Novel serine protease variant

Abstract

The present application relates to a novel serine protease variant.

Description

Novel serine protease variants

[0001] The present application relates to a novel serine protease variant.

[0002]

[0003] Proteases perform various functions in the body, such as digestion, absorption, and defense, and are classified into serine proteases, cysteine ​​proteases, aspartic proteases, and metalloproteases depending on the structure of the active site. Among them, serine proteases (or serine endopeptidases) are characterized by having an active serine residue in common in the active site, and serine acts as a nucleophilic amino acid at the active site of the protease to cleave peptide bonds within proteins (Hedstrom, 2002. Chem Rev 102: 4501-4524).

[0004] Serine proteases have a wide range of uses, including treating human diseases such as dissolving blood clots, as well as being used as ingredients in clothing detergents and contact lens cleaners, and in various other applications such as modification of milk proteins, silk degumming, soaking and unhairing of leather, oligopeptide synthesis, recovery of silver from waste X-ray films, and manufacturing and improvement of feed and food.

[0005]

[0006] In order to secure better industrial economic feasibility and efficacy, a serine protease with improved thermal stability, activity, etc. is required.

[0007]

[0008] One aspect of the present application provides a serine protease variant in which the amino acid corresponding to position 180 of SEQ ID NO: 10 is replaced with another amino acid.

[0009] In one specific example, the other amino acid may be tyrosine (Y), glutamic acid (E), histidine (H), leucine (L), methionine (M), asparagine (N), glutamine (Q), or tryptophan (W).

[0010] In any one of the preceding specific examples, the serine protease variant may comprise an amino acid sequence having at least 75% sequence homology or identity with SEQ ID NO: 10.

[0011] In any one of the preceding specific examples, the serine protease variant may comprise an amino acid sequence having at least 75% sequence homology or identity with SEQ ID NO: 7.

[0012] In any one of the preceding specific examples, the serine protease variant may have the amino acid corresponding to position 331 of SEQ ID NO: 9 or SEQ ID NO: 12 replaced with another amino acid.

[0013] In any one of the preceding specific examples, the serine protease variant may have the amino acid corresponding to position 361 of SEQ ID NO: 8 or SEQ ID NO: 11 replaced with another amino acid.

[0014] Another aspect of the present application provides a polynucleotide encoding the serine protease variant.

[0015] Another aspect of the present application provides a vector comprising the polynucleotide.

[0016] Another aspect of the present application provides a microorganism comprising the serine protease variant, a polynucleotide encoding the variant, or a vector comprising the polynucleotide.

[0017] In one specific example, the microorganism may be a Bacillus sp. microorganism. In any of the preceding specific examples, the microorganism may be Bacillus subtilis.

[0018]

[0019] Another aspect of the present application provides a composition comprising at least one of the serine protease variant; and a microorganism comprising the serine protease variant, a polynucleotide encoding the variant, or a vector comprising the polynucleotide.

[0020] In one specific example, the composition may be a feed composition.

[0021] Another aspect of the present application provides a method for producing the serine protease variant, comprising the step of culturing a microorganism comprising the serine protease variant, a polynucleotide encoding the variant, or a vector comprising the polynucleotide.

[0022] In one specific example, the method for producing the serine protease variant may further comprise a step of recovering the variant polypeptide having the serine protease activity of the present application expressed in the culturing step.

[0023] Another aspect of the present application provides a method for decomposing casein using the serine protease variant.

[0024]

[0025] The serine protease variant of the present application has superior activity compared to existing serine proteases and thus can be usefully utilized industrially.

[0026]

[0027] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions described below.

[0028] Furthermore, those skilled in the art will recognize or be able to ascertain, using only 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.

[0029] Additionally, numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety, thereby providing a clearer understanding of the technical field to which this application pertains and the content of this application.

[0030]

[0031] definition

[0032] 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 the context clearly dictates otherwise, the use of "or" is intended to include "and / or."

[0033]

[0034] 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.

[0035] 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.

[0036] 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.

[0037]

[0038] mutant

[0039] 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."

[0040] 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 to C-terminal orientation, unless otherwise indicated.

[0041] 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.

[0042]

[0043] 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.

[0044] In the present application, the parent sequence or backbone refers to a reference sequence into which a modification is introduced to become a variant. That is, the parent sequence may be a starting sequence and may be a target for introducing mutations such as substitutions, insertions, and / or deletions. 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.

[0045] 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.

[0046]

[0047] In the present application, the term "Nth position" may include the Nth position and an amino acid position corresponding to the Nth position. Specifically, it may include an amino acid position corresponding to any amino acid residue in a mature polypeptide disclosed in a particular amino acid sequence.

[0048]

[0049] As used herein, the term "corresponding to" refers to an amino acid residue at a position listed in a polypeptide, or an amino acid residue that is similar, identical, or homologous to the residue listed in the 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.

[0050] In the present application, SEQ ID NO. 10 can be used as a reference sequence to determine the position of an amino acid in any amino acid sequence.

[0051] That is, SEQ ID NO: 10 disclosed in the present application can be used to determine the corresponding amino acid residue in a polypeptide having any serine protease activity, and unless otherwise indicated in the present application, residues of a particular amino acid sequence are numbered based on SEQ ID NO: 10.

[0052] For example, any amino acid sequence can be aligned with SEQ ID NO: 10, 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: 10. 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”).

[0053] For such alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), Trends Genet. 16: 276-277) can be used, but is not limited thereto, and any sequence alignment program known in the art, pairwise sequence comparison algorithm, etc. can be appropriately used.

[0054] Multiple sequence alignment can also be used to identify corresponding amino acid residues in other polypeptides. 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.

[0055] Additionally, when polypeptides diverge from a mature polypeptide 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 when a polypeptide family or superfamily 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.

