KR20250054900A - A novel serine protease variant - Google Patents

Abstract

The present application relates to novel serine protease variants.

Description

{A novel serine protease variant}

The present application relates to novel serine protease variants.

Proteases perform various functions in the body, such as digestion, absorption, and defense, and are classified into serine protease, cysteine protease, aspartic protease, and metalloprotease depending on the structure of the active site. Among them, serine protease (or serine endopeptidase) is characterized by having an active serine residue in common in the active site, and is an enzyme that cleaves peptide bonds within proteins by having a serine amino acid act as a nucleophilic amino acid in the active site of the protease (Hedstrom, 2002. Chem Rev 102: 4501-4524).

Serine protease has various uses. In addition to treating human diseases such as dissolving blood clots, it is also used as an ingredient in clothing detergents and contact lens cleaners, and is also widely used in the modification of milk proteins, silk degumming, soaking and unhairing of leather, oligopeptide synthesis, recovery of silver from waste X-ray films, and manufacturing and improvement of feed and food. However, in order to secure better industrial economic feasibility and efficacy, improved serine proteases in terms of thermal stability and activity are required.

One embodiment of the present application provides a serine protease variant, wherein the amino acid corresponding to position 15 of SEQ ID NO: 10 is replaced with another amino acid.

In one specific embodiment, the other amino acid can be glycine or serine.

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

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

In any one of the preceding specific examples, the serine protease variant may have the amino acid corresponding to position 166 of SEQ ID NO: 9 substituted with another amino acid.

In any one of the preceding specific examples, the serine protease variant may have the amino acid corresponding to position 196 of SEQ ID NO: 8 substituted with another amino acid.

In any one of the preceding specific examples, the serine protease variant may comprise an amino acid sequence set forth in any one of SEQ ID NOs: 11 to 16.

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

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

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

In one specific embodiment, the microorganism may be a Bacillus sp. microorganism. In any one of the preceding specific embodiments, the microorganism may be Bacillus subtilis.

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

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

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

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

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

This is explained specifically as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in this application fall within the scope of this application. In addition, the scope of this application cannot be considered limited by the specific description described below.

Furthermore, 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 in this application. Such equivalents are also intended to be encompassed by this application.

In addition, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the level of the technical field to which this application belongs and the content of this application.

definition

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

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

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

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

Mutant

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

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

In this application, the term "mature polypeptide" means a polypeptide in a form without a signal sequence or a propeptide sequence. The mature protein/polypeptide/peptide can be a functional form of the protein/polypeptide/peptide. The mature polypeptide can be a final form that has undergone post-translational or post-translational modifications. Examples of post-translational modifications include, but are not limited to, N- or C-terminal modifications, glycosylation, phosphorylation, and leader sequence removal.

In this application, the term "wild type" means naturally-occurring, without any artificial modification. When the term "wild type" is used in relation to a polypeptide, it means a naturally occurring polypeptide, which does not have any artificial modification (substitution, insertion, deletion, etc.) at one or more amino acid positions. Similarly, when the term "wild type" is used in relation to a polynucleotide, it means that it does not have any artificial modification (substitution, insertion, deletion) of one or more nucleotides. However, a polynucleotide encoding a wild type polypeptide is not limited to a naturally occurring polynucleotide, and also includes a sequence encoding any wild type polypeptide.

In the present application, the parent sequence or backbone refers to a reference sequence into which a modification is introduced to become a mutant. That is, the parent sequence may be a starting sequence to which mutations such as substitution, insertion, and/or deletion are introduced. The parent sequence may be a naturally occurring or wild type, or a variant in which one or more substitutions, insertions, or deletions have occurred in the natural or wild type, or may be an artificially synthesized sequence. If the parent sequence is an amino acid sequence that exhibits activity, that is, an amino acid sequence of an enzyme, it may be called a parent enzyme.

In this application, the term "reference sequence" means a sequence used to determine the position of an amino acid in any amino acid sequence. By aligning any amino acid sequence with a reference sequence, the position of an amino acid corresponding to a specific position of the reference sequence in any amino acid sequence can be determined.

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

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

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

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

For example, any amino acid sequence can be aligned with SEQ ID NO: 10, and each amino acid residue of the amino acid sequence can be numbered based on the numerical position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 10. For example, a sequence alignment algorithm such as that described in the present application can identify the position of an amino acid, or the position at which a modification, such as a substitution, insertion, or deletion, occurs, by comparison with a query sequence (also referred to as a “reference sequence”).

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

Multiple sequence alignment can also be used to identify corresponding amino acid residues in other polypeptides. Examples of multiple sequence alignment programs known in the art include MUSCLE (multiple sequence comparison by log-expectation; version 3.5 or higher; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT (version 6.857 or higher; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518; Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009, Methods in Molecular Biology 537: 39-64; Katoh and Toh, 2010, Bioinformatics 26: 1899-1900), and EMBOSS using ClustalW. EMMA (1.83 or higher; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), etc., and the default parameters of each of the above programs can be used, but are not limited thereto.

