucleic acid) being at least substantially free from at least one other component with which it is naturally associated and found in nature, such as a sequence, enzyme, or nucleic acid.

[0041] For example, the isolated sequences, enzymes or nucleic acids provided in the present application may be provided in a form substantially free of one or more contaminants.

[0042] 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 with it in nature have been removed (e.g., an enzyme, variant, nucleic acid, protein, peptide, or cofactor), iii) any substance found in nature that has been artificially modified, or iv) a substance that has been modified to alter the amount of that substance relative to other naturally associated components (e.g., increasing the number of copies of a gene encoding the substance; modifying a promoter naturally associated with a gene encoding the substance to a more active promoter, etc.).

[0043]

[0044] In this application, the term "wild type" means a naturally occurring polypeptide without any artificial modifications. When the term "wild type" is used in relation to a polypeptide, it means a naturally occurring polypeptide that does not have any artificial mutations (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 a polypeptide that does not have any artificial modifications (substitutions, insertions, deletions) at one or more nucleotides. However, a polynucleotide encoding a wild type polypeptide is not limited to a naturally occurring polynucleotide, and also includes a sequence encoding any wild type polypeptide.

[0045]

[0046] In the present application, the parent sequence or backbone refers to a reference sequence into which a modification is introduced to become a mutant polypeptide. That is, the parent sequence may be a starting sequence into which mutations such as substitutions, insertions, and / or deletions are introduced. The parent sequence may be a naturally occurring or wild type, or a variant in which one or more substitutions, insertions, or deletions have occurred in the natural or wild type, or may be 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.

[0047]

[0048] In this application, the term "reference sequence" refers to a sequence used to determine the position of an amino acid within an arbitrary amino acid sequence. By aligning an arbitrary amino acid sequence with a reference sequence, the position of an amino acid corresponding to a specific position of the reference sequence within the arbitrary amino acid sequence can be determined.

[0049]

[0050] In the present application, with respect to an amino acid or nucleic acid sequence, the term "fragment" means a portion of a parent sequence. For example, it may be a polypeptide in which one or more amino acids from the parent sequence are removed from the C or N terminus.

[0051] In the present application, a "fragment" of an enzyme may refer to a "functional fragment." A "functional fragment," also referred to as an "active fragment," refers to a polypeptide that is part of a parent enzyme and possesses the enzymatic activity of the parent enzyme. For example, a functional fragment of an enzyme may include the catalytic site of the enzyme.

[0052] The enzyme fragment may comprise a portion of the full length of the parent enzyme, for example, but not limited to, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or less than 100% of the amino acids of the full length of the parent enzyme.

[0053]

[0054] In this application, "mutating / modifying" means changing or altering. This may be a change from a naturally occurring sequence. For example, an enzyme may be modified in such a way that it is altered from its parent sequence or reference sequence.

[0055] In the present application, the modified enzyme may be a non-naturally occurring enzyme, i.e., an enzyme that does not exist in nature itself.

[0056] The term "modified" in this application means, for example, something that 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 enzymes that are not found in nature. For example, the modified enzymes of this application may be, but are not limited to, enzymes that do not occur spontaneously.

[0057] When 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 the N-terminal and / or C-terminal amino acid sequence or 5' and / or 3' nucleic acid sequence, and any combination thereof.

[0058]

[0059] In the present application, a "modified polypeptide" or "variant" of an enzyme refers to a protein that differs from the parent enzyme in one or more amino acids by conservative substitution and / or modification. "Modified polypeptide" or "variant" may be used interchangeably. The modified polypeptide or variant may be, but is not limited to, a non-naturally occurring one.

[0060] The above mutant polypeptide differs from the sequence of the parent enzyme by one or more modifications, e.g., amino acid substitutions, deletions and / or insertions.

[0061] Such mutant polypeptides can generally be identified by modifying one or more amino acids in the parent enzyme and evaluating the properties of the modified protein. That is, the ability of the mutant polypeptide may be increased, unchanged, or decreased compared to the parent enzyme.

[0062] Additionally, some variant polypeptides may include variant polypeptides in which one or more portions, such as the N-terminal leader sequence or the transmembrane domain, are deleted.

[0063] Other variant polypeptides may include variants in which portions are deleted from the N- and / or C-terminus of the mature protein.

[0064] The term "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 is not limited thereto if the term is used in the meaning of variant.

[0065] Variants may include deletions or additions of amino acids that have minimal impact on the properties and secondary structure of the polypeptide. For example, the polypeptide may be conjugated to a signal (or leader) sequence at the N-terminus of the protein that is involved in co-translational or post-translational protein transfer. Furthermore, the polypeptide may be conjugated to other sequences or linkers to facilitate identification, purification, or synthesis of the polypeptide.

[0066]

[0067] In this application, the term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues.

[0068]

[0069] Throughout this application, the conventional one-letter and three-letter codes for naturally occurring amino acids are used. Furthermore, amino acids referred to by abbreviations in this application are described according to the IUPAC-IUB nomenclature.

[0070]

[0071] Alanine Ala, A

[0072] Arginine Arg, R

[0073] Asparagine Asn, N

[0074] Aspartic acid Asp, D

[0075] Cysteine ​​Cys, C

[0076] Glutamic acid Glu, E

[0077] Glutamine Gln, Q

[0078] Glycine Gly, G

[0079] Histidine His, H

[0080] Isoleucine Ile, I

[0081] Leu, L

[0082] Lysine Lys, K

[0083] Methionine Met, M

[0084] Phenylalanine Phe, F

[0085] Proline Pro, P

[0086] Serin Ser, S

[0087] Threonine Thr, T

[0088] Tryptophan Trp, W

[0089] Tyrosine Tyr, Y

[0090] Valin Val, V

[0091]

[0092] Meanwhile, any amino acid can be written as Xaa, X.

[0093] Additionally, the generally accepted three-letter codes for other amino acids, such as Aib (2-Aminoisobutyric acid), Sar (N-methylglycine), and α-methyl-glutamic acid, may be used, as well as for naturally occurring amino acids.

[0094]

[0095] 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.

[0096] For example, among the 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 the amino acids having uncharged side chains, nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline, and 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.

[0097]

[0098] As used herein, the term "gene" refers to a polynucleotide encoding a polypeptide and a polynucleotide comprising regions preceding and following the coding region. In some embodiments, a gene may have a sequence (intron) inserted between each coding region (exon).

[0099]

[0100] As used herein, the terms "homology" or "identity" refer to the degree of relationship between two given amino acid sequences or base sequences, which may be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0101] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms, and may be combined with default gap penalties established by the program being used. In practice, homologous or identical sequences are generally capable of hybridizing under moderate or high stringency conditions, typically along at least about 50%, 60%, 70%, 80%, or 90% of the entire sequence or its entire length. It should be appreciated that hybridization also encompasses polynucleotides containing common codons or codons that are considered codon degenerate.

[0102] Whether any two polynucleotide or polypeptide sequences are homologous, similar or identical can be determined using known computer algorithms such as the "FASTA" program using default parameters, for example as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) can be determined using the GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48: 1073). For example, homology, similarity, or identity can be determined using, but is not limited to, BLAST of the National Center for Biotechnology Information Database, or ClustalW.

[0103] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information, for example, using a GAP computer program such as that of Needleman et al. (1970), J Mol Biol. 48:443, as disclosed, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In brief, the 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). Default parameters for the GAP program include (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and (2) a comparison matrix as disclosed by Gribskov et al. (1986) Nucl. Acids Res. 48:443, as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979). 14: 6745 weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) permutation 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.

[0104] Additionally, whether any two polynucleotide sequences have homology, similarity or identity can be determined by comparing the sequences by Southern hybridization experiments under defined stringent conditions, and the defined appropriate hybridization conditions are within the scope of the art and can be determined by methods well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York), but are not limited thereto.

[0105]

[0106] 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-translational or post-translational modifications. Examples of post-translational modifications include, but are not limited to, N- or C-terminal modifications, glycosylation, phosphorylation, and leader sequence removal.

[0107]

[0108] The term "nucleic acid construct" in this application means a single or double-stranded nucleic acid molecule that contains one or more regulatory sequences and is artificially synthesized, engineered to contain a specific sequence in a manner that does not exist in nature, or isolated from nature.

[0109]

[0110] As used herein, the term “expression” includes, but is not limited to, any step involved in the production of a polypeptide, such as transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0111] As used herein, the term "expression vector" means a linear or circular nucleic acid molecule comprising a coding sequence and regulatory sequences operably linked thereto for expression thereof.

[0112]

[0113] As used herein, the term "operably linked" refers to a configuration in which a regulatory sequence is positioned appropriately to direct the expression of a coding sequence. Therefore, "operably linked" includes a regulatory region of a functional domain with known or desired activity, such as a promoter, terminator, signal sequence, or enhancer region, attached or linked to a target (gene or polypeptide) so as to regulate the expression, secretion, or function of the target in accordance with the known or desired activity.

[0114]

[0115] As used herein, the term "cDNA" refers to a DNA sequence that can be produced by reverse transcription from a mature, spliced ​​mRNA molecule, which can be obtained from a eukaryotic or prokaryotic cell. The cDNA sequence does not include intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor to mRNA before being processed through a series of steps, including splicing, to form the mature, spliced ​​mRNA.