[0056] 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).

[0057] The above methods are examples and are not limiting.

[0058]

[0059] In the present application, with respect to the amino acid sequence, even if it is described as a polypeptide “comprising” the amino acid sequence set forth in a specific sequence number, a polypeptide “consisting of” the amino acid sequence set forth in a specific sequence number, or a polypeptide or protein “having” the 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 is also included within the scope of the present application, as long as it has the same or corresponding activity as the protein consisting of the amino acid sequence of the corresponding sequence number. For example, if it has the same or corresponding activity as the above-mentioned mutant protein, it does not exclude sequence additions that do not alter the function of the protein before or after the amino acid sequence, mutations that may occur naturally, silent mutations thereof, or conservative substitutions, and it is obvious that even if it has such sequence additions or mutations, it is also within the scope of the present application.

[0060] For example, if the amino acid sequence has sequence additions, naturally occurring mutations, silent mutations or conservative substitutions that do not alter the function of the variant polypeptide of the present application at the N-terminus, C-terminus and / or within the amino acid sequence.

[0061] 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. Typically, conservative substitutions may have little or no effect on the activity of the polypeptide.

[0062] In this application, the term "other amino acid" is not limited to an amino acid different from the amino acid prior to substitution. Furthermore, when this application states that "a specific amino acid has been substituted," it is self-evident that the amino acid has been substituted with an amino acid different from the amino acid prior to substitution, even if it is not specifically stated that it has been substituted with another amino acid.

[0063]

[0064] In this application, "mutating / modifying" means changing or altering. This may be a change from a naturally occurring sequence. For example, a polypeptide may be modified in a way that causes the polypeptide to be altered from its parent sequence or reference sequence.

[0065] In the present application, the modified polypeptide may be a polypeptide that does not exist in nature itself, i.e., a non-naturally occurring polypeptide.

[0066] As used herein, the term "modified" means, for example, altered from its naturally occurring form. Modified polypeptides of the present application include non-naturally occurring polypeptides or naturally occurring variants. For example, modified polypeptides of the present application are modified polypeptides not found in nature. For example, modified polypeptides of the present application may be, but are not limited to, those that do not occur spontaneously.

[0067] 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.

[0068]

[0069] In the present application, the term "variant" or "variant polypeptide" refers to a polypeptide that differs from the parent sequence of the polypeptide by conservative substitution and / or modification of one or more amino acids, but retains the functions or properties of the polypeptide. The variant polypeptide differs from the identified sequence by several amino acid substitutions, deletions, or additions. Such variant polypeptides can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant polypeptide may be increased, unchanged, or decreased compared to the native polypeptide. 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. Other variant polypeptides may include variants in which portions are deleted from the N- and / or C-terminus of the mature protein. The term "variant polypeptide" may be used interchangeably with terms such as variant, modified, mutated protein, variant, and variant (in English, modification, modified protein, mutant, mutein, divergent, variant, etc.), and is not limited thereto as long as the term is used in the meaning of variant.

[0070] Additionally, the variant polypeptide may contain 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 polypeptide that is involved in co-translational or post-translational protein transfer. The polypeptide may also be conjugated to other sequences or linkers to facilitate identification, purification, or synthesis of the polypeptide.

[0071]

[0072] The serine protease variant of the present application may be in isolated form.

[0073] 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 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 or nucleic acid.

[0074] For example, the isolated sequences or nucleic acids provided in the present application may be provided in a form substantially free of one or more contaminants.

[0075] 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.).

[0076] In the present application, with respect to amino acid or nucleic acid sequences, the term "biologically active fragments" or "fragments" may refer to "functional fragments." A "functional fragment," also referred to as an active fragment, refers to a polypeptide that contains fewer amino acids than a full-length protein but retains at least one biological activity of the corresponding full-length protein. For example, a functional fragment of an enzyme may comprise the catalytic site of the enzyme.

[0077] The serine protease variant of the present invention may comprise biologically active fragments of the serine protease variant. The biologically active fragments may comprise a portion of the full length of the native polypeptide.

[0078] For example, it may contain, but is 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 native polypeptide.

[0079]

[0080] 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.

[0081] Alanine Ala, A Arginine Arg, R

[0082] Asparagine Asn, N Aspartic acid Asp, D

[0083] Cysteine ​​Cys, C Glutamic acid Glu, E

[0084] Glutamine Gln, Q Glycine Gly, G

[0085] Histidine His, H Isoleucine Ile, I

[0086] Leucine Leu, L Lysine Lys, K

[0087] Methionine Met, M Phenylalanine Phe, F

[0088] Proline Pro, P Serine Ser, S

[0089] Threonine Thr, T Tryptophan Trp, W

[0090] Tyrosine Tyr, Y Valine 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 their residues.

[0096] Examples of these classifications include positively charged (basic) amino acids such as arginine, lysine, and histidine; negatively charged (acidic) amino acids such as glutamic acid and aspartic acid; amino acids with nonpolar side chains (nonpolar amino acids) such as glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; and amino acids with polar or hydrophilic side chains (polar amino acids) such as serine, threonine, cysteine, tyrosine, asparagine, and glutamine. As another example, amino acids with charged side chains (electrically charged amino acids) include arginine, lysine, histidine, glutamic acid, and aspartic acid; and amino acids with uncharged side chains (also called neutral amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. As another example, phenylalanine, tryptophan, and tyrosine can be classified as aromatic amino acids. As another example, valine, leucine, and isoleucine can be classified as branched amino acids. As another example, the 20 amino acids can be classified by size into five groups: glycine, alanine, and serine; cysteine, proline, threonine, aspartic acid, and asparagine; valine, histidine, glutamic acid, and glutamine; isoleucine, leucine, methionine, lysine, and arginine; and phenylalanine, tryptophan, and tyrosine. However, this is not necessarily limited to these groups.