In addition, when polypeptides diverge from a mature polypeptide and their relationships are not detected by conventional sequence-based comparison, other pairwise sequence comparison algorithms may be used (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615). Higher sensitivity can be achieved in sequence-based searches by using search programs that utilize probabilistic representations of polypeptide families (profiles) to search databases. For example, the PSI-BLAST program generates profiles through an iterative database search process and can detect remote homologs (Atschul et al., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivity can be achieved when a family or superfamily for a polypeptide has more than one representation in a protein structure database. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffin and Jones, 2003, Bioinformatics 19: 874-881) use information from a variety of sources, such as PSI-BLAST, secondary structure predictions, structural alignment profiles, and solvation potentials, as inputs to a neural network that predicts the structural folding of a query sequence. Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919 can be used to align an unknown structure to superfamily models available in the SCOP database. These alignments can in turn be used to build homology models for the polypeptide, and these models can be evaluated for accuracy using a variety of tools developed for the purpose.

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

The above methods are examples and are not limiting.

In the present application, with respect to an amino acid sequence, even if it is described as a polypeptide "comprising" an amino acid sequence set forth in a specific sequence number, a polypeptide "consisting of" an amino acid sequence set forth in a specific sequence number, or a polypeptide or protein "having" an amino acid sequence set forth in a specific sequence number, it is obvious that a protein having an amino acid sequence in which a portion of the sequence is deleted, modified, substituted, conservatively substituted or added is also included within the scope of the present application if it has the same or corresponding activity as a protein consisting of the amino acid sequence of the corresponding sequence number. For example, if it has the same or corresponding activity as the above-mentioned mutant protein, it does not exclude addition of a sequence that does not change the function of the protein before or after the above-mentioned amino acid sequence, a mutation that may occur naturally, a silent mutation or conservative substitution thereof, and it is obvious that even if it has such sequence addition or mutation, it is within the scope of the present application.

For example, in the case where the amino acid sequence has sequence additions, naturally occurring mutations, silent mutations or conservative substitutions that do not alter the function of the variant polypeptide of the present application at the N-terminus, C-terminus and/or internally.

In this application, the term "conservative substitution" means replacing one amino acid with another amino acid having similar structural and/or chemical properties. Such amino acid substitutions may generally occur based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature of the residues. Typically, conservative substitutions may have little or no effect on the activity of the polypeptide.

In the present application, the term "another amino acid" is not limited as long as it is an amino acid different from the amino acid before substitution. Meanwhile, when the present application expresses that "a specific amino acid is substituted", it is self-evident that the amino acid is substituted with an amino acid different from the amino acid before substitution, even if it is not specifically stated that it is substituted with another amino acid.

In this application, "mutating/modifying" means changing or altering. This may be a change from something that occurs naturally. For example, a polypeptide may be altered in such a way that the polypeptide is altered from a parent sequence or a reference sequence.

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

As used herein, the term "modified" means, for example, changed from its naturally occurring form. A modified polypeptide of the present application includes a non-naturally occurring polypeptide or a naturally occurring variant. In one embodiment, a modified polypeptide of the present application is a modified polypeptide that is not found in nature. In one embodiment, a modified polypeptide of the present application may be, but is not limited to, one that does not occur spontaneously.

When the term "modification" is used in relation to an amino acid/nucleic acid sequence in this application, it can include substitution of an amino acid/nucleic acid residue of the parent sequence for a different amino acid/nucleic acid residue at one or more sites in the amino acid sequence, deletion of an amino acid/nucleic acid residue (or a series of amino acid/nucleic acid residues) of the parent sequence at one or more sites, insertion of an amino acid/nucleic acid residue (or a series of amino acid/nucleic acid residues) of the parent sequence at one or more sites, truncation of the N-terminal and/or C-terminal amino acid sequence or 5' and/or 3' nucleic acid sequence, and any combination thereof.

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

Additionally, the variant polypeptide may contain 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 conjugated to a signal (or leader) sequence at the N-terminus of the polypeptide that is involved in the transfer of proteins co-translationally or post-translationally. Additionally, the polypeptide may be conjugated to other sequences or linkers so that the polypeptide can be identified, purified, or synthesized.

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

In this application, the term "isolated" refers to a material that is present in an environment in which it does not occur naturally, or in a form that does not exist naturally. This includes the material (sequence or nucleic acid) being at least substantially free from at least one other component with which it is naturally associated and found in nature, such as a sequence, or nucleic acid.

For example, the isolated sequence or nucleic acid provided in the present application can be provided in a form substantially free of one or more contaminants.

Examples of isolated substances may include, but are not limited to, i) any non-naturally occurring substance, ii) any substance from which one, more, or all of the naturally occurring components associated with the substance have been removed (e.g., an enzyme, mutant, nucleic acid, protein, peptide, or cofactor), iii) any substance that has been artificially modified from a substance found in nature, or iv) a substance that has been modified to alter the amount of the substance relative to other naturally associated components (e.g., increasing the number of copies of a gene encoding the substance; modifying a promoter naturally associated with a gene encoding the substance to be a more active promoter, etc.).

