JPWO2022191653A5 - - Google Patents

Description

The present invention relates to a novel serine protease mutant.

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 their active sites. Among them, serine proteases (or serine endopeptidases) are characterized by having an active serine residue in common mainly in the active site, and are enzymes in which serine acts as a nucleophilic amino acid in the active site of the protease, cleaving peptide bonds in proteins (Non-Patent Document 1).

Serine proteases have a wide range of uses, including the treatment of human diseases such as dissolving blood clots, as well as being used as ingredients in laundry detergents and contact lens cleaners, as well as in the modification of milk proteins, degumming silk fibers, soaking and unhairing leather, synthesizing oligopeptides, recovering silver from waste X-ray films, and producing and improving feed and food (Patent Document 1).

Korean Patent Publication No. 10-2005-0068750 U.S. Pat. No. 7,662,943 U.S. Pat. No. 1,058,438 U.S. Pat. No. 1,027,3491

Hedstrom, 2002. Chem Rev 102: 4501-4524 Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444 Rice et al., 2000, Trends Genet. 16: 276-277 Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 Devereux, J., et al., Nucleic Acids Research 12: 387 (1984) Atschul, S., F., ET AL, J MOLEC BIOL 215: 403 (1990) Guide to Huge Computers, Martin J. Bishop, [Ed.,] Academic Press, San Diego,1994 [CARILLO ETA/.](1988) SIAM J Applied Math 48: 1073 Smith and Waterman, Adv. Appl. Math (1981) 2:482 Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979) Gribskov et al. (1986) Nucl. Acids Res. 14: 6745 Edgar, 2004, Nucleic Acids Research 32: 1792-1797 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 Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680 Perry and Kuramitsu, 1981, Infect. Immun. 32: 1295-1297 Barrett, A. J., Cathepsin G. Methods Enzymol., 80, Pt. C, 561-565, (1981)

To ensure better industrial economics and efficacy, there is a demand for serine proteases with improved thermostability, activity, etc.

The present invention aims to provide a serine protease mutant.

The present invention also aims to provide a polynucleotide encoding the serine protease mutant and a vector containing the polynucleotide.

Furthermore, the present invention aims to provide a microorganism containing at least one of the serine protease mutant, a polynucleotide encoding the mutant, and a vector containing the polynucleotide.

Furthermore, the present invention aims to provide a feed composition containing at least one of the serine protease variants and a microorganism expressing the same.

The serine protease variant of the present invention has superior activity compared to conventional serine proteases and is therefore industrially useful.

FIG. 2 shows the positions of mutated residues in the tertiary structure of a Thermobifida fusca-derived serine protease mutant.

These will be described in detail below. Note that each description and embodiment disclosed in the present invention also applies to the other descriptions and embodiments. In other words, the present invention includes all combinations of the various elements disclosed in the present invention. Furthermore, the present invention is not limited to the specific descriptions below.

Moreover, those of ordinary skill 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.

One aspect of the present invention provides a serine protease variant.

In the present invention, "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 to these.

The serine protease of the present invention is derived from a microorganism of the genus Thermobifida, Nocardiopsis, Actinorugispora, or Spinactinospora, but is not limited thereto. 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.

As an example, the serine protease of the present invention is a polypeptide comprising the amino acid sequence represented by SEQ ID NO:31, a polypeptide consisting essentially of the amino acid sequence represented by SEQ ID NO:31, or a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:31, but is not limited thereto. As an example, 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, but is not limited thereto.

By way of example, the serine protease of the present invention includes, consists essentially of, or consists of an amino acid sequence of any one of SEQ ID NOs: 49-54, but is not limited thereto. By way of example, the amino acid sequence of any one of SEQ ID NOs: 49-54 is derived from, but is not limited to, an amino acid sequence of any one of SEQ ID NOs: 67-72.

The serine protease of the present invention may be any one that contains a sequence having the same activity as the amino acid sequence described above. In addition, the serine protease 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, but is not limited 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 a portion 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 protein.

In other words, 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 containing 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 containing 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.