[0097]

[0098] To describe the variants provided in this application, the following nomenclature is used.

[0099] 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."

[0100] 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 in a specific sequence, is replaced with valine, it can be described as "A6V" or "Ala6Val."

[0101] 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.

[0102]

[0103] polynucleotide

[0104] 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).

[0105]

[0106] In this application, the term "polynucleotide, nucleic acid or nucleic acid molecule" means a polymer of nucleotides in which nucleotide units (monomers) are covalently bonded to form a long chain, and a strand of DNA (e.g., cDNA or genomic DNA) or RNA (e.g., mRNA) of a certain length or longer.

[0107]

[0108] homology, identity

[0109] As used herein, the terms "homology" or "identity" refer to the degree of relationship between two given amino acid sequences or nucleic acid sequences, which may be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0110] 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.

[0111] 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 et al.](1988) SIAM J Applied Math 48: 1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information Database.

[0112] Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using, for example, the GAP computer program disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2:482, or, for example, Needleman et al. (1970), J Mol Biol. 48:443. 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). The default parameters for the GAP program are (1) unitary matrices (containing values ​​of 1 for identity and 0 for non-identity), the PAM Matrix (see the disclosure by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation (1978)), Gribskov et al (1986) Nucl. Acids Res. 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.

[0113] Additionally, whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined by comparing the sequences by Southern hybridization experiments under defined 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; F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York), but are not limited thereto.

[0114] In this application, the term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8). For example, conditions under which polynucleotides having high homology or identity hybridize with each other, polynucleotides having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity hybridize with each other, and polynucleotides having lower homology or identity do not hybridize with each other, or conditions under which washing conditions of typical southern hybridization are 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.

[0115] The above hybridization requires that the two nucleotides 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. Accordingly, the polynucleotide 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.

[0116] The appropriate stringency for hybridizing the polynucleotides depends on the length and degree of complementarity of the polynucleotides, variables which are well known in the art (e.g., J. Sambrook et al., supra).

[0117]

[0118] Nucleic acid structures, vectors, and transformation

[0119] 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.

[0120]

[0121] The term "vector" as used herein refers to a DNA construct comprising a nucleic acid sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to enable expression of the target polypeptide 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 sequences regulating the termination of transcription and translation. After being transformed into a suitable host cell, the vector may replicate or function independently of the host genome, or may be integrated into the genome itself.

[0122] The vector 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 pDZ series, pDC series, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series can be used as plasmid vectors. For example, pDZ, pDC, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors can be used.

[0123] For example, a polynucleotide encoding a target polypeptide 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. 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.

[0124] The term "transformation" in this application refers to introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism, thereby enabling expression of the polypeptide encoded by the polynucleotide in the host cell. The transformed polynucleotide may be located within the chromosome of the host cell or located extrachromosomally, as long as it can be expressed in the host cell. Furthermore, the polynucleotide includes DNA and RNA encoding the target polypeptide. The polynucleotide may be introduced in any form as long as it can be introduced into the host cell and expressed. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for autonomous expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, all of which are operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. Additionally, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.

[0125] 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 said known or desired activity. For example, it refers to a polynucleotide sequence that is operatively linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target variant polypeptide of the present application.

[0126] 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.

[0127] 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.

[0128] 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 sequences that regulate transcription and translation termination. The minimum unit of the regulatory sequence may include a promoter, a transcription and translation termination sequence.

[0129]

[0130] In relation to a cell, polynucleotide, polypeptide, or vector, the term "recombinant" in this application means that the cell, polynucleotide, polypeptide, or vector has been modified by the introduction of a heterologous nucleic acid or polypeptide or by alteration of a native polynucleotide 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.

[0131]

[0132] host cell

[0133] The host cell of the present application may be, but is not limited to, a microorganism comprising at least one of the variant of the present application, the polynucleotide of the present application, and the vector comprising the polynucleotide of the present application; a microorganism modified to express the variant of the present application or the polynucleotide of the present application; a microorganism (e.g., a recombinant strain) expressing the variant of the present application or the polynucleotide of the present application; or a microorganism (e.g., a recombinant strain) having the activity of the variant of the present application.

[0134]

[0135] Specific description of this application

[0136] Hereinafter, specific examples of the present application will be described in more detail as follows.

[0137]

[0138] One aspect of the present application provides a serine protease variant.

[0139]

[0140] The term "serine protease" in the present application refers to an enzyme belonging to a subgroup of proteases and possessing proteolytic activity. Specifically, a serine protease is an enzyme that degrades proteins by hydrolyzing peptide bonds and may be an enzyme that essentially has an active serine residue at its active site, and more specifically, an enzyme that has a spatial arrangement of amino acid residues, histidine, aspartate, and serine, which may be referred to as a catalytic triad.

[0141] The serine protease of the present application may be derived from a microorganism of the genus Thermobifida or the genus Actinorugispora.

[0142] For example, the wild type of serine protease provided in the present application may be a serine protease derived from Thermobifida fusca, Thermobifida cellulosilytica, Thermobifida halotolerans, or Actinorugispora endophytica. Examples thereof include sequences such as GenBank: AAZ54522.1, NCBI Accession number WP_016188200.1, GenBank: KUP96625.1, NCBI Reference Sequence: WP_068687914.1, and NCBI Reference Sequence: WP_133739400.1.

[0143] As an example of a wild-type serine protease, the wild-type serine protease of the present application may have an amino acid sequence derived from SEQ ID NO: 8 or SEQ ID NO: 11. As an example, the wild-type serine protease may have an amino acid sequence derived from SEQ ID NO: 9 or SEQ ID NO: 12. As an example, the wild-type serine protease may be a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 10, but is not limited thereto.