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

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

For example, it may contain at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or less than 100% of the amino acids of the native polypeptide, but is not limited thereto.

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

Alanine Ala, A Arginine Arg, R

Asparagine Asn, N Aspartate Asp, D

Cysteine Cys, C Glutamic acid Glu, E

Glutamine Gln, Q Glycine Gly, G

Histidine His, H Isoleucine Ile, I

Leu, L Lys, K

Methionine Met, M Phenylalanine Phe, F

Proline Pro, P Serine Ser, S

Threonine Thr, T Tryptophan Trp, W

Tyrosine Tyr, Y Valine Val, V

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

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

Amino acids can generally be classified based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature of their residues.

As examples of this classification, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartate; amino acids with nonpolar side chains (nonpolar amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; and amino acids with polar or hydrophilic side chains (polar amino acids) include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. As another example, amino acids with charged side chains can be classified as arginine, lysine, histidine, glutamic acid, and astaxanthin, and amino acids with uncharged side chains (also called neutral amino acids) can be classified as glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. As another example, phenylalanine, tryptophan, and tyrosine can be classified as aromatic amino acids. As another example, valine, leucine, and isoleucine can be classified as branched amino acids. As another example, 20 amino acids can be classified according to size, starting from a group of amino acids with a relatively small volume: glycine, alanine, and serine; Cysteine, proline, threonine, aspartate, asparagine; valine, histidine, glutamic acid, glutamine; isoleucine, leucine, methionine, lysine, arginine; and phenylalanine, tryptophan, tyrosine can be classified into five groups. However, it is not necessarily limited to this.

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

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

Amino acid substitutions can be expressed by listing the amino acid before substitution, the position, and the amino acid being substituted in that order. The amino acids can be expressed using the usual one-letter and three-letter codes. For example, when alanine, the amino acid at position 6 of a specific sequence, is replaced with valine, it can be written as "A8V" or "Ala8Val."

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

polynucleotide

In this application, the term "gene" means a polynucleotide that encodes a polypeptide and a polynucleotide comprising regions preceding and following the coding region. In some embodiments, a gene may have a sequence (intron) inserted between each coding region (exon).

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

Homology, identity

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

Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms, and may be utilized in conjunction with a default gap penalty established by the program being used. In practice, homologous or identical sequences are generally capable of hybridizing under moderate or high stringency conditions along at least about 50%, 60%, 70%, 80% or 90% of the entire or full-length sequence. It will be appreciated that hybridization also includes polynucleotides containing common codons or codons that are considered codon degeneracy in the polynucleotide.

Whether any two polynucleotide or polypeptide sequences are homologous, similar or identical can be determined using well-known computer algorithms such as the "FASTA" program using default parameters as in, for example, Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) can be determined using the GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [Ed.,] Academic Press, San Diego,1994, and [CARILLO et al.](1988) SIAM J Applied Math 48: 1073). For example, homology, similarity or identity can be determined using BLAST from the National Center for Biotechnology Information Database, or ClustalW.

Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information, for example, as disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2:482, or, for example, using a GAP computer program such as Needleman et al. (1970), J Mol Biol. 48:443. In brief, the GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly arranged symbols (i.e., nucleotides or amino acids). The default parameters for the GAP program are (1) unitary matrices (containing values of 1 for identity and 0 for non-identity), the PAM Matrix (as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation (1978)), Gribskov et al(1986) Nucl. Acids Res. 14: 6745 weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or gap opening penalty 10, gap extension penalty 0.5); and (3) no penalty for terminal gaps.

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

In this application, the term "stringent conditions" means conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8). For example, the conditions under which polynucleotides having high homology or identity hybridize with each other, or polynucleotides having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity hybridize, and polynucleotides having lower homology or identity do not hybridize, or the conditions of washing once, specifically twice to three times, at a salt concentration and temperature corresponding to 60°C, 1ХSSC, 0.1% SDS, specifically 60°C, 0.1ХSSC, 0.1% SDS, and more specifically 68°C, 0.1ХSSC, 0.1% SDS, which are washing conditions of conventional southern hybridization, can be listed.

The above hybridization requires that the two nucleotides have complementary sequences, although mismatches between bases are possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the polynucleotides of the present application may also include isolated nucleic acid fragments that are complementary to the entire sequence, as well as substantially similar nucleic acid sequences.

The appropriate stringency to hybridize the above polynucleotides depends on the length and degree of complementarity of the polynucleotides, variables which are well known in the art (e.g., J. Sambrook et al., supra).

Nucleic acid structures, vectors, transformation

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

The term "vector" as used in this application means a DNA construct comprising a nucleic acid sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to enable expression of the target polypeptide in a suitable host. The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences regulating the termination of transcription and translation. The vector, after being transformed into a suitable host cell, may replicate or function independently of the host genome, and may be integrated into the genome itself.

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

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

In this application, the term "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide can include both the position of being inserted into the chromosome of the host cell or being positioned outside the chromosome, as long as it can be expressed in the host cell. In addition, the polynucleotide includes DNA and RNA encoding the target polypeptide. The polynucleotide can be introduced in any form as long as it can be introduced into the host cell and expressed. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for self-expression. The expression cassette can typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal that are operably linked to the polynucleotide. The expression cassette can be in the form of an expression vector capable of self-replication. Additionally, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.

