WO2022191653A1 - 신규 세린 프로테아제 변이체 - Google Patents

신규 세린 프로테아제 변이체 Download PDF

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WO2022191653A1
WO2022191653A1 PCT/KR2022/003415 KR2022003415W WO2022191653A1 WO 2022191653 A1 WO2022191653 A1 WO 2022191653A1 KR 2022003415 W KR2022003415 W KR 2022003415W WO 2022191653 A1 WO2022191653 A1 WO 2022191653A1
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amino acid
seq
serine protease
present application
acid sequence
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English (en)
French (fr)
Korean (ko)
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이주희
조아라
지창준
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CJ CheilJedang Corp
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CJ CheilJedang Corp
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Priority to CA3175414A priority Critical patent/CA3175414A1/en
Priority to EP22767549.3A priority patent/EP4116416A4/en
Priority to US17/997,455 priority patent/US20230174964A1/en
Priority to JP2022562328A priority patent/JP7676439B2/ja
Priority to CN202280004542.9A priority patent/CN115698280A/zh
Publication of WO2022191653A1 publication Critical patent/WO2022191653A1/ko
Anticipated expiration legal-status Critical
Priority to JP2024159122A priority patent/JP2025011087A/ja
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/14—Pretreatment of feeding-stuffs with enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C40B40/08—Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries

Definitions

  • This application relates to novel serine protease variants.
  • Protease has various functions such as digestion, absorption, and defense in vivo, and depending on the structure of the active site, serine protease, cysteine protease, and aspartate proteolysis It is divided into an enzyme (aspartic protease) and a metalloprotease (metalloprotease).
  • serine protease or serine endopeptidase is an enzyme that mainly has an active serine residue at the active site in common, and serine acts as a nucleophilic amino acid at the active site of the protease to cleave a peptide bond in a protein (Hedstrom) , 2002. Chem Rev 102: 4501-4524).
  • Serine protease has a variety of uses, so it is not only used as an ingredient in detergents for clothing and contact lens cleaners, in addition to treating human diseases such as dissolving blood clots, but also as a component of milk protein, silk degumming, and leather. It is widely used for soaking, hair removal, oligopeptide synthesis and recovery of silver from waste Xray films, and for the production and improvement of feed and food (Korean Patent Application Laid-Open No. 10-2005-0068750).
  • Another object of the present application is to provide a polynucleotide encoding the serine protease variant and a vector comprising the same.
  • Another object of the present application is the serine protease variant; a polynucleotide encoding the variant; And it is to provide a microorganism comprising one or more of the vectors comprising the polynucleotide.
  • Another object of the present application is the serine protease variant; And it is to provide a composition for feed, comprising one or more of the microorganisms expressing the same.
  • the serine protease variant of the present application has superior activity compared to the existing serine protease, it can be usefully used industrially.
  • One aspect of the present application provides a serine protease variant.
  • serine protease refers to an enzyme belonging to a subgroup of proteases and having proteolytic activity.
  • the serine protease may be an enzyme that degrades proteins by hydrolyzing peptide bonds and basically has an active serine residue at the active site, more specifically histidine, an amino acid residue that can be referred to as a catalytic triad , an enzyme having a spatial arrangement of aspartate and serine, but is not limited thereto.
  • the serine protease of the present application is, but is not limited to, the genus Thermobifida, the genus Nocardiopsis, the genus Actinorugispora, or the genus Spinactinospora derived from microorganisms. may be Specifically, in the present application, the wild type of the serine protease is Thermobifida fusca , Thermobifida cellulosilytica , Thermobifida halotolerans , Actinolgispora endophyte.
  • Tica Actinorugispora endophytica ), spinactinospora alkalitolerans ), Nocardiopsis composta ), or Nocardiopsis potens ) It may be a serine protease derived from, but is not limited to .
  • the serine protease of the present application may be a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 31, but is not limited thereto.
  • the amino acid sequence of SEQ ID NO: 31 may be an amino acid sequence derived from SEQ ID NO: 40 or SEQ ID NO: 2, but is not limited thereto.
  • the serine protease of the present application may include, consist essentially of, or consist of the amino acid sequence of any one of SEQ ID NOs: 49 to 54, but is not limited thereto.
  • the amino acid sequence of SEQ ID NOs: 49 to 54 may be derived from any one of the amino acid sequences of SEQ ID NOs: 67 to 72, but is not limited thereto.
  • the serine protease of the present application may include, without limitation, a sequence having the same activity as the above-described amino acid sequence. Further, it comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 31 and 49 to 54 or an amino acid sequence having at least 60% homology or identity therewith. may be, but is not limited thereto. Specifically, the amino acid sequence is at least 60%, 61%, 62%, 63 with any one of the amino acid sequences set forth in SEQ ID NOs: 31 and 49 to 54 or any one of the amino acid sequences set forth in SEQ ID NOs: 31 and 49 to 54.
  • a 'polypeptide comprising the amino acid sequence of SEQ ID NO: 31' may belong to a 'polypeptide comprising the amino acid sequence of SEQ ID NO: 31' if it has the same or corresponding activity.
  • the term 'homology' or 'identity' refers to a degree related to two given amino acid sequences or base sequences, and may be expressed as a percentage.
  • the terms homology and identity can often be used interchangeably.
  • Sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, with default gap penalties established by the program used may be used.
  • Substantially homologous or identical sequences generally have moderate or high stringency conditions along at least about 50%, 60%, 70%, 80% or 90% of the entire or full-length sequence. It can hybridize under stringent conditions. Hybridization is also contemplated for polynucleotides containing degenerate codons instead of codons in the polynucleotides.
  • GAP program is defined as the total number of symbols in the shorter of two sequences divided by the number of similarly aligned symbols (ie, nucleotides or amino acids).
  • Default parameters for the GAP program are: (1) a binary comparison matrix (containing values of 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation , pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps.
  • the serine protease variant provided in the present application among the proteins having serine protease activity described above, in which an amino acid at a specific position is substituted, means a variant in which the enzyme activity exceeds 100% compared to the protein before the mutation.
  • the variant provided herein is greater than about 100%, specifically about 110%, about 120%, about 130 compared to a wild-type enzyme comprising the amino acid sequence of any one of SEQ ID NOs: 31 and 49 to 54. %, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about 200% or more of increased enzyme activity.