[0144] In the present application, the parent sequence of the serine protease, i.e. the serine protease to be mutated, may include, without limitation, not only the wild-type serine protease described above, but also a sequence having the same activity as the amino acid sequence of the wild-type serine protease.

[0145] Specifically, the parent sequence of the serine protease may include an amino acid represented by any one of SEQ ID NOs: 8 to 12, or an amino acid having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity with any one of SEQ ID NOs: 8 to 12. In addition, it is obvious that a protein having an amino acid sequence in which a part of the sequence is deleted, modified, substituted, or added is also included within the scope of the present application, as long as it has such homology or identity and exhibits an effect corresponding to the protein.

[0146] For example, the parent sequence of the protease may include an amino acid sequence represented by SEQ ID NO: 6 or 7 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity with SEQ ID NO: 6 or 7.

[0147] As another example, the parent sequence of the protease may comprise an amino acid sequence set forth in SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity with SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

[0148]

[0149] The serine protease variant provided in the present application is a variant having serine protease activity, in which the amino acid corresponding to the 180th position from the N-terminus of SEQ ID NO: 10 in any polypeptide or serine protease having serine protease activity is substituted with another amino acid.

[0150] The serine protease variant of the present application may have enhanced serine protease enzyme activity compared to the polypeptide before mutation, the natural wild-type polypeptide, the unmodified polypeptide, or the polypeptide of the parent sequence.

[0151] For example, the serine protease variant provided in the present application may mean a variant in which, among the proteins having serine protease activity described above, the amino acid corresponding to the 180th position of sequence number 10 is replaced with a different amino acid compared to the parent sequence, and the enzyme activity exceeds 100% compared to the protein of the parent sequence.

[0152] For example, the serine protease variant of the present application may be one in which phenylalanine (F), an amino acid corresponding to position 180 in the amino acid sequence of SEQ ID NO: 10, is substituted with glycine, alanine, arginine, aspartate, cysteine, glutamic acid, glutamine, histidine, proline, serine, tyrosine, isoleucine, leucine, lysine, tryptophan, valine, methionine, threonine, or asparagine.

[0153] For example, the serine protease variant of the present application may be one in which phenylalanine, an amino acid corresponding to position 180 in the amino acid sequence of SEQ ID NO: 10, is substituted with tyrosine (Y), glutamic acid (E), histidine (H), leucine (L), methionine (M), asparagine (N), glutamine (Q), or tryptophan (W).

[0154] For example, the variant may include an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity with SEQ ID NO: 7 or 10, wherein the amino acid at a position corresponding to amino acid position 180 from the N-terminus of SEQ ID NO: 10 is substituted with another amino acid. For example, the other amino acid may be tyrosine (Y), glutamic acid (E), histidine (H), leucine (L), methionine (M), asparagine (N), glutamine (Q), or tryptophan (W).

[0155]

[0156] Meanwhile, the mature region of GenBank: AAZ54522.1 (SEQ ID NO: 8) corresponds to SEQ ID NO: 9, the mature region of NCBI Reference Sequence WP_016188200.1 (SEQ ID NO: 11) corresponds to SEQ ID NO: 12 of the present application, and the sequences excluding only the signal peptide in SEQ ID NO: 9 and SEQ ID NO: 12 correspond to SEQ ID NO: 10 of the present application.

[0157] Additionally, sequence number 7 has improved enzymatic activity compared to the polypeptide of sequence number 10 by substituting amino acids corresponding to positions 12 and 116 in sequence number 10 with other amino acids.

[0158] It is obvious that the serine protease variant of the present application may include deletions or additions of amino acids that have minimal impact on the properties and secondary structure of the serine protease, in which the amino acid at position corresponding to position 180 of SEQ ID NO: 10 is replaced with another amino acid as described above. In addition, a person skilled in the art can determine through sequence alignment known in the art that position 180 from the N-terminus of SEQ ID NO: 7 of the present application corresponds to position 180 of SEQ ID NOs: 10, 13 to 15, position 331 of SEQ ID NOs: 6, 9, and 12, and position 361 of SEQ ID NOs: 8 and 11, and that SEQ ID NO: 7 is included in SEQ ID NO: 6, and that SEQ ID NO: 10 is included in SEQ ID NOs: 8, 9, 11, and 12.

[0159] Accordingly, as an example of a serine protease variant of the present application, for SEQ ID NO: 6 and SEQ ID NO: 8, a variant may be exemplified in which the amino acid corresponding to the 180th position of SEQ ID NO: 10 (the 331st amino acid in SEQ ID NO: 6, the 361st amino acid in SEQ ID NO: 8) is substituted. As another example of a serine protease of the present application, for SEQ ID NO: 9, a variant may be exemplified in which the amino acid corresponding to the 180th position of SEQ ID NO: 10 (the 331st amino acid in SEQ ID NO: 9) is substituted.

[0160] Additionally, the description of the 180th amino acid of SEQ ID NO: 10 in this application can be applied to the 180th amino acid of SEQ ID NOs: 7, 13 to 15, the 331st amino acid of SEQ ID NOs: 6 and 9, and the 361st amino acid of SEQ ID NO: 8.

[0161] Similarly, what has been described for amino acids 12 and 116 of SEQ ID NO: 10 can be applied to amino acids 12 and 116 of SEQ ID NO: 7, amino acids 163 and 267 of SEQ ID NOs: 6, 9 and 12, and amino acids 193 and 297 of SEQ ID NOs: 8 and 11.