In the present invention, "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.

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 length under moderate or high stringent conditions. Hybridization may be such that the polynucleotide has degenerate codons in place of codons.

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 versions), 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.

Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as GAP (1999) (1999) (1999) (1999). 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, non-identity a value of 0) and a weighted comparison matrix (or EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in GAP (1999) (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, gap extension penalty of 0.5), and (3) no penalty for terminal gaps.

In addition, 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 skill in the art.

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 of a protein having the above-mentioned serine protease activity is substituted, and the enzyme activity exceeds 100% of that of the protein before the mutation.

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

The term "about" refers to a range that includes ±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 to these.

In the present invention, a "variant" refers to a polypeptide that differs from the recited sequences by conservative substitution and/or modification of at least one amino acid, but 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 evaluating the properties of the modified polypeptide; that is, the performance of the variant may be improved, unchanged or reduced compared to the native protein.

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

The term "mutant" may refer to a modified/mutated protein, mutant polypeptide, or mutation (in English, modification, modified protein, modified polypeptide, mutant, mutein, divergent, variant, etc.), but any term that means a mutation may be used.

The mutants include, but are not limited to, mutant proteins that have increased activity compared to the native wild-type or unmodified protein.

In the present invention, a "conservative substitution" refers to the replacement 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 may 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. The polypeptide may also be linked to other sequences or linkers that allow the polypeptide to be identified, purified, or synthesized.

In the present invention, a "serine protease variant" refers to a polypeptide having at least one amino acid substitution in the amino acid sequence of a polypeptide having serine protease activity.

The serine protease variants of the present invention include those 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 SEQ ID NO: 31 is replaced with another amino acid. Specifically, the serine protease variants include amino acid sequences that have a substitution of the amino acid at the position 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 with any of the amino acid sequences of SEQ ID NOs: 31 and 49 to 54.

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 any of the amino acid sequences of SEQ ID NOs:31 and 49 to 54, but is not limited thereto.

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, so the contents described regarding the positions of amino acids based on SEQ ID NO:31 also apply to the 12th and 116th amino acids of any of the amino acid sequences of SEQ ID NO:49 to 54.

As an example, the serine protease variants of the present invention include amino acid sequences having substitutions of amino acids at positions 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, e.g., 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 any of the amino acid sequences 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.

As an example, the serine protease variant 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 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.

As an example, the serine protease variant 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 substituted with glycine, alanine, arginine, aspartate, cysteine, glutamate, asparagine, glutamine, histidine, proline, serine, tyrosine, or isoform. Substitutions of isoleucine, leucine, lysine, tryptophan, valine, methionine, or threonine and/or 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 are included, but are not limited to these.

As an example, the serine protease variant of the present invention is an amino acid sequence selected from SEQ ID NOs: 52 to 54, in which the proline at the 12th position is substituted with phenylalanine, glycine, alanine, arginine, aspartate, cysteine, glutamate, asparagine, glutamine, histidine, serine, tyrosine, or isoloamine. Substitutions of isoleucine, leucine, lysine, tryptophan, valine, methionine, or threonine and/or 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 are included, but are not limited to these.

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

Needless to say, in any of the amino acid sequences selected from SEQ ID NOs: 31 and 49 to 54, mutants in which the amino acids at positions 12 and/or 116 are replaced with other amino acids include mutants in which the amino acids at the above positions are replaced with other amino acids.

In addition, the mutant has an amino acid sequence represented by any one of SEQ ID NOs: 31 and 49 to 54, or an amino acid sequence selected from SEQ ID NOs: 31 and 49 to 54 that is 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% or 99% or more homology or identity, and 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 replaced with another amino acid.

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: 31 is replaced with another amino acid comprises, essentially consists of, or consists of an amino acid sequence represented by any one of SEQ ID NOs: 32 to 39, but is not limited thereto.

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 comprises, essentially consists of, or consists of the amino acid sequence of SEQ ID NO: 55 or 56, but is not limited to these.