  • variant means that one or more amino acids differ from the recited sequence in conservative substitution and/or modification, but the function of the protein ( functions) or properties (properties) are maintained.
  • a variant differs from an identified sequence by several amino acid substitutions, deletions or additions.
  • Such variants can generally be identified by modifying one of the polypeptide sequences and evaluating the properties of the modified polypeptide. That is, the ability of the variant may be increased, unchanged, or decreased compared to the native protein.
  • variants may include variants in which one or more portions, such as an N-terminal leader sequence or a transmembrane domain, have been removed.
  • variants may include variants in which a portion is removed from, or added to, the N- and/or C-terminus of the mature protein.
  • variant may include terms such as modified/mutated protein, mutated polypeptide, and mutant (in English, modified, modified protein, modified polypeptide, mutant, mutein, divergent, variant, etc.) The term is not limited thereto.
  • the mutant may have increased activity of the mutated protein compared to the natural wild-type or unmodified protein, but is not limited thereto.
  • conservative substitution means substituting one amino acid with another amino acid having similar structural and/or chemical properties. Such variants may have, for example, one or more conservative substitutions while still retaining one or more biological activities. Such amino acid substitutions may generally occur based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues. For example, among amino acids with electrically charged amino acids, positively charged (basic) amino acids are arginine, lysine, and histidine, and negatively charged (acidic) amino acids are glutamic acid and aspartate.
  • nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan and proline
  • polar or hydrophilic ( hydrophilic) amino acids include serine, threonine, cysteine, tyrosine, asparagine and glutamine
  • aromatic amino acids among the amino acids include phenylalanine, tryptophan and tyrosine.
  • variants may include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide.
  • polypeptide can be conjugated with a signal (or leader) sequence at the N-terminus of the protein that is involved in the transfer of the protein either co-translationally or post-translationally.
  • the polypeptide may also be conjugated with other sequences or linkers to enable identification, purification, or synthesis of the polypeptide.
  • amino acid protease variant refers to a polypeptide comprising one or more amino acid substitutions in the amino acid sequence of a polypeptide having serine protease activity.
  • the serine protease variant of the present application may include an amino acid in which an amino acid at a position corresponding to amino acid 12 and/or amino acid 116 from the N-terminus of SEQ ID NO: 31 is substituted with another amino acid.
  • the serine protease variant comprises a substitution of amino acids corresponding to amino acids 12 and/or 116 of SEQ ID NO: 31, and is at least 60% 100% with the amino acid sequence of any one of SEQ ID NOs: 31 and 49 to 54 It may include an amino acid sequence having less than homology or identity.
  • the serine protease variant of the present application comprises a substitution of an amino acid at a position corresponding to amino acid 12 and/or amino acid 116 of SEQ ID NO: 31, and the amino acid sequence of any one of SEQ ID NOs: 31 and 49 to 54 and 60% or more, for example 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, but may have less than 100% homology or identity, but is not limited thereto.
  • amino acids 12 and 116 from the N-terminus of SEQ ID NO: 31 correspond to amino acids 12 and 116 from the N-terminus of SEQ ID NOs: 49 to 54, with respect to the position of amino acids based on SEQ ID NO: 31
  • the description can be equally applied to amino acids 12 and 116 of the amino acid sequence of any one of SEQ ID NOs: 49 to 54.
  • the serine protease variant of the present application comprises a substitution of an amino acid at a position corresponding to amino acid 12 and/or amino acid 116 of SEQ ID NO: 54, and the amino acid sequence of any one of SEQ ID NOs: 52 to 54 and 60 % or more, e.g.
  • the serine protease variant comprises a substitution of an amino acid at a position corresponding to amino acid 12 of SEQ ID NO: 54, and 60% or more of the amino acid sequence of SEQ ID NO: 54, for example, 61%, 62%, 63%, 64 %, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, or 99% or more, and less than 100% homology or identity, but is not limited thereto.
  • the serine protease variant of the present application corresponds to position 12, position 116, or position 12 and 116 from the N-terminus in any one amino acid sequence selected from SEQ ID NOs: 31 and 49 to 54. It may be a protein in which all amino acids are substituted with other amino acids.
  • the 'other amino acid' means an amino acid different from that before the substitution, and is not limited as long as it is an amino acid other than the amino acid before the substitution.
  • phenylalanine at position 12 in any one amino acid sequence selected from SEQ ID NOs: 31 and 49 to 51 is glycine, alanine, arginine ), aspartate, cysteine, glutamate, asparagine, glutamine, histidine, proline, serine, tyrosine, isoleucine ( isoleucine), leucine, lysine, tryptophan, valine, methionine or threonine, and/or asparagine at position 116 is replaced with glycine, alanine, It may be substituted with arginine, aspartate, cysteine, glutamic acid, glutamine, histidine, proline, serine, tyrosine, isoleucine, leucine, lysine, phenylalanine, tryptophan, valine, methionine or threonine, but is not limited thereto.
  • proline at position 12 in any one amino acid sequence selected from SEQ ID NOs: 52 to 54 is phenylalanine, glycine, alanine, Arginine, aspartate, cysteine, glutamate, asparagine, glutamine, histidine, serine, tyrosine, isoleucine , leucine, lysine, tryptophan, valine, methionine or threonine is substituted and / and the asparagine at position 116 is glycine, alanine, arginine, It may be substituted with aspartate, cysteine, glutamic acid, glutamine, histidine, proline, serine, tyrosine, isoleucine, leucine, lysine, phenylalanine, tryptophan, valine, methionine or threonine, but is not limited thereto.
  • the amino acid corresponding to position 12 in any one amino acid sequence selected from SEQ ID NOs: 31 and 49 to 54 is tyrosine (Y), serine (S), alanine (A) or arginine (R). is substituted, or the amino acid corresponding to position 116 is substituted with aspartate (D), serine (S), threonine (T) or glycine (G), or positions 12 and 116 in the amino acid sequence of SEQ ID NO: 31
  • the amino acids corresponding to the positions are tyrosine (Y) and aspartate (D), respectively; tyrosine (Y) and serine (S); serine (S) and aspartate (D); serine (S) and threonine (T); Alternatively, it may be a protein comprising a substituted with alanine (A) and glycine (G), but is not limited thereto.
  • the serine protease variant may be one in which proline at position 12 in any one amino acid sequence selected from SEQ ID NOs: 52 to 54 is substituted with tyrosine, alanine, serine or arginine, but is not limited thereto.