[0162] For example, the serine protease variant of the present application may include an amino acid sequence in which the amino acid at the position corresponding to the 180th amino acid of SEQ ID NO: 10 is substituted with another amino acid, and may have at least 75% but less than 100% sequence homology with SEQ ID NO: 6. In another embodiment, the serine protease variant of the present application may include an amino acid sequence in which the amino acid at the position corresponding to the 180th amino acid of SEQ ID NO: 10 is substituted with another amino acid, and may have at least 75% but less than 100% sequence homology with SEQ ID NO: 8.

[0163]

[0164] For example, the serine protease variant of the present application may include a substitution of the amino acid at a position corresponding to amino acid position 12 and / or amino acid position 116 from the N-terminus of SEQ ID NO: 10 with another amino acid.

[0165] Specifically, the serine protease variant of the present application is a variant in which phenylalanine at position 12 in the amino acid sequence of SEQ ID NO: 10 is replaced with glycine, alanine, arginine, aspartate, cysteine, glutamic acid, asparagine, glutamine, histidine, proline, serine, tyrosine, isoleucine, leucine, lysine, tryptophan, valine, methionine or threonine, and / or asparagine at position 116 is replaced with glycine, alanine, arginine, aspartate, cysteine, glutamic acid, glutamine, It may be substituted with, but is not limited to, histidine, proline, serine, tyrosine, isoleucine, leucine, lysine, phenylalanine, tryptophan, valine, methionine or threonine.

[0166] Specifically, the variant may be a protein comprising an amino acid corresponding to position 12 in the amino acid sequence of SEQ ID NO: 10 substituted with tyrosine (Y), serine (S), alanine (A), or arginine (R), an amino acid corresponding to position 116 substituted with aspartate (D), serine (S), threonine (T), or glycine (G), or an amino acid corresponding to positions 12 and 116 in the amino acid sequence of SEQ ID NO: 10 substituted with tyrosine (Y) and aspartate (D); tyrosine (Y) and serine (S); serine (S) and aspartate (D); serine (S) and threonine (T); or alanine (A) and glycine (G).

[0167] For example, the serine protease variant of the present application is one in which (i) the amino acid corresponding to position 12 of SEQ ID NO: 10 is tyrosine (Y, tyrosine), alanine (A, Alanine), serine (S, Serine), or arginine (R, Arginine); or (ii) the amino acid corresponding to position 116 of SEQ ID NO: 10 is aspartate (D, aspartate), serine (S, Serine), threonine (T, Threonine), or glycine (G, Glycine); Or (iii) the amino acid corresponding to the 12th position of SEQ ID NO: 10 may be tyrosine (Y, tyrosine), alanine (A, Alanine), serine (S, Serine), or arginine (R, Arginine), and the amino acid corresponding to the 116th position of SEQ ID NO: 10 may be aspartate (D, aspartate), serine (S, Serine), threonine (T, Threonine), or glycine (G, Glycine). For example, the serine protease variant of the present application may be one in which the amino acid corresponding to the 12th position of SEQ ID NO: 10 is tyrosine, and the amino acid corresponding to the 116th position of SEQ ID NO: 10 is aspartate.

[0168] For example, the serine protease variant of the present application may be such that (i) the amino acid corresponding to the 12th position of SEQ ID NO: 10 is phenylalanine (F, phenylalanine); (ii) the amino acid corresponding to the 116th position of SEQ ID NO: 10 is asparagine (N, asparagine); or (iii) the amino acid corresponding to the 12th position of SEQ ID NO: 10 is phenylalanine (F, phenylalanine) and the amino acid corresponding to the 116th position is asparagine (N, asparagine). For example, the serine protease variant of the present application may be such that the amino acid corresponding to the 12th position of SEQ ID NO: 10 is phenylalanine and the amino acid corresponding to the 116th position is asparagine.

[0169]

[0170] Another aspect of the present application provides a polynucleotide encoding a serine protease variant of the present application.

[0171] The polynucleotide encoding the serine protease variant of the present application may include, without limitation, any polynucleotide sequence encoding the serine protease variant of the present application having enhanced activity.

[0172] The polynucleotide of the present application may undergo various modifications in the coding region without altering the amino acid sequence of the polypeptide, due to codon degeneracy or in consideration of codons preferred by the organism to which the polypeptide is to be expressed. Specifically, the polynucleotide may include, without limitation, a polynucleotide sequence encoding a variant in which the amino acid corresponding to the 180th position from the N-terminus of SEQ ID NO: 10 is substituted with a different amino acid.

[0173] In addition, as described above, the serine protease variant of the present application includes a variant in which the amino acid corresponding to the 180th position from the N-terminus of SEQ ID NO: 10, i.e., the amino acid corresponding to the 180th position from the N-terminus of SEQ ID NO: 10 (the 331st amino acid in SEQ ID NO: 9 or 12, the 361st amino acid in SEQ ID NO: 8 or 11) is substituted for SEQ ID NO: 8, 9, 11 or 12, which is an amino acid sequence including SEQ ID NO: 10, and therefore, the polynucleotide sequence encoding the serine protease variant is also included in the scope of the present application.

[0174] In addition, a probe that can be prepared from a known genetic sequence, for example, a sequence that hybridizes under stringent conditions with a complementary sequence to all or part of the base sequence, and encodes a protein having the activity of a variant in which the amino acid corresponding to the 180th position from the N-terminus of SEQ ID NO: 10 is substituted with another amino acid, may be included without limitation.

[0175] For example, the polynucleotide sequence encoding the serine protease variant of the present application may include a sequence encoding amino acid sequences at positions 152 to 338 in the amino acid sequence encoded by SEQ ID NO: 16, and may be a sequence in which the codon encoding the amino acid corresponding to position 361 in the amino acid sequence encoded by the polynucleotide of SEQ ID NO: 16 is substituted with an amino acid other than phenylalanine, for example, tyrosine (Y), glutamic acid (E), histidine (H), leucine (L), methionine (M), asparagine (N), glutamine (Q), or tryptophan (W).

