Novel serine protease mutants

Abstract

The present invention relates to novel serine protease variants.

Description

[Technical field]

[0001] The present invention relates to novel serine protease variants. [Background technology]

[0002] Proteases fulfill various functions in the body, such as digestion, absorption, and defense, and are classified into serine proteases, cysteine ​​proteases, aspartic proteases, and metalloproteases according to the structure of their active sites. Among them, serine proteases (or serine endopeptidases) are mainly characterized by having an active serine residue in common in the active site, and are enzymes that cleave peptide bonds in proteins by acting as a nucleophilic amino acid in the active site of the protease (Non-Patent Document 1).

[0003] Serine proteases have a wide variety of uses. In addition to treating human diseases such as dissolving blood clots, they are also used as ingredients in laundry detergents and contact lens cleaners, as well as in the modification of milk proteins, degumming of silk fibers, soaking and unhairing of leather, synthesis of oligopeptides, recovery of silver from waste X-ray films, and the manufacture and improvement of feed and food (Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Korean Patent Publication No. 10-2005-0068750 [Patent Document 2] U.S. Patent No. 7,662,943 [Patent Document 3] U.S. Pat. No. 1,058,438 [Patent Document 4] U.S. Patent No. 10,273,491 Specification

Non-Patent Literature

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[0006] To ensure better industrial economy and efficacy, there is a need for serine proteases with improved thermostability, activity, etc. [Means for solving the problem]

[0007] An object of the present invention is to provide a serine protease mutant.

[0008] Another object of the present invention is to provide a polynucleotide encoding the serine protease mutant and a vector comprising the same.

[0009] A further object of the present invention is to provide a microorganism comprising at least one of the serine protease variant, a polynucleotide encoding the variant, and a vector comprising the polynucleotide.

[0010] A further object of the present invention is to provide a feed composition comprising at least one of the serine protease variant and a microorganism expressing the same. Effect of the Invention

[0011] The serine protease mutant of the present invention has superior activity compared to conventional serine proteases and is therefore industrially useful. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows the positions of mutated residues in the tertiary structure of a Thermobifida fusca serine protease mutant. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] These will be described in detail below. Note that each description and embodiment disclosed in the present invention is also applicable to other descriptions and embodiments. In other words, all combinations of various elements disclosed in the present invention are included in the present invention. In addition, the present invention is not limited to the following specific description.

[0014] Additionally, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the present invention.

[0015] One aspect of the present invention provides serine protease variants.

[0016] In the present invention, the term "serine protease" refers to an enzyme that belongs to a subgroup of proteases and has proteolytic activity. Specifically, serine proteases are enzymes that degrade proteins by hydrolyzing peptide bonds and basically have an active serine residue at the active site, more specifically, enzymes that have a spatial arrangement of histidine, aspartic acid, and serine, which are amino acid residues called the catalytic triad, but are not limited thereto.

[0017] The serine protease of the present invention is derived from, but is not limited to, a microorganism of the genus Thermobifida, Nocardiopsis, Actinorugispora, or Spinactinospora. Specifically, the wild-type serine protease in the present invention is a serine protease derived from Thermobifida fusca, Thermobifida cellulosilytica, Thermobifida halotolerans, Actinorugispora endophytica, Spinactinospora alkalitolerans, Nocardiopsis composta, or Nocardiopsis potens, but is not limited thereto.

[0018] By way of example, and not limitation, the serine protease of the present invention is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 31, a polypeptide consisting essentially of the amino acid sequence set forth in SEQ ID NO: 31, or a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 31. By way of example, and not limitation, the amino acid sequence of SEQ ID NO: 31 is an amino acid sequence derived from SEQ ID NO: 40 or SEQ ID NO: 2.

[0019] As an example, but not limited to, the serine protease of the present invention comprises any of the amino acid sequences set forth in SEQ ID NOs: 49 to 54, consists essentially of any of the amino acid sequences set forth in SEQ ID NOs: 49 to 54, or consists of any of the amino acid sequences set forth in SEQ ID NOs: 49 to 54. As an example, but not limited to, the amino acid sequences set forth in SEQ ID NOs: 49 to 54 are derived from any of the amino acid sequences set forth in SEQ ID NOs: 67 to 72.

[0020] The serine protease of the present invention may be any one that contains a sequence having the same activity as the above-mentioned amino acid sequence, and is not limited thereto, and may contain any one of the amino acid sequences of SEQ ID NO: 31 and 49 to 54, or an amino acid sequence having 60% or more homology or identity thereto, or may consist essentially of any one of the amino acid sequences of SEQ ID NO: 31 and 49 to 54, or an amino acid sequence having 60% or more homology or identity thereto, or may consist of any one of the amino acid sequences of SEQ ID NO: 31 and 49 to 54, or an amino acid sequence having 60% or more homology or identity thereto, Specifically, the amino acid sequence includes any of the amino acid sequences represented by SEQ ID NOs: 31 and 49 to 54, or an amino acid sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to any of the amino acid sequences represented by SEQ ID NOs: 31 and 49 to 54. Furthermore, it goes without saying that the present invention also includes proteins having an amino acid sequence in which part of the sequence has been deleted, modified, substituted or added, so long as the amino acid sequence has such homology or identity and exhibits efficacy equivalent to that of the above-mentioned protein.

[0021] That is, even if the present invention describes "a protein or polypeptide having an amino acid sequence represented by a specific SEQ ID NO" or "a protein or polypeptide comprising an amino acid sequence represented by a specific SEQ ID NO," it goes without saying that a protein having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted or added can be used in the present invention as long as it has the same or corresponding activity as a polypeptide consisting of the amino acid sequence of the SEQ ID NO. For example, it goes without saying that a "polypeptide comprising the amino acid sequence of SEQ ID NO: 31" belongs to the "polypeptide consisting of the amino acid sequence of SEQ ID NO: 31" as long as it has the same or corresponding activity.

[0022] As used herein, "homology" or "identity" refers to the degree to which two given amino acid or nucleotide sequences are related, and is expressed as a percentage. Homology and identity are often used interchangeably.

[0023] Sequence homology or identity of conserved polynucleotides or polypeptides may be determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences will generally hybridize over at least about 50%, 60%, 70%, 80% or 90% of the entire sequence or full length under moderate or high stringent conditions. Hybridization may also be such that the polynucleotide has degenerate codons in place of codons.

[0024] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms such as the "FASTA" program with default parameters as in, for example, Non-Patent Document 2. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 4), as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 3) (version 5.0.0 or later), which includes the GCG program package (Non-Patent Document 5), BLASTP, BLASTN, FASTA (Non-Patent Documents 6, 7 and 8). For example, BLAST or Clustal W from the National Center for Biotechnology Information can be used to determine homology, similarity or identity.

[0025] Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as that disclosed in, for example, J. Biol. 1999, 143:1311-1354 (2001). Briefly, the GAP program defines the number of similar sequence symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program include (1) a binary comparison matrix (identity takes a value of 1 and non-identity takes a value of 0) and a weighted comparison matrix (or EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in, (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap open penalty of 10 and a gap extension penalty of 0.5), and (3) no penalty for terminal gaps.

[0026] Additionally, whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be ascertained by comparing the sequences in a Southern hybridization experiment under defined stringent conditions, with suitable defined hybridization conditions being within the skill of the art and determined by methods well known to those of ordinary skill in the art.

[0027] As an example, the serine protease mutant provided by the present invention refers to a mutant in which an amino acid at a specific position in the protein having the above-mentioned serine protease activity is substituted, and the enzyme activity exceeds 100% of that of the protein before the mutation.