  • a variant in which the amino acid at positions 12 and/or 116 in any one amino acid sequence selected from SEQ ID NOs: 31 and 49 to 54 is substituted with another amino acid is a variant in which the amino acid corresponding to the position is substituted with another amino acid. It is self-evident to include
  • the variant is at least 60% or more, 60%, 61 with an amino acid sequence described in any one selected from SEQ ID NOs: 31 and 49 to 54 described above or an amino acid sequence selected from any one of SEQ ID NOs: 31 and 49 to 54 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , in the amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% homology or identity, amino acid 12 from the N-terminus of any one amino acid sequence selected from SEQ ID NOs: 31 and 49 to 54 and/or the amino acid at the position corresponding to amino acid 116 is substituted with another amino acid.
  • the variant 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 is any one selected from SEQ ID NOs: 32 to 39 It may include, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO:, but is not limited thereto.
  • the variant in which the amino acid at the position corresponding to amino acid 12 and/or amino acid 116 in the amino acid sequence of SEQ ID NO: 49 is substituted with another amino acid comprises the amino acid sequence of SEQ ID NO: 55 or 56, It may consist essentially of or consist of, but is not limited thereto.
  • the variant in which the amino acid at the position corresponding to amino acid 12 and/or amino acid 116 in the amino acid sequence of SEQ ID NO: 50 is substituted with another amino acid comprises the amino acid sequence of SEQ ID NO: 57 or 58, It may consist essentially of or consist of, but is not limited thereto.
  • the variant in which the amino acid at the position corresponding to the 12th amino acid and / or the 116th amino acid in the amino acid sequence of SEQ ID NO: 51 is substituted with another amino acid comprises the amino acid sequence of SEQ ID NO: 59 or 60, It may consist essentially of or consist of, but is not limited thereto.
  • the variant in which the amino acid at the position corresponding to amino acid 12 and/or amino acid 116 in the amino acid sequence of SEQ ID NO: 52 is substituted with another amino acid among the variants comprises the amino acid sequence of SEQ ID NO: 61 or 62, It may consist essentially of or consist of, but is not limited thereto.
  • the variant in which the amino acid at the position corresponding to amino acid 12 and/or amino acid 116 in the amino acid sequence of SEQ ID NO: 53 is substituted with another amino acid comprises the amino acid sequence of SEQ ID NO: 63 or 64, It may consist essentially of or consist of, but is not limited thereto.
  • the variant in which the amino acid at the position corresponding to amino acid 12 and/or amino acid 116 in the amino acid sequence of SEQ ID NO: 54 is substituted with another amino acid comprises the amino acid sequence of SEQ ID NO: 65 or 66, It may consist essentially of or consist of, but is not limited thereto.
  • the serine protease variant of the present application comprises a substitution with another amino acid at the position corresponding to the 12th and / or 116th position of any one amino acid sequence selected from SEQ ID NOs: 31 and 49 to 54, Any one amino acid sequence selected from SEQ ID NOs: 31 and 49 to 54 and 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75 %, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, but less than 100% sequence homology, and may have serine protease activity.
  • the serine protease variant of the present application may have enhanced activity compared to the unmodified polypeptide, the native wild-type polypeptide, or the unmodified polypeptide, but is not limited thereto.
  • it is an amino acid sequence having such homology and exhibiting efficacy corresponding to the protein, it is apparent that a protein having an amino acid sequence in which some sequences are deleted, modified, substituted or added is also included within the scope of the present application.
  • the mature region of the NCBI Reference Sequence WP_016188200.1 corresponds to SEQ ID NO: 31 of the present application, and the sequence except for the signal peptide in SEQ ID NO: 40 corresponds to SEQ ID NO: 2 of the present application.
  • the serine protease variant of the present application is a serine protease in which amino acids at positions 12 and 116 of SEQ ID NO: 31 are substituted with other amino acids as described above. Deletion or addition of amino acids having minimal effect on the properties and secondary structure of the serine protease It is obvious that it may include In addition, those skilled in the art through sequence alignment known in the art, positions 12 and 116 from the N-terminus of SEQ ID NO: 31 of the present application are positions 193 and 297 of SEQ ID NO: 40, positions 163 and 267 of SEQ ID NO: 2 It can be seen that they correspond, and SEQ ID NO: 31 is included in SEQ ID NO: 2 and SEQ ID NO: 40.
  • the serine protease variant of the present application is, with respect to SEQ ID NO: 2 and SEQ ID NO: 40, including the amino acid sequence of SEQ ID NO: 31, amino acids at positions 12 and 116 of SEQ ID NO: 31 (163 and / in SEQ ID NO: 2) or amino acid at position 267, 193 and/or amino acid at position 297 in SEQ ID NO: 40) is substituted.
  • the description of SEQ ID NO: 31 and its 12th and 116th amino acids in the present application can be applied to SEQ ID NO: 2 and its 163, 267th amino acids, and SEQ ID NO: 40 and its 193, 297th amino acids.
  • the serine protease variant of the present application comprises an amino acid sequence in which the amino acids at positions corresponding to 12 and / or 116 of SEQ ID NO: 31 are substituted with other amino acids, and at least 60% or more from SEQ ID NO: 2, e.g.
  • the serine protease variant of the present application may be one in which the 163 and / or 267 amino acids of SEQ ID NO: 2 are substituted with other amino acids, and have 60% or more and less than 100% sequence homology with SEQ ID NO: 2, It may have a sequence homology of 60% or more with any one amino acid sequence selected from SEQ ID NOs: 3 to 10. However, it is not limited thereto.
  • variants containing the substitution of amino acids corresponding to positions 12 and/or 116 from the N-terminus of SEQ ID NOs: 49 to 54 are also serine of the present application. It is obvious that it is included within the scope of proteases.
  • sequence of the polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 49 to 54 is, for example, GenBank Accession: KUP96625.1 (SEQ ID NO: 67), NCBI Reference Sequence: WP_068687914.1 (SEQ ID NO: 68), NCBI Reference Sequence: WP_133739400.1 (SEQ ID NO: 69), NCBI Reference Sequence: WP_179641868.1 (SEQ ID NO: 70), NCBI Reference Sequence: WP_184391208.1 (SEQ ID NO: 71), or NCBI Reference Sequence: WP_017594871.1 (SEQ ID NO: 72) It may be an amino acid sequence described in the like.