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 comprises, essentially consists of, or consists of the amino acid sequence of SEQ ID NO:57 or 58, but is not limited to these.

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:51 is replaced with another amino acid comprises, essentially consists of, or consists of the amino acid sequence of SEQ ID NO:59 or 60, but is not limited thereto.

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 comprises, essentially consists of, or consists of the amino acid sequence of SEQ ID NO:61 or 62, but is not limited to these.

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 comprises, essentially consists of, or consists of the amino acid sequence of SEQ ID NO:63 or 64, but is not limited to these.

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 comprises, essentially consists of, or consists of the amino acid sequence of SEQ ID NO:65 or 66, but is not limited to these.

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, and 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% with any amino acid sequence selected from SEQ ID NOs: 31 and 49 to 54, and have serine protease activity.

The serine protease mutant 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 efficacy equivalent to that of the protein.

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 corresponding 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.

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.

As described above, the serine protease variant of the present invention may include deletions or additions 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.

Therefore, the serine protease variants of the present invention include variants in which the amino acids at positions corresponding to the 12th and 116th positions of SEQ ID NO:31 (the 163rd and/or 267th amino acids in SEQ ID NO:2, and the 193rd and/or 297th amino acids in SEQ ID NO:40) in SEQ ID NO:31, which contain the amino acid sequence of SEQ ID NO:31, have been substituted. Furthermore, in the present invention, the contents described for SEQ ID NO:31 and the 12th and 116th amino acids also apply to SEQ ID NO:2 and the 163rd and 267th amino acids, and SEQ ID NO:40 and the 193rd and 297th amino acids.

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 replaced with other amino acids, and may have a sequence identity of at least 60%, e.g., 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 variant 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 sequence homology of 60% or more but less than 100% with SEQ ID NO:2, or may have sequence homology of 60% or more with any amino acid sequence selected from SEQ ID NOs:3 to 10. However, the variant is not limited to these.

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

The sequence of a polypeptide containing any one of the amino acid sequences of SEQ ID NOs: 49 to 54 can be, for example, 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_184391208.1 (SEQ ID NO: 72), NCBI Reference Sequence: WP_184391208.1 (SEQ ID NO: 73), NCBI Reference Sequence: WP_184391208.1 (SEQ ID NO: 74), NCBI Reference Sequence: WP_184391208.1 (SEQ ID NO: 75), NCBI Reference Sequence: WP_184391208.1 (SEQ ID NO: 76), NCBI Reference Sequence: WP_184391208.1 (SEQ ID NO: 77), NCBI Reference Sequence: WP_184391208.1 (SEQ ID NO: 78), NCBI Reference Sequence: WP_184391208.1 (SEQ ID NO: 79), NCBI Reference Sequence: WP_184391208.1 (SEQ ID NO: 80), NCBI Reference Sequence: WP_184391208.1 (SEQ ID NO: 81), NCBI Reference Sequence: WP_184391208.1 (SEQ ID NO: 82), NCBI Reference It may be an amino acid sequence represented by Sequence:WP_017594871.1 (SEQ ID NO:72), etc.

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

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 any of the amino acid sequences of SEQ ID NOs: 49-54 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 any of the amino acid sequences of SEQ ID NOs: 67-72.

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 60% or more homology or identity, 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 homology or identity to the amino acid sequence represented by any 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 represented by SEQ ID NO:54.

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 variant may comprise an amino acid sequence in which the amino acid at the position corresponding to the 12th amino acid in SEQ ID NO: 49 is substituted with another amino acid and has 70% or more sequence identity with SEQ ID NO: 49. As a specific example, the serine protease variant may further comprise a substitution of an amino acid at the 302nd position based on the amino acid sequence represented by SEQ ID NO: 67.

As an example, a serine protease variant of the present invention may have a substitution of an amino acid corresponding to position 178 based on the amino acid sequence represented by SEQ ID NO: 68. The variant may have 60% or more homology or identity to SEQ ID NO: 68, 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 variant may comprise an amino acid sequence in which the amino acid at the position corresponding to amino acid position 12 of SEQ ID NO: 50 is substituted with another amino acid and has 70% or more sequence identity with SEQ ID NO: 50. As a specific example, the serine protease variant may further comprise a substitution of an amino acid at position 282 based on the amino acid sequence represented by SEQ ID NO:68.

