DK2011864T3 - Polypeptider med alkalisk alfa-amylaseaktivitet og nukleinsyrer, der koder for dem - Google Patents

Polypeptider med alkalisk alfa-amylaseaktivitet og nukleinsyrer, der koder for dem Download PDF

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DK2011864T3
DK2011864T3 DK08075617.4T DK08075617T DK2011864T3 DK 2011864 T3 DK2011864 T3 DK 2011864T3 DK 08075617 T DK08075617 T DK 08075617T DK 2011864 T3 DK2011864 T3 DK 2011864T3
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polypeptide
alpha
seq
nucleic acid
amylase
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DK08075617.4T
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Helle Outtrup
Lisbeth Hedegaard Hoeck
Bjarne Rönfeldt Nielsen
Vibeke Skovgaard Nielsen
Henrik Bisgard-Frantzen
Allan Svendsen
Carsten Andersen
Torben Vedel Borchert
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Novozymes As
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    • 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)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • 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
    • 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
    • C11D3/38609—Protease or amylase in solid compositions only

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
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  • Medicinal Chemistry (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Detergent Compositions (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Claims (10)

  1. rM i c in i rvrcAV
    1. Isoleret polypeptid med alfa-amylaseaktivitet, som omfatter aminosyresekvensen ifølge SEQ ID NO: 2, og varianter deraf med en aminosyresekvens, som udviser mindst 96% identitet med aminosyrerne 1 til 485 i SEQ ID NO: 2, hvor varianten har en forbedret vaskeevne i sammenligning med stam-alfa-amylasen, hvor vaskeevnen er bestemt under følgende vaskebetingelser: teststofprøver tilsmudset med orange risstivelse vaskes i 15 minutter ved 25°C ved en pH på 10,5, en vandhårdhed på 6 °dH og et Ca:Mg-forhold på 2:1 i en opløsning med 3 g/l af A/P Model-detergent bestående af: 20% natriumtripolyphosphat, 25% Na2S04, 15% Na2C03, 20% lineært alkylbenzensulfonat, 5% (Ci2-Ci5)-alkoholethoxylat, 5% Na2Si205 og 0,3% NaCI, som indeholder 1 mg/l af polypeptidet med alfa-amylaseaktivitet; efter vask vurderes stofprøverne ved at måle remissionen ved 460 nm, og vaskeevnen bestemmes som remissionen for de vaskede stofprøver minus remissionen for en stofprøve vasket under de samme betingelser, men uden et polypeptid med alfa-amylaseaktivitet.
  2. 2. Polypeptid ifølge krav 1, som består af aminosyrerne 1 til 485 ifølge SEQ ID NO: 2.
  3. 3. Isoleret nukleinsyresekvens, som omfatter en nukleinsyresekvens, der koder for polypeptidet ifølge et hvilket som helst af kravene 1-2.
  4. 4. Isoleret nukleinsyresekvens ifølge krav 3, som omfatter en nukleinsyresekvens med mindst én mutation i det modne polypeptids kodningssekvens ifølge SEQ ID NO: 1, hvor den muterede nukleinsyresekvens koder for et polypeptid, som består af aminosyrerne 1 til 485 ifølge SEQ ID NO: 2.
  5. 5. Rekombinant ekspressionsvektor, som omfatter nukleinsyrekonstruktionen ifølge krav 3-4.
  6. 6. Rekombinant værtscelle, som omfatter nukleinsyrekonstruktionen ifølge krav 5.
  7. 7. Fremgangsmåde til frembringelse af en muteret nukleinsyresekvens ifølge krav 3 eller 4, hvilken fremgangsmåde omfatter (a) indføring af mindst én mutation i det modne polypeptids kodningssekvens ifølge SEQ ID NO: 1, hvor den muterede nukleinsyresekvens koder for et polypeptid, som består af aminosyrerne 1 til 485 ifølge SEQ ID NO: 2; og (b) indvinding af den muterede nukleinsyresekvens. o. i-remgangsmaae tu TremDringeise aT poiypepnaet iTøige et ηνιικβι som neist at kravene 1-2, hvilken fremgangsmåde omfatter (a) dyrkning af en værtscelle ifølge krav 6 under betingelser, som er egnede til frembringelse af polypeptidet; og (b) indvinding af polypeptidet.
  8. 9. Fremgangsmåde ifølge krav 8 til frembringelse af et polypeptid ifølge et hvilket som helst af kravene 1-2, hvilken fremgangsmåde omfatter (a) dyrkning af en værtscelle ifølge krav 6 under betingelser, som er befordrende for frembringelse af polypeptidet, hvor værtscellen omfatter en muteret nukleinsyresekvens med mindst én mutation i det modne polypeptids kodningssekvens ifølge SEQ ID NO: 1, hvor den muterede nukleinsyresekvens koder for et polypeptid, som består af aminosyrerne 1 til 485 ifølge SEQ ID NO: 2, og (b) indvinding af polypeptidet.
