Novel serine protease mutants

Abstract

The present application relates to novel serine protease variants.

Description

[Technical field]

[0001] The present application 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, and 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). However, in order to ensure better industrial economy and efficacy, there is a demand for serine proteases with improved thermal stability, activity, etc. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Korean Patent Publication No. 10-2005-0068750 [Patent Document 2] Republic of Korea Patent No. 10-0620092 [Patent Document 3] Korea Registration No. 10-1783170 [Patent Document 4] Korea Registration Patent No. 10-1632642

Non-licensed documents

[0005]

Non-patent document 1

Non-patent document 2

Non-patent document 3

Non-patent document 4

Non-patent document 5

Non-patent document 6

Non-patent document 7

Non-patent document 8

Non-patent document 9

Non-patent document 10

[0006] The present inventors have developed a novel mutant of a serine protease derived from Thermobifida fusca and confirmed that the activity of the mutant is significantly superior to that of existing serine proteases, thereby completing the present application. [Means for solving the problem]

[0007] The present application aims to provide serine protease variants.

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

[0009] A further object of the present application is to provide a microorganism expressing the serine protease variant.

[0010] A further object of the present application 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 variant of the present application has superior activity as well as high-temperature activity and heat resistance compared to existing serine proteases, and is therefore industrially useful. [Brief description of the drawings]

[0012] [Figure 1] This shows the positions of the mutated residues in the tertiary structure of the 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 this application also applies to other descriptions and embodiments. In other words, all combinations of various elements disclosed in this application are included in this application. In addition, this application 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 present application described herein which equivalents are intended to be encompassed by this application.

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

[0016] In the present application, 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 essentially have an active serine residue at the active site, and more specifically, may be enzymes that have a spatial arrangement of histidine, aspartic acid, and serine, which are amino acid residues called the catalytic triad.

[0017] The serine protease of the present application may be derived from a microorganism of the genus Thermobifida. Specifically, the wild-type serine protease of the present application may be a serine protease derived from Thermobifida fusca, specifically, an amino acid sequence derived from SEQ ID NO: 40, an amino acid sequence derived from SEQ ID NO: 2, or more specifically, a polypeptide including the amino acid sequence represented by SEQ ID NO: 31, but is not limited thereto. The amino acid sequence of SEQ ID NO: 2, 31, or 40 can be obtained from GenBank of NCBI, a publicly known database.

[0018] The serine protease of the present application may be any one that contains a sequence having the same activity as the amino acid sequence. It may also contain the amino acid sequence of SEQ ID NO: 31 or an amino acid sequence having 80% or more homology or identity thereto, but is not limited thereto. Specifically, the amino acids may contain amino acids represented by SEQ ID NO: 31 or amino acids having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to the above-mentioned SEQ ID NO: 31. It goes without saying that the present application 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 or identity and shows an effect equivalent to that of the above-mentioned protein.

[0019] That is, even if the present application 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 also be used in the present application, so 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" if it has the same or corresponding activity.

[0020] By "homology" or "identity" in this application is meant the degree to which two given amino acid or nucleotide sequences are related, expressed as a percentage. Homology and identity are often used interchangeably.

[0021] 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 over the entire length under moderate or high stringent conditions. Hybridization also takes into account polynucleotides in which the polynucleotide contains degenerate codons in place of codons.

[0022] 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 (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 3) (version 5.0.0 or later) of the EMBOSS package (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.

[0023] 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.

[0024] 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.

[0025] The serine protease variant provided by the present application means a variant in which an amino acid at a specific position in the above-mentioned protein having serine protease activity is substituted, and the enzyme activity is more than 100% of that of the protein before the mutation.

[0026] By "variant" in this application is meant a polypeptide that differs from the recited sequences by at least one conservative substitution and / or modification of an amino acid, but that 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; i.e., the performance of the variant may be improved, unchanged or decreased compared to the native protein.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] In the present application, "conservative substitution" means that an amino acid is replaced with another amino acid having similar structural and / or chemical properties. The variant has, for example, at least one conservative substitution while still having at least one biological activity. Such amino acid substitutions can generally be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; aromatic amino acids include phenylalanine, tryptophan, and tyrosine, and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Amino acids can also be classified as those with electrically charged side chains and those with uncharged side chains, with the charged side chains including aspartic acid, glutamic acid, lysine, arginine, and histidine, and the uncharged side chains can also be classified as nonpolar or polar, with the nonpolar amino acids being classified as glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline, and the polar amino acids being classified as serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Typically, conservative substitutions have little or no effect on the activity of the resulting polypeptide.

[0031] 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.