  • positions 12 and/or 116 from the N-terminus of SEQ ID NOs: 49 to 54 in SEQ ID NOs: 67 to 72 through sequence alignment known in the art, and N- The description for positions 12 and/or 116 from the end may be applied.
  • the serine protease variant of the present application comprises a substitution of an amino acid corresponding to positions 12 and/or 116 from the N-terminus of the amino acid sequence of any one of SEQ ID NOs: 49 to 54 with other amino acids, an amino acid sequence of any one of Nos.
  • the serine protease variant of the present application comprises a substitution of the amino acid corresponding to position 12 based on the amino acid sequence set forth in SEQ ID NO: 54, and the amino acid sequence described in any one of SEQ ID NOs: 70 to 72 and 60 % or more, e.g.
  • the serine protease variant may further include a substitution of the amino acid corresponding to position 116 based on the amino acid sequence set forth in SEQ ID NO: 54.
  • the serine protease variant of the present application may include a substitution of the amino acid corresponding to position 198 based on the amino acid sequence set forth in SEQ ID NO: 67.
  • the variant comprises SEQ ID NO: 67 and at least 60%, for example 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology or identity.
  • the variant may include an amino acid sequence in which an amino acid at a position corresponding to amino acid 12 of SEQ ID NO: 49 is substituted with another amino acid, and has an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 49.
  • the serine protease variant may further include a substitution of the amino acid corresponding to position 302 based on the amino acid sequence set forth in SEQ ID NO: 67.
  • the serine protease variant of the present application may include a substitution of the amino acid corresponding to position 178 based on the amino acid sequence set forth in SEQ ID NO: 68.
  • the variant comprises SEQ ID NO: 68 and at least 60%, for example 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology or identity.
  • the variant may include an amino acid sequence in which an amino acid at a position corresponding to amino acid 12 of SEQ ID NO: 50 is substituted with another amino acid, and has an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 50.
  • the serine protease variant may further include a substitution of an amino acid corresponding to position 282 based on the amino acid sequence set forth in SEQ ID NO: 68.
  • the serine protease variant of the present application may include a substitution of the amino acid corresponding to position 207 based on the amino acid sequence set forth in SEQ ID NO: 69.
  • the variant is SEQ ID NO: 69 and at least 60%, for example 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology or identity.
  • the variant may include an amino acid sequence in which an amino acid at a position corresponding to amino acid 12 of SEQ ID NO: 51 is substituted with another amino acid, and has an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 51.
  • the serine protease variant may further include a substitution of the amino acid corresponding to position 311 based on the amino acid sequence set forth in SEQ ID NO: 69.
  • the serine protease variant of the present application may include a substitution of the amino acid corresponding to position 203 based on the amino acid sequence set forth in SEQ ID NO: 70.
  • the variant is SEQ ID NO: 70 and at least 60%, for example 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology or identity.
  • the variant may include an amino acid sequence in which an amino acid at a position corresponding to amino acid 12 of SEQ ID NO: 52 is substituted with another amino acid, and has an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 52.
  • the serine protease variant may further include a substitution of the amino acid corresponding to position 303 based on the amino acid sequence set forth in SEQ ID NO: 70.
  • the serine protease variant of the present application may include a substitution of the amino acid corresponding to position 201 based on the amino acid sequence set forth in SEQ ID NO: 71.
  • the variant is SEQ ID NO: 71 and at least 60%, for example 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology or identity.
  • the variant may include an amino acid sequence in which an amino acid at a position corresponding to amino acid 12 of SEQ ID NO: 53 is substituted with another amino acid, and has an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 53.
  • the serine protease variant may further include a substitution of the amino acid corresponding to position 304 based on the amino acid sequence set forth in SEQ ID NO: 71.
  • the serine protease variant of the present application may include a substitution of the amino acid corresponding to position 201 based on the amino acid sequence set forth in SEQ ID NO: 72.
  • the variant is SEQ ID NO: 72 and at least 60%, for example 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology or identity.
  • the variant may include an amino acid sequence in which an amino acid at a position corresponding to amino acid 12 of SEQ ID NO: 54 is substituted with another amino acid, and has 70% or more sequence identity to SEQ ID NO: 54.
  • the serine protease variant may further include a substitution of the amino acid corresponding to position 304 based on the amino acid sequence set forth in SEQ ID NO: 72.
  • corresponding to refers to an amino acid residue at a listed position in a protein or polypeptide, or to an amino acid residue that is similar, identical to, or homologous to a residue listed in a protein or polypeptide. Identifying an amino acid at a corresponding position may be determining a specific amino acid in a sequence that refers to a specific sequence.
  • corresponding region generally refers to a similar or corresponding position in a related protein or reference protein. For example, any amino acid sequence is aligned with SEQ ID NO: 31, and based on this, each amino acid residue of the amino acid sequence can be numbered with reference to the numerical position of the amino acid residue corresponding to the amino acid residue of SEQ ID NO: 31. .
  • a sequence alignment algorithm such as that described in this application can identify the position of an amino acid, or a position at which modifications, such as substitutions, insertions, or deletions, occur compared to a query sequence (also referred to as a "reference sequence").
  • Such alignments include the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000). , Trends Genet. 16: 276-277), but is not limited thereto.
  • EMBOSS European Molecular Biology Open Software Suite, Rice et al., 2000).
  • Trends Genet. 16: 276-277 but is not limited thereto.
  • the corresponding amino acid residues can be identified through multiple sequence alignment. 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).
  • Another aspect of the present application is to provide a polynucleotide encoding the serine protease variant.
  • polynucleotide refers to a DNA or RNA strand of a certain length or longer as a polymer of nucleotides in which nucleotide monomers are linked in a long chain by covalent bonds, and more specifically, encoding the variant. polynucleotide fragments.
  • 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 gene encoding the wild-type serine protease in the present application is the genus Thermobifida, the genus Nocardiopsis, the genus Actinorugispora, or the genus Spinactinospora.
  • Thermobifida fusca May be derived from microorganisms of the genus, specifically Thermobifida fusca , Thermobifida cellulosilytica , Thermobifida halotolerans , Actinorugispora . endophytica ), spinactinospora alkalitolerans , Nocardiopsis composta , or Nocardiopsis potens ), but may be derived from, but not limited to.