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, 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: 69. For example, the variant may comprise 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 which has 70% or more sequence identity with SEQ ID NO: 51. As a specific example, the serine protease variant may further comprise a substitution of an amino acid at the 311th position based on the amino acid sequence represented by SEQ ID NO: 69.

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 60% or more homology or identity 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 variant may comprise an amino acid sequence in which the amino acid at the position corresponding to the 12th amino acid in SEQ ID NO: 52 is replaced with another amino acid and which has 70% or more sequence identity with SEQ ID NO: 52. As a specific example, the serine protease variant may further comprise a substitution of an amino acid at the 303rd position based on the amino acid sequence represented by SEQ ID NO: 70.

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 variant may comprise an amino acid sequence in which the amino acid at the position corresponding to the 12th amino acid in SEQ ID NO: 53 is substituted with another amino acid and has 70% or more sequence identity with SEQ ID NO: 53. As a specific example, the serine protease variant may further comprise a substitution of an amino acid at the 304th position based on the amino acid sequence represented by SEQ ID NO: 71.

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, 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: 72. For example, the variant may comprise an amino acid sequence in which the amino acid at the position corresponding to the 12th amino acid in SEQ ID NO: 54 is substituted with another amino acid and has 70% or more sequence identity with SEQ ID NO: 54. As a specific example, the serine protease variant may further comprise a substitution of an amino acid at the 304th position based on the amino acid sequence represented by SEQ ID NO: 72.

However, this is not limited to these.

In the present invention, "corresponding to" refers to an amino acid residue at a recited position in a protein or polypeptide, or an amino acid residue similar, identical or corresponding to a recited residue in a protein or polypeptide. Identifying an amino acid at a corresponding position determines the specific amino acid in the sequence that references the particular sequence. In the present invention, "corresponding region" generally refers to a similar or corresponding position in a related or reference protein. For example, an arbitrary amino acid sequence can be aligned with SEQ ID NO: 31, and based thereon, 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").

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. Corresponding amino acid residues can also 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.

Another aspect of the present invention provides a polynucleotide encoding the serine protease variant.

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, a DNA or RNA chain longer than a certain length, and more specifically, a polynucleotide fragment that codes for the mutant.

The polynucleotide encoding the 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, from Thermobifida fusca, Thermobifida cellulosilytica, Thermobifida halotolerans, Actinorugispora endophytica, Spinactinospora alkalitolerans, Nocardiopsis composta, or Nocardiopsis potens, but is not limited thereto.

The polynucleotide of the present invention may be modified in various ways in the coding region, taking into consideration codon degeneracy or preferred codons in the organism in which the polypeptide is to be expressed, as long as the amino acid sequence of the polypeptide is not changed. 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 replaced with another amino acid.

For example, the polynucleotide of the present invention is a polynucleotide sequence that encodes a variant 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 thereto.

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 any one of SEQ ID NOs: 41 to 48, but is not limited thereto.

As described above, the serine protease variants of the present invention include variants in which the amino acids at the 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 are substituted, and it goes without saying that polynucleotide sequences encoding such serine protease variants are also included in the present invention.

For example, variants in which the amino acids at positions corresponding to the 12th and/or 116th amino acids of SEQ ID NO:31 (the 163rd and/or 267th amino acids in SEQ ID NO:2, and the 193rd and/or 297th amino acids 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 proteases of the present invention, and therefore the polynucleotide sequences encoding them are also included in the present invention. For example, the 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.

Also, any sequence may be used as long as it hybridizes under stringent conditions with a probe prepared from a known gene sequence, for example, a sequence complementary to all or part of the base sequence, to encode 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 of any of SEQ ID NOs: 31 and 49 to 54 is replaced with another amino acid.