[0116]

[0117] As used herein, the term "regulatory sequence" refers to a polynucleotide sequence necessary for the expression of a coding sequence. Each regulatory sequence may be native to the coding sequence (having the same origin) or foreign (derived from another gene). Examples of the regulatory sequence include a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, an operator sequence, a sequence encoding a ribosome binding site, and a sequence that regulates transcription and translation termination. The minimum unit of the regulatory sequence may include a promoter, a transcription and translation termination sequence.

[0118]

[0119] To describe the mutant polypeptides provided in this application, the following nomenclature is used.

[0120] In this application, reference to a specific position in an amino acid sequence may include reference to an amino acid present or substituted at that position. Reference to an amino acid at a specific position may be described in various ways. For example, "position 003" may be described as "position 3," "amino acid 3," or "the third amino acid." Furthermore, for example, if the amino acid at position 3 is serine (S), it may be described as "S3" or "Ser3."

[0121] Amino acid substitutions can be expressed by listing the amino acid before substitution, the position, and the amino acid being replaced. These amino acids can be expressed using conventional one-letter and three-letter codes. For example, if alanine, the amino acid at position 6 of a specific sequence, is replaced with valine, it can be written as "A8V" or "Ala8Val."

[0122] Any amino acid at a particular position can be designated as "X". For example, X6 refers to any amino acid at position 6. Also, when a substituted amino acid is designated as X, it means that it is replaced with an amino acid different from the amino acid present before the substitution. For example, "V6X" indicates that V is replaced with any amino acid other than V at position 6.

[0123] Different alternations can be expressed by listing multiple amino acids simultaneously using the " / " or "," symbol. For example, a substitution of the amino acid (D) at position 12 with S or K can be written interchangeably as D12S / K or D12S,K. As another example, P / S197K means that the amino acid P or S at position 197 before substitution is replaced with K.

[0124] Multiple mutations can be described using a "+" sign. For example, a description such as "G2A+ M8V" means that the amino acid at position 2, glycine, is replaced with alanine, and the amino acid at position 8, methionine, is replaced with valine, respectively.

[0125] Deletions of amino acids can be expressed by listing the amino acid before the deletion, the position, and then *. For example, if alanine, the amino acid corresponding to position 8 of a specific sequence, is deleted, it can be expressed as A8* or (Ala8*).

[0126] Insertion of an amino acid may be described as, for example, Gly8GlyLys or G8GK when a lysine is inserted between the glycine at position 8 and the amino acid at position 9 in a specific sequence. Insertion of more than one amino acid, for example, insertion of a lysine and a valine between the glycine at position 8 and the amino acid at position 9 in a specific sequence, may be described as Gly8GlyLysVal or G8GKV. In this case, the position of the inserted amino acid may be indicated using the amino acid number and the alphabet preceding the inserted amino acid, and for example, in the above case, the inserted lysine and valine may be numbered as 8aK and 8bV.

[0127]

[0128] As used herein, the term "corresponding to" refers to an amino acid residue at a position listed in a protein or polypeptide, or an amino acid residue that is similar, identical, or homologous to the residue listed in the protein or polypeptide. Identifying an amino acid at a corresponding position may be determining a specific amino acid in a sequence that references a particular sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

[0129] In the present application, SEQ ID NO. 1 can be used as a reference sequence to determine the position of an amino acid in any amino acid sequence.

[0130] That is, SEQ ID NO: 1 disclosed in the present application can be used to determine the corresponding amino acid residue in a polypeptide having any protease activity, and unless otherwise indicated in the present application, residues of a specific amino acid sequence are numbered based on SEQ ID NO: 1.

[0131] For example, any amino acid sequence can be aligned with SEQ ID NO: 1, and based on this, each amino acid residue of the amino acid sequence can be numbered by referring to the numerical position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 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 at which a modification such as a substitution, insertion, or deletion occurs, by comparing it to a query sequence (also referred to as a “reference sequence”).

[0132] These alignments can be performed using, but are not limited to, 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), and Trends Genet. 16: 276-277).

[0133] Multiple sequence alignment can also be used to identify corresponding amino acid residues in other proteases. Examples of multiple sequence alignment programs known in the art include MUSCLE (multiple sequence comparison by log-expectation; version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT (version 6.857 or later; 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 using ClustalW. EMMA (1.83 or higher; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), etc., and the default parameters of each of the above programs can be used, but are not limited thereto.

[0134] Additionally, if enzymes diverged from the mature polypeptide of SEQ ID NO: 1 and their relationships cannot be detected by conventional sequence-based comparison, other pairwise sequence comparison algorithms can be used (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615). Higher sensitivity can be achieved in sequence-based searches by using search programs that utilize probabilistic representations of polypeptide families (profiles) to search databases. For example, the PSI-BLAST program generates profiles through an iterative database search process and can detect remote homologs (Atschul et al., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivity can be achieved if the family or superfamily for the polypeptide has more than one representation 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) use information from a variety of sources, such as PSI-BLAST, secondary structure predictions, structural alignment profiles, and solvation potentials, as input to a neural network that predicts the structural folding of a query sequence. Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919 can be used to align an unknown sequence with superfamily models available in the SCOP database. These alignments can in turn be used to build homology models for the polypeptide, and these models can be evaluated for accuracy using a variety of tools developed for this purpose.

[0135] For proteins with known structures, several tools and resources are available for searching and generating structural alignments. For example, the SCOP superfamily of proteins is structurally aligned, and these alignments are accessible and downloadable. Two or more protein structures can be aligned using various algorithms, such as distance alignment matrix alignment (Holm and Sander, 1998, Proteins 33: 88-96) or Combinatorial extension (CE) (Shindyalov and Bourne, 1998, Protein Engineering 11: 739-747). Implementations of these algorithms can additionally be used to query structural databases containing the target structure to discover possible structural homologues (Holm and Park, 2000, Bioinformatics 16: 566-567).

[0136] The above methods are examples and are not limiting.

[0137]

[0138] Hereinafter, specific examples of the present application will be described in more detail as follows.

[0139]

[0140] One aspect of the present application is a mutant polypeptide having aspartic acid protease activity, which is at least one of the following i) to iii):

[0141] i) a polypeptide having a sequence identity of 70% or more and less than 100% with the amino acid sequence of sequence number 1 or an amino acid sequence having a sequence identity of 90% or more therewith;

[0142] ii) a polypeptide encoded by a polynucleotide having a sequence identity of at least 70% and less than 100% with a sequence encoding a mature polypeptide of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% sequence identity therewith;

[0143] iii) a polypeptide encoded by a polynucleotide that hybridizes to (a) a mature polypeptide coding sequence of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% sequence identity thereto, (b) a cDNA thereof, or (c) a full-length complement of (a) or (b) under low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions,

[0144] A variant polypeptide is provided comprising any one of the following modifications:

[0145] 65, 66, 77, 78, 80, 85, 87, 110, 118, 125, 128, 132, 143, 146, 158, 160, 161, 163, 172, 179, 187, 191, 193, 197, 209, 217, 223, 236, 241, 242, 261, 266, 288, 296, 298, 304, 307, 309, 312, 314, 315, Deletion, insertion, substitution of another amino acid, or a combination thereof at one or more of positions 316, 319, 327, 335, 339, 342, and 380;

[0146] Here, the position number corresponds to the position of the polypeptide of sequence number 1.

[0147]

[0148] In the present application, aspartic protease refers to an enzyme belonging to a subgroup of proteases and possessing proteolytic activity. This enzyme may be classified under EC 3.4.23. This enzyme is a type of acidic protease with optimal activity at acidic pH. Specifically, aspartic protease is an enzyme that degrades proteins by hydrolyzing peptide bonds and essentially has an active aspartic acid residue at its active site.

[0149] Aspartic protease activity in the present application can be measured and evaluated using methods known in the art, including the embodiments described in the present application.

[0150]

[0151] In this application, the term "parent aspartic protease" refers to an aspartic protease that is modified to produce a mutant polypeptide or variant of the present application. Specifically, the parent aspartic protease, parent enzyme, or parent sequence may be a naturally occurring polypeptide or a wild-type polypeptide, may be a mature polypeptide thereof, may include a variant or functional fragment thereof, but is not limited to any polypeptide that has aspartic protease activity and can be the parent of the variant.

[0152] The parent aspartic protease provided in the present application may be, but is not limited to, a polypeptide having SEQ ID NO: 1. In addition, as long as it has aspartic protease activity, it may be a polypeptide having a sequence identity of at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% with the polypeptide having SEQ ID NO: 1, and as long as it has the same or corresponding activity as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, it may be included in the scope of the parent aspartic protease without limitation.

[0153] In the present application, the parent aspartic protease may include an amino acid sequence having sequence number 1 and a sequence identity of 90% or more therewith.

[0154] The parent aspartic protease of the mutant polypeptide provided in the present application may be derived from a microorganism of the genus Trichoderma, specifically, from Trichoderma reesei, but is not limited thereto.

[0155] Meanwhile, the above-mentioned microorganism is an example of a microorganism from which the parent aspartic acid protease provided in the present application can be derived, and includes a microorganism derived from a taxonomically homologous microorganism, regardless of the name of the microorganism.

[0156] In the present application, a sequence "derived from" a particular microorganism is not limited to a sequence that is naturally produced or can be produced in the microorganism, but also includes a sequence encoded by a gene that is produced and isolated from the microorganism containing the gene.

[0157] For example, an aspartic protease from the genus Trichoderma includes not only enzymes having aspartic protease activity that are naturally produced in the genus Trichoderma, but also those produced from Trichoderma sources, and those produced in other host cells through genetic modification known in the art (e.g., transformation with a sequence encoding the enzyme).