  9. 10. Polypeptid ifølge krav 1, hvor polypeptidet har en eller flere mutationer, navnlig substitution eller deletioner, i følgende stillinger (i forhold til SEQ ID NO: 2): 1: R181*, G182*, D183*, G184*; 2: N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 3:1206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V; 4: E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 5: E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 6: K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V; 7: R181A,N,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V, hvor polypeptidet fortrinsvis er valgt fra gruppen bestående af mutanter med følgende mutationer: -R1817G1827N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; -G1827D1837N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; -D1837G1847R181A,N,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; -D1837G1847N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; -R1817G1827I206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y; -G1827D1837I206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y; -D1837G1847I206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y; -R1817G1827E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; -G1827D1837E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; -D1837G1847E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; -R1817G1827E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; -G1827D1837E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; -D1837G1847E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; -R1817G1827K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V; -U1b27mbd7K2byA,K,U,N,U,t,U,lj,H,l,L,IVI,|-,M,S, i ,νν,γ,ν; -D183YG184YK269A,R,D,N,C,EAG,H,I,L,M,F,P,S,T,W,Y,V; -N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; /I206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y; -N195A,R,D,C,EAG,H,I,L,K,M,F,P,S,T,W,Y,V /E212A,R,D,N,CAG,H,I,L,K,M,F,P,S,T,W,Y,V; -N195A,RAC,EAG,H,I,L,K,M,F,P,S,T,W,Y,V/E216A,R,D,N,CAG,H,I,L,K,M,F,P,S,T,W,Y,V; -N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V /K269A,R,D,N,C,EAG,H,I,L,M,F,P,S,T,W,Y,V; -I206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y /E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; -I206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; -I206A,R,D,N,C,EAG,H,I,L,K,M,F,P,S,T,W,Y/K269A,R,D,N,C,EAG,H,I,L,M,F,P,S,T,W,Y,V; -E212A,R,D,N,CA,G,H,I,L,K,M,F,P,S,T,W,Y,V /E216A,R,D,N,CAG,H,I,L,K,M,F,P,S,T,W,Y,V; E212A,R,D,N,CAG,H,I,L,K,M,F,P,S,T,W,Y,V/K269A,R,D,N,C,EAG,H,I,L,M,F,P,S,T,W,Y,V; -E216A,R,D,N,CAG,H,I,L,K,M,F,P,S,T,W,Y,V /K269A,R,D,N AEAG,H,I,L,M,F,P,S,T,W,Y,V, og hvor polypeptidet fortrinsvis endvidere omfatter en substitution i stilling E216Q, og hvor * angiver en deletion.
  10. 11. Anvendelse af polypeptidet ifølge et hvilket som helst af kravene 1-2 eller en variant ifølge krav 10 i: (a) en detergentsammensætning, navnlig en detergentsammensætning til tøjvask og en detergentsammensætning til opvask; eller (b) en afsletningssammensætning; eller (c) til forflydning af stivelse; eller (d) til ethanolfremstilling.
DK08075617.4T 1999-03-31 2000-03-28 Polypeptider med alkalisk alfa-amylaseaktivitet og nukleinsyrer, der koder for dem DK2011864T3 (da)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DKPA199900439 1999-03-31
US09/290,734 US6361989B1 (en) 1997-10-13 1999-04-13 α-amylase and α-amylase variants
DKPA199900490 1999-04-13
EP00912416A EP1173554A2 (en) 1999-03-31 2000-03-28 Polypeptides having alkaline alpha-amylase activity and nucleic acids encoding same

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EP2011864B1 (en) 2014-12-31
EP1173554A2 (en) 2002-01-23
CA2365446A1 (en) 2000-10-12
EP2889375A1 (en) 2015-07-01
WO2000060060A2 (en) 2000-10-12
CN1234854C (zh) 2006-01-04
AU3419400A (en) 2000-10-23
BR0009392A (pt) 2002-01-08
AU781258B2 (en) 2005-05-12
BR0009392B1 (pt) 2012-06-12
KR20020002415A (ko) 2002-01-09
JP2002540786A (ja) 2002-12-03
JP4745503B2 (ja) 2011-08-10
EP2889375B1 (en) 2019-03-20
WO2000060060A3 (en) 2001-04-19
EP2011864A1 (en) 2009-01-07
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AR023229A1 (es) 2002-09-04
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CN1367824A (zh) 2002-09-04
CA2365446C (en) 2012-07-10

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