The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in known literature. For example, conditions in 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, are hybridized with each other, and genes with lower homology are not hybridized with each other, or conditions in which washing is performed once, specifically 2 to 3 times, at a salt concentration and temperature equivalent to 60°C, 1xSSC, 0.1% SDS, specifically 60°C, 0.1xSSC, 0.1% SDS, which are the washing conditions for normal Southern hybridization, specifically 68°C, 0.1xSSC, 0.1% SDS. However, the conditions are not limited to these and can be appropriately adjusted by a person skilled in the art depending on the purpose.

Hybridization requires that two polynucleotides have complementary sequences, even though mismatches between bases are possible depending on the stringency of the hybridization. "Complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, in DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Thus, the invention may include isolated polynucleotide fragments that are complementary to an entire sequence, as well as substantially similar polynucleotide sequences.

Specifically, a polynucleotide having homology can be detected using the hybridization conditions described above, 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 can be appropriately adjusted by a person skilled in the art depending on the purpose.

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

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

In the present invention, a "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 as to enable expression of the target protein 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.

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

The vector used in the present invention is not particularly limited, and any vector known in the art may 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, pUC, pBluescriptII, pGEM, pTZ, pCL, pET, pUB110, etc. can be used as plasmid vectors. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pSM704 vectors, etc. can be used. There are no particular limitations on the vectors that can be used in the present invention, and any known expression vector can be used.

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 it 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.

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 the variant, and a vector comprising the polynucleotide.

The host cell is specifically a microorganism.

The 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.

The microorganism may be a microorganism that expresses a serine protease variant.

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

Specifically, "introduction of a protein" means causing the activity of a specific protein that the microorganism does not originally possess to appear, or causing the activity of the protein to appear that is improved compared to the endogenous activity of the protein or the activity before modification. For example, it may mean introducing a polynucleotide encoding a specific protein into a chromosome in a microorganism, or introducing a vector containing a polynucleotide encoding a specific protein into a microorganism to cause this activity to appear.

The microorganism of the present invention may be a recombinant microorganism. The recombination may be performed by genetic modification such as transformation.

In the present invention, "transformation" means expressing a protein encoded by a polynucleotide in a host cell by introducing a vector containing a polynucleotide encoding a 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(H ₂ PO ₄ ) ₂ , CaHPO ₄ or Ca ₃ (PO ₄ ) ₂ ) precipitation, calcium chloride (CaCl ₂ ) 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.

The recombinant microorganism may have enhanced serine protease activity according to the present invention.

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. "Endogenous activity" means the activity of a specific protein that was originally possessed by the parent strain before the trait change when the trait of a microorganism changes due to genetic mutation caused by natural or artificial factors.

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

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.

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

In addition, the method of modifying a polynucleotide sequence on a chromosome is not particularly limited to the following: to further enhance the activity of the polynucleotide sequence, a mutation in the expression regulatory sequence can be induced by deletion, insertion, non-conservative or conservative substitution of the nucleic acid sequence, or a combination thereof, or by replacing the polynucleotide sequence with an improved polynucleotide sequence that has stronger activity.

Introduction and enhancement of such protein activity generally means, but is not limited to, that the activity or concentration of the corresponding protein is increased 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.

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 a 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. coli), Corynebacterium glutamicum, Aspergillus oryzae, and more specifically, Bacillus subtilis, but are not limited to these.

Yet another aspect of the present invention provides a method for producing the serine protease variant of the present invention.

The method for producing the mutant of the present invention may include a step of culturing a microorganism containing at least one of the serine protease mutant of the present invention, a polynucleotide encoding the mutant, and a vector containing the polynucleotide.

In the present invention, "culturing" means growing the host cells 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 and used by a person skilled in the art depending on the selected strain. Specifically, the culturing is batch, continuous, or fed-batch culture, but is not limited thereto.

In the present invention, the term "culture medium" refers to a mixture of nutrients necessary for culturing the host cells, including water, which are essential for survival and growth, and growth factors. Specifically, the culture medium and other culture conditions used for culturing the host cells of the present invention may be any medium used for culturing normal host cells, and the host cells of the present invention can be cultured under aerobic conditions in a normal culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and/or vitamins, by adjusting the temperature, pH, etc.