  • polynucleotide of the present application various modifications are made in the coding region within the range that does not change the amino acid sequence of the polypeptide due to codon degeneracy or considering codons preferred in the organism to express the polypeptide.
  • the polynucleotide of the present application is a variant of the present application, specifically, a polypeptide consisting of an amino acid sequence described in any one of SEQ ID NOs: 32 to 39 and SEQ ID NOs: 55 to 66, or homology thereto.
  • the branch may be a polynucleotide sequence encoding a polypeptide, but is not limited thereto.
  • polynucleotide sequence encoding a polypeptide consisting of an amino acid sequence described in any one of SEQ ID NOs selected from SEQ ID NOs: 32 to 39 is described in any one of SEQ ID NOs selected from SEQ ID NOs: 41 to 48 It may be composed of a polynucleotide sequence, but is not limited thereto.
  • the serine protease variant of the present application is a mutant in which the amino acid at positions 12 and/or 116 of the sequence is substituted in a polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 31 and 49 to 54. Therefore, it is obvious that polynucleotide sequences encoding such serine protease variants are also included in the scope of the present application.
  • amino acids at positions 12 and/or 116 of SEQ ID NO: 31 (163 and/or 267 amino acids in SEQ ID NO: 40, 193 and/or 297 in SEQ ID NO: 40)
  • Amino acid substituted variants are also included in the scope of the serine protease of the present application, and therefore a polynucleotide sequence encoding the same is also included in the scope of the present application.
  • the polynucleotide sequence encoding the serine protease variant may encode an amino acid sequence described in any one of SEQ ID NOs: 3 to 10, specifically selected from SEQ ID NOs: 23 to 30 It may be composed of a polynucleotide sequence described in any one of the SEQ ID NOs, but is not limited thereto.
  • any one N- selected from SEQ ID NOs: 31 and 49 to 54 Any sequence encoding a protein having the activity of a mutant in which the amino acid at the position corresponding to amino acid 12 and/or amino acid 116 from the terminal is substituted with another amino acid may be included without limitation.
  • stringent conditions means conditions that allow specific hybridization between polynucleotides. These conditions are specifically described in the known literature. For example, genes having high homology between genes having homology of 40% or more, specifically 90% or more, more specifically 95% or more, still more specifically 97% or more, and particularly specifically 99% or more homology. Conditions that hybridize with each other and do not hybridize with genes with lower homology than that, or wash conditions for general Southern hybridization at 60°C, 1X SSC, 0.1% SDS, specifically 60°C, 0.1X SSC, 0.1% SDS, more specifically, at a salt concentration and temperature equivalent to 68° C., 0.1X SSC, 0.1% SDS, washing conditions once, specifically 2 to 3 times, can be enumerated. However, the present invention is not limited thereto, and may be appropriately adjusted by those skilled in the art according to the purpose.
  • Hybridization requires that two polynucleotides have complementary sequences, although mismatch between bases is possible depending on the stringency of hybridization.
  • complementary is used to describe the relationship between nucleotide bases capable of hybridizing to each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the present application may also include substantially similar polynucleotide sequences as well as isolated polynucleotide fragments complementary to the overall sequence.
  • polynucleotides having homology can be detected using hybridization conditions including a hybridization step at a Tm value of 55° C. and using the conditions described above.
  • the Tm value may be 60° C., 63° C. or 65° C., but is not limited thereto and may be appropriately adjusted by those skilled in the art according to the purpose.
  • the appropriate stringency for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides, and the parameters are well known in the art.
  • Another aspect of the present application is to provide a vector comprising a polynucleotide encoding the serine protease variant of the present application.
  • the term "vector” refers to a DNA preparation containing the nucleotide sequence of a polynucleotide encoding the target protein operably linked to a suitable regulatory sequence so that the target protein can be expressed in a suitable host.
  • suitable regulatory sequences 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 a sequence regulating the termination of transcription and translation.
  • the vector After transformation into an appropriate host cell, the vector can replicate or function independently of the host genome, and can be integrated into the genome itself.
  • operably linked means that a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a target protein of the present application and the polynucleotide sequence are functionally linked.
  • the operable linkage may be prepared using a genetic recombination technique known in the art, and site-specific DNA cleavage and ligation may be made using a cleavage and ligation enzyme in the art, but is not limited thereto.
  • the vector used in the present application is not particularly limited, and any vector known in the art may be used.
  • Examples of commonly used vectors include plasmids, cosmids, viruses and bacteriophages in a natural or recombinant state.
  • pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage vectors or cosmid vectors
  • pBR-based, pUC-based, and pBluescriptII-based plasmid vectors may be used as plasmid vectors.
  • pGEM-based, pTZ-based, pCL-based, pET-based, pUB110-based and the like can be used.
  • pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pSM704 vectors and the like can be used.
  • the vector usable in the present application is not particularly limited and a known expression vector may be used.
  • a polynucleotide encoding a target mutant in a chromosome may be replaced with a mutated polynucleotide through a vector for intracellular chromosome insertion.
  • the insertion of the polynucleotide into the chromosome may be performed by any method known in the art, for example, homologous recombination, but is not limited thereto. It may further include a selection marker (selection marker) for confirming whether the chromosome is inserted.
  • the selectable marker is used to select cells transformed with the vector, that is, to determine whether a target nucleic acid molecule is inserted, and selectable phenotypes such as drug resistance, auxotrophicity, resistance to cytotoxic agents, or expression of a surface variant polypeptide. Markers that give ? may be used. In an environment treated with a selective agent, only the cells expressing the selectable marker survive or exhibit other expression traits, so that the transformed cells can be selected.
  • Another aspect of the present application is a serine protease variant of the present application; a polynucleotide encoding the variant; And it provides a host cell comprising one or more of the vector containing the polynucleotide.
  • the host cell specifically, may be a microorganism.
  • the microorganism comprising at least one of the serine protease variant, the polynucleotide encoding the variant, and a vector including the same may be a microorganism produced by transformation with a vector comprising a polynucleotide encoding the variant, but not limited
  • the microorganism may be a microorganism expressing a serine protease variant.
  • the term "to be/are/are” a protein may refer to a state in which a target protein is introduced into a microorganism or modified to be expressed in the microorganism.
  • the “protein of interest” may be the aforementioned serine protease variant.