[0028] As a specific example, the mutant provided by the present invention has improved enzymatic activity of more than about 100% of that of a wild-type enzyme comprising any one of the amino acid sequences of SEQ ID NOs: 31 and 49 to 54, specifically about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190% or about 200% or more, but is not limited to these.

[0029] The term "about" refers to a range including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all numerical values ​​that are equal to or in a similar range to the numerical value following the term "about," but is not limited thereto.

[0030] By "variant" in the present invention is meant a polypeptide that differs from the recited sequences by at least one conservative substitution and / or modification of an amino acid, but which maintains the functions or properties of the protein. A variant differs from an identified sequence by a few amino acid substitutions, deletions or additions. Such variants can generally be identified by modifying one of the polypeptide sequences and assessing the properties of the modified polypeptide; i.e., the performance of the variant may be improved, unchanged or reduced compared to the native protein.

[0031] Some variants also include variants in which at least one portion has been deleted, such as the N-terminal leader sequence, the transmembrane domain, etc. Other variants include variants in which portions have been deleted or added to the N- and / or C-terminus of the mature protein.

[0032] The "mutant" may be an altered / mutated protein, a mutant polypeptide, or a mutation (in English, the term "modification," "modified protein," "modified polypeptide," "mutant," "mutein," "divergent," "variant," etc.), but any term meaning a mutation may be used.

[0033] The variants include, but are not limited to, variants in which the activity of the mutated protein is increased compared to the native wild-type or unmodified protein.

[0034] In the present invention, a "conservative substitution" refers to a substitution of an amino acid with another amino acid having similar structural and / or chemical properties. The variant has, for example, at least one conservative substitution while still retaining at least one biological activity. Such amino acid substitutions can generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids with electrically charged side chains include arginine, lysine, and histidine, negatively charged (acidic) amino acids include glutamic acid and aspartic acid, nonpolar amino acids with uncharged side chains include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline, polar or hydrophilic amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine, and aromatic amino acids include phenylalanine, tryptophan, and tyrosine. Variants may also include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the polypeptide may be linked to an N-terminal signal (or leader) sequence of a protein involved in co-translationally or post-translationally protein transfer, or to other sequences or linkers that allow the polypeptide to be identified, purified or synthesized.

[0035] The term "serine protease variant" as used herein means a polypeptide having at least one amino acid substitution in the amino acid sequence of a polypeptide having serine protease activity.

[0036] The serine protease variants of the present invention include those in which the amino acids at the positions corresponding to the 12th amino acid and / or the 116th amino acid from the N-terminus of SEQ ID NO: 31 are substituted with other amino acids. Specifically, the serine protease variants include amino acid sequences that have a substitution of the amino acid at the positions corresponding to the 12th amino acid and / or the 116th amino acid of SEQ ID NO: 31 and have 60% or more and less than 100% homology or identity to any of the amino acid sequences of SEQ ID NOs: 31 and 49 to 54.

[0037] As an example, the serine protease variant of the present invention has an amino acid substitution at a position corresponding to the 12th amino acid and / or the 116th amino acid of SEQ ID NO: 31, and has a homology or identity of 60% or more, for example, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more, but less than 100%, to the amino acid sequence of any of SEQ ID NOs: 31 and 49 to 54, but is not limited thereto.

[0038] On the other hand, the 12th and 116th amino acids from the N-terminus of SEQ ID NO: 31 correspond to the 12th and 116th amino acids from the N-terminus of SEQ ID NO: 49 to 54, and therefore the description of the amino acid positions based on SEQ ID NO: 31 also applies to the 12th and 116th amino acids of any of the amino acid sequences of SEQ ID NO: 49 to 54.

[0039] As an example, the serine protease variant of the present invention includes an amino acid sequence having an amino acid substitution at a position corresponding to the 12th amino acid and / or the 116th amino acid of SEQ ID NO:54, and having a homology or identity of 60% or more, for example, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more, but less than 100%, to the amino acid sequence of any of SEQ ID NOs:52 to 54. Specifically, the serine protease variant has an amino acid substitution at the position corresponding to the 12th amino acid of SEQ ID NO:54, and has a homology or identity of 60% or more, for example, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more but less than 100%, to the amino acid sequence of SEQ ID NO:54, but is not limited thereto.

[0040] As an example, the serine protease mutant of the present invention may be a protein in which all of the amino acids corresponding to the 12th position, the 116th position, or the 12th and 116th positions from the N-terminus in any one of the amino acid sequences selected from SEQ ID NOs: 31 and 49 to 54 are substituted with other amino acids. The "other amino acids" refer to amino acids different from those before substitution, and may be any amino acids other than the amino acid before substitution.

[0041] As an example, the serine protease mutant of the present invention is an amino acid sequence selected from SEQ ID NOs: 31 and 49 to 51, in which phenylalanine at the 12th position is replaced with glycine, alanine, arginine, aspartate, cysteine, glutamate, asparagine, glutamine, histidine, proline, serine, tyrosine, isoform, erythritol, arginine, cysteine, glutamic acid, isoform, erythritol, arginine, cysteine, glutamic acid, isoform, erythritol, erythritol, isoform ... and / or a substitution of asparagine at position 116 with glycine, alanine, arginine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, proline, serine, tyrosine, isoleucine, leucine, lysine, phenylalanine, tryptophan, valine, methionine or threonine, but is not limited to these.

[0042] As an example, the serine protease mutant of the present invention has an amino acid sequence selected from SEQ ID NOs: 52 to 54, in which proline at position 12 is substituted with phenylalanine, glycine, alanine, arginine, aspartate, cysteine, glutamate, asparagine, glutamine, histidine, serine, tyrosine, isopropylamino, or propylamino. and / or a substitution of asparagine at position 116 with glycine, alanine, arginine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, proline, serine, tyrosine, isoleucine, leucine, lysine, phenylalanine, tryptophan, valine, methionine or threonine, but is not limited to these.

[0043] Specifically, the mutant is a protein in which the amino acid corresponding to position 12 in any one of the amino acid sequences selected from SEQ ID NOs: 31 and 49 to 54 is substituted with tyrosine (Y), serine (S), alanine (A), or arginine (R), or the amino acid corresponding to position 116 is substituted with aspartic acid (D), serine (S), threonine (T), or glycine (G), or in which the amino acids corresponding to positions 12 and 116 in the amino acid sequence of SEQ ID NO: 31 are substituted with tyrosine (Y) and aspartic acid (D), tyrosine (Y) and serine (S), serine (S) and aspartic acid (D), serine (S) and threonine (T), or alanine (A) and glycine (G), respectively, but is not limited thereto. As an example, the serine protease mutant has an amino acid sequence selected from SEQ ID NOs: 52 to 54 in which the proline at the 12th position is replaced with tyrosine, alanine, serine, or arginine, but is not limited thereto.

[0044] Needless to say, a mutant in which the amino acid at the 12th and / or 116th position in any of the amino acid sequences selected from SEQ ID NOs: 31 and 49 to 54 is replaced with another amino acid includes a mutant in which the amino acid corresponding to the above positions is replaced with another amino acid.

[0045] The mutant also has an amino acid sequence selected from the above-mentioned SEQ ID NOs: 31 and 49 to 54, or a sequence similar to any of the amino acid sequences selected from SEQ ID NOs: 31 and 49 to 54 at least 60%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155 , 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more homology or identity, in which the amino acid at the position corresponding to the 12th amino acid and / or the 116th amino acid from the N-terminus of any amino acid sequence selected from SEQ ID NOs: 31 and 49 to 54 is replaced with another amino acid.

[0046] As an example, among the above-mentioned mutants, a mutant in which the amino acid at the position corresponding to the 12th amino acid and / or the 116th amino acid in the amino acid sequence of SEQ ID NO: 31 is replaced with another amino acid is an amino acid sequence represented by any one of SEQ ID NOs: 32 to 39. consisting essentially of, or It consists of, but is not limited to, these.