As an example, the method for producing a mutant of the present invention may further include a step of recovering the mutant of the present invention expressed in the culturing step.

In another example, the variants expressed during the culturing step can be recovered using methods well known in the art. For example, the variants can be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, and precipitation.

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.

In yet another example, the variant expressed by the host cells during the culturing step may not need to be recovered. In this example, the host cells expressing the variant may themselves be used as the source of the variant.

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 the same.

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

In the present invention, a "feed composition" is any natural or artificial diet, meal, etc., or components 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.

The feed composition may be a feed additive.

The type of feed is not particularly limited, and feeds commonly used in the relevant technical field can be used. Examples of the feed include, but are not limited to, 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, zooplankton, and food and drink. These can be used alone or in a mixture of two or more types.

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., and natural antioxidants.

The feed composition of the present invention may further comprise one or more of the following additives: supplemental ingredients such as amino acids, inorganic salts, vitamins, antibiotics, antibacterial substances, antioxidants, antifungal enzymes, and other live microbial preparations; 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 such as sugar and dairy products such as various milk powders and whey powders; lipids such as 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.

The feed composition of the present invention may be in a dry or liquid form and may contain a feed additive excipient. Examples of the feed additive excipient include, but are not limited to, zeolite, corn meal, and rice bran.

The feed composition of the present invention may further contain an enzyme preparation other than the serine protease variant. For example, the composition 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, an enzyme that hydrolyzes α-1,4-glycoside bonds contained in potato starch, glycogen, etc., phosphatase, an enzyme that hydrolyzes organic phosphate esters, maltase that hydrolyzes maltose into two glucose molecules, and a conversion enzyme that hydrolyzes sucrose to produce a glucose-fructose mixture. However, the present invention is not limited to these.

The feed compositions of the present invention may be administered to animals alone or in combination with other feed additives in an edible carrier. Additionally, the feed compositions may 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. Additionally, single daily intakes or divided daily intakes may be used, as is well known in the art.

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

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 is adjusted appropriately depending on the purpose. As an example, as is well known in the technical field to which the present invention pertains, the variant is contained in an amount suitable for surviving for a long period of time in the digestive tract of livestock and breaking down protein source materials, but is not limited thereto.

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.

As an 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.

As another example, the protease variant may be included in various food compositions and used as a food solubilizer, softener, or meat quality modifier. As yet another example, the protease variant may be added to a bread-making process and used in a step to break down the gluten network. Alternatively, the protease variant may be used for hydrolysis of food proteins (e.g., proteins in milk). Alternatively, the protease variant may be included in various food compositions and used for rendering, flavoring, bitterness reduction, emulsification property change, bioactive peptide production, and reduction of protein allergens, but these are merely examples and are not limited to the above uses.

The amount of serine protease variant in the food composition can be adjusted as appropriate by those skilled in the art depending on the purpose.

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 the same.

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 slurry forms. The detergent compositions may be used for removing food soils, food stains and other minor food compositions.

The detergent compositions according to the present invention may be provided in the form of, but are not limited to, a hard surface cleaning detergent composition, a fabric cleaning detergent composition, a kitchen detergent composition, a mouthwash composition, a denture cleaner, a contact lens cleaning solution, and the like.

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 the same.

The pharmaceutical composition of the present invention is used as a pharmaceutical composition for digestive enzymes to improve digestive diseases, digestive disorders, and abnormal diseases after digestive surgery, as a thrombolytic agent or antithrombotic composition that acts directly on thrombi to dissolve fibrin, as an anti-inflammatory agent that acts as an in vivo defense system to remove inflammatory substances or necrotic tissue, or as an anti-inflammatory agent that reduces edema after surgery or trauma.

The pharmaceutical composition may further comprise 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 saline, but is not limited thereto.

