DK2510093T3 - Proteasevarianter - Google Patents

Proteasevarianter Download PDF

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DK2510093T3
DK2510093T3 DK10803307.7T DK10803307T DK2510093T3 DK 2510093 T3 DK2510093 T3 DK 2510093T3 DK 10803307 T DK10803307 T DK 10803307T DK 2510093 T3 DK2510093 T3 DK 2510093T3
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protease
polypeptide
variant
ala
protease activity
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DK10803307.7T
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Tomoko Matsui
Allan Noergaard
Thomas Agersten Poulsen
John Matthews
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Novozymes North America Inc
Novozymes As
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/48Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/58Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00Preparation of oxygen-containing organic compounds
    • C12P7/02Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06Ethanol, i.e. non-beverage
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00Preparation of oxygen-containing organic compounds
    • C12P7/02Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06Ethanol, i.e. non-beverage
    • C12P7/08Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y304/00Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24Metalloendopeptidases (3.4.24)
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02EREDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00Technologies for the production of fuel of non-fossil origin
    • Y02E50/10Biofuels, e.g. bio-diesel

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Mycology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Claims (14)

1. Fremgangsmåde ti! fremstilling af en proteasevariant med proteaseaktivitet og forbedret termostabilitet i sammenligning med sfamproteasen, hvilken fremgangsmåde omfatter dyrkning af en celle, hvori der er indført en ekspressionsvektor, som omfatter følgende operativt forbundne elementer: føj en transskriptionspromofor, fbj et poiynukleotidmoiekyie, der koderfor en proteasevariani, som udviser mindst SS% identitet medgroteasén Vist i aminosyrerne Ϊ til 177 ifølge SEQID NO: 2, og som omfatter mindst én modifikation i sammenligning med aminosyrerne 1 til 177 ifølge SEQ ID NO: 2 i mindst én position vaigt blandt de følgende: 27,79,82,87,112,142, 2, 5,6, 8,26,41,43,46, 49, 63, 64, 73, 88, 104,114, 115,118,126,152,157,158 og173, hvilket pøtynukleotidmotekyfe er fremstillet ved at indføre mindst én mutation i et DNA-molekyie, der koder for en protease, og (c) en fm nssiy ptiemstermi nater, hvorved cellen udtrykker proteasevarianten, der kodes for af polynukleoisdmolekylet; og indvinding af proteasevarianten.
2. Fremgangsmåde ifølge krav 1, hvor proteasevarianten, der kodes for af poHmuk eofidmolekylet, omfatter mindst én modifikation valgt fra gruppen bestående af A27K, A27G, A27V, Q53K, Q53R, T54R, D79K, D79L D79M, Y82F, S87P, S87G. A112P, D142L R2P, AS5, C6R, AGS, N26R, S41R, Y43F, T46R, S49R, A73C. P81R, N88R, D104R, D104P, T114P, S115R, T118V, T124L, T124V, A126V, M152R, S157K, Q158Wog M73V.