[0047] As an example, among the above mutants, a mutant in which the amino acid at the position corresponding to the 12th amino acid and / or the 116th amino acid in the amino acid sequence of SEQ ID NO: 49 is replaced with another amino acid is an amino acid sequence of SEQ ID NO: 55 or 56. consisting essentially of, or It consists of, but is not limited to, these.

[0048] As an example, among the above mutants, a mutant in which the amino acid at the position corresponding to the 12th amino acid and / or the 116th amino acid in the amino acid sequence of SEQ ID NO: 50 is replaced with another amino acid is an amino acid sequence of SEQ ID NO: 57 or 58. consisting essentially of, or It consists of, but is not limited to, these.

[0049] As an example, among the above mutants, a mutant in which the amino acid at the position corresponding to the 12th amino acid and / or the 116th amino acid in the amino acid sequence of SEQ ID NO: 51 is replaced with another amino acid is an amino acid sequence of SEQ ID NO: 59 or 60. consisting essentially of, or It consists of, but is not limited to, these.

[0050] As an example, among the above mutants, a mutant in which the amino acid at the position corresponding to the 12th amino acid and / or the 116th amino acid in the amino acid sequence of SEQ ID NO: 52 is replaced with another amino acid is an amino acid sequence of SEQ ID NO: 61 or 62. consisting essentially of, or It consists of, but is not limited to, these.

[0051] As an example, among the above mutants, a mutant in which the amino acid at the position corresponding to the 12th amino acid and / or the 116th amino acid in the amino acid sequence of SEQ ID NO: 53 is replaced with another amino acid is an amino acid sequence of SEQ ID NO: 63 or 64. consisting essentially of, or It consists of, but is not limited to, these.

[0052] As an example, among the above mutants, a mutant in which the amino acid at the position corresponding to the 12th amino acid and / or the 116th amino acid in the amino acid sequence of SEQ ID NO: 54 is replaced with another amino acid is an amino acid sequence of SEQ ID NO: 65 or 66. consisting essentially of, or It consists of, but is not limited to, these.

[0053] As an example, the serine protease variant of the present invention may have a substitution with another amino acid at a position corresponding to the 12th and / or 116th position of any amino acid sequence selected from SEQ ID NOs: 31 and 49 to 54, have a sequence homology of 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more but less than 100% to any amino acid sequence selected from SEQ ID NOs: 31 and 49 to 54, and have serine protease activity.

[0054] The serine protease variant of the present invention has enhanced activity compared to the polypeptide before mutation, the natural wild-type polypeptide, or the unmodified polypeptide, but is not limited thereto. It goes without saying that the present invention also includes proteins having an amino acid sequence in which a part of the sequence has been deleted, modified, substituted, or added, so long as the amino acid sequence has such homology and exhibits an efficacy equivalent to that of the above protein.

[0055] Furthermore, in addition to the mutations at the 12th and / or 116th amino acids, or mutations at positions corresponding thereto, meaningless sequence additions before or after the amino acid sequence of the SEQ ID NO:, naturally occurring mutations, or silent mutations thereof are not excluded, so long as they have the same or equivalent activity as the mutants of the present invention, and it goes without saying that those having such sequence additions or mutations are also included in the present invention.

[0056] On the other hand, the mature region of NCBI Reference Sequence WP_016188200.1 (SEQ ID NO: 40) corresponds to SEQ ID NO: 31 of the present invention, and the sequence obtained by removing only the signal peptide from SEQ ID NO: 40 corresponds to SEQ ID NO: 2 of the present invention.

[0057] As described above, the serine protease variant of the present invention may include deletion or addition of amino acids that have minimal effects on the properties and secondary structure of the serine protease, in which the amino acids at positions corresponding to positions 12 and 116 of SEQ ID NO: 31 are replaced with other amino acids. Furthermore, a person skilled in the art can understand by sequence alignment known in the art that positions 12 and 116 from the N-terminus of SEQ ID NO: 31 of the present invention correspond to positions 193 and 297 of SEQ ID NO: 40 and positions 163 and 267 of SEQ ID NO: 2, and can understand that SEQ ID NO: 31 is included in SEQ ID NO: 2 and SEQ ID NO: 40.

[0058] Thus, the serine protease mutants of the present invention include mutants in which amino acids at positions corresponding to positions 12 and 116 of SEQ ID NO: 31 (amino acids 163 and / or 267 of SEQ ID NO: 2, and amino acids 193 and / or 297 of SEQ ID NO: 40) are substituted in SEQ ID NO: 31, which contains the amino acid sequence of SEQ ID NO: 31. Furthermore, in the present invention, the details explained regarding SEQ ID NO: 31 and the amino acids at positions 12 and 116 also apply to SEQ ID NO: 2 and the amino acids at positions 163 and 267, and SEQ ID NO: 40 and the amino acids at positions 193 and 297.

[0059] As an example, the serine protease variant of the present invention may comprise an amino acid sequence in which the amino acids at positions corresponding to 12 and / or 116 of SEQ ID NO:31 are substituted with other amino acids, and may have a sequence identity of at least 60%, for example 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more, but less than 100%, to SEQ ID NO:2. As another example, the serine protease mutant of the present invention may have the amino acids at positions 163 and / or 267 of SEQ ID NO: 2 substituted with other amino acids and have a sequence homology of 60% or more but less than 100% with SEQ ID NO: 2, or may have a sequence homology of 60% or more with any amino acid sequence selected from SEQ ID NOs: 3 to 10. However, the mutant is not limited thereto.

[0060] On the other hand, it goes without saying that mutants having substitutions of amino acids at positions 12 and / or 116 from the N-terminus of SEQ ID NOs: 49 to 54 in a polypeptide comprising any of the amino acid sequences of SEQ ID NOs: 49 to 54 are also included within the scope of the serine proteases of the present invention.

[0061] The sequence of a polypeptide containing any of the amino acid sequences of SEQ ID NOs: 49 to 54 may be, for example, an amino acid sequence represented by GenBank Accession: KUP96625.1 (SEQ ID NO: 67), NCBI Reference Sequence: WP_068687914.1 (SEQ ID NO: 68), NCBI Reference Sequence: WP_133739400.1 (SEQ ID NO: 69), NCBI Reference Sequence: WP_179641868.1 (SEQ ID NO: 70), NCBI Reference Sequence: WP_184391208.1 (SEQ ID NO: 71), NCBI Reference Sequence: WP_017594871.1 (SEQ ID NO: 72), etc.

[0062] A person skilled in the art can confirm the amino acids in SEQ ID NOs: 67 to 72 that correspond to the 12th and / or 116th positions from the N-terminus of SEQ ID NOs: 49 to 54 by sequence alignment known in the art, and apply the description regarding the 12th and / or 116th positions from the N-terminus of SEQ ID NOs: 49 to 54.

[0063] As an example, the serine protease variant of the present invention may have a substitution of an amino acid corresponding to the 12th and / or 116th position from the N-terminus of the amino acid sequence of any of SEQ ID NOs: 49 to 54 with another amino acid, and may have a homology or identity of 60% or more, for example, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more to the amino acid sequence of any of SEQ ID NOs: 67 to 72.

[0064] As an example, the serine protease variant of the present invention may have an amino acid substitution corresponding to the 12th position based on the amino acid sequence represented by SEQ ID NO:54, and may have a homology or identity of 60% or more, for example, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more to the amino acid sequence represented by any one of SEQ ID NOs:70 to 72. As a specific example, the serine protease variant may further include a substitution of an amino acid corresponding to position 116 based on the amino acid sequence shown in SEQ ID NO:54.