As used herein, the term "operably linked" means a configuration in which a regulatory sequence is positioned at an appropriate location such that the regulatory sequence directs the expression of a coding sequence. Accordingly, "operably linked" includes attachment or linkage of a regulatory region of a functional domain having a known or desired activity, such as a promoter, a terminator, a signal sequence, or an enhancer region, to a target (gene or polypeptide) so as to regulate the expression, secretion, or function of the target according to the known or desired activity. For example, it means that a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a target variant polypeptide of the present application is functionally linked to the polynucleotide sequence.

The term "expression" as used herein includes any step involved in the production of a polypeptide, such as, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

As used herein, the term “expression vector” means a linear or circular nucleic acid molecule comprising a coding sequence and regulatory sequences operably linked to it for expression.

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

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

host cell

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

Specific description of this application

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

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

The term "serine protease" of the present application refers to an enzyme belonging to a subgroup of proteases and having protein decomposition activity. Specifically, a serine protease may be an enzyme that decomposes proteins by hydrolyzing peptide bonds and basically has an active serine residue at the active site, and more specifically, may be an enzyme having a spatial arrangement of amino acid residues histidine (His, H), aspartate (Asp, D) and serine (Ser, S), which may be referred to as a three-functional catalyst (catalytic triad).

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

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

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

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

Specifically, the parent sequence of the serine protease may include an amino acid represented by any one of SEQ ID NOs: 8 to 10, or an amino acid having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity with any one of SEQ ID NOs: 8 to 10. In addition, it is obvious that a protein having an amino acid sequence in which a part of the sequence is deleted, modified, substituted, or added is also included within the scope of the present application, as long as it has such homology or identity and exhibits an effect corresponding to the protein.

For example, the parent sequence of the protease may comprise an amino acid sequence set forth in any one of SEQ ID NOs: 4 to 7 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity with any one of SEQ ID NOs: 4 to 7.

The serine protease variant provided in the present application is a variant having serine protease activity, wherein in any polypeptide or serine protease having serine protease activity, the amino acid corresponding to the 15th position from the N-terminus of SEQ ID NO: 10 is substituted with another amino acid.

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

For example, the serine protease variant provided in the present application may mean a variant in which, among the proteins having the serine protease activity described above, the amino acid corresponding to the 15th position of the sequence number 10 is replaced with a different amino acid compared to the parent sequence, so that the enzyme activity exceeds 100% compared to the protein of the parent sequence.

For example, the serine protease variant of the present application may be one in which tyrosine (Y), an amino acid corresponding to position 15 in the amino acid sequence of SEQ ID NO: 10, is substituted with glycine, alanine, arginine, aspartate, cysteine, glutamic acid, glutamine, histidine, proline, serine, phenylalanine, isoleucine, leucine, lysine, tryptophan, valine, methionine, threonine or asparagine.

For example, the serine protease variant of the present application may have tyrosine (Y), an amino acid corresponding to position 15 in the amino acid sequence of SEQ ID NO: 10, substituted with alanine, glycine, serine, cysteine, proline, threonine, aspartate, or asparagine.

For example, the serine protease variant of the present application may have tyrosine (Y), an amino acid corresponding to position 15 in the amino acid sequence of SEQ ID NO: 10, substituted with alanine (A), histidine (H), arginine (R), glycine (G), or serine (S).

For example, the serine protease variant of the present application may have tyrosine (Y), an amino acid corresponding to position 15 in the amino acid sequence of SEQ ID NO: 10, substituted with glycine or serine.

For example, the serine protease variant of the present application may be one in which tyrosine (Y), an amino acid corresponding to position 15 in the amino acid sequence of SEQ ID NO: 10, is substituted with serine.

For example, the variant may include an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity with SEQ ID NO: 7 or 10, wherein the amino acid corresponding to the 15th position from the N-terminus of SEQ ID NO: 10 is substituted with another amino acid. For example, the other amino acid may be glycine or serine.

Meanwhile, the mature region of NCBI Reference Sequence WP_016188200.1 (SEQ ID NO: 8) corresponds to SEQ ID NO: 9 of the present application, and the sequence of SEQ ID NO: 9 excluding only the signal peptide corresponds to SEQ ID NO: 10 of the present application. In addition, SEQ ID NO: 7 has improved enzymatic activity compared to the polypeptide of SEQ ID NO: 10, since amino acids corresponding to positions 12, 116, and 180 in SEQ ID NO: 10 are substituted with other amino acids.

It is obvious that the serine protease variant of the present application may include deletions or additions of amino acids having minimal influence on the properties and secondary structure of the serine protease, in which the amino acid at the position corresponding to position 15 of SEQ ID NO: 10 is replaced with another amino acid as described above. In addition, a person skilled in the art can know through sequence alignment known in the art that position 15 from the N-terminus of SEQ ID NO: 5 and SEQ ID NO: 7 of the present application corresponds to position 15 of SEQ ID NO: 10, position 166 of SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 9, and position 196 of SEQ ID NO: 8. In addition, a person skilled in the art can know through sequence alignment that SEQ ID NO: 5 is included in SEQ ID NO: 4, SEQ ID NO: 7 is included in SEQ ID NO: 6, and SEQ ID NO: 10 is included in SEQ ID NO: 9 and SEQ ID NO: 8.

