DK2194133T3 - Amylaser, nukleinsyrer, som koder for dem, og fremgangsmåder til fremstilling og anvendelse deraf - Google Patents

Amylaser, nukleinsyrer, som koder for dem, og fremgangsmåder til fremstilling og anvendelse deraf Download PDF

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DK2194133T3
DK2194133T3 DK09180956.6T DK09180956T DK2194133T3 DK 2194133 T3 DK2194133 T3 DK 2194133T3 DK 09180956 T DK09180956 T DK 09180956T DK 2194133 T3 DK2194133 T3 DK 2194133T3
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polypeptide
nucleic acid
sequence
amylase
mutagenesis
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Walter Callen
Toby Richardson
Gerhard Frey
Kevin Gray
Janne S Kerovuo
Matgorzata Slupska
Nelson Barton
Eileen O'donoghue
Carl Miller
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Basf Enzymes Llc
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Priority claimed from EP04718513A external-priority patent/EP1727898A4/en
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    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT OF FLOUR OR DOUGH FOR BAKING, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS
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    • 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
    • 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)
    • C12N9/24—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.)
    • 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
    • C12N9/2428—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/16—Preparation of compounds containing saccharide radicals produced by the action of an alpha-1, 6-glucosidase, e.g. amylose, debranched amylopectin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/20—Preparation of compounds containing saccharide radicals produced by the action of an exo-1,4 alpha-glucosidase, e.g. dextrose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • Coloring Foods And Improving Nutritive Qualities (AREA)
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Claims (15)