[0065] As an example, a serine protease variant of the present invention may have a substitution of an amino acid corresponding to position 198 based on the amino acid sequence represented by SEQ ID NO: 67. The variant may have a homology or identity of 60% or more, for example, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more to SEQ ID NO: 67. For example, the mutant may have an amino acid sequence in which the amino acid at the position corresponding to the 12th amino acid of SEQ ID NO:49 is replaced with another amino acid and has a sequence identity of 70% or more with SEQ ID NO:49. As a specific example, the serine protease variant may further include a substitution of an amino acid at position 302 based on the amino acid sequence set forth in SEQ ID NO:67.

[0066] As an example, a serine protease variant of the present invention may have a substitution of an amino acid at position 178 based on the amino acid sequence represented by SEQ ID NO: 68. The variant may have a homology or identity of 60% or more, for example, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more to SEQ ID NO: 68. For example, the mutant may have an amino acid sequence in which the amino acid at the position corresponding to the 12th amino acid of SEQ ID NO:50 is replaced with another amino acid and has a sequence identity of 70% or more with SEQ ID NO:50. As a specific example, the serine protease variant may further include a substitution of an amino acid at position 282 based on the amino acid sequence set forth in SEQ ID NO:68.

[0067] As an example, a serine protease variant of the present invention may have a substitution of an amino acid corresponding to position 207 based on the amino acid sequence represented by SEQ ID NO: 69. The variant may have a homology or identity of 60% or more to SEQ ID NO: 69, for example, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more. For example, the mutant may have an amino acid sequence in which the amino acid at the position corresponding to the 12th amino acid in SEQ ID NO:51 is replaced with another amino acid and has a sequence identity of 70% or more with SEQ ID NO:51. As a specific example, the serine protease variant may further include a substitution of an amino acid at position 311 based on the amino acid sequence set forth in SEQ ID NO:69.

[0068] As an example, a serine protease variant of the present invention may have a substitution of an amino acid corresponding to position 203 based on the amino acid sequence represented by SEQ ID NO: 70. The variant may have a homology or identity of 60% or more to SEQ ID NO: 70, for example, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more. For example, the mutant may have an amino acid sequence in which the amino acid at the position corresponding to the 12th amino acid of SEQ ID NO:52 is replaced with another amino acid and has a sequence identity of 70% or more with SEQ ID NO:52. As a specific example, the serine protease variant may further include a substitution of an amino acid at position 303 based on the amino acid sequence set forth in SEQ ID NO:70.

[0069] As an example, a serine protease variant of the present invention may have a substitution of an amino acid corresponding to position 201 based on the amino acid sequence represented by SEQ ID NO: 71. The variant may have a homology or identity of 60% or more, for example, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more to SEQ ID NO: 71. For example, the mutant may have an amino acid sequence in which the amino acid at the position corresponding to the 12th amino acid of SEQ ID NO:53 is replaced with another amino acid and has a sequence identity of 70% or more with SEQ ID NO:53. As a specific example, the serine protease variant may further include a substitution of an amino acid at position 304 based on the amino acid sequence shown in SEQ ID NO:71.

[0070] As an example, a serine protease variant of the present invention may have a substitution of an amino acid corresponding to position 201 based on the amino acid sequence represented by SEQ ID NO: 72. The variant may have a homology or identity of 60% or more to SEQ ID NO: 72, for example, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more. For example, the mutant may have an amino acid sequence in which the amino acid at the position corresponding to the 12th amino acid of SEQ ID NO:54 is replaced with another amino acid and has a sequence identity of 70% or more with SEQ ID NO:54. As a specific example, the serine protease variant may further include a substitution of an amino acid at position 304 based on the amino acid sequence set forth in SEQ ID NO:72.

[0071] However, the present invention is not limited to these.

[0072] "Corresponding to" in the present invention means an amino acid residue at a recited position in a protein or polypeptide, or an amino acid residue similar, identical or equivalent to a recited residue in a protein or polypeptide. Identifying an amino acid at a corresponding position will determine the specific amino acid of the sequence to which the particular sequence refers. "Corresponding region" in the present invention generally means a similar or corresponding position in a related or reference protein. For example, based on aligning any amino acid sequence with SEQ ID NO: 31, each amino acid residue in the amino acid sequence can be numbered with reference to the number and position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 31. For example, the sequence alignment algorithm in the present invention can identify the position of the amino acid or the position where a modification such as a substitution, insertion, deletion occurs when compared to a query sequence (also called a "reference sequence").

[0073] For such alignment, the Needleman-Wunsch algorithm (Non-Patent Document 4), the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 3), etc. can be used, but are not limited to these. Also, corresponding amino acid residues can be identified by multiple sequence alignment. Examples of multiple sequence alignment programs known in the art include programs such as MUSCLE (multiple sequence comparison by log-expectation; version 3.5 or later; Non-Patent Document 12), MAFFT (version 6.857 or later; Non-Patent Document 13; Non-Patent Document 14; Non-Patent Document 15; Non-Patent Document 16; Non-Patent Document 17), and EMBOSS EMMA using Clustal W (1.83 or later; Non-Patent Document 18), and the default parameters of each of the above programs can be used, but are not limited to these.

[0074] Another aspect of the invention provides a polynucleotide encoding said serine protease variant.

[0075] In the present invention, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide monomers are linked in a long chain by covalent bonds, and is a DNA or RNA chain longer than a certain length, and more specifically, refers to a polynucleotide fragment encoding the mutant.

[0076] A polynucleotide encoding a serine protease variant of the present invention may be any polynucleotide sequence that encodes a serine protease variant having enhanced activity of the present invention. As an example, in the present invention, the gene encoding the wild-type serine protease is derived from a microorganism of the genus Thermobifida, Nocardiopsis, Actinorugispora, or Spinactinospora, specifically, Thermobifida fusca, Thermobifida cellulosilytica, Thermobifida halotolerans, Actinorugispora endophytica, Spinactinospora alkalitolerans, Nocardiopsis composta, Nocardiopsis potens, or the like. potens), but is not limited to these.

[0077] The polynucleotide of the present invention may be modified in various ways in the coding region within the scope of the amino acid sequence of the polypeptide, taking into consideration codon degeneracy or preferred codons in an organism in which the polypeptide is to be expressed. Specifically, any polynucleotide sequence may be used as long as it encodes a mutant in which the amino acid at the position corresponding to the 12th amino acid and / or the 116th amino acid from the N-terminus of any of the amino acid sequences selected from SEQ ID NOs: 31 and 49 to 54 is substituted with another amino acid.

[0078] For example, the polynucleotide of the present invention is a polynucleotide sequence that encodes a mutant of the present invention, specifically, a polypeptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 32 to 39 and SEQ ID NOs: 55 to 66, or a polypeptide having homology thereto, but is not limited to these.

[0079] As an example, a polynucleotide sequence encoding a polypeptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 32 to 39 is a polynucleotide sequence consisting of a polynucleotide sequence represented by any one of SEQ ID NOs: 41 to 48, but is not limited to these.

[0080] Furthermore, as described above, the serine protease mutants of the present invention include mutants in which amino acids at positions corresponding to the 12th and / or 116th amino acids in a polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 31 and 49 to 54 have been substituted, and it goes without saying that polynucleotide sequences encoding such serine protease mutants are also included in the present invention.

[0081] For example, variants in which amino acids at positions corresponding to 12th and / or 116th in SEQ ID NO:31 (amino acids at positions 163rd and / or 267th in SEQ ID NO:2, and amino acids at positions 193rd and / or 297th in SEQ ID NO:40) are substituted in SEQ ID NO:2 and SEQ ID NO:40 are also included in the scope of the serine protease of the present invention, and therefore polynucleotide sequences encoding them are also included in the present invention. For example, a polynucleotide sequence encoding the serine protease variant encodes an amino acid sequence represented by any one of SEQ ID NOs:3 to 10, and specifically, a polynucleotide sequence represented by any one of SEQ ID NOs:23 to 30, but is not limited thereto.