Accordingly, as an example of the serine protease variant of the present application, for SEQ ID NOs: 4, 6 and 9, there may be exemplified a variant in which the amino acid corresponding to the 15th position of SEQ ID NO: 10 (the 166th amino acid in SEQ ID NOs: 4, 6 and 9, and the 196th amino acid in SEQ ID NO: 8) is substituted. As another example of the serine protease of the present application, for SEQ ID NOs: 5 and 7, there may be exemplified a variant in which the amino acid corresponding to the 15th position of SEQ ID NO: 10 (i.e., the 15th amino acid in SEQ ID NOs: 5 and 7) is substituted.

Additionally, the description of the 15th amino acid of SEQ ID NO: 10 in the present application can be applied to the 15th amino acid of SEQ ID NO: 5 and SEQ ID NO: 7, the 166th amino acid of SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 9, and the 196th amino acid of SEQ ID NO: 8.

Similarly, what has been described for amino acids 12, 116 and 180 of SEQ ID NO: 10 can be applied to amino acids 12, 116 and 180 of SEQ ID NO: 5 and SEQ ID NO: 7, amino acids 163, 267 and 331 of SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 9, and amino acids 193, 297 and 361 of SEQ ID NO: 8.

For example, the serine protease variant of the present application comprises an amino acid sequence in which an amino acid at a position corresponding to the 15th amino acid of SEQ ID NO: 10 is substituted with a different amino acid, and may have at least 75% to less than 100% sequence homology or identity with any one of SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 9. In another embodiment, the serine protease variant of the present application comprises an amino acid sequence in which an amino acid at a position corresponding to the 15th amino acid of SEQ ID NO: 10 is substituted with a different amino acid, and may have at least 75% to less than 100% sequence homology or identity with SEQ ID NO: 8. In another embodiment, the serine protease variant of the present application comprises an amino acid sequence in which an amino acid at a position corresponding to the 15th amino acid of SEQ ID NO: 10 is substituted with a different amino acid, and may have at least 75% to less than 100% sequence homology or identity with any one of SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 10.

For example, the serine protease variant of the present application may include one or more amino acids selected from positions corresponding to amino acids 12, 116, and 180 from the N-terminus of SEQ ID NO: 10, in which any one or more amino acids are substituted with another amino acid.

Specifically, the serine protease variant of the present application has phenylalanine at position 12 in the amino acid sequence of SEQ ID NO: 10 replaced with glycine, alanine, arginine, aspartate, cysteine, glutamate, asparagine, glutamine, histidine, proline, serine, tyrosine, isoleucine, leucine, lysine, tryptophan, valine, methionine or threonine; The asparagine at position 116 may be replaced with glycine, alanine, arginine, aspartate, cysteine, glutamic acid, glutamine, histidine, proline, serine, tyrosine, isoleucine, leucine, lysine, phenylalanine, tryptophan, valine, methionine, or threonine; the phenylalanine at position 180 may be replaced with glycine, alanine, arginine, aspartate, cysteine, glutamic acid, glutamine, histidine, proline, serine, tyrosine, isoleucine, leucine, lysine, asparagine, tryptophan, valine, methionine, or threonine; or a combination of the above substitutions, but is not limited thereto.

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

For example, the serine protease variant of the present application comprises: (i) an amino acid corresponding to position 12 of SEQ ID NO: 10 is tyrosine (Y, tyrosine), alanine (A, Alanine), serine (S, Serine), or arginine (R, Arginine); or (ii) an amino acid corresponding to position 116 of SEQ ID NO: 10 is aspartate (D, aspartate), serine (S, Serine), threonine (T, Threonine), or glycine (G, Glycine); Or (iii) the amino acid corresponding to the 12th position of SEQ ID NO: 10 may be tyrosine (Y, tyrosine), alanine (A, Alanine), serine (S, Serine), or arginine (R, Arginine), and the amino acid corresponding to the 116th position of SEQ ID NO: 10 may be aspartate (D, aspartate), serine (S, Serine), threonine (T, Threonine), or glycine (G, Glycine). In the variant, the amino acid corresponding to the 180th position of SEQ ID NO: 10 may be tyrosine (Y) or phenylalanine (F). For example, the serine protease variant of the present application may be one in which the amino acid corresponding to the 12th position of SEQ ID NO: 10 is tyrosine, the amino acid corresponding to the 116th position is aspartate, and the amino acid corresponding to the 180th position is tyrosine.