3. Fremgangsmåde ifølge et hvilket som helst af kravene 1 eller 2. hvor proteasevarianten, dér: kodes for af poiynuk|eotidmd!éky|et5 drnfader m én af følgende kombinationer af modifikationer: AS5/D7S^S8lRi Ii5/D79L/S37P/A112P/D142L. AS6/N26R/D79L/S87P/A112P/D142L, C6R/D79L/S87P, AG8/D79L./S87P. N26R/D79L/S87P, N28R/r46R/D79L/S87P/A1 12P/D142L, A27G/D79L/S87P/A112P/D142L, M7K/D7^ 12P/D142L, A27K/D73L/S87R/A112P/T124V7D142L, A27V/079IJG87P/A112P/D142U S41R/D79L/S87P, S41R/Q79L/S87P/A112P/D142L, S41R/ppUS87PfA112P/D142L/S157K, Y43F/D79L/S87P/A112P/D142L, T48R/D79L/S87P, Y48Rd379L/S87P/A112P/D142L, T46R/D79L/S87P/T116V/D142L, S49R/D79L/S87P, Q53l®79L/S87P/i 173Y, G53R/D79L/S87P, 1fS41^p79y867P, D79L/S87P/A112P, D79hP81Ra87P/A112P/pi42t, D79L/S87P, D79L/S87P/A112P, D79lp87PiAi:12P7D142L, D79L/S87P/A112P/D142L/S157K, ; D79L/S87P/A412P/T 1 24l/D1 42L, P79L/S87P/A1 1 2P/T124Y«28WD 142 L, mmsm>w 12P7T1 24\//D142L. D79L/S87P/D104R, D79L/S87P/D142L, rørø87R/(488Rs D79L/S87P/Q158W, P79t,/S87P/S11$R( D79US87P/S157K, TOi^S87P7ri 14P, P79ySl7P/T118V, D79L/Y82F/SS7P/A112P/T124V/D142L, D79L/Y82F/S87P/A112P/T124V/P142L, A27K/D79L/Y82F/S87G/D104P/A112P/A128¥/D142L, A27K/Y82F/S87G/D1Q4P/A112P/A126V/D142L. A27K/D79L/Y82F/D104P/A112P/A126V/D142L, A27K/Y82F/0! 04P/A112P/A126V/D142L.
4. Fremgangsmåde ifølge krav 1, hvor proteasevarianfen omfatter mindst Ih higdiffkétipn i mindst én position valgt fra gruppen bestående afpositionerne: 27, 79, 82, 87, 1Q4A112,126 og 142.
5. Fremgangsmåde ifølge krav 4, hvor proteasevarianten omfatter mindst én af modifikationerne valgt fra gruppen bestående af: A27K, D79L, Y82F, S87G, S87P, D1Q4P, A112P, A126V og D142L. 8. iPeiypeptid med proteaseaktivitet, som udviser mindst 85% kfehtitet med proteasen vist tiamihosyferne 1 til 177 ifølge SEQ ID NG: 2, og som omfatter mindst én modifikation i sammenligning med aminosyreme 1 til 177 ifølge SEQ ID NO. 2 i mindst én position våigt blandt de følgende: 27, 79, 82, 87,112, 142. 2, 5, 6, 8, 26, 41,43,46, 49, 53, 54, 73,88,104, 144, 415 116, 126, 152, 157, 158 og 173, hvor poiypeptidet har forbedret termostabilitet i i«^meRiigning:med'proteas!!^''Yiétéi8mthosyrerhe 1 til 177 ifølge SEQ ID NO: 2.
7. Pøiypeptid med proteaseaktivitet ifølge krav 6, som omfatter mindst én modifikation valgt blandt de følgende: A27K, A27G, A27V, Q53K, G53R, T54R, D79K. D79L, D79M, Y82F, S87P, S87G, A112P, D142L, R2P, Δ85, C6R, Δ08, N26R, S41R. Y43F, T46R. S49R, A73C, P81R, N88R, D104R, D104P, T114P, S115R, T116V, T124U, T124V, A126V, M152R, S157K, Q158W og I173V. 8: Pøiypeptid med proteaseaktivitet iføige krav 6, hlpr poiypeptidet omfattet rhindst én modifikation i mindst én position valgt fra gruppen bestående af posttfonerne: 27, 79, 82, 87, 104, 112, 126 og 142,
9. Poiypeptid med proteaseaktivitet iføige krav 8, hvor poiypeptidet omfatter mindst én af rhddifikatiohérhé bestående af: A27R, D79L, Y82F, S87G, S87P, D4G4P,