[0082] Furthermore, the sequence may be any sequence that, when hybridized under stringent conditions with a probe prepared from a known gene sequence, for example a sequence complementary to all or a part of the base sequence, encodes a protein having the activity of a mutant in which the amino acid at the position corresponding to the 12th amino acid and / or the 116th amino acid from the N-terminus selected from SEQ ID NOs: 31 and 49 to 54 is substituted with another amino acid.

[0083] The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in known literature. For example, the conditions include conditions under which genes with high homology, 40% or more, specifically 90% or more, more specifically 95% or more, even more specifically 97% or more, and particularly specifically 99% or more, hybridize with each other, and genes with lower homology do not hybridize with each other, or conditions under which washing is performed once, specifically 2 to 3 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, more specifically 68°C, 0.1×SSC, 0.1% SDS, which are normal washing conditions for Southern hybridization. However, the conditions are not limited to these, and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0084] Hybridization requires that two polynucleotides have complementary sequences, even though mismatches between bases are possible depending on the stringency of hybridization. "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. Thus, the present invention may include isolated polynucleotide fragments that are complementary to the entire sequence, as well as substantially similar polynucleotide sequences.

[0085] Specifically, the homologous polynucleotides can be detected using the above-mentioned hybridization conditions, in which the hybridization step is performed at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and may be appropriately adjusted by those skilled in the art depending on the purpose.

[0086] The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementation, variables known in the art.

[0087] Yet another aspect of the present invention provides a vector comprising a polynucleotide encoding the serine protease variant of the present invention.

[0088] In the present invention, the term "vector" refers to a DNA product containing a base sequence of a polynucleotide encoding a target protein operably linked to a suitable regulatory sequence so that the target protein can be expressed in a suitable host. The regulatory sequence includes a promoter for initiating transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating the termination of transcription and translation. When transformed into a suitable host cell, the vector can replicate or function independently of the host genome, or may be integrated into the genome itself.

[0089] The term "operably linked" means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target protein of the present invention. The operable linkage can be prepared using recombinant gene technology known in the art, and the site-specific DNA cleavage and ligation can be prepared using cleavage and ligation enzymes known in the art, but is not limited thereto.

[0090] The vector used in the present invention is not particularly limited, and any vector known in the art can be used. Examples of vectors that are commonly used include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used as phage vectors or cosmid vectors, and pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, pET series, pUB110 series, etc. can be used as plasmid vectors. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pSM704 vectors, etc. can be used. The vector that can be used in the present invention is not particularly limited, and any known expression vector can be used.

[0091] As an example, a polynucleotide encoding a target mutant in a chromosome can be replaced with a mutated polynucleotide by a vector for intracellular chromosome insertion. The polynucleotide can be inserted into a chromosome by any method known in the art, such as, but not limited to, homologous recombination. A selection marker for confirming whether or not the target nucleic acid molecule has been inserted into the chromosome may be further included. The selection marker is used to select cells transformed with the vector, i.e., to confirm whether or not the target nucleic acid molecule has been inserted, and a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface mutant polypeptide is used. In an environment treated with a selection agent, only cells expressing the selection marker survive or show a different phenotype, so that transformed cells can be selected.

[0092] Yet another aspect of the present invention provides a host cell comprising at least one of the serine protease variants of the present invention, a polynucleotide encoding said variant, and a vector comprising said polynucleotide.

[0093] Said host cell is in particular a microorganism.

[0094] A microorganism containing at least one of the serine protease mutant, the polynucleotide encoding the mutant, and the vector containing the same is specifically, but not limited to, a microorganism produced by transformation with a vector containing a polynucleotide encoding the mutant.

[0095] The microorganism may be a microorganism expressing a serine protease variant.

[0096] In the present invention, "to be expressed" a protein means that the target protein is introduced into a microorganism or modified to be expressed in the microorganism. For the purposes of the present invention, the "target protein" may be the serine protease variant described above.

[0097] Specifically, "introduction of a protein" means making the activity of a specific protein that the microorganism does not originally have appear, or making the activity of the protein appear to be improved compared to the endogenous activity or the activity before modification. For example, it may be that a polynucleotide encoding a specific protein is introduced into a chromosome in a microorganism, or that a vector containing a polynucleotide encoding a specific protein is introduced into a microorganism to make the activity appear.

[0098] The microorganism of the present invention may be a recombinant microorganism. The recombination may be achieved by genetic modification such as transformation.

[0099] In the present invention, "transformation" means expressing a protein encoded by a polynucleotide in a host cell by introducing a vector containing the polynucleotide encoding the target protein into the host cell. The transformed polynucleotide may be any polynucleotide that is expressed in the host cell, regardless of whether it is inserted into the chromosome of the host cell or located extrachromosomally. The polynucleotide may include DNA or RNA that encodes the target protein. The polynucleotide may be introduced in any form as long as it is 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 structure containing all elements necessary for its own expression. Typically, the expression cassette contains a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. The polynucleotide may be introduced into the host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not limited thereto. The transformation method may be any method for introducing a polynucleotide into a cell, and may be performed by selecting a standard technique suitable for the host cell, as known in the art, such as, but not limited to, electroporation, calcium phosphate (Ca(H2PO4)2, CaHPO4, or Ca3(PO4)2) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, natural competence (see, for example, Non-Patent Document 19), lithium acetate-DMSO method, etc.

[0100] The recombinant microorganism may have enhanced serine protease activity of the present invention.

[0101] The term "enhanced activity" means that the activity is improved compared to the endogenous activity of a specific protein possessed by a microorganism or the activity before modification. The term "endogenous activity" means the activity of a specific protein that a parent strain originally had before the trait change when the trait of a microorganism is changed due to genetic mutation caused by natural or artificial factors.

[0102] Specifically, in the present invention, the activity of the protein variant is enhanced by at least one of the following methods: increasing the intracellular copy number of a gene encoding the protein variant; introducing a mutation into an expression regulatory sequence of a gene encoding the protein variant; substituting an expression regulatory sequence of a gene encoding the protein variant with a sequence having stronger activity; replacing a gene encoding a natural protein having serine protease activity on a chromosome with a gene encoding the protein variant; and further introducing a mutation into the gene encoding the variant so that the activity of the protein variant is enhanced, but the methods are not limited to these.

[0103] Next, the method of modifying the expression control sequence to increase the expression of the polynucleotide can be, but is not limited to, by inducing a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of the nucleic acid sequence so that the activity of the expression control sequence is further enhanced, or by replacing it with a nucleic acid sequence having stronger activity. The expression control sequence can include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating the termination of transcription and translation, and the like.

[0104] A strong promoter is linked upstream of the polynucleotide expression unit in place of the original promoter, but is not limited thereto. Examples of known strong promoters include cj1 to cj7 promoters (Patent Document 2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, P LExamples of such promoters include, but are not limited to, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13 (sm3) promoter (Patent Document 3), the O2 promoter (Patent Document 4), the tkt promoter, and the yccA promoter.

[0105] Furthermore, the method of modifying a polynucleotide sequence on a chromosome is not particularly limited to the above, but can be performed by inducing a mutation in an expression control sequence by deletion, insertion, non-conservative or conservative substitution of a nucleic acid sequence, or a combination thereof, so that the activity of the polynucleotide sequence is further enhanced, or by replacing it with an improved polynucleotide sequence so as to have stronger activity.

