DK2510094T3 - Sammensætninger og fremgangsmåder omfattende proteasevarianter - Google Patents

Sammensætninger og fremgangsmåder omfattende proteasevarianter Download PDF

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DK2510094T3
DK2510094T3 DK10836666.7T DK10836666T DK2510094T3 DK 2510094 T3 DK2510094 T3 DK 2510094T3 DK 10836666 T DK10836666 T DK 10836666T DK 2510094 T3 DK2510094 T3 DK 2510094T3
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bpn
variant
protease
amino acid
cleaning
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Joshua Roy Basler
Luis Gustavo Cascao-Pereira
David A Estell
James T Kellis
Alexander Pisarchik
Ayrookaran J Poulose
Daniel Torres-Pazmino
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Danisco Us Inc
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    • 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
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • 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)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • 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)
    • C12Y304/21062—Subtilisin (3.4.21.62)

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Claims (15)

  1. SAMMENSÆTNINGER OG FREMGANGSMÅDER OMFATTENDE PROTEASEVARIANTER PATENTKRAV
    1. Isoleret proteasevariant, hvilken variant omfatter en aminosyresekvens med mindst 80 % sekvensidentitet med aminosyresekvensen ifølge SEQ ID NO: 2 og omfatter aminosyresubstitutioneme X024G/R, X053G, X078N, X101N, X128A/S og X217Q/L, hvor varianten har forbedret proteolytisk aktivitet og/eller rensningsaktivitet sammenlignet med den proteolytiske aktivitet og/eller rensningsaktivitet for BPN'-proteasen med sekvensen ifølge SEQ ID NO: 2, og hver aminosyreposition af varianten er nummereret i forhold til en aminosyreposition i aminosyresekvensen ifølge SEQ ID NO: 2 som bestemt ved alignment af aminosyresekvensen for varianten med SEQ ID NO: 2.
  2. 2. Variant ifølge krav 1, hvor aminosyresekvensvarianten endvidere omfatter aminosyresubstitutionen X097A.
  3. 3. Variant ifølge krav 1, hvor aminosyresekvensvarianten omfatter aminosyresubstitutioneme S024G+S053G+S078N+S101N+G128S+Y217Q eller S024G+S053G+S078N+S101N+G128A+Y217Q.
  4. 4. Variant ifølge krav 3, hvor aminosyresekvensvarianten endvidere omfatter (i) en substitution udvalgt fra gruppen bestående af N109G, N076D, S033T, N243V, S248A, A088T og S063G; eller (ii) et sæt af aminosyresubstitutioner udvalgt fra gruppen bestående af: A08 8T+N109G+A116T+G13111+N243V+L257G, S033T+N076D, S009T+N109G+K141R+N243V, S162G+K256R, N109G+A116T, N109G+L257G, S162G+L257G, N061G+N109G+N243V, N 109G+N243V+S248A, S03 3T+N076D+N109G+N218S+N243V+S248N+K256R, N109G+A116T+N243V+K256R, A088T+N109G+A116T+G131H+N243V, A088T+N109G, N109G+N243V, T158S+L257G, N061 S+Nl 09G+N243V, P040A+N109G+N243V+S248N+K256R, S009T+S018T+Y021N+N109G+K14 IR, A088T+N109G+A116T+T158S+N243V+K256R, A088T +N109G+A116T+T15 8 S+N218 S+L257G, N109G+K256R, N109G+N243V+K256R, S063G+K256R, S063G+N109G, S063G, S063G+N076D, S033T+N076D+N218S og N076D+N218S
  5. 5. Variant ifølge et hvilket som helst af de foregående krav, hvor amino syre sekvensvarianten har mindst 85 % sekvensidentitet med amino syre sekvensen ifølge SEQ ID NO: 2 eller SEQ ID NO: 6.
  6. 6. Variant ifølge krav 1, hvor varianten har aminosyresekvensen ifølge SEQ ID NO: 6 eller har sekvensen: ai) BPN' S024G-S053G-S078N-S101N-G128S-Y217Q; a) BPN'-S024G+S053G+S078N+S101N+G128A+Y217Q+A088T+N109G+A116T+ G131H+N243V+L257G; b) BPN'-S024G+S053G+S078N+S101N+G128A+Y217Q+S033T+N076D; c) BPN-S024G+S053G+S078N+S101N+G128S+Y217Q+S009T+N109G+ K141R+N243V; d) BPN-S024G+S053G+S078N+S101N+G128A+Y217Q+S162G+K256R; e) BPN-S024G+S053G+S078N+S101N+G128A+Y217Q+N109G+A116T; f) BPN’-S024G+S053G+S078N+S 101N+G128A+Y217Q+N109G+L257G; g) BPN'-S024G+S053G+S078N+S101N+G128A+Y217Q+S162G+L257G; h) BPN-S024G+S053G+S078N+S101N+G128A+Y217Q+N061G+N109G+N243V; i) BPN'-S024G+S053G+S078N+S101N+G128S+Y217Q+N109G+N243V+S248A; j) BPN’-S024G+S053G+S078N+S101N+G128S+Y217Q+S033T+N076D+N109G+N218S+ N243V+S248N+K256R; k) BPN-S024G+S053G+S078N+S101N+G128A+Y217Q+N109G+A116T+N243V+ K256R; l) BPN’-S024G+S053G+S078N+S101N+G128A+Y217Q+A088T+N109G+A116T+G131H+ N243V; m) BPN'-S024G+S053G+S078N+S 101N+G128A+Y217Q+A088T+N109G; n) BPN'-S024G+S053G+S078N+S 101N+G128A+Y217Q+N109G+N243V; o) BPN'-S024G+S053G+S078N+S 101N+G128A+Y217Q+T158S+L257G; p) BPN-S024G+S053G+S078N+S101N+G128A+Y217Q+N061S+N109G+N243V; q) BPN'-S024G+S053G+S078N+S101N+G128S+Y217Q+P040A+N109G+N243V+S248N+ K256R; r) BPN'-S024G+S053G+S078N+S101N+G128S+Y217Q+S009T+S018T+Y021N+N109G+ K141R; s) BPN'-S024G+S053G+S078N+S101N+G128A+Y217Q+A088T+N109G+A116T+T158S+ N243V+K256R; t) BPN'-S024G+S053G+S078N+S101N+G128A+Y217Q+A088T+N109G+A116T+T158S+ N218S+L257G; u) BPN'-S024G+S053G+S078N+S 101N+G128A+Y217Q+N109G+K256R; v) BPN-S024G+S053G+S078N+S101N+G128S+Y217Q+N109G+N243V+K256R, w) BPN'-S024G+S053G+S078N+S 101N+G128 A+Y217Q+S063G+K256R; x) BPN-S024G+S053G+S078N+S101N+G128A+Y217Q+S063G+N109G; y) BPN'-S024G+S053G+S078N+S 101N+G128S+Y217Q+S063G; z) BPN-S024G+S053G+S078N+S101N+G128A+Y217Q+S063G+N076D; aa) BPN'-S024G+S053G+S078N+S101N+G128S+Y217Q+S033T+N076D+N218S; bb) BPN'-S024G+S053G+S078N+S101N+G12 8 A+Y217Q+N076D+N218 S eller cc) BPN'-S024G+S053G+S078N+S 101N+G128A+Y217Q.
  7. 7. Variant ifølge krav 4, hvor varianten har forbedret proteolytisk aktivitet sammenlignet med den proteolytiske aktivitet for proteasen med sekvensen ifølge SEQ ID NO: 6.
  8. 8. Isoleret nukleinsyre, der omfatter en polynukleotidsekvens, der koder for varianten ifølge et hvilket som helst af kravene 1 til 7, der f.eks. har mindst 80 % sekvensidentitet med polynukleotidsekvensen ifølge SEQ ID NO: 3 eller SEQ ID NO: 5, eller en komplementær polynukleotidsekvens deraf.
  9. 9. Ekspressionsvektor, der omfatter mindst én nukleinsyre ifølge krav 8, eventuelt hvor den mindst ene nukleinsyre er operativt bundet til en promoter.
  10. 10. Rekombinant værtscelle, der omfatter: (a) en nukleinsyre ifølge krav 8, eller (b) en ekspressionsvektor ifølge krav 9; hvor værtscellen eventuelt er en bakteriecelle, f.eks. en Bacillus-celle, såsom en Bacillus subtilis-celle.
  11. 11. Fremgangsmåde til fremstilling af en proteasevariant, hvilken fremgangsmåde omfatter dyrkning af en rekombinant værtscelle ifølge krav 10 under forhold, der bidrager til fremstilling af varianten, og eventuelt indvinding af varianten fra cellekulturen.
  12. 12. Sammensætning omfattende varianten ifølge et hvilket som helst af kravene 1 til 7, hvilken sammensætning ikke er et stof- eller rengøringsprodukt; f.eks. hvor sammensætningen er en rensemiddelsammensætning til rensning af en kontaktlinse eller en rensemiddelsammensætning, der kan anvendes til personlig pleje.
  13. 13. Sammensætning ifølge krav 12, hvor sammensætningen omfatter et supplerende enzym, f.eks. hvor det supplerende enzym er udvalgt fra gmppen bestående af en hemicellulase, cellulase, amylase, peroxidase, protease, xylanase, lipase, phospholipase, esterase, cutinase, pectinase, pectatlyase, mannanase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase, pullulanase, tannase, pentosanase, malanase, B-glucanase, arabinosidase, hyaluronidase, chondroitinase og laccase.
  14. 14. Sammensætning ifølge krav 12 eller krav 13, hvilken sammensætning endvidere omfatter mindst én adjuvans og/eller mindst ét overfladeaktivt middel.
  15. 15. Fremgangsmåde til rensning af et emne eller en overflade, der har behov for at blive renset, hvilken fremgangsmåde omfatter etablering af kontakt mellem emnet og overfladen med en variant ifølge et hvilket som helst af kravene 1 til 7, eller en sammensætning ifølge krav 12 til 14, og eventuelt skylning af emnet eller overfladen med vand.
DK10836666.7T 2009-12-09 2010-12-09 Sammensætninger og fremgangsmåder omfattende proteasevarianter DK2510094T3 (da)

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