[0176]

[0177] Another aspect of the present application provides a vector comprising a polynucleotide encoding a serine protease variant of the present application.

[0178]

[0179] Another aspect of the present application provides a microorganism comprising one or more of the serine protease variant, a polynucleotide encoding the same, and a vector comprising the same.

[0180] The microorganism comprising at least one of the above serine protease variant, a polynucleotide encoding the variant, and a vector comprising the same may be a microorganism produced by transformation with a vector comprising a polynucleotide encoding the variant, but is not limited thereto.

[0181] The above microorganism may be a microorganism expressing a serine protease variant.

[0182] In this application, the term "protein to be / is / is expressed" may refer to a state in which the target protein is introduced into a microorganism or modified to be expressed within the microorganism. For the purposes of this application, the "target protein" may be the aforementioned serine protease variant.

[0183] Specifically, "introduction of a protein" may mean that a microorganism exhibits activity of a specific protein that it did not originally possess, or exhibits activity that is enhanced compared to the protein's inherent activity or activity prior to modification. For example, a polynucleotide encoding a specific protein may be introduced into the chromosome of a microorganism, or a vector containing a polynucleotide encoding a specific protein may be introduced into the microorganism, resulting in its activity.

[0184]

[0185] The above microorganism may be a recombinant microorganism, and the recombination may be achieved by genetic modification such as transformation.

[0186]

[0187] The above recombinant microorganism may have enhanced serine protease activity of the present application.

[0188] The above "enhanced activity" may refer to enhanced activity of a specific protein of a microorganism compared to its inherent activity or activity prior to modification. "Intrinsic activity" may refer to the activity of a specific protein originally possessed by the parent strain prior to a change in phenotype, when the phenotype of a microorganism changes due to genetic mutation caused by natural or artificial factors.

[0189] Specifically, in the present application, the enhancement of the activity of the protein variant may be achieved by one or more of, but is not limited to, increasing the intracellular copy number of the gene encoding the protein variant, introducing a mutation into the expression control sequence of the gene encoding the protein variant, replacing the expression control sequence of the gene encoding the protein variant with a sequence having strong activity, replacing a gene encoding a natural protein having serine protease activity on a chromosome with a gene encoding the protein variant, and additionally introducing a mutation into the gene encoding the variant so as to enhance the activity of the protein variant.

[0190] Next, modifying the expression control sequence to increase the expression of the polynucleotide may be performed by, but is not particularly limited to, inducing a sequence mutation in the nucleic acid sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof to further enhance the activity of the expression control sequence, or by replacing the nucleic acid sequence with a nucleic acid sequence having stronger activity. The expression control sequence may include, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating the termination of transcription and translation, etc.

[0191] A strong promoter may be linked upstream of the above polynucleotide expression unit instead of the original promoter, but is not limited thereto. Examples of known strong promoters include, but are not limited to, cj1 to cj7 promoters (Korean Patent No. 10-0620092), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (Korean Patent No. 10-1783170), O2 promoter (Korean Patent No. 10-1632642), tkt promoter, and yccA promoter.

[0192] In addition, modification of a polynucleotide sequence on a chromosome may be performed, but is not particularly limited thereto, by inducing mutations in the expression regulatory sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof of the nucleic acid sequence to further enhance the activity of the polynucleotide sequence, or by replacing it with a polynucleotide sequence that has been improved to have stronger activity.

[0193] Such introduction and enhancement of protein activity may be, but is not limited to, an increase in the activity or concentration of the corresponding protein, typically by at least 1%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, or up to 1000% or 2000%, relative to the activity or concentration of the protein in a wild-type or unmodified microbial strain.

[0194]

[0195] The host cell or microorganism of the present application may be any microorganism that expresses a serine protease variant, including the polynucleotide of the present application or the vector of the present application.

[0196] Specifically, it may include microbial strains of the genus Escherichia, the genus Serratia, the genus Erwinia, the genus Enterobacteria, the genus Providencia, the genus Salmonella, the genus Streptomyces, the genus Pseudomonas, the genus Brevibacterium, the genus Corynebacterium, or the genus Bacillus, and specifically, Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus velezensis, Escherichiacoli, Corynebacterium It may be a strain of Corynebacterum glutamicum, Aspergillus oryzae, or more specifically, Bacillus subtilis, but is not limited thereto.

[0197]

[0198] Another aspect of the present application provides a composition comprising a serine protease variant of the present application; and one or more microorganisms expressing the same.

[0199] The serine protease variant included in the composition of the present application may be, but is not limited to, a microorganism expressing the same, or may be in a form isolated and purified from a microorganism expressing the same.

[0200] The composition of the present invention may further include any component suitable for application in various industrial fields. Examples of materials that may be added include, but are not limited to, stabilizers, surfactants, builders, chelating agents, dispersants, enzymes, enzyme stabilizers, catalysts, activators, carriers, mixing agents, lubricants, disintegrants, excipients, solubilizers, suspending agents, colorants, fragrances, buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, diluents, lubricants, preservatives, and the like.

[0201] For 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 variant provided in the present application.

[0202]

[0203] For example, the composition according to the present application may be a feed composition.

[0204] 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.

[0205] The above feed composition may be a feed additive.

[0206] 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.

[0207] 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.

[0208] 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.

[0209] 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.

[0210] The feed composition of the present application may further include an enzyme preparation other than the serine protease variant. For example, the feed composition may further include one or more selected from 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. However, the present invention is not limited thereto.

[0211] 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.

[0212] 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.

[0213] The amount of the serine protease variant of the present application included in the feed composition of the present application is not particularly limited and may be appropriately adjusted depending on the intended purpose. In one embodiment, as is commonly known in the art to which the present application pertains, the serine protease variant 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.