[0158]

[0159] In the present application, the “variant polypeptide having aspartic protease activity” may be a variant of the parent aspartic protease.

[0160] In the present application, the term "variant of parent aspartic protease" or "aspartic protease variant" refers to a protein having one or more amino acids different from the amino acid sequence of the parent aspartic protease and having the activity of aspartic protease.

[0161] The above “mutant polypeptide having aspartic acid protease activity”, “mutant of parent aspartic acid protease” and “mutant aspartic acid protease” can be used interchangeably.

[0162] The variant polypeptide provided in the present application may have aspartic protease activity and include modifications of one or more amino acids in a parent aspartic protease sequence. In addition, the variant polypeptide is i) a polypeptide having a sequence identity of at least 70% and less than 100% with SEQ ID NO: 1; and / or ii) the variant polypeptide is a polypeptide encoded by a polynucleotide having a sequence identity of at least 70% and less than 100% with a sequence encoding a mature polypeptide of SEQ ID NO: 1; And / or iii) the variant polypeptide is a polypeptide encoded by (a) a mature polypeptide coding sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% sequence identity thereto, (b) a cDNA thereof, or (c) a polynucleotide that hybridizes to the full-length complement of (a) or (b) under low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions; and / or iv) the variant polypeptide may be a functional fragment of i), ii) or ii) polypeptide having aspartic protease activity.

[0163] Specifically, the variant polypeptide provided in the present application may have aspartic protease activity, and may have one or more altered functions or properties compared to the parent aspartic protease, including modification of one or more amino acids in the parent aspartic protease sequence.

[0164] In one specific example, the variant polypeptide provided in the present application has aspartic protease activity, and may have one or more altered functions or properties compared to the parent aspartic protease, including modification of one or more amino acids in the parent aspartic protease sequence, and may have one or more conservative substitutions.

[0165]

[0166] The mutant polypeptide provided in the present application may be a mutant of a parent aspartic protease and may be a polypeptide having aspartic protease activity.

[0167] In one specific example, the mutant polypeptide provided in the present application is selected from the group consisting of SEQ ID NO: 65, 66, 77, 78, 80, 85, 87, 110, 118, 125, 128, 132, 143, 146, 158, 160, 161, 163, 172, 179, 187, 191, 193, 197, 209, 217, 223, 236, 241, 242, 261, 266, 288, 296, 298, 304, 307, 309, It may include modifications at one or more positions corresponding to positions 312, 314, 315, 316, 319, 327, 335, 339, 342 and 380.

[0168] 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.

[0169] The variant polypeptides provided in this application include all possible combinations of the above-described modifications.

[0170]

[0171] As a specific example, the mutant polypeptide provided in the present application is a mutant polypeptide having aspartic acid protease activity, which is at least one of the following i) to iii):

[0172] i) a polypeptide having the amino acid sequence of sequence number 1 or a sequence identity of at least 90% thereto;

[0173] ii) a polypeptide encoded by a polynucleotide encoding a mature polypeptide having the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% sequence identity thereto;

[0174] iii) a polypeptide encoded by (a) a mature polypeptide coding sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% sequence identity thereto, (b) a cDNA thereof, or (c) a polynucleotide that hybridizes to the full-length complement of (a) or (b) under low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions;

[0175] It may be a mutant polypeptide comprising a substitution of an amino acid at one or more positions selected from the following:

[0176] 65, 66, 77, 78, 80, 85, 87, 110, 118, 125, 128, 132, 143, 146, 158, 160, 161, 163, 172, 179, 187, 191, 193, 197, 209, 217, 223, 236, 241, 242, 261, 266, 288, 296, 298, 304, 307, 309, 312, 314, 315, 316, 319, 327, 335, 339, 342 and 380 (wherein the position numbers correspond to the positions of the polypeptide of SEQ ID NO: 1).

[0177] In one specific example, the amino acids before modification of the variant polypeptide provided in the present application may be any one or more selected from the following: amino acids 65, 160, 288, 296 and 304 are asparagine (N); amino acids 66 and 197 are histidine (H); amino acids 77, 118, 161, 172 and 339 are threonine (T); amino acids 78, 80, 110, 132, 143, 158, 209, 261, 319 and 327 are serine (S); amino acids 85, 187, 217 and 242 are alanine (A); Amino acids 87, 128, 179, 236, 241, and 309 are valine (V); amino acids 163 and 191 are glycine (G); amino acids 193, 223, and 298 are glutamine (Q); amino acids 307, 315, 342, and 380 are glutamic acid (E); amino acids 146, 312, 314, and 316 are aspartic acid (D); amino acid 266 is lysine (K); and amino acid 335 is leucine (L).

[0178] In one specific example, the variant polypeptide of the present application may comprise an amino acid substitution at any one or more positions selected from the following: 65; 66; 77; 78; 80; 85; 87; 110; 118; 125; 128; 132; 143; 146; 158; 160; 161; 163; 172; 179; 187; 191; 193; 197; 209; 217; 223; 236; 241; 242; 261; 266; 288; 296; 298; 304; 307; 309; 312; 314; 315; 316; 319; 327; 335; 339; 342 and 380 (wherein the position numbers correspond to the positions of the polypeptide of SEQ ID NO: 1).

[0179] In one specific example of the above-described specific examples, the mutant polypeptide of the present application may include one or more amino acid substitutions selected from the following: substitution of amino acid 65 with alanine; substitution of amino acid 66 with tryptophan; substitution of amino acid 77 with valine; substitution of amino acid 78 with alanine; substitution of amino acid 80 with glutamine; substitution of amino acid 85 with proline; substitution of amino acid 87 with threonine; substitution of amino acid 110 with glutamine; substitution of amino acid 118 with aspartic acid; substitution of amino acid 125 with alanine; substitution of amino acid 128 with leucine; substitution of amino acid 132 with threonine; substitution of amino acid 143 with alanine; substitution of amino acid 146 with threonine; substitution of amino acid 158 with threonine; Substitution of amino acid 160 with threonine; substitution of amino acid 161 with asparagine; substitution of amino acid 163 with alanine; substitution of amino acid 172 with aspartic acid; substitution of amino acid 179 with asparagine; substitution of amino acid 187 with glycine; substitution of amino acid 191 with leucine; substitution of amino acid 193 with threonine; substitution of amino acid 197 with glutamine; substitution of amino acid 209 with glutamine; substitution of amino acid 217 with valine; substitution of amino acid 223 with lysine; substitution of amino acid 236 with lysine; substitution of amino acid 241 with isoleucine; substitution of amino acid 242 with cysteine; substitution of amino acid 261 with alanine; substitution of amino acid 266 with serine; Substitution of amino acid 288 with aspartic acid; substitution of amino acid 296 with glutamine; substitution of amino acid 298 with proline; substitution of amino acid 304 with proline; substitution of amino acid 307 with glycine; substitution of amino acid 309 with tryptophan; substitution of amino acid 312 with proline; substitution of amino acid 314 with serine; substitution of amino acid 315 with serine;Substitution of amino acid 316 with serine; substitution of amino acid 319 with aspartic acid; substitution of amino acid 327 with cysteine; substitution of amino acid 335 with tyrosine; substitution of amino acid 339 with alanine; substitution of amino acid 342 with aspartic acid; and substitution of amino acid 380 with asparagine.

[0180] In one specific example of the above-described specific examples, the mutant polypeptide of the present application may include one or more amino acid substitutions selected from, but not limited to: N65A; H66W; T77V; S78A; S80Q; A85P; V87T; S110Q; T118D; S125A; V128L; S132T; S143A; D146T; S158T; N160T; T161N; G163A; T172D; V179N; A187G; G191L; Q193T; H197Q; S209Q; A217V; Q223K; V236K; V241I; A242C; S261A; K266S; N288D; N296Q; Q298P; N304P; E307G; V309W; D312P; D314S; E315S; D316S; S319D; S327C; L335Y; T339A; E342D; and E380N.

[0181] In one specific example, the variant polypeptide of the present application may include, but is not limited to, a substitution of an amino acid at any of the following positions with another amino acid:

[0182] 65, 66, 77, 78, 80, 85, 87, 110, 118, 125, 128, 132, 143, 146, 158, 160, 161, 163, 172, 179, 187, 191, 193, 197, 209, 217, 223, 236, 241, 242, 261, 266, 288, 296, 298, 304, 307, 309, 312, 314, 315, 316, 319, 327, 335, 339, 342 and 380 (wherein the position numbers correspond to the positions of the polypeptide of SEQ ID NO: 1).

[0183]

[0184] In one specific example, the variant polypeptide of the present application may comprise, consist of, consist essentially of, or have an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0185] As a specific example, in the present application, in SEQ ID NO: 1, N65A; H66W; T77V; S78A; S80Q; A85P; V87T; S110Q; T118D; S125A; V128L; S132T; S143A; D146T; S158T; N160T; T161N; G163A; T172D; V179N; A187G; G191L; Q193T; H197Q; S209Q; A217V; Q223K; V236K; V241I; A242C; S261A; K266S; N288D; N296Q; Q298P; N304P; E307G; V309W; D312P; A variant polypeptide comprising all of the D314S; E315S; D316S; S319D; S327C; L335Y; T339A; E342D; and E380N substitutions may be represented by SEQ ID NO: 2. A variant polypeptide comprising all of the 48 substitutions may be referred to as “48-fold variant,” “48-fold multiple variant,” “48-fold substitution aspartic protease variant,” or “48-fold multiple substitution aspartic protease variant” in the present application.