For example, the serine protease variant of the present application may be such that (i) the amino acid corresponding to position 12 of SEQ ID NO: 10 is phenylalanine (F); (ii) the amino acid corresponding to position 116 of SEQ ID NO: 10 is asparagine (N); (iii) the amino acid corresponding to position 180 of SEQ ID NO: 10 is phenylalanine (F); or (iv) the amino acid corresponding to position 12 of SEQ ID NO: 10 is phenylalanine (F), the amino acid corresponding to position 116 is asparagine (N), and the amino acid corresponding to position 180 is phenylalanine (F). For example, the serine protease variant of the present application may be such that the amino acid corresponding to position 12 of SEQ ID NO: 10 is phenylalanine, the amino acid corresponding to position 116 is asparagine, and the amino acid corresponding to position 180 is phenylalanine.

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

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

The polynucleotide of the present application may have various modifications in the coding region within a range that does not change the amino acid sequence of the polypeptide due to the degeneracy of the codon or in consideration of the codon preferred in an organism that is intended to express the polypeptide. Specifically, the polynucleotide sequence may include, without limitation, a polynucleotide sequence encoding a variant in which the amino acid corresponding to the 15th position from the N-terminus of SEQ ID NO: 10 is substituted with another amino acid.

For example, the polynucleotide of the present application may be, but is not limited to, a polynucleotide sequence encoding a variant of the present application, specifically a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 to 16, or a polypeptide homologous thereto.

In addition, as described above, the serine protease variant of the present application includes a variant in which the amino acid corresponding to the 15th position from the N-terminus of SEQ ID NO: 10 (the 166th amino acid in SEQ ID NO: 9, the 196th amino acid in SEQ ID NO: 8) is substituted for SEQ ID NO: 9 and SEQ ID NO: 8, which are amino acid sequences including SEQ ID NO: 10, and also includes a variant in which the amino acid corresponding to the 15th position from the N-terminus of SEQ ID NO: 10 is substituted for SEQ ID NOs: 4 to 7, and therefore, a polynucleotide sequence encoding the serine protease variant is also included in the scope of the present application.

In addition, a probe that can be prepared from a known genetic sequence, for example, a sequence that hybridizes under stringent conditions with a complementary sequence to all or part of the base sequence, and encodes a protein having the activity of a variant in which the amino acid corresponding to the 15th position from the N-terminus of SEQ ID NO: 10 is substituted with another amino acid, may be included without limitation.

For example, a polynucleotide encoding a polypeptide of the present application may be a polynucleotide sequence having a codon encoding an amino acid other than tyrosine corresponding to the amino acid at the 15th position from the N-terminus of SEQ ID NO: 10 in the polynucleotide sequence of SEQ ID NO: 17 to 19, and having a sequence homology or identity of about 75% or more with any one of SEQ ID NOs: 17 to 19.

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

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

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

The above microorganism may be a microorganism expressing a serine protease variant.

In the present application, the term "protein to be/is/is expressed" may mean a state in which the 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.

Specifically, "introduction of a protein" may mean that a microorganism exhibits activity of a specific protein that it did not originally possess, or exhibits activity that is enhanced compared to the intrinsic activity or activity of the protein before modification. For example, a polynucleotide encoding a specific protein may be introduced into a chromosome in a microorganism, or a vector containing a polynucleotide encoding a specific protein may be introduced into a microorganism and its activity may be exhibited.

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

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

The above "enhancement of activity" may mean that the activity is enhanced compared to the intrinsic activity or activity before modification of a specific protein possessed by a microorganism. "Intrinsic activity" may mean the activity of a specific protein originally possessed by a parent strain before the trait change, when the trait of a microorganism changes due to genetic mutation caused by natural or artificial factors.

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

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

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

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

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

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

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

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

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

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

For example, the composition provided in the present application may further include a naturally occurring substance or a non-naturally occurring substance in addition to the variant provided in the present application.

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

The "feed composition" of the present application is any natural or artificial diet, meal, etc. or a component of said meal, which is intended to be eaten, ingested, or digested by an animal or suitable therefor, and the feed can be manufactured into various forms of feed known in the art.

The above feed composition may be a feed additive.

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

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

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

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

The feed composition of the present application may further include an enzyme preparation other than the serine protease variant. For example, the feed composition may further include one or more selected from a fat-decomposing enzyme such as lipase, a phytase that decomposes phytic acid to create phosphate and inositol phosphate, an amylase that hydrolyzes an α-1,4-glycoside bond contained in starch and glycogen, a phosphatase that hydrolyzes an organophosphate ester, a maltase that hydrolyzes maltose into two molecules of glucose, and a converting enzyme that hydrolyzes saccharose to create a glucose-fructose mixture. However, the present invention is not limited thereto.

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

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

The amount of the serine protease variant of the present application included in the feed composition of the present application is not particularly limited and may be appropriately adjusted depending on the purpose. In one embodiment, as is commonly known in the technical field to which the present application belongs, it may be included in an amount suitable for surviving in the digestive tract of livestock for a long period of time and decomposing a protein source material, but is not limited thereto.

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

The above protease protein can be used in a food composition in a liquid or solid form. Additionally, the food can be a powder, a pill, a beverage, a tea, or an additive to a general food.

As an example, the food may be a food group requiring protease, such as dairy products, health functional foods for bowel movements or diets, and functional health functional foods for preventing hypertension.