[0214]

[0215] For example, the composition according to the present application may be a food composition.

[0216] 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.

[0217] In one embodiment, the food may be a food group requiring protease, such as dairy products, health functional foods for bowel movements or dieting, or functional health functional foods for preventing hypertension.

[0218] In other embodiments, the protease variant may be incorporated into various food compositions and used as a food solubilizer or tenderizer, or a meat texture modifier. In other embodiments, the protease variant may be added to a baking process and used in a step of breaking down the gluten network. Alternatively, the protease variant may be used to hydrolyze food proteins (e.g., proteins in milk). Alternatively, the protease variant may be incorporated into various food compositions and used for purposes such as rendering, flavoring, reducing bitterness, changing emulsifying properties, producing bioactive peptides, and reducing protein allergens, but these are merely examples and are not limited to the aforementioned uses.

[0219] The amount of the serine protease variant in the above food composition can be appropriately adjusted by a person skilled in the art depending on the purpose.

[0220]

[0221] For example, the composition according to the present application may be a detergent composition.

[0222] The detergent composition according to the present application may be in the form of a one-part and 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 residue films, and other small amounts of food compositions.

[0223] 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.

[0224]

[0225] For example, the composition according to the present application may be a pharmaceutical composition.

[0226] 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 tissue, or an anti-inflammatory agent for reducing swelling after surgery or trauma.

[0227] 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 purpose of 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.

[0228]

[0229] In addition to the above-described uses, the serine protease variant of the present application or the microorganism expressing the same can be used for purposes such as manufacturing cosmetics, leather processing, pharmaceuticals, diagnostic agents, waste management, and chemicals for academic research.

[0230] However, the above use is only an example, and it may be used for any other purpose related to denaturation, decomposition, or removal of protein substances known in the art.

[0231]

[0232] Another aspect of the present application provides a method for producing a serine protease variant, comprising the step of culturing a microorganism comprising the serine protease variant of the present application, a polynucleotide encoding the variant, or a vector comprising the polynucleotide.

[0233] As an example, the method for producing the serine protease variant may further include a step of recovering a variant polypeptide having the serine protease activity of the present application expressed in the culturing step of the microorganism.

[0234] As another example, variants expressed during the culture stage of a microorganism may not be recovered. The microorganism itself expressing the variant can be used as a source of the variant.

[0235]

[0236] Another aspect of the present application provides a method for decomposing casein using the serine protease variant. An example of the decomposition product is tyrosine.

[0237] Casein can be degraded using the variant of the present invention, a host cell expressing the variant, or a composition comprising the variant and / or the host cell. Cofactors, coenzymes, and the like may be added during this degrading step. The substrate hydrolysis step can be performed under optimal pH, temperature, and other conditions, and those skilled in the art can select appropriate conditions.

[0238]

[0239] 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.

[0240]

[0241] Example 1. Screening of serine protease mutants derived from Thermobifida fusca

[0242] Example 1-1. Construction of a serine protease library derived from Thermobifida fusca

[0243] Random mutations were introduced into the gene encoding the mature region of a serine protease from Thermobifida fusca using error-prone PCR. The PCR template in this study used the mutant sequences (F12Y, N116D) protected by a prior patent (PCT / KR2020 / 016775). Error-prone PCR was performed using the Diversify™ PCR Random Mutagenesis Kit (Clontech, Cat# 630703), and the PCR conditions are shown in [Table 1]. Under the following conditions, mutations were inserted at a frequency of 6.2 mutations / Kb.

[0244]

[0245] Composition Contents: Template DNA 0.5 ng, Sequence ID 1, Primer_Forward 5 uM, Sequence ID 2, Primer_Reverse 5 uM, Sequence ID 3, 10X Titanium Taq buffer 5 uL, nSO 4 6 40 uM, dGTP 40 uM, 50X Diversify dNTP mix 1 uL, 50X dNTP mix 1 uL, Titanium Taq Polymerase 1 uL, Total 50 uL, Adjust to 50 ul with DW, PCR Conditions: I. 94 ℃ 30 sec, II, III 50 cycles, II. 94 ℃ 30 sec, III. 68 ℃ 1 min, IV. 68 ℃ 1 min, V. 4 ℃ ∞

[0246] The PCR fragment obtained through the above process was ligated into the vector amplified using the primers listed in [Table 2] using the In-FusionR HD cloning kit (clontech), and then transformed into DH5α to obtain colonies. The plasmid was purified from the obtained colonies and approximately 5*10 4 A library of sizes has been secured.

[0247] Template DNA (pBE-S-TFP) Sequence number 1 Primer_Forward Sequence number 4 Primer_Reverse Sequence number 5

[0248] Example 1-2, Screening of a serine protease library derived from Thermobifida fusca

[0249] The protease library produced in Example 1-1 was transformed into the Bacillus subtilis LB700 strain, which is easy to secrete proteins, and screening was performed. The screening was performed in two stages. In the first stage, the Bacillus subtilis strain transformed with the library was spread on a 2% skim milk plate, and selection was performed based on the formed halo size. Bacillus subtilis transformation was performed according to the Groningen method, and the composition of the skim milk plate used for screening is as shown in [Table 3].