[0106] Such introduction and enhancement of protein activity generally means, but is not limited to, an increase in the activity or concentration of the corresponding protein by at least 1%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to 1000% or 2000%, compared to the activity or concentration of the protein in a wild-type or unmodified microbial strain.

[0107] The host cell or microorganism of the present invention may be any microorganism that contains the polynucleotide of the present invention or the vector of the present invention and expresses the serine protease variant. Specifically, the present invention includes microbial strains of the genera Escherichia, Serratia, Erwinia, Enterobacteria, Providencia, Salmonella, Streptomyces, Pseudomonas, Brevibacterium, Corynebacterium, and Bacillus, and more specifically, Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus velezensis, Escherichia coli, and the like. Examples of suitable strains of the bacteria include, but are not limited to, strains of Bacillus subtilis, Corynebacterium glutamicum, and Aspergillus oryzae, and more specifically, strains of Bacillus subtilis.

[0108] Yet another aspect of the present invention provides a method for producing the serine protease variants of the present invention.

[0109] A method for producing a variant of the invention may comprise culturing a microorganism comprising at least one of a serine protease variant of the invention, a polynucleotide encoding said variant, and a vector comprising said polynucleotide.

[0110] In the present invention, "culturing" means growing the host cell under appropriately adjusted environmental conditions. The culturing process of the present invention can be carried out in a suitable medium and under suitable culture conditions known in the art. Such a culturing process can be easily adjusted by a person skilled in the art depending on the strain selected. Specifically, the culturing can be batch, continuous, or fed-batch culture, but is not limited thereto.

[0111] In the present invention, the term "medium" refers to a mixture of nutrients necessary for culturing the host cells as the main components, and supplies nutrients such as water essential for survival and growth, growth factors, etc. Specifically, the medium and other culture conditions used for culturing the host cells of the present invention may be any medium used for culturing ordinary host cells, and the host cells of the present invention can be cultured in an ordinary medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids and / or vitamins under aerobic conditions by controlling the temperature, pH, etc.

[0112] By way of example, the method for producing a variant of the invention may further comprise the step of recovering the variant of the invention expressed in said culturing step.

[0113] Alternatively, the variant expressed during the culturing step can be recovered using methods well known in the art, for example, the variant can be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, and precipitation.

[0114] The recovery may involve collecting the mutant using a suitable method known in the art depending on the culture method of the host cell of the present invention, such as batch, continuous, or fed-batch culture. For example, centrifugation, filtration, crystallization, treatment with a protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatographies such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, and combinations thereof may be used to recover the mutant from the medium or host cell using a suitable method known in the art.

[0115] As yet another example, the variant expressed by the host cells during the culturing step may not be recovered - in this example, the host cells expressing the variant may themselves be used as the source of the variant.

[0116] Yet another aspect of the present invention provides a feed composition comprising at least one of the serine protease variants of the present invention and a microorganism expressing same.

[0117] The serine protease variant contained in the feed composition of the present invention may be in a form that contains the microorganism that expresses it, or in a form that has been separated and purified from the microorganism that expresses it, but is not limited to these.

[0118] A "feed composition" in the present invention is any natural or artificial diet, meal, etc., or component of said meal, intended or suitable for consumption, ingestion and digestion by an animal, and the feed can be manufactured into various forms of feed known in the art.

[0119] The feed composition may be a feed additive.

[0120] The type of the feed is not particularly limited, and feeds commonly used in the technical field can be used. Examples of the feed include vegetable feeds such as grains, nuts, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, meals, and grain by-products, and animal feeds such as proteins, inorganic substances, oils and fats, minerals, single-cell proteins, animal plankton, and food and drink, but are not limited thereto. These can be used alone or in combination of two or more types.

[0121] The feed composition of the present invention may further contain one or more selected from the group consisting of organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid, etc.; phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphates (polymerized phosphates), etc.; polyphenols, catechin, α-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid, etc.

[0122] The feed composition of the present invention may further comprise one or more selected from the group consisting of supplementary ingredients such as amino acids, inorganic salts, vitamins, antibiotics, antibacterial substances, antioxidants, antifungal enzymes, other live microbial preparations, etc.; cereals such as crushed or crushed wheat, oats, barley, corn and rice, vegetable protein feeds such as those based on rapeseed, beans and sunflower, animal protein feeds such as blood meal, meat meal, bone meal and fish meal, dry ingredients consisting of sugars and dairy products such as various milk powders and whey powders; main ingredients such as lipids, for example animal fats or vegetable fats optionally liquefied by heating; and additives such as nutritional supplements, digestive and absorption promoters, growth promoters and disease preventive agents.

[0123] The feed composition of the present invention may be in a dry or liquid form and may contain a feed additive excipient, such as, but not limited to, zeolite, corn meal, rice bran, etc.

[0124] The feed composition of the present invention may further contain an enzyme preparation other than the serine protease variant. For example, it may further contain one or more selected from the group consisting of lipase and other fat-decomposing enzymes, phytase that decomposes phytic acid to produce phosphate and inositol phosphate, amylase that is an enzyme that hydrolyzes α-1,4-glycoside bonds contained in potato starch, glycogen, etc., phosphatase that is an enzyme that hydrolyzes organic phosphate esters, maltase that hydrolyzes maltose into two glucose molecules, and conversion enzymes that hydrolyze sucrose to produce a glucose-fructose mixture. However, the present invention is not limited to these.

[0125] The feed compositions of the present invention may be administered to animals alone or in combination with other feed additives in an edible carrier. Also, the feed compositions can be readily administered as feed additives, either as top dressings or by mixing them directly with livestock feed, or in separate oral dosage forms, or in combination with other ingredients. Furthermore, single daily intakes or divided daily intakes can be used, as is well known in the art.

[0126] Examples of animals for which the feed composition of the present invention can be used include livestock such as beef cattle, dairy cows, calves, pigs, piglets, sheep, goats, horses, rabbits, dogs, and cats, and poultry such as chicks, egg-laying hens, chickens, roosters, ducks, geese, turkeys, quails, and small birds, but are not limited to these.

[0127] The amount of the serine protease variant of the present invention contained in the feed composition of the present invention is not particularly limited and may be appropriately adjusted depending on the purpose. For example, as is well known in the technical field to which the present invention pertains, the amount of the serine protease variant of the present invention may be an amount suitable for surviving for a long period of time in the digestive tract of livestock and decomposing protein source materials, but is not limited thereto.

[0128] Yet another aspect of the present invention provides a food composition comprising at least one of the serine protease variants of the present invention and a microorganism expressing the same. The protease protein can be used in a liquid or solid food composition. The food can also be a powder, pill, beverage, tea or general food additive.

[0129] For example, the food may be a food group that requires proteases, such as dairy products, functional foods for bowel movement or dieting, and functional foods for preventing high blood pressure.

[0130] As another example, the protease variants may be included in various food compositions and used as food solubilizers, softeners, or meat quality modifiers. As yet another example, the protease variants may be added to bread-making processes and used in the step of breaking down gluten networks. Alternatively, the protease variants may be used for hydrolysis of food proteins (e.g., proteins in milk). Alternatively, the protease variants may be included in various food compositions and used for rendering, flavoring, bitterness reduction, emulsification property change, bioactive peptide production, and protein allergy-inducing antigen reduction, but these are merely examples and are not limited to the above uses.

[0131] The amount of the serine protease variant in the food composition can be appropriately adjusted by those skilled in the art depending on the purpose.

[0132] Yet another aspect of the present invention provides a detergent composition comprising at least one of the serine protease variants of the present invention and a microorganism expressing same.