  • introduction of protein may mean that the microorganism exhibits the activity of a specific protein that it did not originally have, or exhibits improved activity compared to the intrinsic activity or activity before modification of the protein.
  • a polynucleotide encoding a specific protein may be introduced into a chromosome in a microorganism, or a vector including a polynucleotide encoding a specific protein may be introduced into the microorganism to exhibit its activity.
  • the microorganism of the present application may be a recombinant microorganism.
  • the recombination may be accomplished by genetic modification such as transformation.
  • the term "transformation" refers to introducing a vector including a polynucleotide encoding a target protein into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell.
  • the transformed polynucleotide may include all of them regardless of whether they are inserted into the chromosome of the host cell or located outside the chromosome, as long as they can be expressed in the host cell.
  • the polynucleotide includes DNA and RNA encoding a target protein. As long as the polynucleotide can be introduced and expressed into a host cell, it may be introduced in any form.
  • the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct including all elements necessary for self-expression.
  • the expression cassette may include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal.
  • the expression cassette may be in the form of an expression vector capable of self-replication.
  • the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.
  • the transformation method includes any method of introducing a polynucleotide into a cell, and may be performed by selecting a suitable standard technique as known in the art depending on the host cell. For example, electroporation, calcium phosphate (Ca(H 2 PO 4 ) 2 , CaHPO 4 , or Ca 3 (PO 4 ) 2 ) precipitation, calcium chloride (CaCl 2 ) precipitation, microinjection, Polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, natural competence (see, eg, Perry and Kuramitsu, 1981, Infect. Immun . 32: 1295-1297) and acetic acid Lithium-DMSO method and the like, but is not limited thereto.
  • a suitable standard technique as known in the art depending on the host cell. For example, electroporation, calcium phosphate (Ca(H 2 PO 4 ) 2 , CaHPO 4 , or Ca 3 (PO 4 ) 2 ) precipitation, calcium chlor
  • the recombinant microorganism may have enhanced serine protease activity of the present application.
  • the “enhancement of activity” may mean that the activity is improved compared to the intrinsic activity or activity before modification of a specific protein of the microorganism.
  • “Intrinsic activity” may refer to the activity of a specific protein originally possessed by the parent strain prior to transformation when the trait of a microorganism is changed due to genetic variation caused by natural or artificial factors.
  • the enhancement of the activity of the protein variant includes increasing the intracellular copy number of the gene encoding the protein variant, a method of introducing a mutation into the expression control sequence of the gene encoding the protein variant, and the protein variant A method of replacing a gene expression control sequence coding for a sequence with strong activity, a method of replacing a gene encoding a native protein having serine protease activity on a chromosome with a gene encoding the protein variant, and enhancing the activity of the protein variant Any one or more of the methods of additionally introducing a mutation into the gene encoding the mutant as possible may be used, but the present invention is not limited thereto.
  • modifying the expression control sequence to increase the expression of the polynucleotide is not particularly limited thereto, but deletion, insertion, non-conservative or conservative substitution of the nucleic acid sequence to further enhance the activity of the expression control sequence, or their It can be carried out by inducing a mutation in the sequence in combination, or by replacing it with a nucleic acid sequence having a stronger activity.
  • the expression control sequence is not particularly limited thereto, but may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence for regulating the termination of transcription and translation, and the like.
  • a strong promoter may be linked to the upper portion of the polynucleotide expression unit instead of the original promoter, but is not limited thereto.
  • Examples of known strong promoters include cj1 to cj7 promoter (US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) ) promoter (US 10584338 B2), O2 promoter (US 10273491 B2), tkt promoter, and yccA promoter, but are not limited thereto.
  • the modification of the polynucleotide sequence on the chromosome is not particularly limited thereto, but a mutation in the expression control sequence by deletion, insertion, non-conservative or conservative substitution of a nucleic acid sequence or a combination thereof to further enhance the activity of the polynucleotide sequence. It can be carried out by inducing and replacing the polynucleotide sequence with an improved polynucleotide sequence to have stronger activity.
  • Incorporation and enhancement of such protein activity is generally performed such that the activity or concentration of the corresponding protein is at least 1%, 10%, 25%, 50%, based on the activity or concentration of the protein in the wild-type or unmodified microbial strain. It may be increased by 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to 1000% or 2000%, but is not limited thereto.
  • the host cell or microorganism of the present application may be any microorganism expressing a serine protease variant including the polynucleotide of the present application or the vector of the present application.
  • Another aspect of the present application provides a method for preparing a serine protease variant of the present application.
  • the method for preparing the variant of the present application includes the serine protease variant of the present application; a polynucleotide encoding the variant; and culturing a microorganism comprising at least one of the vectors including the polynucleotide.
  • the term "culturing” means growing the host cell in an appropriately regulated environmental condition.
  • the culture process of the present application may be performed according to a suitable medium and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art according to the selected strain. Specifically, the culture may be batch, continuous, and fed-batch, but is not limited thereto.
  • the term "medium” refers to a material in which nutrients required for culturing the host cells are mixed as a main component, and supplies nutrients and growth factors, including water, which are essential for survival and growth.
  • any medium and other culture conditions used for culturing the host cells of the present application may be used without particular limitation as long as they are conventional media used for culturing host cells. It can be cultured under aerobic conditions in a conventional medium containing phosphorus, inorganic compounds, amino acids and/or vitamins, while controlling temperature, pH, and the like.
  • the method for preparing the variant of the present application may further include the step of recovering the variant of the present application expressed in the culturing step.
  • the mutant expressed in the culturing step may be recovered using a method known in the art to which the present invention pertains.
  • variants can be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation or precipitation.
  • the recovery method may be to collect mutants using a suitable method known in the art according to the culture method of the host cell of the present application, for example, a batch, continuous, or fed-batch culture method.
  • a suitable method known in the art for example, centrifugation, filtration, treatment with a crystallized protein precipitating agent (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity
  • chromatography methods such as island chromatography, HPLC, and methods thereof may be used in combination, and mutants may be recovered from the medium or host cells using a suitable method known in the art.
  • the mutant expressed by the host cell in the culture step may not be recovered.
  • the host cell itself expressing the variant may be used as a source of the variant.
  • Another aspect of the present application is a serine protease variant of the present application; And it provides a composition for feed, comprising one or more of the microorganisms expressing the same.