[0032] In the present application, the term "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 variant of the present application includes one 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 substituted with another amino acid. Specifically, the variant may be a protein in which the amino acids at the 12th position, the 116th position, or all of the amino acids at the 12th and 116th positions from the N-terminus of the amino acid sequence of SEQ ID NO: 31 are substituted with another amino acid. The term "another amino acid" refers to an amino acid different from the amino acid before substitution, and may be any amino acid other than the amino acid before substitution.

[0033] Specifically, the serine protease mutant of the present application is a mutant in which phenylalanine at the 12th position in the amino acid sequence of SEQ ID NO: 31 is substituted with any of glycine, alanine, arginine, aspartate, cysteine, glutamate, asparagine, glutamine, histidine, proline, serine, tyrosine, or isoleucine. and / or the asparagine at position 116 may be replaced by, but is not limited to, glycine, alanine, arginine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, proline, serine, tyrosine, isoleucine, leucine, lysine, phenylalanine, tryptophan, valine, methionine, or threonine.

[0034] Specifically, the mutant is a protein in which the amino acid corresponding to the 12th position in the amino acid sequence of SEQ ID NO: 31 is replaced with tyrosine (Y), serine (S), alanine (A) or arginine (R), the amino acid corresponding to the 116th position is replaced with aspartic acid (D), serine (S), threonine (T) or glycine (G), or 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. Needless to say, the mutant in which the amino acid at the 12th and / or 116th position in the amino acid sequence of SEQ ID NO: 31 is replaced with another amino acid includes a mutant in which the amino acid corresponding to the above position is replaced with another amino acid.

[0035] The mutants also include those in which the amino acid sequence represented by the aforementioned SEQ ID NO:31 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more homology or identity to the aforementioned SEQ ID NO:31, 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.

[0036] Specifically, among the above mutants, the 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 substituted with another amino acid has an amino acid sequence represented by any one of SEQ ID NOs: 32 to 39, but is not limited thereto. The mutant may contain a substitution at the position corresponding to the 12th and / or the 116th position of SEQ ID NO: 31 with another amino acid, have at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more but less than 100% sequence homology with the amino acid sequence of SEQ ID NO: 31, and have serine protease activity.

[0037] The serine protease variant of the present application is one having 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 application 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.

[0038] Furthermore, in addition to the mutations at the 12th and / or 116th amino acids, or mutations at positions corresponding thereto, which confer a specific activity, 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 variants of the present application, and it goes without saying that those having such sequence additions or mutations are also included in the present application.

[0039] 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 application, and the sequence obtained by removing only the signal peptide from SEQ ID NO: 40 corresponds to SEQ ID NO: 2 of the present application.

[0040] As described above, the serine protease variant of the present application 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 application 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.

[0041] Thus, the serine protease variants of the present application include variants in which amino acids at positions corresponding to positions 12 and 116 of SEQ ID NO: 31 (amino acids 163 and / or 267 in SEQ ID NO: 2, and amino acids 193 and / or 297 in SEQ ID NO: 40) are substituted in SEQ ID NO: 31, which contains the amino acid sequence of SEQ ID NO: 31. Furthermore, the contents explained in the present application 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.

[0042] As one example, the serine protease variant of the present application may comprise an amino acid sequence in which the amino acids at positions corresponding to positions 12 and / or 116 of SEQ ID NO: 31 are substituted with other amino acids, and may have at least 80% or more and less than 100% sequence homology with SEQ ID NO: 2. As another example, the serine protease variant of the present application may comprise an amino acid sequence in which the amino acids at positions 163 and / or 267 of SEQ ID NO: 2 are substituted with other amino acids, and may have 80% or more and less than 100% sequence homology with SEQ ID NO: 2, or may have 80% or more sequence homology with any amino acid sequence selected from SEQ ID NOs: 3 to 10. However, the present application is not limited thereto.

[0043] Another aspect of the present application provides a polynucleotide encoding said serine protease variant.

[0044] In this application, 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.

[0045] The polynucleotide encoding the serine protease variant of the present application may be any polynucleotide sequence that encodes the serine protease variant of the present application having enhanced activity. In the present application, the gene encoding the wild-type serine protease may be derived from a microorganism of the genus Thermobifida, specifically, but not limited to, Thermobifida fusca.

[0046] The polynucleotide of the present application may be modified in various ways in the coding region, taking into consideration codon degeneracy or preferred codons in an 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 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.