In other embodiments, the protease variant may be included in various food compositions and used as a food solubilizer or tenderizer, a meat texture modifier. In other various embodiments, it may be added to a baking process and used in a step of destroying a gluten network. Or, it may be used to hydrolyze food proteins (e.g., proteins in milk). Or, it may be included in various food compositions and used for purposes such as rendering, flavoring, reducing bitterness, changing emulsifying properties, producing bioactive peptides, reducing allergens of proteins, etc., but this is only one example and is not limited to the above-mentioned uses.

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

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

The detergent composition according to the present application can be in the form of a one-part and two-part aqueous detergent composition, a non-aqueous liquid detergent composition, a cast solid, a granule, a particle, a compressed tablet, a gel, a paste or a slurry. The detergent composition can be used for removing food stains, food residue films and other small amounts of food compositions.

The detergent composition according to the present application may be provided in the form of, but is not limited to, a detergent composition for cleaning a hard surface, a detergent composition for cleaning a fabric, a detergent composition for washing dishes, a detergent composition for cleaning an oral cavity, a detergent for cleaning dentures, or a contact lens cleaning solution.

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

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

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

The serine protease variant of the present invention or a microorganism expressing the same can be used for purposes other than those described above, for example, in the manufacture of cosmetics, leather processing, pharmaceuticals, diagnostic agents, waste management, and chemicals for academic research.

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

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

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

As another example, a variant expressed in the culture stage of a microorganism may not be recovered. The microorganism expressing the variant itself may be used as a source of the variant.

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

The variant of the present invention, the host cell expressing the same, and the composition comprising the variant and/or the host cell can be used to decompose casein. In this decomposition step, cofactors, coenzymes, etc. can be added together. The hydrolysis step of the substrate can be performed under optimal pH, temperature conditions, etc., and those skilled in the art can select appropriate conditions.

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

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

Example 1. Making a Template

The Thermobifida fusca serine protease (TAP) gene was cloned into the pBE-S vector and used as a template. The PCR template in this study used the wild type or F12Y and N116D disclosed in prior patent PCT/KR2020/016775. Specifically, the method and primers described in Example 2-1 of PCT/KR2020/016775 were used to produce the F12Y and N116D mutants, and the F12Y and N116D mutants (F163Y and N267D based on SEQ ID NO: 9) were introduced into the pBE-S-TAP plasmid through site-directed mutagenesis based on the mature polypeptide (SEQ ID NO: 10) and used as a template (SEQ ID NO: 1).

In addition, through additional research, it was confirmed that the activity increased when the F180Y mutation was introduced in addition to the F12Y/N116D mutation, and the F12Y/N116D/F180Y sequence (SEQ ID NO: 20) was used as a template. The primers used to introduce the F180Y mutation are as follows.

F180Y (Forward): ccaaccaatcaatccattgcttagctattacggccttcaattagtg

F180Y (Reverse): cactaattgaaggccgtaatagctaagcaatggattgattggttgg

Example 2. Production of point mutants

A single mutation was introduced into the gene encoding the mature region of serine protease from Thermobifida fusca . Specifically, the single mutation site was selected based on rational design for improving casein activity of TAP, and primers were created as follows to mutate/substitute amino acids into desired amino acids through Site Directed Mutagenesis (SDM) (Table 1). In the table below, the mutation locations are listed based on SEQ ID NO: 10.

Protease Variant Primer List Template Mutation location Primer Sequence (5’-3’) F12Y/N116D
/F180Y (sequence number 7) Y15A Y15A_F GTGGAAACCCGTACTATTACGGAAATGCCAGATGCAGTATCG Y15A_R CGATACTGCATCTGGCATTTCCGTAATAGTACGGGTTTCCAC Y15G Y15G_F GTGGAAACCCGTACTATTACGGAAATGGCAGATGCAGTATCG Y15G_R CGATACTGCATCTGCCATTTCCGTAATAGTACGGGTTTCCAC Y15H Y15H_F GTGGAAACCCGTACTATTACGGAAATCACAGATGCAGTATC Y15H_R GATACTGCATCTGTGATTTCCGTAATAGTACGGGTTTCCAC Y15R Y15R_F GTGGAAACCCGTACTATTACGGAAATCGCAGATGCAGTATCG Y15R_R CGATACTGCATCTGCGATTTCCGTAATAGTACGGGTTTCCAC Y15S Y15S_F GTGGAAACCCGTACTATTACGGAAATAGCAGATGCAGTATCG Y15S_R CGATACTGCATCTGCTATTTCCGTAATAGTACGGGTTTCCAC WT (SEQ ID NO: 10) Y15S Y15S_F CCCTACTATTTCGGGAACAGCCGGCTGCTCTATCGGATT Y15S_R AATCCGATAGAGCAGCGGCTGTTCCCGAAATAGTAGGG

Single mutants were generated by PCR using Template, Primer, and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, La Jolla, CA, USA). PCR was performed using Eppendorf Mastercycler Nexus GX2, and the reaction conditions were as follows.