[0186]

[0187] In one specific example, the variant polypeptide of the present application may be a variant polypeptide further comprising a substitution of an amino acid at any one or more positions selected from the following: positions 71, 339, 163 and 199; positions 71, 339, 110 and 175; positions 163, 199, 110 and 175; positions 71, 339, 163, 199, 110 and 175; positions 163 and 199; positions 71 and 339; and positions 110 and 175 (wherein the position numbers correspond to positions of the polypeptide of SEQ ID NO: 1).

[0188] In one specific example, the amino acids before modification of the mutant polypeptide may be any one or more selected from the following: amino acids 71, 163, and 339 are alanine; amino acids 110 and 199 are glutamine; and amino acid 175 is valine.

[0189] In one specific example, amino acids 71, 339, 163 and 199; 71, 339, 110 and 175; 163, 199, 110 and 175; 71, 339, 163, 199, 110 and 175; 163 and 199; 71 and 339; and 110 and 175 may comprise substitutions with hydrophilic amino acids, specifically cysteine.

[0190] In one specific example, the variant polypeptide may further comprise a substitution of an amino acid at one or more positions selected from the following:

[0191] Substitution of amino acids 71, 339, 163 and 199 with cysteine; substitution of amino acids 71, 339, 110 and 175 with cysteine; substitution of amino acids 163, 199, 110 and 175 with cysteine; substitution of amino acids 71, 339, 163, 199, 110 and 175 with cysteine; substitution of amino acids 163 and 199 with cysteine; substitution of amino acids 71 ​​and 339 with cysteine; and substitution of amino acids 110 and 175 with cysteine ​​(wherein the position numbers correspond to the positions of the polypeptide of SEQ ID NO: 1).

[0192] In one specific example of the above-described specific example, the mutant polypeptide may include one or more amino acid substitutions selected from, but not limited to:

[0193]

[0194] In one specific example, in the variant polypeptide of SEQ ID NO: 2 in the present application, the variant polypeptide further comprising the A71C+A339C+A163C+Q199C substitution is SEQ ID NO: 3; the variant polypeptide further comprising the A71C+A339C+Q110C+V175C substitution is SEQ ID NO: 4; the variant polypeptide further comprising the A163C+Q199C+Q110C+V175C substitution is SEQ ID NO: 5; the variant polypeptide further comprising the A71C+A339C+A163C+Q199C+Q110C+V175C substitution is SEQ ID NO: 6; the variant polypeptide further comprising the A163C+Q199C substitution is SEQ ID NO: 7; the variant polypeptide further comprising the A71C+A339C substitution is SEQ ID NO: 8; And a variant polypeptide further comprising the Q110C+V175C substitution may be represented by the amino acid sequence of SEQ ID NO: 9. These variant polypeptides may comprise, consist of, consist essentially of, or have a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 1 or 2 while including the above-described substitutions.

[0195]

[0196] In one specific example, the variant polypeptide provided in the present application may have a sequence identity of at least about 60%, for example, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% and less than 100% to a parent aspartic protease; its mature polypeptide or a functional fragment thereof.

[0197] In one specific example, the variant polypeptide provided in the present application may have a sequence identity of at least about 60%, for example, at least 65%, at least 70%, at least 75%, 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 polypeptide having the sequence of SEQ ID NO: 1.

[0198] In one specific example, the variant polypeptide provided in the present application may be a polypeptide encoded by a polynucleotide having a sequence identity of at least about 60%, for example, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% and less than 100%, with a sequence encoding a polypeptide of the sequence of SEQ ID NO: 1.

[0199] In one specific example, the variant polypeptide provided in the present application may have a sequence identity of at least about 60%, for example, at least 65%, at least 70%, at least 75%, 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 a functional fragment of a polypeptide having the sequence of SEQ ID NO: 1.

[0200]

[0201] The mutant polypeptides provided in the present application may have one or more altered characteristics or properties of the polypeptide that can be selected or detected as compared to other aspartic proteases, such as wild-type aspartic proteases, parent aspartic proteases, other aspartic protease mutant polypeptides, etc.

[0202] The above properties or attributes include, but are not limited to, oxidative stability, substrate specificity, catalytic activity, thermal stability, alkaline stability, pH activity profile, resistance to proteolysis, Km, kcat, kcat / Km ratio, protein folding, induction of an immune response, ability to bind a ligand, ability to bind a receptor, ability to be secreted, ability to be displayed on the surface of cells, ability to form oligomers, ability to signal, 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 a disease.

[0203]

[0204] Specifically, the variant polypeptides provided in the present application may have one or more of the following altered activities compared to the parent sequence:

[0205] i) Increase or decrease in enzyme activity;

[0206] ii) increase or decrease in specific activity; and

[0207] iii) Increase or decrease in heat resistance or heat stability; but is not limited thereto.

[0208]

[0209] In the present application, "enzymatic activity" refers to at least one catalytic activity. Specifically, it may be, but is not limited to, the conversion efficiency of an enzyme, which is mainly expressed as kcat / Km.

[0210] kcat is the catalytic constant for the conversion of a single enzyme into a product per unit time when the enzyme is completely saturated with substrate, and is also called the turnover number. Km is the substrate concentration when the reaction rate is half of the maximum value (Vmax).

[0211] Examples of ways to express enzyme activity include 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).

[0212] However, defining enzyme activity is not limited to the above-mentioned content, and can be defined and evaluated based on known content 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 ef a / ., "Biochemie" [Biochemistry], 1992, VCH-Verlag, Chapter 13, pages 331-332 with respect to enzymatic activity.

[0213]

[0214] In one specific example, the variant polypeptide provided in the present application may have an increased enzymatic activity of about 101%, or about 110% or more compared to the parent enzyme.

[0215] In another specific embodiment, the variant polypeptide provided in the present application may have a reduced enzymatic activity of about 99%, about 95%, or about 90% or less compared to the parent enzyme.

[0216]

[0217] The term "specific activity" in this application refers to the activity of an enzyme per unit weight of protein, expressed as unit / mg. Protein quantification can be performed using, for example, SDS-PAGE or the Bradford assay.

[0218]

[0219] Enzyme stability refers to the preservation of enzyme activity during storage or reaction time. To measure changes in this stability, the initial enzyme activity is measured at time zero (100%) and after a specified time (x%) under defined conditions. This comparison allows for the expression of enzyme stability, or the level at which enzyme activity is lost.

[0220] Factors that affect enzyme activity include, for example, pH, heat, and the presence of other substances (e.g., oxidizing agents, chelating agents).

[0221]

[0222] In this application, the term "thermal stability" means the ability of a protein to function over a specific temperature range. In one specific example, the mutant polypeptide provided in the present application may have activity in the range of about 20°C to about 120°C, and more specifically, in the range of about 30°C to 100°C, 30°C to 90°C, 30°C to 80°C, 30°C to 75°C, 30°C to 70°C, 30°C to 65°C, 40°C to 100°C, 40°C to 90°C, 40°C to 80°C, 40°C to 75°C, 40°C to 70°C, 40°C to 65°C, 50°C to 100°C, 50°C to 90°C, 50°C to 80°C, 50°C to 75°C, 50°C to 70°C, 50°C to 65°C, 55°C to 100°C, 55°C to It may be active in the range of 90°C, 55°C to 80°C, 55°C to 75°C, 55°C to 70°C, or 55°C to 65°C, but is not limited thereto.

[0223]

[0224] As used herein, the term "thermal tolerance" refers to the ability of a protein to function after exposure to a specific temperature, such as high or low temperatures. For example, a protein that is thermostable may not function at the temperature to which it is exposed, but may regain function when returned to its optimal temperature environment.

[0225]

[0226] Increased stability includes maintaining high enzymatic activity compared to other enzymes, e.g., wild-type enzymes, parent enzymes, and / or other mutant polypeptides; increasing the range of pH, temperature, and / or time over which the protein remains functional.

[0227] Decreased stability includes lower retention of enzyme activity compared to other enzymes, e.g., wild-type enzymes, parent enzymes, and / or other mutant polypeptides; a decrease in the range of pH, temperature, and / or time over which the protein remains functional.

[0228]

[0229] The altered properties of the mutant polypeptides provided in the present application may be activities, improved activities, suitable for application in various compositions including foods, feeds, pharmaceuticals, and detergents.

[0230]

[0231] A polynucleotide encoding a variant polypeptide of the present invention may comprise the coding sequence of the variant polypeptide described above. The polynucleotide may undergo various modifications to the coding region within a range that does not alter the amino acid sequence of the polypeptide, due to codon degeneracy or in consideration of codons preferred by the organism intended to express the polypeptide.

[0232] In addition, the polynucleotide of the present application may include, without limitation, a probe that can be prepared from a known gene sequence, for example, a sequence that hybridizes under stringent conditions with a complementary sequence to all or part of the base sequence, so long as it encodes a variant polypeptide of the present application.

[0233] The above "stringent conditions" refer to conditions that allow 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).

[0234] For example, conditions under which polynucleotides having a high degree of homology or identity hybridize with each other, specifically at least 40%, specifically at least 90%, more specifically at least 95%, at least 96%, at least 97%, at least 98%, and even more specifically at least 99%, and polynucleotides having a lower degree of homology or identity than that hybridize, or conditions under which washing is performed once, specifically twice or three times, at a salt concentration and temperature equivalent to 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, and more specifically 68°C, 0.1×SSC, 0.1% SDS, which are washing conditions of typical southern hybridization, can be listed.