[0047] For example, the polynucleotide of the present application may be a polynucleotide sequence encoding a variant of the present application, specifically, a polypeptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 32 to 39, or a polypeptide having homology thereto, but is not limited thereto. More specifically, the polynucleotide may be a polynucleotide sequence represented by any one of SEQ ID NOs: 41 to 48, but is not limited thereto. As described above, the serine protease variant of the present application also includes variants of SEQ ID NOs: 2 and 40, which are amino acid sequences including SEQ ID NO: 31, in which amino acids at positions corresponding to 12th and / or 116th of 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, and therefore polynucleotide sequences encoding the serine protease variants are also included in the present application. 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.

[0048] 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 part of the base sequence, encodes a protein having the activity of a mutant 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.

[0049] 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.

[0050] 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 present application may include isolated polynucleotide fragments that are complementary to the entire sequence, as well as substantially similar polynucleotide sequences.

[0051] 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.

[0052] The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementation, variables known in the art.

[0053] Yet another aspect of the present application provides a vector comprising a polynucleotide encoding the serine protease variant of the present application.

[0054] In this application, 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.

[0055] 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 application. 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.

[0056] The vector used in the present application 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. may be used as phage or cosmid vectors, and pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pET, pUB110, etc. may be used as plasmid vectors. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pSM704 vectors, etc. may be used. The vector that can be used in the present application is not particularly limited, and any known expression vector may be used.

[0057] 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.

[0058] Yet another aspect of the present application provides a microorganism expressing the serine protease variant of the present application.

[0059] In the present application, the term "to be expressed" may refer to a state in which a target protein is introduced into a microorganism or modified to be expressed in a microorganism. For the purposes of the present application, the "target protein" may be the serine protease variant described above.

[0060] 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.

[0061] The microorganism expressing the serine protease variant may contain one or more of the serine protease variant of the present application, a polynucleotide encoding the same, and a vector containing the same.

[0062] The microorganism containing one or more of the serine protease variant, the polynucleotide encoding the variant, and the vector containing the same may specifically be a microorganism prepared by transformation with a vector containing a polynucleotide encoding the variant, but is not limited thereto.

[0063] The microorganism may be a recombinant microorganism, and the recombination may be achieved by genetic modification, such as transformation.

[0064] In the present application, "transformation" means introducing a vector containing a polynucleotide encoding a target protein into a host cell, thereby expressing the protein encoded by the polynucleotide in 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 12), lithium acetate-DMSO method, etc.

[0065] The recombinant microorganism may have enhanced serine protease activity according to the present application.

[0066] 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.

[0067] Specifically, in the present application, 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] A host cell or microorganism of the present application may be any microorganism that contains a polynucleotide of the present application or a vector of the present application 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. 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.

[0073] Yet another aspect of the present application provides a feed composition comprising at least one of the serine protease variants of the present application and a microorganism expressing same.

[0074] The serine protease variant contained in the feed composition of the present application 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.

[0075] A "feed composition" in this application is any natural or artificial diet, meal, etc., or component of said meal, intended or suitable for consumption and digestion by an animal, and the feed can be manufactured into various forms of feed known in the art.

[0076] The feed composition may be a feed additive.

[0077] 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.

[0078] The feed composition of the present application 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.

[0079] The feed composition of the present application 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 broken 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 sugar 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.

[0080] The feed composition of the present application 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.

[0081] The feed composition of the present application 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 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.

[0082] The feed compositions of the present application 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.

[0083] Examples of animals for which the feed composition of the present application 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, layers, chickens, roosters, ducks, geese, turkeys, quails, and small birds, but are not limited to these.

[0084] The amount of the serine protease variant of the present application contained in the feed composition of the present application 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 application pertains, the amount of the serine protease variant of the present application contained in the feed composition of the present application is 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.

[0085] Yet another aspect of the present application provides a food composition comprising at least one of the serine protease variants of the present application 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] Yet another aspect of the present application provides a detergent composition comprising at least one of the serine protease variants of the present application and a microorganism expressing same.

[0090] The detergent compositions according to the present application 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.

[0091] The detergent compositions according to the present application 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.

[0092] Yet another aspect of the present application provides a pharmaceutical composition comprising at least one of the serine protease variants of the present application and a microorganism expressing same.

[0093] The pharmaceutical composition of the present application is used as a pharmaceutical composition for digestive enzymes for improving digestive diseases, digestive disorders, and disorders following 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 following surgery or trauma.

[0094] 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.