The serine protease variants of the present invention or microorganisms expressing the same are used in applications other than those mentioned above, such as cosmetics, leather processing, pharmaceuticals, diagnostic agents, waste management, and the manufacture of chemical agents for academic research. However, the above applications are merely examples, and the variants can be used in any application related to the denaturation, decomposition, or removal of protein substances known in the art.

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.

Selection of Thermobifida fusca-derived serine protease mutants Example 1-1: Preparation of Thermobifida fusca-derived serine protease library Random mutations were introduced by error-prone PCR into a gene encoding amino acids (SEQ ID NO: 31) corresponding to the mature region of Thermobifida fusca-derived serine protease. Diversify ^™ PCR Random Mutagenesis Kit (Clontech, Cat# 630703) was used for error-prone PCR. PCR conditions are shown in Table 1. It was confirmed that mutations were inserted at a frequency of 6.2 mutations/Kb when performed under the following conditions.

The PCR fragment obtained in the above process was ligated to a vector amplified with the primers shown in Table 2 using In-Fusion® HD cloning kit (Clontech), and then transformed into DH5α to obtain colonies. Plasmids were purified from the resulting colonies to obtain a library of approximately 5× ¹⁰⁴ in size.

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.

(in 1L)

In the second step, the colonies selected in the first step were selected by azocasein coloring. 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) in a three-fold amount to the enzyme reaction solution, and the coagulated protein was removed by centrifugation. The same amount of NaOH was mixed to perform a coloring reaction, and the degree of coloring 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.

Preparation and activity evaluation of selected mutants Example 2-1: Preparation 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. Figure 1 shows the positions of the above mutations based on the amino acid sequence shown in SEQ ID NO: 2. The two selected mutations (F12, N116) were reintroduced into the pBE-S-TAP plasmid in single mutation form by site directed mutagenesis, and the activities of the double mutation and single mutation forms were then compared and evaluated with the activity of the wild type. The primers used in the preparation of the mutants are shown in Table 4.

Example 2-2: Activity evaluation The plasmid prepared as described above was transformed into Bacillus subtilis LB700 strain and expressed, and then activity evaluation was performed using N-SUCCINYL-ALA-ALA-PRO-PHE-P-NITROANILIDE (Sigma, cat#S7388, hereinafter referred to as SUC-AAPF-pNA) peptide as a substrate. The transformed Bacillus subtilis strain was inoculated into a BHI medium containing kanamycin antibiotic and cultured at 37°C for 20 to 24 hours, after which the cells were removed and a part 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, reported in the literature is 8,800 M ^-1 cm ^-1 at 410 nm, and the enzyme units were calculated based on this (Non-Patent Document 20). The measured activities are shown in Table 5.

The results showed that at pH 7.5 and 37°C, the F12Y and F12YN116D mutations increased activity by approximately 2.1 and 3.9 times, respectively, compared to the wild type.

Example 2-3: Evaluation of thermostability In order to confirm the effect of introducing a mutation on thermostability, the following experiment was carried out.

Specifically, the same sample that was used in the activity evaluation in Example 2-2 was left 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.

The measurement results 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. Therefore, it was confirmed that the serine protease mutant of the present invention retains high activity even at high temperatures, making it industrially useful.

Example 3-1. Construction of a saturation mutagenesis library of F12 and N116 residues As described above, the N116 residue of F12, a mutant selected as above, was substituted with a residue other than tyrosine or aspartic acid, and a saturation mutagenesis library of two residues was constructed to confirm the effect on activity.

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 a library of approximately 4 x ¹⁰³ in size was obtained.

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 by screening, and the mutants were subjected to sequence analysis and activity evaluation using Suc-AAPF-pNA as a substrate.

The results showed that activity increased when the F12 and N116 residues were replaced with other amino acids, such as F12S, F12A, F12R, N116S, N116T, and N116G, in addition to the tyrosine and aspartic acid confirmed in Example 2.

Example 3-3: Production 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 assessed in comparison with that of the wild type.

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

As a result of the measurement, it was confirmed that the activity of the F12S mutant was increased by about 1.5 times.