[0235] Hybridization requires that two nucleic acids have complementary sequences, although mismatches between bases are possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of the present application may also include isolated nucleic acid fragments that are complementary in their entirety, as well as substantially similar nucleic acid sequences.

[0236] 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. In addition, the Tm value may be, but is not limited to, 60°C, 63°C, or 65°C and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0237] The appropriate stringency for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides, variables well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

[0238] For example, “high stringency” may occur at about 5 to 10°C below the Tm of the probe; “medium stringency” may occur at about 10 to 20°C below the Tm of the probe; and “low stringency” may occur at about 20 to 25°C below the Tm, but is not limited thereto.

[0239] For example, “low stringency conditions” can be prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms / ml of sheared and denatured salmon sperm DNA, and 25% formamide for 12-24 hours, for a probe of at least 100 nucleotides in length, according to standard Southern blotting procedures. The carrier material can be finally washed two to three times for 15 minutes each with 2 X SSC, 0.1 to 0.2% SDS at 50°C.

[0240] For example, “medium stringency conditions” may be prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms / ml of sheared and denatured salmon sperm DNA, and 35% formamide for 12-24 hours, for a probe of at least 100 nucleotides in length, according to standard Southern blotting procedures. The carrier material can be finally washed two to three times for 15 minutes each at 55°C with 2 X SSC, 0.1 to 0.2% SDS. As an example, “medium-high stringency conditions” can be prehybridization and hybridization at 42°C in 5 X SSPE, 0.3% SDS, 200 micrograms / ml of sheared and denatured salmon sperm DNA and 35% formamide for a probe of at least 100 nucleotides in length for 12-24 hours according to standard Southern blotting procedures. The carrier material can be finally washed two to three times for 15 minutes each at 60°C with 1 to 2 X SSC, 0.1 to 0.2% SDS.

[0241] For example, “high stringency conditions” can be prehybridization and hybridization at 42°C in 5 X SSPE, 0.3% SDS, 200 micrograms / ml of sheared and denatured salmon sperm DNA, and 35% formamide for 12-24 hours, for a probe of at least 100 nucleotides in length, according to standard Southern blotting procedures. The carrier material can be finally washed two to three times for 15 minutes each with 2 X SSC, 0.1 to 0.2% SDS at 65°C.

[0242]

[0243] In one specific example, the polynucleotide sequence encoding the variant polypeptide of the present application may comprise, consist of, consist essentially of, or have a polynucleotide sequence of SEQ ID NO: 11 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or may be a degenerate sequence thereof, but is not limited thereto.

[0244] As a specific example, the sequence encoding the amino acid sequence of SEQ ID NO: 2 of the present application may be, but is not limited to, the polynucleotide sequence of SEQ ID NO: 11. Furthermore, it is obvious that a polynucleotide sequence encoding the variant polypeptide of the present application can be obtained based on the technical common sense of a person skilled in the art.

[0245]

[0246] Another aspect of the present application provides a nucleic acid construct comprising a polynucleotide of the present application.

[0247] Another aspect of the present application provides a vector comprising a polynucleotide of the present application or a nucleic acid construct of the present application.

[0248] The above polynucleotide is as described in other aspects of the present application.

[0249] The nucleic acid construct provided in the present application comprises a polynucleotide encoding a variant polypeptide provided in the present application, operably linked to one or more regulatory sequences that direct expression of the coding sequence in a suitable host cell under conditions suitable for the regulatory sequences.

[0250] Polynucleotides can be manipulated in a variety of ways to enable the expression of variant polypeptides. Depending on the expression vector, it may be desirable or necessary to manipulate the polynucleotide before inserting it into the vector. Such manipulations can be performed using methods known in the art.

[0251]

[0252] The "vector" provided in the present application refers to a DNA construct containing a base sequence of a polynucleotide encoding a variant polypeptide of the present application, operably linked to a suitable expression control region (or expression control sequence) so as to enable expression of the variant polypeptide of the present application in a suitable host. The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding 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 can replicate or function independently of the host genome, and can be integrated into the genome itself.

[0253] The vector that can be used in the present application is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series can be used as plasmid vectors. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors can be used.

[0254] For example, a polynucleotide encoding a variant polypeptide provided in the present application can be inserted into a chromosome via a vector for intracellular chromosomal insertion. The insertion of the polynucleotide into the chromosome can be achieved by any method known in the art, for example, homologous recombination, but is not limited thereto. A selection marker for confirming the chromosomal insertion can be additionally included. The selection marker is used to select cells transformed with the vector, i.e., to confirm the insertion of the target nucleic acid molecule, and markers that confer a selectable phenotype, such as drug resistance, nutrient requirement, cytotoxic agent resistance, or expression of a surface polypeptide, can be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypic traits, so that transformed cells can be selected.

[0255]

[0256] Another aspect of the present application provides a host cell comprising at least one of the variant polypeptide of the present application, the polynucleotide of the present application, the nucleic acid construct of the present application, and the vector of the present application.

[0257] The above mutant polypeptides, polynucleotides, nucleic acid structures, and vectors are as described in other aspects.

[0258] The host cell of the present application may include, without limitation, any cell capable of expressing the mutant polypeptide of the present application.

[0259] The host cell of the present application may comprise the above-described mutant polypeptide, a polynucleotide encoding the mutant polypeptide, a nucleic acid construct comprising the same, and / or a vector.

[0260] The nucleic acid construct or vector may be integrated into a chromosome as described above, or may be maintained as an extrachromosomal vector that replicates autonomously.

[0261] The host cell of the present invention includes any progeny of the parent cell that are not identical to the parent cell due to mutations that occur during replication.

[0262] The host cell can be any cell useful for the recombinant production of a variant polypeptide, e.g., a prokaryotic or eukaryotic cell.

[0263] The prokaryotic host cell can be any gram-positive or gram-negative bacterium.

[0264] Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces.

[0265] Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Iliobacter, Neisseria, Pseudomonas, Salmonella, Vibrio (e.g., Vibrio natriegens), and Ureaplasma.

[0266] In one specific example, the bacterial host cell can be a Bacillus genus host cell, 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.

[0267] In one specific example, the bacterial host cell can be a Streptococcus genus host cell, specifically including but not limited to Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis and Streptococcus equi subspecies Zooepidemicus cells.

[0268] In one specific example, the bacterial host cell can be a host cell of the genus Streptomyces, specifically including but not limited to Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicol, Streptomyces griseus, and Streptomyces lividans cells.

[0269] In one specific example, the bacterial host cell may be a host cell of the genus Corynebacterium, such as Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes. ammoniagenes), Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens.

[0270] The host cell may be a eukaryote, such as a mammalian, insect, plant, or fungal cell.

[0271] The host cell may be a fungal cell. In the present application, "fungus" includes the Ascomycota, Basidiomycota, Fasciomycota, and Zygomycota, as well as the Oomycota and all imperfect fungi.

[0272] The fungal host cell may be a yeast cell. The term "yeast" in the present application includes yeasts belonging to the order ascosporogenous yeasts (Endomycetales), basidiosporogenous yeasts, and Fungi imperfecti (Blastomycetes). However, this classification may vary and may be defined according to the Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0273] Yeast host cells include Candida, Hansenula, Kluyveromyces, Pichia, Komagataella, Saccharomyces, Schizosaccharomyces or Yarrowia cells, for example, Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces These may be cells of Saccharomyces norbensis, Saccharomyces oviformis, Komagataella phaffii or Yarrowia lipolytica.

[0274] The fungal host cell may be a filamentous fungal cell. "Filamentous fungi" include all filamentous forms of the phylum Eumycota and the subphylum Oomycota (as defined in the above reference (Hawksworth et al., 1995)). Filamentous fungi are typically characterized by a hyphal wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation, and carbonation is strictly aerobic. In contrast, vegetative growth in yeasts, such as Saccharomyces cerevisiae, is by germination of a unicellular thallus, and carbonation may be fermentative.

[0275] The filamentous fungal host cells are Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Mycellioptora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, It may be a cell of Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes or Trichoderma.

[0276] For example, the 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, Ceriporiopsis 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, Mysellioptora 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 a cell of Trichoderma reesei or Trichoderma viride, but is not limited thereto.

[0277]

[0278] Another aspect of the present application provides a composition comprising a variant polypeptide of the present application.

[0279] Another aspect of the present application provides a mutant polypeptide of the present application for reaction with a protein or a reaction composition comprising the mutant polypeptide.

[0280] The above mutant polypeptide is as described in other aspects.

[0281] The composition of the present application (including both the composition and the composition for reaction; the same applies hereinafter) can be used to break down proteins into polypeptides or amino acids.

[0282] The composition of the present application may further comprise other components in addition to the mutant polypeptide provided in the present application. Those skilled in the art can appropriately select the components to be added to the composition of the present application.

[0283] In one specific example, the composition of the present application may further comprise any component suitable for use in decomposing a protein into polypeptides or amino acids.

[0284] In one specific example, the composition of the present application may further comprise any component suitable for application to various compositions including foods, feeds, detergents, pharmaceuticals, paper and cosmetics (e.g., detergents).

[0285] 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, compounding agents, lubricants, disintegrants, excipients, solubilizers, suspending agents, colorants, flavorings, buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, diluents, lubricants, preservatives, and the like.

[0286] In one specific example, the composition provided in the present application may further include a naturally occurring substance or a non-naturally occurring substance in addition to the mutant polypeptide provided in the present application.

[0287] In one specific example, the composition provided in the present application may further comprise, in addition to the mutant polypeptide provided in the present application, an additional enzyme used in various compositions including foods, feeds, pharmaceuticals, paper, and cosmetics (e.g., detergents).