[0133] The detergent compositions according to the present invention may be one-part and two-part aqueous detergent compositions, non-aqueous liquid detergent compositions, cast solids, granular forms, particulate forms, compressed tablets, gels, pastes or slurries. The detergent compositions may be used for removing food soils, food stains and other minor food compositions.

[0134] The detergent compositions according to the present invention may be provided in the form of, but are not limited to, hard surface cleaning compositions, fabric cleaning compositions, dishwashing detergent compositions, mouthwash compositions, denture cleaners, contact lens cleaning solutions, and the like.

[0135] Yet another aspect of the present invention provides a pharmaceutical composition comprising at least one of the serine protease variants of the present invention and a microorganism expressing same.

[0136] The pharmaceutical composition of the present invention is used as a pharmaceutical composition for digestive enzymes for improving digestive diseases, digestive disorders, and abnormal diseases after digestive surgery; a thrombolytic agent or antithrombotic composition that acts directly on thrombi 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 that relieves edema after surgery or trauma.

[0137] The pharmaceutical composition may further include a pharma- ceutically or nutritionally acceptable carrier, excipient, diluent, or accessory ingredient depending on the method and purpose of use. The carrier, excipient, or diluent is at least one selected from the group consisting of lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin, and physiological saline, but is not limited thereto.

[0138] In addition to the uses mentioned above, the serine protease variants of the present invention or microorganisms expressing the same can be used in other applications, such as cosmetics, leather processing, medicines, diagnostic agents, waste management, and the production of chemical agents for academic research, etc. However, the above uses are merely examples, and the variants can be used in any other application related to the denaturation, decomposition, or removal of protein substances known in the art. EXAMPLES

[0139] The present invention will be described in more detail below with reference to examples and experimental examples. However, these examples and experimental examples are merely illustrative of the present invention, and the present invention is not limited to these examples and experimental examples. EXAMPLES

[0140] Selection of mutants of serine proteases from Thermobifida fusca Example 1-1: Construction of a serine protease library derived from Thermobifida fusca Random mutations were introduced by error-prone PCR into the gene encoding the amino acid sequence (SEQ ID NO: 31) corresponding to the mature region of the serine protease derived from Thermobifida fusca. TM A PCR Random Mutagenesis Kit (Clontech, Cat# 630703) was used. The PCR conditions are shown in Table 1. When performed under the following conditions, it was confirmed that mutations were inserted at a frequency of 6.2 mutations / Kb.

[0141] [Table 1]

[0142] The PCR fragment obtained in the above process was ligated to a vector amplified with the primers shown in Table 2 using an In-Fusion® HD cloning kit (Clontech), and then transformed into DH5α to obtain colonies. Plasmids were purified from the obtained colonies, and approximately 5 × 10 4 We secured a library of sizes.

[0143] [Table 2]

[0144] Example 1-2: Screening of Thermobifida fusca-derived serine protease library The protease library prepared in Example 1-1 was transformed into Bacillus subtilis LB700 strain, which is easy to secrete proteins, and screening was performed. Screening was performed in two steps. In the first step, the Bacillus subtilis strain transformed with the library was spread on a 2% skim milk plate, and then a selection method was used based on the size of the halo formed. Bacillus subtilis transformation was performed according to the Groningen method. The composition of the skim milk plate used for screening is shown in Table 3.

[0145] [Table 3] (in 1L)

[0146] In the second step, the colonies selected in the first step were selected by azocasein coloration. A 96-deep well plate was filled with a BHI (Brain Heart Infusion, bd, cat#53286) liquid medium containing kanamycin antibiotic, and the colonies selected in the first step were inoculated and cultured at 37°C for 20-24 hours. After the culture, the supernatant containing the enzyme was obtained by centrifugation, and the supernatant was mixed with the substrate 2% (w / v) azocasein in equal amounts, and then reacted at 37°C for 1 hour. The reaction was stopped by adding 10% TCA (Trichloro acetic acid) to the enzyme reaction solution in a three-fold amount, and the coagulated protein was removed by centrifugation. The same amount of NaOH was mixed to perform a color reaction, and the degree of coloration was compared by measuring the absorbance at 440 nm. In this process, colonies with an absorbance increased by 150% or more compared to the wild-type serine protease were selected. EXAMPLES

[0147] Production and activity evaluation of selected mutants Example 2-1: Construction of mutants As a result of sequence analysis of the mutants selected by screening, it was finally confirmed that the 12th amino acid (phenylalanine, Phe) and the 116th amino acid (asparagine, Asn) of SEQ ID NO: 31 were replaced with tyrosine (Tyr) and aspartic acid (Asp), respectively. Based on the amino acid sequence shown in SEQ ID NO:2, The positions of the above mutations are shown. The two selected mutations (F12, N116) were reintroduced into the pBE-S-TAP plasmid in single mutant form by site directed mutagenesis, and the activities of the double and single mutant forms were then compared with the wild type activity. The primers used to generate the mutants are shown in Table 4.

[0148] [Table 4]

[0149] Example 2-2: Activity evaluation The plasmid prepared as described above was transformed into Bacillus subtilis LB700 strain and expressed, and then activity was evaluated using N-SUCCINYL-ALA-ALA-PRO-PHE-P-NITROANILIDE (Sigma, cat#S7388, hereafter referred to as SUC-AAPF-pNA) peptide as a substrate. The transformed Bacillus subtilis strain was inoculated into a kanamycin antibiotic-containing BHI medium and cultured at 37°C for 20 to 24 hours, after which the cells were removed and a portion of the culture solution was mixed with 25 mM Tris-HCl (pH 7.5) buffer and 1 mM Suc-AAPF-pNA, and then reacted at 37°C for 30 minutes. The absorbance of the reaction solution described above was measured at 410 nm. The extinction coefficient of the enzyme product, para-nitroaniline, as reported in the literature is 8,800 M at 410 nm. -1 cm -1 The enzyme units were calculated based on this (Non-Patent Document 20). The measured activities are shown in Table 5.

[0150] [Table 5]

[0151] As a result of the measurements, it was confirmed that under conditions of pH 7.5 and 37°C, the F12Y and F12YN116D mutations had activities that were approximately 2.1-fold and 3.9-fold increased compared to the wild type, respectively.

[0152] Example 2-3: Evaluation of thermal stability To confirm the effect of introducing the mutation on thermostability, the following experiment was carried out.

[0153] Specifically, the same samples as those used in the activity evaluation in Example 2-2 were allowed to stand for 5 minutes at room temperature, 70° C., 80° C., and 90° C., and then the enzyme activity was measured. The measured activities are shown in Table 6.

[0154] [Table 6]

[0155] As a result of the measurements, it was confirmed that the F12Y and F12YN116D mutations retained approximately two and four times the enzyme activity of the wild type, respectively, even at 80° C. Thus, it was confirmed that the serine protease mutants of the present invention retain high activity even at high temperatures, and are therefore industrially useful. EXAMPLES

[0156] Construction and selection of saturation mutagenesis libraries Example 3-1. Construction of F12, N116 residue saturation mutation library As described above, the N116 residue of the selected mutant, F12, was replaced with a residue other than tyrosine or aspartic acid, and a saturation mutation library of the two residues was prepared to confirm the effect on activity.

[0157] Using pBE-S-TAP as a template, two PCR fragments were obtained using primers of SEQ ID NOs: 11 and 12, and primers of SEQ ID NOs: 13 and 14, respectively, and ligated using the In-Fusion HD cloning kit. Then, the fragments were transformed into DH5α to obtain colonies. Plasmids were purified from the obtained colonies, and approximately 4 × 10 3 We secured a library of sizes.

[0158] [Table 7]

[0159] Example 3-2: Screening and activity evaluation of saturation mutation library The saturation mutation library prepared in Example 3-1 was screened in the same manner as in Example 1-2. Mutants with the same or increased activity as the F12YN116D mutant were selected through screening, and the mutants were subjected to sequence analysis and activity evaluation using Suc-AAPF-pNA as a substrate.