  • the serine protease variant included in the composition for feed of the present application may include the microorganism itself expressing it, or may be in a form separated and purified from the microorganism expressing it, but is not limited thereto.
  • composition for feed is any natural or artificial diet, one meal, etc. or a component of the one meal meal for animals to eat, ingest, and digest or suitable for, and the feed may be prepared in various forms known in the art. It can be manufactured as feed.
  • composition for feed may be a feed additive.
  • the type of feed is not particularly limited, and feed commonly used in the art may be used.
  • Non-limiting examples of the feed include plant feeds such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, gourds or grain by-products; and animal feeds such as proteins, inorganic materials, oils and fats, minerals, oils and fats, single cell proteins, zooplankton, or food. These may be used alone or in combination of two or more.
  • composition for feed of the present application includes organic acids such as citric acid, humic acid, adipic acid, lactic acid and malic acid; phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate (polyphosphate) and the like; Polyphenol, catechin, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, natural antioxidants such as chitosan, tannic acid, phytic acid, etc.; may further include one or more selected from have.
  • organic acids such as citric acid, humic acid, adipic acid, lactic acid and malic acid
  • phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate (polyphosphate) and the like
  • composition for feed of the present application includes auxiliary components such as amino acids, inorganic salts, vitamins, antibiotics, antibacterial substances, antioxidants, antifungal enzymes, and other live cell types of microbial preparations; grains such as milled or shredded wheat, oats, barley, corn and rice; plant protein feeds, such as those based on rape, soybean and sunflower; animal protein feeds such as blood meal, meat meal, bone meal and fish meal; Dry ingredients consisting of sugar and dairy products, for example, various milk powders and whey powder; lipids, for example main components such as animal fats and vegetable fats optionally liquefied by heating; It may further include one or more selected from; additives such as nutritional supplements, digestion and absorption enhancers, growth promoters, and disease prevention agents.
  • auxiliary components such as amino acids, inorganic salts, vitamins, antibiotics, antibacterial substances, antioxidants, antifungal enzymes, and other live cell types of microbial preparations
  • grains such as milled or shredded wheat,
  • composition for feed of the present application may be in the form of a dry or liquid formulation, and may include an excipient for adding feed.
  • the excipient for adding the feed includes, for example, zeolite, jade powder or rice bran, but is not limited thereto.
  • composition for feed of the present application may further include an enzyme preparation other than the serine protease variant.
  • lipolytic enzymes such as lipase, phytase that breaks down phytic acid to produce phosphate and inositol phosphate, alpha-1,4-glycosidic bonds contained in starch and glycogen, etc.
  • Amylase an enzyme that hydrolyzes ( ⁇ -1,4-glycoside bond), phosphatase, an enzyme that hydrolyzes organophosphate, maltase, which hydrolyzes maltose into two molecules of glucose, and saccharose It may further include one or more selected from a converting enzyme and the like to make a glucose-fructose mixture. However, it is not limited thereto.
  • composition for feed of the present application may be administered to an animal alone or in combination with other feed additives in an edible carrier.
  • the composition for feed can be easily administered as a feed additive, as a top dressing, directly mixing them into livestock feed, or separately from the feed, in a separate oral formulation, or in combination with other ingredients. It is also possible to use a single daily intake or divided daily intake as is commonly known in the art.
  • Animals to which the feed composition of the present application can be used include, for example, livestock such as edible cattle, dairy cows, calves, pigs, piglets, sheep, goats, horses, rabbits, dogs, cats, chicks, roosters, domestic chickens, roosters, including, but not limited to, poultry such as ducks, geese, turkeys, quails, small birds, and the like.
  • livestock such as edible cattle, dairy cows, calves, pigs, piglets, sheep, goats, horses, rabbits, dogs, cats, chicks, roosters, domestic chickens, roosters, including, but not limited to, poultry such as ducks, geese, turkeys, quails, small birds, and the like.
  • the amount of the serine protease variant of the present application included in the composition for feed of the present application is not particularly limited and may be appropriately adjusted according to the purpose. In one embodiment, as commonly known in the art to which the present application pertains, it may be included in an amount suitable for decomposing the protein source material while surviving for a long time in the digestive tract of livestock, but is not limited thereto.
  • Another aspect of the present application is a serine protease variant of the present application; And it provides a food composition comprising one or more of the microorganisms expressing the same.
  • the protease protein may be used in a food composition in liquid or solid form.
  • the food may be an additive of powder, pills, beverages, tea or general food.
  • the food may be a food group requiring a protease, such as dairy products, health functional food for defecation or diet, and functional health functional food for preventing hypertension.
  • a protease such as dairy products, health functional food for defecation or diet, and functional health functional food for preventing hypertension.
  • the protease variant may be included in various food compositions and used as a solubilizing or softening agent of food, or a meat quality modifier.
  • it may be added to the baking process and used in the step of breaking the gluten network.
  • it can be used to hydrolyze food proteins (eg, proteins in milk).
  • it may be included in various food compositions for purposes such as rendering, fragrance preparation, bitterness reduction, emulsification characteristic change, bioactive peptide production, protein allergen reduction, etc., but this is an example, It is not limited to one use.
  • the amount of the serine protease variant in the food composition can be appropriately adjusted by those skilled in the art according to the purpose.
  • Another aspect of the present application is a serine protease variant of the present application; And it provides a detergent composition comprising one or more of the microorganisms expressing the same.
  • the detergent composition according to the present application may be in the form of one-part and two-part aqueous detergent compositions, non-aqueous liquid detergent compositions, cast solids, granules, particles, compressed tablets, gels, pastes or slurries.
  • the detergent composition may be used to remove food residues, food residues, and other small amounts of food composition.
  • the detergent composition according to the present application may be provided in the form of 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 mouth washing, a detergent for cleaning dentures, or a contact lens cleaning solution. .
  • 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 washing dishes
  • a detergent composition for mouth washing a detergent for cleaning dentures
  • a contact lens cleaning solution .
  • Another aspect of the present application is a serine protease variant of the present application; And it provides a pharmaceutical composition comprising one or more of the microorganisms expressing the same.