[0288] For example, the additional enzyme may further comprise one or more enzymes selected from the group consisting of beta-amylase, cellulase (beta-glucosidase, cellobiohydrolase and endoglucanase), glucoamylase, hemicellulase (e.g., xylanase), isoamylase, isomerase, lipase, phytase, protease, pullulanase and / or alpha-amylase, together with other enzymes useful in commercial processes.

[0289]

[0290] Another aspect of the present application provides a feed composition comprising at least one of the variant polypeptides of the present application and a host cell expressing the variant polypeptide.

[0291] The mutant polypeptide included in the feed composition of the present application may be, but is not limited to, a form that includes the host cell expressing it itself or is purified and separated from the host cell expressing it.

[0292] The "feed composition" of the present application is any natural or artificial diet, meal, etc. or a component of the meal for eating, ingesting, and digesting by an animal or suitable therefor, and the feed can be manufactured into various types of feed known in the art.

[0293] The above feed composition may be a feed additive.

[0294] The type of the above feed is not particularly limited, and feed commonly used in the relevant technical field can be used. Non-limiting examples of the above feed include plant feed such as grains, roots, food processing by-products, algae, fiber, pharmaceutical by-products, oils, starches, meal, or grain by-products; and animal feed such as proteins, inorganic substances, oils, minerals, oils, single-cell proteins, zooplankton, or food. These may be used alone or in combination of two or more.

[0295] The feed composition of the present application may further include one or more selected from organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, and malic acid; phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, and polyphosphate (polyphosphate); and natural antioxidants such as polyphenol, catechin, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, and phytic acid.

[0296] The feed composition of the present application may further include one or more selected from the following: auxiliary ingredients such as amino acids, inorganic salts, vitamins, antibiotics, antimicrobial substances, antioxidants, antifungal enzymes and other microbial preparations in the form of live bacteria; grains such as ground or crushed wheat, oats, barley, corn and rice; plant protein feeds such as those mainly composed of rapeseed, soybeans and sunflower; animal protein feeds such as blood meal, meat meal, bone meal and fish meal; dry ingredients composed of sugars and dairy products such as various types of milk powder and whey powder; lipids such as animal fats and plant fats optionally liquefied by heating; and additives such as nutritional supplements, digestion and absorption enhancers, growth promoters and disease preventive agents.

[0297] The feed composition of the present application may be in the form of a dry or liquid formulation and may include an excipient for feeding. Examples of such excipients for feeding include, but are not limited to, zeolite, corn meal, or rice bran.

[0298] The above-mentioned feed composition may further include an enzyme preparation other than the mutant polypeptide of the present application. For example, the feed composition may further include one or more selected from, but is not limited to, a fat-decomposing enzyme such as lipase, a phytase that breaks down phytic acid to produce phosphate and inositol phosphate, an amylase that hydrolyzes α-1,4-glycoside bonds contained in starch and glycogen, a phosphatase that hydrolyzes organic phosphate esters, a maltase that hydrolyzes maltose into two molecules of glucose, and a converting enzyme that hydrolyzes saccharose to produce a glucose-fructose mixture.

[0299] The feed composition of the present invention can be administered to animals alone or in combination with other feed additives in an edible carrier. Furthermore, the feed composition can be readily administered as a feed additive, as a top dressing, or by mixing it directly into livestock feed, or separately from the feed, in a separate oral formulation, or in combination with other ingredients. Furthermore, as is commonly known in the art, a single daily intake or divided daily intake can be used.

[0300] Animals for which the feed composition of the present application can be used include, but are not limited to, livestock such as edible cattle, dairy cows, calves, pigs, piglets, sheep, goats, horses, rabbits, dogs, cats, etc., and poultry such as chicks, egg hens, domestic chickens, roosters, ducks, geese, turkeys, quail, small birds, etc.

[0301] The amount of the mutant polypeptide of the present application included in the feed composition is not particularly limited and can be appropriately adjusted depending on the intended purpose. In one embodiment, as is commonly known in the technical field to which the present application pertains, the mutant polypeptide may be included in an amount suitable for long-term survival in the digestive tract of livestock and for decomposing protein sources, but is not limited thereto.

[0302]

[0303] Another aspect of the present application provides a food composition comprising at least one of a variant polypeptide of the present application and a host cell expressing the variant polypeptide.

[0304] The above protease protein may be used in a food composition in liquid or solid form. Furthermore, the food may be a powder, pill, beverage, tea, or an additive to general food.

[0305] The above foods may be food groups that require protease, such as dairy products, health functional foods for bowel movements or dieting, and functional health functional foods for preventing high blood pressure.

[0306] The mutant polypeptide having aspartic acid protease activity of the present invention can be incorporated into various food compositions and used as a food solubilizer or tenderizer, or a meat quality modifier. In other embodiments, it can be added to a baking process and used in a step to break down the gluten network. Alternatively, it can be used to hydrolyze food proteins (e.g., proteins in milk). Alternatively, it can be incorporated into various food compositions and used for purposes such as rendering, flavoring, reducing bitterness, altering emulsifying properties, producing bioactive peptides, and reducing protein allergens. However, these are merely examples and are not limited to the aforementioned uses.

[0307] The amount of the mutant polypeptide in the above food composition can be appropriately adjusted by a person skilled in the art depending on the purpose.

[0308]

[0309] Another aspect of the present application provides a detergent composition comprising at least one of a variant polypeptide of the present application and a host cell expressing the variant polypeptide.

[0310] The detergent composition according to the present application may be in the form of a one-part or two-part aqueous detergent composition, a non-aqueous liquid detergent composition, a cast solid, a granule, a particle, a compressed tablet, a gel, a paste, or a slurry. The detergent composition may be used to remove food stains, food residues, and other small amounts of food compositions.

[0311] The detergent composition according to the present application may be provided in the form of a detergent composition for cleaning hard surfaces, a detergent composition for cleaning fabrics, a detergent composition for washing dishes, a detergent composition for cleaning mouths, a detergent for cleaning dentures, or a contact lens cleaning solution, but is not limited thereto.

[0312]

[0313] Another aspect of the present application provides a pharmaceutical composition comprising at least one of a variant polypeptide of the present application and a host cell expressing the variant polypeptide.

[0314] The pharmaceutical composition of the present application includes a pharmaceutical composition for digestive enzymes for improving digestive diseases, digestive disorders, and abnormal diseases after digestive surgery, a thrombolytic or antithrombotic composition that acts directly on blood clots to dissolve fibrin, an anti-inflammatory agent that acts as an in vivo defense system to remove inflammatory substances or necrotic tissues, or an anti-inflammatory agent for reducing swelling after surgery or trauma.

[0315] The pharmaceutical composition may further comprise a pharmaceutically acceptable or nutritionally acceptable carrier, excipient, diluent or auxiliary ingredient depending on the method of use and the intended use. The carrier, excipient or diluent may be at least one selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin and saline solution, but is not limited thereto.

[0316] In addition to the aforementioned uses, the mutant polypeptide of the present invention or a host cell expressing the mutant polypeptide may be used for purposes such as manufacturing cosmetics, leather processing, pharmaceuticals, diagnostic agents, waste management, and chemicals for academic research. However, the aforementioned uses are merely examples, and the invention may be used for any other purpose known in the art related to the denaturation, degradation, or removal of proteinaceous substances.

[0317]

[0318] Another aspect of the present application provides a method for preparing a protein hydrolyzate, comprising contacting a substrate with at least one of a variant polypeptide of the present application and a host cell expressing the variant polypeptide.

[0319] Another aspect of the present application provides a method of degrading a protein, comprising contacting a substrate with at least one of a variant polypeptide of the present application and a host cell expressing the variant polypeptide.

[0320] The above substrate may include a protein (e.g., a protein-containing material), but is not limited to a protein.

[0321] The term “protein-containing material” in the present application includes any material that can be decomposed by a protease, and any material that can be decomposed by a protease is included without limitation in type.

[0322] The term “protein degradation” in this application means a chemical reaction that hydrolyzes peptide bonds within a protein molecule to produce a mixture of amino acids or peptides, and may be used interchangeably with “protein denaturation” or “protein removal.”

[0323] The above mutant polypeptide and host cell, etc. are as described in other aspects.

[0324]

[0325] Another aspect of the present application provides a method for producing a variant polypeptide having aspartic protease activity, comprising the step of culturing a host cell expressing the variant polypeptide of the present application.

[0326] The above mutant polypeptide, host cell, and aspartic protease are as described in other aspects.

[0327] The method for producing a mutant polypeptide of the present application may include a step of culturing a host cell; and a step of recovering a mutant polypeptide expressed in the culturing step.

[0328] In this application, the term "cultivation" refers to growing the host cells under appropriately controlled environmental conditions. The culturing process of this application can be performed using appropriate media and culture conditions known in the art. This culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing process may be batch, continuous, or fed-batch, but is not limited thereto.

[0329] In this application, the term "medium" refers to a material containing nutrients as a main component necessary for culturing the host cells, and supplies nutrients and growth factors, including water, which is essential for survival and development. Specifically, the medium and other culture conditions used for culturing the host cells of the present application may be any medium used for culturing conventional host cells without particular limitation, but the host cells of the present application may be cultured under aerobic conditions while controlling temperature, pH, etc. in a conventional medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid, and / or vitamin.