Confirmation of the effect of the 12th and 116th residues of the serine protease-like protein derived from Thermobifida fusca Example 4-1: Preparation of wild type and mutants 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 in Table 10 .

The 12th and 116th residues of each serine protease were replaced with tyrosine (Y) and aspartic acid (D) to create mutants of SEQ ID NOs: 55 to 66.

Example 4-2: Activity evaluation The plasmid prepared as described above was transformed into Bacillus subtilis LB700 strain and expressed, and then activity evaluation was performed in the same manner as in Example 2-2. The measured activities are shown in Table 11 .

The results of the measurements confirmed that, like the Thermobifida fusca serine protease, the activity of six proteins that share sequence homology with it increased when a mutation was introduced into the 12th residue.

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

From the above description, a person skilled in the art of the present invention 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 construed as including all modifications and alterations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

The present disclosure relates, for example, to the following:
[1]
A serine protease variant having an amino acid substitution corresponding to the 12th position based on the amino acid sequence represented by 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 represented by SEQ ID NO:54.
[2]
The serine protease mutant according to [1] above, further comprising an amino acid substitution at position 116 based on the amino acid sequence represented by 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 represented by SEQ ID NO:54.
[4]
The serine protease mutant according to the above-mentioned [1], 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 corresponding to 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 the 201st position based on the amino acid sequence represented by SEQ ID NO: 71 or 72.
[7]
The serine protease variant according to [1] above, which has an amino acid substitution at position 203 based on the amino acid sequence represented by SEQ ID NO:70.
[8]
The serine protease variant 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 at 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, wherein the amino acid at position 116 is substituted with aspartic acid (D, aspartate), serine (S, serine), threonine (T, threonine), or glycine (G, glycine).
[11]
A composition comprising the serine protease variant according to any one of [1] to [10] above.
[12]
A polynucleotide encoding any one of the serine protease mutants according to [1] to [10] above.
[13]
A vector comprising the polynucleotide of [12].
[14]
A host cell comprising at least one of the serine protease mutant 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 (13)

A protein mutant having serine protease activity, which has an amino acid substitution corresponding to the 12th position based on the amino acid sequence shown in SEQ ID NO:54, and which comprises an amino acid sequence having an identity of 60 % or more but less than 100% with the amino acid sequence shown in SEQ ID NO:54 , in which the amino acid corresponding to the 12th position is substituted with tyrosine (Y) . The protein mutant having serine protease activity according to claim 1 , wherein the mutant has an identity of 75% or more but less than 100% with the amino acid sequence represented by SEQ ID NO:54. The protein mutant having serine protease activity according to claim 1 , wherein the mutant has an identity of 80 % or more but less than 100% with the amino acid sequence represented by SEQ ID NO:54. The protein mutant having serine protease activity according to claim 1 , wherein the mutant has an identity of 85% or more but less than 100% with the amino acid sequence represented by SEQ ID NO:54. The protein mutant having serine protease activity according to claim 1 , wherein the mutant has an identity of 90% or more but less than 100% with the amino acid sequence represented by SEQ ID NO:54. 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: 71 or 72. 2. The protein mutant having serine protease activity according to claim 1, wherein the mutant has an amino acid substitution corresponding to position 203 based on the amino acid sequence represented by SEQ ID NO:70. The protein mutant having serine protease activity according to claim 1, wherein the mutant has an identity of at least 60% but less than 100% with an amino acid sequence represented by any one of SEQ ID NOs: 70 to 72. A composition comprising a protein variant having serine protease activity according to any one of claims 1 to 8 . A polynucleotide encoding a protein variant having serine protease activity according to any one of claims 1 to 8 . A vector comprising the polynucleotide of claim 10 . A host cell comprising at least one of a protein variant having serine protease activity according to any one of claims 1 to 8 , a polynucleotide encoding said variant, and a vector comprising said polynucleotide. A composition comprising at least one of the protein variants having serine protease activity according to any one of claims 1 to 8 and a microorganism expressing the same.