[0095] In addition to the uses mentioned above, the serine protease variants of the present application or microorganisms expressing the same are also used in, for example, cosmetics, leather processing, medicines, diagnostic agents, waste management, and chemical agent production for academic research, etc. However, the above uses are merely examples, and the variants may also be used in any other uses related to the denaturation, degradation, or removal of proteinaceous substances known in the art. The present disclosure relates, for example, to the following: [1] A serine protease variant in which one or more amino acids selected from the amino acid corresponding to position 12 and the amino acid corresponding to position 116 of SEQ ID NO:31 are replaced with other amino acids, and which has a sequence identity of at least 80% and less than 100% with the amino acid sequence of SEQ ID NO:31. [2] The serine protease mutant according to [1] above, in which the amino acid corresponding to the 12th position is substituted with tyrosine (Y), alanine (A), serine (S), or arginine (R). [3] The serine protease mutant according to [1] above, in which the amino acid at position 116 is substituted with aspartic acid (D, aspartate), serine (S, serine), threonine (T, threonine) or glycine (G, glycine). [4] A serine protease variant comprising the amino acid sequence of the serine protease variant described in [1] above, and having a sequence identity of at least 80% and less than 100% with SEQ ID NO:2. [5] The serine protease mutant according to [1] above, wherein the mutant is composed of an amino acid sequence represented by any one of SEQ ID NOs: 3 to 10 and 32 to 39. [6] A polynucleotide encoding the serine protease mutant according to any one of [1] to [5] above. [7] A vector comprising the polynucleotide described in [6] above. [8] A microorganism expressing the serine protease variant according to any one of [1] to [5] above. [9] The microorganism described in [8] above, wherein the microorganism is a Bacillus genus.

[10] The microorganism according to [9], wherein the microorganism is Bacillus subtilis.

[11] A feed composition comprising one or more of the serine protease variants according to [1] above and microorganisms expressing the same. EXAMPLES

[0096] The present application 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 application, and the present application is not limited to these examples and experimental examples. EXAMPLES

[0097] 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.

[0098] [Table 1]

[0099] 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.

[0100] [Table 2]

[0101] 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.

[0102] [Table 3] (in 1L)

[0103] 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

[0104] 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. The positions of the mutations are shown in Figure 1. 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 mutant and single mutant forms were then compared and evaluated with the activity of the wild type. The primers used for generating the mutants are shown in Table 4.

[0105] [Table 4]

[0106] 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 13). The measured activities are shown in Table 5.

[0107] [Table 5]

[0108] 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.

[0109] Example 2-3: Evaluation of thermal stability Since thermostability is a very important characteristic of the enzyme, the following experiment was carried out to confirm the effect of introducing a mutation on thermostability.

[0110] 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.

[0111] [Table 6]

[0112] 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 mutant of the present application retains high activity even at high temperatures, and is therefore industrially useful. EXAMPLES

[0113] 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.

[0114] 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.

[0115] [Table 7]

[0116] 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.

[0117] [Table 8]

[0118] 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.

[0119] Thus, it has been confirmed that the residues F12 and N116 are important residues for serine protease enzyme activity, and that the enzyme activity is increased by substituting them with other amino acids. Therefore, the serine protease mutant of the present application has increased enzyme activity and can be usefully used industrially.

[0120] From the above description, a person skilled in the art to which the present application pertains will understand that the present application 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 application should be interpreted as including all modifications and variations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. A protein variant having serine protease activity, Any one or more amino acids selected from the amino acid corresponding to the 12th position and the amino acid corresponding to the 116th position of SEQ ID NO: 31 are substituted with other amino acids, and have a sequence identity of at least 90% and less than 100% with the amino acid sequence of SEQ ID NO: 31; the amino acid at position 12 is substituted with tyrosine (Y), alanine (A), serine (S), or arginine (R); the amino acid at position 116 is substituted with aspartic acid (D), serine (S), threonine (T) or glycine (G); A protein variant having superior enzymatic activity compared to the wild-type serine protease shown in SEQ ID NO:

31.

2. A protein variant having serine protease activity, comprising the amino acid sequence of the protein variant described in claim 1 and having a sequence identity of at least 90% but less than 100% with SEQ ID NO:

2.

3. The protein mutant of claim 1, wherein the protein mutant is composed of an amino acid sequence represented by any one of SEQ ID NOs: 3 to 10 and 32 to 39.

4. A polynucleotide encoding the protein mutant according to any one of claims 1 to 3.

5. A vector comprising the polynucleotide of claim 4.

6. A microorganism expressing the protein variant according to any one of claims 1 to 3.

7. The microorganism according to claim 6 , wherein the microorganism is of the genus Bacillus.

8. The microorganism of claim 7, wherein the microorganism is Bacillus subtilis.

9. A feed composition comprising one or more of the protein variant according to any one of claims 1 to 3 and a microorganism expressing the same.

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