[0330] 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 pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane bagasse, and corn steep liquor may be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugar) may be used, and other appropriate amounts of carbon sources may be used in various ways without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0331] The nitrogen source may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; 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 liquor, casein hydrolysate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0332] The above-mentioned components may include potassium phosphate monobasic, potassium phosphate dibasic, or their corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins, and / or suitable precursors may be included. These components or precursors may be added to the medium in batch or continuous manner, but are not limited thereto.

[0333] In addition, during the cultivation of the host cells, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. can be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during the cultivation, foaming can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. In addition, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas can be injected into the medium, or in order to maintain the anaerobic and microaerobic state, nitrogen, hydrogen, or carbon dioxide gas can be injected without injecting gas, but is not limited thereto.

[0334] The temperature of the medium may be, but is not limited to, 20°C to 55°C, specifically 25°C to 45°C. The incubation period may continue until the desired amount of useful material is produced, and specifically may be, but is not limited to, 10 hours to 196 hours.

[0335]

[0336] In one specific example, the method for producing a mutant polypeptide having aspartic protease activity of the present application may further include a step of recovering the mutant polypeptide having aspartic protease activity of the present application expressed in the culturing step.

[0337] In another specific embodiment, the mutant polypeptide expressed in the above-described culturing step can be recovered using methods known in the art. For example, the mutant polypeptide can be recovered from the nutrient medium by conventional procedures, including but not limited to collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.

[0338] The above recovery method may be to collect the mutant polypeptide using a suitable method known in the art according to the culture method of the host cell of the present application, for example, a batch, continuous or fed-batch culture method. For example, various chromatographies such as centrifugation, filtration, treatment with a crystallizing protein precipitant (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC and a combination of these methods may be used, and the mutant polypeptide may be recovered from the medium or host cell using a suitable method known in the art.

[0339] In another specific embodiment, the mutant polypeptide expressed by the host cell during the culture step may not be recovered. In this specific embodiment, the host cell itself expressing the mutant polypeptide may be used as a source of the mutant polypeptide.

[0340]

[0341] The mutant polypeptide of the present invention can be used to treat feed containing proteins. Such feed can be used by mixing one or more of organic acids such as citric acid, fumaric acid, adipic acid, and lactic acid for administration; phosphates such as potassium phosphate, sodium phosphate, and polymeric phosphate; and natural antioxidants such as polyphenols, catechins, tocopherols, vitamin C, green tea extract, chitosan, and tannic acid. Other conventional additives such as anti-influenza agents, buffers, and bacteriostatic agents can also be added as needed. Furthermore, diluents, dispersants, surfactants, binders, or lubricants can be additionally added to formulate the feed into injectable formulations such as aqueous solutions, suspensions, and emulsions, capsules, granules, or tablets. In addition, the above feed can be used with various auxiliary ingredients such as amino acids, minerals, vitamins, antioxidants, antifungals, antibacterial agents, etc., as auxiliary ingredients, and plant-based protein feed such as ground or crushed wheat, barley, corn, etc., animal-based protein feed such as blood meal, meat meal, fish meal, etc., animal fats and plant-based fats, in addition to the main ingredients, nutritional supplements, growth promoters, digestion and absorption promoters, and disease preventive agents. The treatment can provide mineral sufficiency for humans and livestock through decomposition in the digestive tract of single-animal animals, and prevent eutrophication and water pollution in rivers and lakes in densely populated areas.

[0342]

[0343] Another aspect of the present application provides a use for producing a feed composition, a food composition, or an industrial composition of the mutant polypeptide or a host cell expressing the mutant polypeptide.

[0344] The contents of the above mutant polypeptide, the host cell expressing the above mutant polypeptide, and the composition are as described in other aspects.

[0345]

[0346] The mutant polypeptide possessing aspartic acid protease activity of the present invention exhibits superior thermal stability compared to the wild-type polypeptide. Therefore, the mutant polypeptide of the present invention can be usefully applied in various industrial fields requiring thermal stability.

[0347]

[0348] Figure 1 is a diagram showing the results of thermal stability evaluation of aspartic protease (TrAsp) wild type (WT) and 48 mutants of aspartic protease at 60°C and 70°C.

[0349] Figure 2 is a diagram showing the results of thermal stability evaluation at 70°C of aspartic protease (TrAsp) wild type (WT), aspartic protease 48 mutants, aspartic protease 48 multiple mutations, and mutants introducing all disulfide bond mutations (DS1, DS2, DS3, DS4, DS5, DS6, and DS7).

[0350] Figure 3 is a diagram showing the results of thermal stability evaluation at 75°C of mutants (DS1, DS2, and DS3) that were confirmed to have excellent thermal stability among the wild type (WT) of aspartic protease (TrAsp) and mutants that introduced both multiple mutations and disulfide bond mutations among 48 aspartic proteases.

[0351] Figure 4 is a diagram showing the results of thermal stability evaluation at 80°C of mutants (DS1, DS2, and DS3) that were confirmed to have excellent thermal stability among the wild type (WT) of aspartic protease (TrAsp) and mutants that introduced both multiple mutations and disulfide bond mutations among 48 aspartic proteases.

[0352]

[0353] Hereinafter, this application will be described in more detail through examples and experimental examples. However, these examples and experimental examples are intended to exemplify this application and the scope of this application is not limited to these examples and experimental examples.

[0354]

[0355] <Experimental methods and materials: Template, host cells, and medium>

[0356] To construct recombinant mutant strains producing wild-type aspartic protease (TrAsp) derived from Trichoderma reesei and its mutants developed in the present application, a recombinant plasmid pPICZαA based on the E. coli-pichiashuttle vector was used. E. coli XL-10 strain was used as a host cell for gene cloning, and low salt LB (Luria Bertani) broth; 1% tryptone, 0.5% yeast extract, 0.5% sodium chloride, or solid medium supplemented with zeocin (final concentration 25 μg / mL) as an antibiotic was used for selection of transformants. Yeast Pichia pastoris X-33 strain (Invitrogen, USA) was used as a host cell for wild-type and mutant protein expression and secretion. The pPICZαA plasmid, in which the coding polynucleotide of wild-type aspartic protease (TrAsp) or its mutant was cloned, was linearized by digestion with the restriction enzyme pmeI and integrated into the genome AOXI region of the yeast P. pastoris X-33 host cell by electroporation. The transformants were selected on YPDS (1% yeast extract, 2% peptone, 2% dextrose, 1 M sorbitol, 2% agar) solid medium containing zeocin (100 μg / mL) and incubated with 20 mL of BMMY (1% yeast extract, 2% peptone, 100 mM potassium phosphate pH 6.0, 1.34% YNB, 4X 10 -5 After induction by adding % biotin, 2% (v / v) methanol, expression was performed at 28°C, 250 rpm, for 72 hours.

[0357] When using the pPICZαA expression plasmid, the aspartic acid protease of the present application is expressed in the form of a fusion protein with a peptide encoding the secretion signal of Saccharomyces cerevisiaeα-factor, thereby improving the secretion of the target protein and facilitating its recovery.

[0358]

[0359] Example 1. Construction of plasmid vectors for producing 48 mutants

[0360] In order to diversify the sequence of aspartic protease and improve its thermal stability, we selected mutation introduction sites for the wild-type aspartic protease (SEQ ID NO: 1) derived from Trichoderma reesei using an in silico method, and attempted to produce 48 mutants in which a total of 48 amino acids were site-specifically substituted with other amino acids.

[0361] The site-directed mutagenesis (SDM) method was utilized to generate mutants. Specifically, the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, La Jolla, CA, USA) was used to introduce mutations encoding a total of 48 mutant amino acids into the wild-type aspartic protease coding sequence (SEQ ID NO: 10). The base sequence substituted by SDM was finally confirmed through base sequence analysis (Macrogen, Seoul).

[0362] The mutation locations of the 48 mutants are shown in Table 1 below.

[0363] 48 Multiple Mutation Location No. Mutation Information No. Mutation Information No. Mutation Information 1N65A17T161N33N288D2H66W18G163A34N296Q3T77V19T172D35Q298P4S78A20V179N36N304P5S80Q21A187G37E307G6A85P22G191L38V309W7V87T23Q193T39D312P8S110Q24H197Q40D314S9T118D 25S209Q41E315S10S125A26A217V42D316S11V128L27Q223K43S319D12S132T28V236K44S327C13S 143A29V241I45L335Y14D146T30A242C46T339A15S158T31S261A47E342D16N160T32K266S48E380N

[0364] Example 2. Production and thermal stability evaluation of 48 mutants

[0365] To evaluate the thermal stability of the 48-fold mutant, the following experiments were performed.

[0366] Specifically, the pichia pastoris X-33 strain, each transformed with the pPICZαA expression plasmid in which the polynucleotide encoding the wild-type aspartic protease or the 48 mutants of Example 1 was cloned, was inoculated into a sterilized LB kanamycin medium (BD Difco) in a 96 Deep well Plate (Bioneer, 90060), cultured at 37°C and 800 rpm for 24 hours, and then inoculated into the main culture in a 96 Deep Well Plate. Then, the cells were collected by centrifugation, and the collected cells were disrupted by treating with BugBuster (Merck, 70584). Only the supernatant was collected from the disrupted enzyme solution after centrifugation, and heat-treated for 5 minutes at temperature conditions of 60°C and 70°C to evaluate thermal stability. The protein decomposition (protease) activity before and after heat treatment was evaluated and shown in Figure 1.