[0250]

[0251] Composition content Remarks M9 minimal salts11.28 gBD, cat#248510Agar20 gSkim milk20 gBD, cat#23210050%Glucose20 g1M MgSO42 mM1M CaCl20.1mMKanamycin50ug / ml

[0252] (For 1 L) Step 2 is a method of selecting the colonies primarily selected in Step 1 through Azocasein color development. BHI (Brain Heart Infusion, bd, cat#53286) liquid medium containing kanamycin antibiotic was added to a 96 deep well plate, and the colonies selected in Step 1 were inoculated and cultured at 37℃ for 20-24 hours. After culture, the supernatant containing the enzyme was obtained through centrifugation, and the supernatant and the substrate 2% (w / v) Azocasein were mixed in equal amounts, and the reaction was performed at 37℃ for 1 hour. The reaction was stopped by adding 3 times the volume of 10% TCA (Trichloro acetic acid) to the enzyme reaction solution, and the coagulated protein was removed through centrifugation. After mixing an equal amount of NaOH and performing the color development reaction, the degree of color development was compared by measuring the absorbance at 440 nm. Through this process, colonies with an absorbance increased by more than 130% compared to the serine protease of sequence number 6 were selected.

[0253]

[0254] Example 2. Preparation and activity evaluation of selected variants

[0255] Example 2-1. Production of mutants

[0256] The sequence analysis results of the mutants selected through screening finally confirmed that in addition to the F12Y and N116D mutations based on sequence number 10, position 180 (phenylalanine, Phe) was substituted with tyrosine (Tyr).

[0257] Example 2-2. Activity evaluation

[0258] In this study, the activity of protease was measured by checking the amount of tyrosine released from casein (from bovine milk) through an enzymatic reaction using the substrate. The F12Y / N116D / F180Y mutant obtained above was transformed into the Bacillus subtilis LB700 strain, inoculated into BHI medium containing kanamycin antibiotic, and cultured at 37°C for 20-24 hours, and a portion of the culture medium was used for the enzymatic reaction. The culture medium (enzyme) diluted in 0.1 mL of 50 mM sodium borate (pH 10.5 at room temperature) buffer and 0.1 mL of substrate 1.0% casein (Sigma-Aldrich Co.) were mixed and reacted at 40°C for 10 minutes. The reaction was terminated by the addition of 0.2 mL of trichloroacetic acid (TCA, Sigma-Aldrich Co.) solution. 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℃ 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. The activities measured in this way are shown in [Table 4].

[0259] Enzyme activity (unit / ml) F12Y / N116D142 F12Y / N116D / F180 Y312

[0260] As a result of the measurement, it was confirmed that when the F180Y mutation was added compared to the F12Y / N116D mutant under pH 10.5 and 40℃ conditions, the activity increased by approximately 2.2 times.

[0261]

[0262] Example 3. Preparation and activity evaluation of the 180 residue mutant.

[0263] From the results of Example 2, it was confirmed that position 180 of sequence number 10 was effective in improving serine protease activity, and additional experiments were performed to introduce F180Y based on saturation mutagenesis and the wild-type sequence (SEQ ID NO: 10).

[0264] Template DNA sequence number 1 Primer_Forward sequence number 17, 19, 21, 23, 25, 27, 29, 31 Primer_Reverse sequence number 18, 20, 22, 24, 26, 28, 30, 32

[0265] The obtained mutants were evaluated for activity using the same method as described in Example 2-2. The experimental results are shown in Table 6 below.

[0266] Mutant enzyme activity (Unit / ml) Enzyme relative activity ratio (%) Wild type 94 100 F180 Y 14 1 50 F12Y / N116D / F180 Y 27 6 294 F12Y / N116D / F180 E 15 4 164 F12Y / N116D / F180 H 16 7 178 F12Y / N116D / F180 L 12 4 132 F12Y / N116D / F180 M 17 4 185 F12Y / N116D / F180 N 15 2 162 F12Y / N116D / F180 Q 13 1 39 F12Y / N116D / F180 W 23 4 249

[0267] As described above, it was confirmed that activity was improved by introducing a mutation at position 180.

[0268]

[0269] Example 4. Activity evaluation after introducing mutations at position 180 in various serine proteases.

[0270] The activity was evaluated by introducing a substitution at the amino acid corresponding to position 180 of SEQ ID NO: 10 by modifying the parent sequence. The serine protease from Thermobifida halotolerans (SEQ ID NO: 13) and the serine protease from Actinorugispora endophytica (SEQ ID NO: 14) were used in the experiment.

[0271] The homology comparison of each sequence with sequence number 10 and the results of introducing the F180Y mutation are shown in the table below.

[0272] Derived variant Enzyme activity (Unit / ml) Enzyme relative activity % homology Thermobifidida halotolerans Wild type 111100% 81.8 % F180Y133120% Actinorugispora endophytica Wild type 421100% 81.3 % F180Y651155%

[0273] Through this, it was confirmed that the activity was greatly improved by substituting the amino acid residue at position 180 with Y.

[0274]

[0275] 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 serine protease mutant in which the amino acid corresponding to position 180 of sequence number 10 is replaced with another amino acid.

2. A serine protease variant in the first paragraph, wherein the other amino acid is tyrosine (Y), glutamic acid (E), histidine (H), leucine (L), methionine (M), asparagine (N), glutamine (Q), or tryptophan (W).

3. A serine protease mutant in which the amino acid corresponding to position 331 of sequence number 9 is replaced with another amino acid.

4. A serine protease mutant in which the amino acid corresponding to position 361 of sequence number 8 is replaced with another amino acid.

5. A serine protease variant according to claim 1, wherein the serine protease variant comprises an amino acid sequence having at least 75% sequence homology or identity with SEQ ID NO:

10.

6. A polynucleotide encoding a serine protease variant of any one of claims 1 to 5.

7. A vector comprising the polynucleotide of paragraph 6.

8. A microorganism comprising a serine protease variant of any one of claims 1 to 5, a polynucleotide encoding the variant, or a vector comprising the polynucleotide.

9. A serine protease variant of any one of claims 1 to 5; and A composition comprising at least one of the serine protease variant, a polynucleotide encoding the variant, or a microorganism comprising a vector comprising the polynucleotide.

10. A composition according to claim 9, wherein the composition is a feed composition.