[0160] [Table 8]

[0161] As a result of checking, it was found that the activity increased when the F12 and N116 residues were substituted with other amino acids, such as F12S, F12A, F12R, N116S, N116T, and N116G, in addition to tyrosine and aspartic acid confirmed in Example 2.

[0162] Example 3-3: Preparation and activity evaluation of F12S mutant The mutant (F12S) was reintroduced in a single mutant form into the pBE-S-TAP plasmid by site directed mutagenesis, and the activity of the mutant was then evaluated in comparison with that of the wild type.

[0163] The activity of this mutant (F12S) was evaluated using Suc-AAPF-pNA as a substrate.

[0164] [Table 9]

[0165] As a result of the measurement, it was confirmed that the activity of the F12S mutant was increased by approximately 1.5 times. EXAMPLES

[0166] Confirmation of the effect of residues 12 and 116 of the serine protease-like protein from Thermobifida fusca Example 4-1: Generation of wild type and mutant In order to confirm whether the amino acid residues corresponding to the 12th and 116th residues of SEQ ID NO:31 affect the increase in activity in other serine proteases having sequence homology with SEQ ID NO:31, the 12th and 116th residues of serine proteases having sequence homology of 87.2%, 81.8%, 81.3%, 73.8%, 69.9%, and 66.7%, respectively, were replaced with tyrosine (Y) and aspartic acid (D), and the activity was then compared with that of the wild type. The origin and sequence information of each serine protease are shown. 10 As shown in.

[0167] [Table 10]

[0168] The 12th and 116th residues of each serine protease were substituted with tyrosine (Y) and aspartic acid (D) to prepare mutants represented by SEQ ID NOs: 55 to 66.

[0169] Example 4-2: Activity evaluation The plasmid prepared as described above was transformed into Bacillus subtilis LB700 strain and expressed, and then the activity was evaluated in the same manner as in Example 2-2. The measured activity is shown in Table 1. 11 As shown in.

[0170] [Table 11]

[0171] As a result of the measurement, it was confirmed that the activity of the Thermobifida fusca serine protease as well as six proteins that have sequence homology with it increased when a mutation was introduced into the 12th residue.

[0172] Thus, it has been confirmed that residues 12 and 116 are important residues for serine protease enzyme activity, and that the enzyme activity is increased by substituting these residues with other amino acids, as confirmed in SEQ ID NO: 31. Thus, the serine protease mutants of the present invention can increase enzyme activity and are industrially useful.

[0173] From the above description, a person skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical ideas or essential features. It should be understood that the above examples are merely illustrative and not limiting. The present invention should be interpreted as including all modifications and alterations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

[0174] The present disclosure relates, for example, to the following: [1] A serine protease variant having an amino acid substitution at the 12th position based on the amino acid sequence shown in SEQ ID NO:54, and comprising an amino acid sequence having 70% or more but less than 100% homology or identity to the amino acid sequence shown in SEQ ID NO:54. [2] The serine protease mutant according to [1] above, further comprising a substitution of an amino acid at position 116 based on the amino acid sequence shown in SEQ ID NO:54. [3] The serine protease mutant according to [1] above, which has a homology or identity of 75% or more but less than 100% with the amino acid sequence shown in SEQ ID NO:54. [4] The serine protease mutant according to [1] above, wherein the amino acid corresponding to the 12th position is substituted with a hydrophilic amino acid, a nonpolar amino acid, or a basic amino acid. [5] The serine protease mutant according to [1] above, wherein the amino acid at the 12th position is substituted with tyrosine (Y), alanine (A), serine (S), or arginine (R). [6] The serine protease variant according to [1] above, which has an amino acid substitution at position 201 based on the amino acid sequence shown in SEQ ID NO: 71 or 72. [7] The serine protease mutant according to [1] above, which has an amino acid substitution at position 203 based on the amino acid sequence shown in SEQ ID NO:70. [8] The serine protease mutant according to [1] above has a sequence homology of at least 60% but less than 100% with an amino acid sequence represented by any one of SEQ ID NOs: 70 to 72. [9] The serine protease mutant according to [2] above, wherein the amino acid corresponding to position 116 is substituted with a hydrophilic amino acid, a nonpolar amino acid, or an acidic amino acid.

[10] The serine protease mutant according to [2] above, in which the amino acid at position 116 is substituted with aspartic acid (D), serine (S), threonine (T), or glycine (G).

[11] A composition comprising any one of the serine protease variants [1] to

[10] above.

[12] A polynucleotide encoding any one of the serine protease mutants [1] to

[10] above.

[13] A vector comprising the polynucleotide of

[12] .

[14] A host cell comprising at least one of the serine protease mutants according to any one of [1] to

[10] above, a polynucleotide encoding the mutant, and a vector comprising the polynucleotide.

[15] A composition comprising at least one of the serine protease variants according to any one of [1] to

[10] above and a microorganism expressing the same.

Claims

1. A protein mutant having serine protease activity, which has an amino acid substitution corresponding to the 12th position based on the amino acid sequence represented by SEQ ID NO:54, and which comprises an amino acid sequence having an identity of 90% or more but less than 100% with the amino acid sequence represented by SEQ ID NO:54, in which the amino acid corresponding to the 12th position is substituted with tyrosine (Y).

2. 2. The protein mutant having serine protease activity according to claim 1, wherein the mutant has an amino acid substitution corresponding to the 201st position based on the amino acid sequence represented by SEQ ID NO:

72.

3. The protein mutant having serine protease activity according to claim 1 , wherein the mutant has at least 90% but less than 100% identity with the amino acid sequence represented by SEQ ID NO:

72.

4. A protein mutant having serine protease activity, comprising an amino acid sequence having an amino acid substitution corresponding to the 12th position based on the amino acid sequence represented by SEQ ID NO:53, and having an identity of 90% or more but less than 100% with the amino acid sequence represented by SEQ ID NO:53, in which the amino acid corresponding to the 12th position is substituted with tyrosine (Y).

5. A protein mutant having serine protease activity as described in claim 4, wherein the mutant has an amino acid substitution corresponding to the 201st position based on the amino acid sequence represented by SEQ ID NO:

71.

6. A protein mutant having serine protease activity as described in claim 4, wherein the mutant has at least 90% but less than 100% identity with the amino acid sequence represented by SEQ ID NO:

71.

7. A protein mutant having serine protease activity, comprising an amino acid sequence having an amino acid substitution corresponding to the 12th position based on the amino acid sequence represented by SEQ ID NO:52, and having an identity of 90% or more but less than 100% with the amino acid sequence represented by SEQ ID NO:52, in which the amino acid corresponding to the 12th position is substituted with tyrosine (Y).

8. A protein mutant having serine protease activity as described in claim 7, wherein the mutant has an amino acid substitution corresponding to the 203rd position based on the amino acid sequence represented by SEQ ID NO:

70.

9. A protein mutant having serine protease activity as described in claim 7, wherein the mutant has at least 90% but less than 100% identity with the amino acid sequence represented by SEQ ID NO:

70.

10. A composition comprising a protein variant having serine protease activity according to any one of claims 1 to 9.

11. A polynucleotide encoding the protein variant having serine protease activity according to any one of claims 1 to 9.

12. A vector comprising the polynucleotide of claim 11.

13. A host cell comprising at least one of a protein variant having serine protease activity according to any one of claims 1 to 9, a polynucleotide encoding said variant, and a vector comprising said polynucleotide.

14. A composition comprising at least one of the protein variants having serine protease activity according to any one of claims 1 to 9 and a microorganism expressing the same.

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