[0367] Specifically, protein decomposition activity was measured by detecting tyrosine, an amino acid released from the substrate casein by enzyme activity. 0.1 mL of enzyme solution diluted in 50 mM lactic acid buffer (pH 3.0 at room temperature) and 0.1 mL of substrate 1.0% casein (Sigma-Aldrich Co.) were added to 0.2 mL of the reaction solution, and the reaction was incubated at 40°C for 10 minutes, after which 0.2 mL of trichloroacetic acid (TCA, Sigma-Aldrich Co.) solution was added to terminate the reaction. After centrifugation (10,000 X g, 10 min), 0.75 mL of 0.4 M sodium carbonate anhydrous and 0.15 mL of 3-fold diluted Folin Ciocalteu (Sigma-Aldrich Co.) solution were added to 0.15 mL of the supernatant, and the color reaction was performed at 40°C for 20 min, and the absorbance was measured at 680 nm using a spectrophotometer. Protease activity was calculated from a standard curve quantifying tyrosine (Sigma-Aldrich Co.), and an activity unit was defined as the amount of enzyme that produces 1 μg of tyrosine per min.

[0368] As a result, as shown in Fig. 1, it was confirmed that the wild type maintained 0.9% residual activity at 60°C, and the 48 mutants maintained 92.8% residual activity (see Fig. 1).

[0369]

[0370] Example 3. Production of a mutant with an additional disulfide bond-forming mutation introduced into the 48-double mutant.

[0371] In addition to the 48 mutants manufactured in Example 2, a total of 7 improved mutants (DS1, DS2, DS3, DS4, DS5, DS6, and DS7) were produced by introducing disulfide bonds.

[0372] To produce additional improved mutants, the site-directed mutagenesis (SDM) method was utilized in the same manner as described in Example 1, and mutations were introduced into the 48 mutant coding sequences using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, La Jolla, CA, USA). The gene sequences substituted by SDM were confirmed through base sequence analysis (Macrogen, Seoul).

[0373] The positions of the disulfide bonds additionally introduced in the 48-fold mutant are shown in Table 2 below.

[0374] Information on disulfide bond position mutations with improved thermal stability: DS1A71C+A339C, A163C+Q199CDS2A71C+A339C, Q110C+V175CDS3A163C+Q199C, Q110C+V175CDS4A71C+A339C, A163C+Q199C, Q110C+V175CDS5A163C+Q199CDS6A71C+A339CDS7Q110C+V175C

[0375] Evaluation of thermal stability of a mutant in which a disulfide bond-forming mutation was additionally introduced into the mutant in Example 4.48.

[0376] To evaluate the thermal stability of a mutant that introduced an additional disulfide bond mutation to the 48-fold mutant, the following experiments were performed.

[0377] Specifically, heat stability was evaluated using the pichia pastoris X-33 strain, each transformed with a pPICZαA expression plasmid in which polynucleotides encoding wild-type aspartic protease, the 48 mutants of Example 1, and the mutants in which a disulfide bond mutation was additionally introduced to the 48 mutants of Example 3 were cloned, using the same method as in Example 2. The results of comparing the residual activity of the TrAsp wild type (WT), 48 mutants, and mutants (DS1, DS2, DS3, DS4, DS5, DS6, and DS7) introduced with both 48 multiple mutations and disulfide bond mutations after treatment at 70°C for 5 or 10 minutes are shown in Fig. 2, and the results of comparing the residual activity of the mutants (DS1, DS2, and DS3) introduced with both 48 multiple mutations and disulfide bond mutations over time with the wild type (WT) at 75°C or 80°C are shown in Figs. 3 and 4, respectively.

[0378] As a result, as shown in FIGS. 2 to 4, in the case of the mutants in which a disulfide bond mutation was additionally introduced to the 48 mutants, more than 30% of the residual activity was maintained under the condition of heat treatment at 70°C for 5 minutes (see FIG. 2), and in the case of the DS1, DS2, and DS3 mutants with the best heat stability, it was confirmed that the activity was maintained even at 75°C and 80°C (see FIGS. 3 and 4).

[0379]

[0380] From the above description, those skilled in the art will understand that the present application can be implemented in other specific forms without altering its technical concept or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of this application should be interpreted to include all changes or modifications derived from the meaning and scope of the following claims and their equivalents, rather than the detailed description above.

Claims

1. A mutant polypeptide having aspartic acid protease activity, which is at least one of the following i) to iii): i) a polypeptide having the amino acid sequence of sequence number 1 or a sequence identity of at least 90% thereto; ii) a polypeptide encoded by a polynucleotide encoding a mature polypeptide having the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% sequence identity thereto; iii) a polypeptide encoded by a polynucleotide that hybridizes to (a) a mature polypeptide coding sequence of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% sequence identity thereto, (b) a cDNA thereof, or (c) a full-length complement of (a) or (b) under low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions, A variant polypeptide comprising a substitution of an amino acid at any of the following positions with another amino acid: 65, 66, 77, 78, 80, 85, 87, 110, 118, 125, 128, 132, 143, 146, 158, 160, 161, 163, 172, 179, 187, 191, 193, 197, 209, 217, 223, 236, 241, 242, 261, 266, 288, 296, 298, 304, 307, 309, 312, 314, 315, No. 316, No. 319, No. 327, No. 335, No. 339, No. 342, and No. 380; Here, the position number corresponds to the position of the polypeptide of sequence number 1.

2. In the first paragraph, the mutant polypeptide comprises all of the following amino acid substitutions: Substitution of amino acid 65 with alanine; substitution of amino acid 66 with tryptophan; substitution of amino acid 77 with valine; substitution of amino acid 78 with alanine; substitution of amino acid 80 with glutamine; substitution of amino acid 85 with proline; substitution of amino acid 87 with threonine; substitution of amino acid 110 with glutamine; substitution of amino acid 118 with aspartic acid; substitution of amino acid 125 with alanine; substitution of amino acid 128 with leucine; substitution of amino acid 132 with threonine; substitution of amino acid 143 with alanine; substitution of amino acid 146 with threonine; substitution of amino acid 158 with threonine; substitution of amino acid 160 with threonine; substitution of amino acid 161 with asparagine; Substitution of amino acid 163 with alanine; substitution of amino acid 172 with aspartic acid; substitution of amino acid 179 with asparagine; substitution of amino acid 187 with glycine; substitution of amino acid 191 with leucine; substitution of amino acid 193 with threonine; substitution of amino acid 197 with glutamine; substitution of amino acid 209 with glutamine; substitution of amino acid 217 with valine; substitution of amino acid 223 with lysine; substitution of amino acid 236 with lysine; substitution of amino acid 241 with isoleucine; substitution of amino acid 242 with cysteine; substitution of amino acid 261 with alanine; substitution of amino acid 266 with serine; substitution of amino acid 288 with aspartic acid; substitution of amino acid 296 with glutamine; Substitution of amino acid 298 with proline; substitution of amino acid 304 with proline; substitution of amino acid 307 with glycine; substitution of amino acid 309 with tryptophan; substitution of amino acid 312 with proline; substitution of amino acid 314 with serine; substitution of amino acid 315 with serine; substitution of amino acid 316 with serine; substitution of amino acid 319 with aspartic acid; substitution of amino acid 327 with cysteine;Substitution of amino acid 335 with tyrosine; substitution of amino acid 339 with alanine; substitution of amino acid 342 with aspartic acid; and substitution of amino acid 380 with asparagine; Here, the position number corresponds to the position of the polypeptide of sequence number 1.

3. A mutant polypeptide according to claim 1, wherein the mutant polypeptide has at least 90% identity with the amino acid sequence of SEQ ID NO:

2.

4. In the first paragraph, the mutant polypeptide further comprises one substitution selected from the following: Substitution of amino acids 71, 339, 163 and 199 with cysteine; substitution of amino acids 71, 339, 110 and 175 with cysteine; substitution of amino acids 163, 199, 110 and 175 with cysteine; substitution of amino acids 71, 339, 163, 199, 110 and 175 with cysteine; substitution of amino acids 163 and 199 with cysteine; substitution of amino acids 71 ​​and 339 with cysteine; and substitution of amino acids 110 and 175 with cysteine; Here, the position number corresponds to the position of the polypeptide of sequence number 1.

5. A mutant polypeptide according to any one of claims 1 to 4, wherein the mutant polypeptide has increased heat resistance compared to a polypeptide consisting of the amino acid sequence of SEQ ID NO:

1.

6. A method for producing a protein hydrolyzate, comprising the step of contacting a substrate with at least one of the mutant polypeptides of any one of claims 1 to 4 and a host cell expressing the mutant polypeptide.

7. A method for producing a protein hydrolyzate, wherein the substrate comprises a protein in the sixth paragraph.

8. A method for decomposing a protein, comprising the step of contacting a substrate with at least one of the mutant polypeptides of any one of claims 1 to 4 and a host cell expressing the mutant polypeptide.

9. A polynucleotide encoding a mutant polypeptide of any one of claims 1 to 4.

10. A host cell comprising at least one of a mutant polypeptide according to any one of claims 1 to 4, a polynucleotide encoding the mutant polypeptide, a nucleic acid structure comprising the polynucleotide, and a vector comprising the nucleic acid structure.

11. A method for producing a mutant polypeptide having aspartic acid protease activity, comprising the step of culturing the host cell of claim 10.

12. A method for producing a mutant polypeptide having aspartic acid protease activity, further comprising a step of recovering a mutant polypeptide expressed in the culturing step, in claim 11.

13. A composition comprising a mutant polypeptide of any one of claims 1 to 4.

14. A composition according to claim 13, wherein the composition is a feed composition, a food composition, a paper-making composition, or a composition for manufacturing cosmetics.

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