10, Pdiyp^gifcf ifhéd proteaseaktivitet ifølge ét hvilket som helst af kravene 6-9, hvilket poiypeptib omfatter et sæt af modifikationer valgt blandt de følgende; AS5/Q79US87P, AS5/D79L/S87P/A112P/D142L &S5/N2SR/D79L/S87P/A112P/D1421, C6R/P791/S87P, &G8/079yS87P, M26R/D791/S87P, N26R/T46R/079I/S87P/A112P/P142L, A27G/D7917S87P/A112P/0142LA27K/D79L/S87P/A112P/D142L, A27K/D79t,7S87p/Ai 12P/T124V/D142L, A27V/D79US87P/A112P/D142L, S41R/D79I/S87P, S41R/D79US87P/A112P/D1421, S41R/D79L/S87 P/A112P/D142L/S157K, Y43F/D7SL/S87FÉM 12P/D142L. T46R/D79US87P, T46R/D79L/S87P/A112P/D142L, T46R'D79L/S87P/T 116V/D142L, S49R/D79L/S87 P, Q53K/D79L/S 87P/1173V. G53R/D79L/S87P, T54R/D79L/S87P, D79U S87P/A112P, D79UP81R/S87P/A112P/D142L, Q7917S87F, D79k/i87P/Ai 12P, D79i../S87FfA112P/D142L, 079L/S87P/A112PVQ142kmS7K, D79L/S87P/A112P/T124L/D142L, D79US87P/A 112P/T124V/A126V/D142L, D79L/S87P/A112P/T124V/D142L. D79US87P/D104R, D79L/S87P/D142L D79L/S87P/N88R, D79L/S87P/G158W. D79L/S87P/S115R D79L/S87P/S157K, D79L/S87P/T114P, D79L/S87P/T116V/, P79L/^82R/S87P/A112P/T124V/0102L 079t/Y82Fi^ 24W0142L A27K/D79UY82F/S87G/D104P/A112P/A126V/D1421, A27K/Y82P/S87G/D104P/A112P/A126V/D142L, A2 7 K7 D7 9L/Y82F/D104 P/A112P/A126V/D142L, A27K/Y82F/D104P/A112P/A126V/D142L. 11 løøifø^r^NkiSid^resekvenSi; som; omfatter en nukleinsyresekvens, Jef koder for poiypeptidet sned protea.seaktivitet ifsige et hvilket som helst af kravene 6-10.
12, Nukieinsyrekonstruktionr som omfatter nuklemsyresekvensen følge krav 11. der er operativt forbundet tji en eller flere kontrolsekvenser, som styret frem^ltlllngéh af proteasen i en egnet ekspressionsvært.
13. Rekombinant ekspressionsvektor, som omfatter nukleinsyrekonstruktionen ifølge krav 12,
14. Rékomøihsht værtscelle, som omfatter nukleinsyrekonstruktionen ifølge krav 12 og/e!!er ekspressiGnsvektoren tfølge krav 13.
15, Fremgangsmåde til fremstilling af poiypeptidet røéd proteaseaktivitet ifølge et hvilket som helst af kravene 6-10, hvilken fremgangsmåde omfatter: (a) dyrkning af vasøseSIføb ifølge kfp 14: for at fremstille en supernatant, som omfatter polypeptidet med proteaseaktivitet; og (b) indvinding af polypeptidet med proteaseaktivitet. li, Tfipogen plante ellerplantedel, som kan udtrykkes! poiypeptld med proteaseaktivitet ifølge oi P^iikét sorr? helst af kravene 6Ί0. 17. fremgangsmidé fffremstilling åf et ferroenteringsprodukt, hvilken fremgangsmåde omfatter (a) fermentering ved anvendelse af en fermenterende mikroorganisme og et inærværelse af et polypeptid med proteaseaktivitet ifølge et hvilket som helst af kravene 6-10 og {fe} fremstilling af fermenteringsproduktet ud fra det fermenterede carbohydratholdige materiale.
18. Fremgangsmåde ifølge krav l 1, hvor fermenteringsproduktet er ethanol og/eilertørret bærm e idlstlSlers dri ed gralns - O DG).
19. Anvendelse af mindst ét polypeptid med proteaseaktivitetifølge et hvilket som helst af kravene 6-10 ved en fremgangsmåde til fremstilling af et fermenieringsprodukt fortrinsvis ethano!.
DK10803307.7T 2009-12-11 2010-12-10 Proteasevarianter DK2510093T3 (da)

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CN102753680B (zh) 2015-05-13
AU2010328033B2 (en) 2014-12-04
CN104774823A (zh) 2015-07-15
EP2510093B1 (en) 2018-02-21
CA2783820A1 (en) 2011-06-16
EP2510093A2 (en) 2012-10-17
US10138473B2 (en) 2018-11-27
CN104774823B (zh) 2020-04-10
CN102753680A (zh) 2012-10-24
US20120309067A1 (en) 2012-12-06
CA2783820C (en) 2018-10-23
US9040280B2 (en) 2015-05-26
AU2010328033A1 (en) 2012-06-07
WO2011072191A2 (en) 2011-06-16
ES2668202T3 (es) 2018-05-17
MX355362B (es) 2018-04-17
MX2012006548A (es) 2012-07-10

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