  • the pharmaceutical composition of the present application is a pharmaceutical composition for digestive enzymes for improving digestive diseases, digestive abnormalities, and abnormal diseases after digestive surgery, a thrombolytic agent or antithrombotic composition that dissolves fibrin by acting directly on a blood clot, an in vivo defense system and use as an anti-inflammatory agent to remove inflammatory substances or necrotic tissue by acting as an anti-inflammatory agent or to relieve edema after surgery or trauma.
  • the pharmaceutical composition may further include a pharmaceutically acceptable or nutritionally acceptable carrier, excipient, diluent or sub-component depending on the method and purpose of use.
  • the carrier, excipient or diluent is lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose 1 selected from the group consisting of oil, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin, and physiological saline It may be above, but is not limited thereto.
  • the serine protease mutant or microorganism expressing the serine protease of the present application may be used for, for example, cosmetics, leather processing, pharmaceuticals, diagnostics, waste management, and chemical preparation for academic research in addition to the above-mentioned uses.
  • the above use is an example, and other uses may be used for any purpose related to denaturation, decomposition or removal of proteinaceous substances known in the art.
  • Example 1-1 Thermobifida fusca- derived serine protease library construction
  • a random mutation was introduced through error-prone PCR into a gene encoding an amino acid (SEQ ID NO: 31) corresponding to the mature region of the Thermobifida fusca -derived serine protease.
  • SEQ ID NO: 31 amino acid sequence corresponding to the mature region of the Thermobifida fusca -derived serine protease.
  • DiversifyTM PCR Random Mutagenesis Kit (Clontech, Cat# 630703) was used, and PCR conditions are as shown in [Table 1]. When proceeding under the following conditions, it was confirmed that mutations were inserted at a frequency of 6.2 mutations/Kb.
  • the PCR fragment obtained through the above process was ligated to the vector amplified using the primers shown in [Table 2] using the In-FusionR HD cloning kit (clontech), and then transformed into DH5 ⁇ to obtain colonies.
  • the plasmid was purified from the obtained colony to obtain a library of about 5*10 4 size.
  • Example 1-2 Thermobifida fusca-derived serine protease library screening
  • Step 1 is a method of selecting the library-transformed Bacillus subtilis strain based on the size of the formed halo after spreading it on a 2% skim milk plate.
  • Bacillus subtilis transformation was carried out according to the Groningen method, and the composition of the skim milk plate used for screening is shown in [Table 3].
  • Step 2 is a method of screening the colonies primarily selected in step 1 through Azocasein color development.
  • a BHI Brain Heart Infusion, bd, cat#53286) liquid medium containing kanamycin antibiotics was put in a 96 deep well plate, and the colonies selected in step 1 were inoculated and cultured at 37°C for 20-24 hours. After incubation, the supernatant containing the enzyme was obtained through centrifugation, and the supernatant and the substrate, 2% (w/v) Azocasein, were mixed in equal amounts, followed by reaction at 37° C. for 1 hour.
  • the reaction was stopped by adding 10% trichloro acetic acid (TCA) to the enzyme reaction solution in three volumes, and the coagulated protein was removed by centrifugation.
  • TCA trichloro acetic acid
  • the color development was compared by measuring the absorbance at 440 nm after the color reaction was carried out by mixing the same amount of NaOH. Through this process, colonies whose absorbance was increased by 150% or more compared to the wild-type serine protease were selected.
  • TAP_F12Y_F SEQ ID NO: 15 TAP_F12Y_R SEQ ID NO: 16 TAP_N116D_F SEQ ID NO: 17 TAP_N116D_R SEQ ID NO: 18
  • N-SUCCINYL-ALA-ALA-PRO-PHE-P-NITROANILIDE (Sigma, cat#S7388, hereinafter referred to as SUC-AAPF-pNA) Activity was evaluated using the peptide as a substrate.
  • the transformed Bacillus subtilis strain was inoculated in BHI medium containing kanamycin antibiotic and cultured at 37°C for 20-24 hours. After mixing with AAPF-pNA, it was reacted at 37°C for 30 minutes. The absorbance of the reaction solution was measured at 410 nm.
  • Example 2-2 the same sample subjected to the activity evaluation in Example 2-2 was left at room temperature, 70°C, 80°C, and 90°C for 5 minutes, respectively, and then the enzyme activity was measured. The measured activity is shown in [Table 6].
  • the F12Y and F12YN116D mutations maintained about 2 times and about 4 times the enzymatic activity compared to the wild type, respectively, even at 80 °C.
  • the serine protease variant of the present application maintains high activity even at high temperatures, and thus can be usefully used industrially.
  • Two PCR fragments were obtained using pBE-S-TAP as a template and primers of SEQ ID NOs: 11 and 12 and SEQ ID NOs: 13 and 14, respectively, ligated using In-Fusion HD cloning kit, and transformed into DH5 ⁇ colonies were obtained.
  • the plasmid was purified from the obtained colony to obtain a library having a size of about 4x10 3 .
  • Example 3-2 Saturated mutant library screening and activity evaluation
  • the saturated mutant library prepared in Example 3-1 was screened in the same manner as described in Example 1-2. Through screening, mutants having the same or increased activity compared to the F12YN116D mutant were selected, and the mutants were subjected to sequence analysis and activity evaluation using Suc-AAPF-pNA as a substrate.
  • the F12 and N116 residues showed an increase in activity even when substituted with other amino acids such as F12S, F12A, F12R, N116S, N116T, and N116G in addition to tyrosine and aspartate confirmed in Example 2.
  • Suc-AAPF-pNA was used as a substrate to evaluate the activity of the F12S mutant.
  • Example 4-1 wild-type and mutant construction
  • Residues 12 and 116 of each serine protease were substituted with tyrosine (Y) and aspartate (D) to prepare variants of SEQ ID NOs: 55-66.
  • Thermobifida cellulosilytica WT 13.6 F12Y 16.3 F12YN116D 29.1
  • Thermobifida halotolerans WT 4.0 F12Y 6.3 Actinorugispora endophytica WT 10.4
  • F12Y 14.0 Spinactinospora alkalitolerans WT 2.0 P12Y 2.6 Nocardiopsis composta WT 12.1 P12Y 26.5 Nocardiopsis potens WT 33.1 P12Y 86.3
  • residues 12 and 116 are important residues for serine protease enzyme activity, and as confirmed through SEQ ID NO: 31, it was confirmed that the enzyme activity can be increased by substituting it with other amino acids, the serine protease variant of the present application has increased enzymatic activity and can be usefully used industrially.

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