Initial denaturation - 95℃, 30sec

Denaturation - 95℃, 30sec

Annealing - 55℃, 1min

Extension - 68℃, 7min (18 cycles from denaturation to extension)

Final Extension - 68℃, 5min

The generated mutants were transformed into E. coli Dh5α strain after removing the original template by treatment with 1 μL Dpn I restriction enzyme (10 U/μL, 37°C, 1 hr), and then confirmed through sequencing (Macrogen, Seoul).

Example 3. Activity Evaluation of Site Directed Mutagenesis Variants

In this study, the activity of protease was measured by checking the amount of tyrosine released from casein (from bovine milk) through an enzymatic reaction as a substrate. After transforming the constructed plasmid into Bacillus subtilis LB700 strain, it was inoculated into BHI medium (Table 2) containing kanamycin antibiotic and cultured at 37℃ for ~96 hours, and a portion of the culture solution was used for the enzymatic reaction. The culture solution (enzyme) diluted in 0.1 mL of 50 mM sodium borate (pH 10.5 at room temperature) buffer and 0.1 mL of substrate 1.0% casein (Sigma-Aldrich Co.) were mixed and reacted at 40℃ for 10 minutes. Thereafter, 0.2 mL of trichloroacetic acid (TCA, Sigma-Aldrich Co.) solution was added to terminate the reaction. After centrifugation (10,000 × g, 10 minutes), 0.75 mL of 0.4 M sodium carbonate anhydrous and 0.15 mL of 3-fold diluted Folin Ciocalteu (Sigma-Aldrich Co.) solution were added to 0.15 mL of the supernatant, and the color reaction was performed at 40℃ for 20 minutes, and the absorbance was measured at 680 nm using a spectrophotometer. The protease activity was calculated from a standard curve quantifying tyrosine (Sigma-Aldrich Co.), and one unit of activity was defined as the amount of enzyme that produces 1 μg of tyrosine per minute. The activities measured in this way are as follows (Tables 3 and 4).

Bacillus BHI medium composition Badge ingredients unit g or mg Sucrose(98.5%) g/L 20.0 MgSO4 _· 7H2O g/L 2.0 CSL g/L 30.00 Yeast Extract g/L 20.00 Na ₂ SO ₄ g/L 2.00 (NH ₄ ) ₂ SO ₄ g/L 2.68 NaH ₂ PO ₄ 2H ₂ O g/L 4.00 K ₂ HPO ₄ g/L 14.60 CAPA g/L 0.5 NCA g/L 0.5 Thiamine g/L 0.5 CaCl ₂ mg/L 1.50 ZnSO _4· 7H ₂ O mg/L 0.54 MnSO ₄ H ₂ O mg/L 0.30 FeSO _4· 7H ₂ O mg/L 42.94 CuSO _4· 5H ₂ O mg/L 0.48 CoCl ₂ H ₂ O mg/L 0.54

Evaluation of mutant enzyme activity Template Additional mutations Enzyme activity (unit/ml) Relative activity (%) F12Y/N116D/F180Y - 219.5 100.0 F12Y/N116D/F180Y Y15A 256.5 116.8 Y15G 271.7 123.8 Y15H 253.2 115.4 Y15R 226.7 103.3 Y15S 315.2 143.6

Evaluation of mutant enzyme activity Template Additional mutations Enzyme activity (unit/ml) Relative activity (%) WT 148.7 100.0 WT Y15S 181.7 122.2

The measurement results showed that when the Y15S mutation was added compared to the F12Y/N116D/F180Y mutant, the activity increased by approximately 140% under pH 10.5 and 40℃ conditions.

From the above description, those skilled in the art to which the present application belongs will be able to understand that the present application can be implemented in other specific forms without changing the technical idea or essential features thereof. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. The scope of the present application should be interpreted as including all changes or modified forms derived from the meaning and scope of the patent claims described below rather than the above detailed description and their equivalent concepts within the scope of the present application.

Attach electronic file of sequence list

Claims (10)

A serine protease mutant in which the amino acid corresponding to position 15 of sequence number 10 is substituted with another amino acid.
A serine protease variant in claim 1, wherein the other amino acid is alanine (A), histidine (H), arginine (R), glycine (G), or serine (S).
A serine protease mutant in which the amino acid corresponding to position 166 of sequence number 9 is substituted with another amino acid.
A serine protease mutant in which the amino acid corresponding to position 196 of sequence number 8 is substituted with another amino acid.
A serine protease variant according to any one of claims 1 to 4, wherein the variant comprises an amino acid sequence represented by any one of SEQ ID NOs: 11 to 16.
A polynucleotide encoding a serine protease variant of any one of claims 1 to 4.
A vector comprising the polynucleotide of claim 6.
A microorganism comprising a serine protease variant according to any one of claims 1 to 4, a polynucleotide encoding the variant, or a vector comprising the polynucleotide.
A serine protease variant of any one of claims 1 to 4; and
A composition comprising at least one of the serine protease variant, a microorganism comprising a polynucleotide encoding the variant, or a vector comprising the polynucleotide.
A composition in claim 9, wherein the composition is a feed composition.