1. Isoleret eller rekombinant nukleinsyre, som koder for et polypeptid med alpha-amylase-aktivitet, omfattende (a) en nukleinsyresekvens, der har mindst 90%, eller mere, eller har 100% sekvensidentitet med SEQ ID NO: 418 over hele længden af genet; (b) en nukleinsyre, som koder for et polypeptid, der har mindst 90%, eller mere, eller har 100% sekvensidentitet med SEQ ID NO:419, eller et enzymatisk aktivt fragment deraf, hvor fragmentet har alpha-amylase-aktivitet, (c) nukleinsyren af (a) eller (b), som mangler en signalsekvens eller et kulhydratbindende modul; (d) nukleinsyren af (a), (b) eller (c), som yderligere omfatter en heterolog sekvens, (e) nukleinsyresekvensen af (d), hvor den heterologe sekvens omfatter en sekvens, som koder for en heterolog signalsekvens, et kulhydratbindende modul, katalytisk domæne (CD), eller en kombination deraf, eller hvor den heterologe signalsekvens, det kulhydratbindende modul eller den katalytiske domæne (CD) er afledt fra et amylase-enzym eller et ikke-amylase-enzym; eller (f) en nukleinsyre, der er komplementær til (a), (b), (c), (d) eller (e).
2. Ekspressionskassette, en vektor eller en kloningsvehikel, omfattende en nukleinsyre omfattende en sekvens ifølge krav 1, hvor ekspressionskassetten, vektoren eller kloningsvehiklen omfatter en viral vektor, en plasmid, en fag, en fagemid, en kosmid, en fosmid, en bakteriofag eller en kunstig kromosom.
3. Transformeret værtscelle, omfattende en nukleinsyre, omfattende en sekvens ifølge krav 1, eller en ekspressionskassette, en vektor eller en kloningsvehikel ifølge krav 2, hvor den transformerede værtscelle er en bakteriecelle, en svampecelle, en gærcelle, en insektcelle eller en plantecelle.
4. Transgen ikke-humant dyr eller transgen plante eller transgen såsæd omfattende en sekvens ifølge krav 1, eller en ekspressionskassette, en vektor eller en kloningsvehikel ifølge krav 2.
5. Isoleret eller rekombinant polypeptid, som har en alpha-amylase-aktivitet bestående af (i) en aminosyresekvens, der har mindst 90 %, eller mere, eller har 100% sekvensidentitet med SEQ ID NO:419, eller et enzymatisk aktivt fragment deraf; (ii) et polypeptid, som kodes af en nukleinsyre, der har en sekvens ifølge krav 1; (iii) et polypeptid, som har aminosyresekvensen af (i) eller (ii), og omfatter mindst én konservativ substitution af aminosyrerest, hvor den konservative substitution omfatter udskiftning af en alifatisk aminosyre med en anden alifatisk aminosyre; udskiftning af en serin med en thre-onin eller omvendt; udskiftning af en syrerest med en anden syrerest; udskiftning af en rest med en amidgruppe med en anden rest med en amidgruppe; udskiftning af en basisk rest med en anden basisk rest; eller udskiftning af en aromatisk rest med en anden aromatisk rest eller en kombination deraf, og den alifatiske rest omfatter Alanin, Valin, Leucin, Isoleucin eller en syntetisk ækvivalent deraf; syreresten omfatter asparaginsyre, glutaminsyre eller en syntetisk ækvivalent deraf; hvor resten, som omfatter en amidgruppe, omfatter Asparagin, Glutamin eller en syntetisk ækvivalent deraf; hvor den basiske rest omfatter Lysin, Arginin eller en syntetisk ækvivalent deraf; eller den syntetiske rest omfatter Phenylalanin, Tyrosin en syntetisk ækvivalent deraf; (iv) polypeptidet af (i), (ii) eller (iii), som har en amylase, men mangler en signalsekvens eller et kulhydratbindende modul; (v) polypeptidet af (i), (ii), (iii) eller (iv) med en amylase-aktivitet og yderligere omfattende en eller flere heterologe sekvenser; (vi) polypeptidet af (v), hvor den ene eller flere heterologe sekvenser omfatter en heterolog signalsekvens, et kulhydratbindende modul, et katalytisk domæne (CD), eller en kombination deraf, eller den heterologe sekvens omfatter en heterolog epitop, rensningstag eller etikette; eller (vii) polypeptidet af (i), (ii), (iii), (iv), (v) eller (vi), yderligere omfattende mindst én glykosyleringsplads, hvor polypeptidet glykosyleres efter at være ekspri-meret i P. pastoris eller S. pombe, og polypeptidet bevarer en amylase-aktivitet under betingelser omfattende ca. pH 6,5, pH 6,0, pH 5,5, 5,0, pH 4,5 eller 4,0, og polypeptidet bevarer en amylase-aktivitet under betingelser omfattende ca. pH 8,0, pH 8,5, pH 9, pH 9,5, pH 10 eller pH 10,5.
6. Homodimer omfattende polypeptid ifølge krav 5 eller en heterodimer omfattende et polypeptid ifølge krav 5 og et andet domæne, hvor det andet domæne er et polypeptid, og heterodimeren er et fusionsprotein eller en epitop eller tag.
7. Immobiliseret polypeptid, hvor polypeptidet omfatter en sekvens ifølge krav 5, hvor polypeptidet er immobiliseret en celle, et metal, en harpiks, en polymer, en ceramik, et glas, en mikroelektrode, en grafitpartikel, en lille kugle, en gel, en plade, et array eller et kapillærrør.
8. Isoleret eller rekombinant antistof, som specifikt binder til et polypeptid ifølge krav 5 eller et polypeptid, der kodes af en nukleinsyre ifølge krav 1; hvor antistoffet er et monoklonalt eller et polyklonalt antistof.
9. Fremgangsmåde til fremstilling af et rekombinant, omfattende de følgende trin: (a) tilvejebringelse af en nukleinsyre, der er operabelt forbundet med en promotor, hvor nukleinsyren omfatter en sekvens ifølge krav 1; og (b) ek-sprimering af nukleinsyren i trin (a) under betingelser, som tillader eksprime-ring af polypeptidet, hvorved der produceres et rekombinant polypeptid, hvor fremgangsmåden yderligere omfatter en værtscelle med nukleinsyren i trin (a), efterfulgt af eksprimering af nukleinsyren i trin (a), hvorved der produceres et rekombinant polypeptid i en transformeret celle.
10. Fremgangsmåde til generering af en variant af en nukleinsyre, som koder for et polypeptid med en amylase-aktivitet, som omfatter følgende trin: (a) tilvejebringelse af en skabelonnukleinsyre omfattende en sekvens ifølge krav 1; og (b) modificere, slette eller tilføje en eller flere nukleotider i skabelonsekvensen, eller en kombination deraf, for at generere en variant af skabelonnukle-insyren; hvor fremgangsmåden yderligere omfatter eksprimering af variantnukleinsy-ren eller at generere en variant af skabelonnukleinsyren, og modificeringerne, tilføjelserne og sletningerne indføres med en fremgangsmåde, som omfatter fejltilbøjelig PCR, shuffling, oligonukleotid-styret mutagenese, assembly PCR, sexuel PCR-mutagenes, in vivo mutagenese, kassette-mutagenese, rekursiv ensemble-mutagenese, exponential-ensemble-mutagenese, sted-specifik mutagenese, gen-gensamling, gene-site-saturation-mutagenese (GSSM), syntetisk ligation-gensamlilng (SLR), rekombination, rekursiv sekvensrekombination, phosphothioat-modificeret DNA-mutagenese, uracil-indeholdende skabelon-mutagenese, gapped-duplex-mutagenese, point-mismatch-repair-mutagenese, reparatur-deficient-værtsstamme-mutagenes, kemisk mutagenese, radiomutagenese, deletion-smutagenese, restriktions-selektions-mutagenese, restriktions-rensnings-mutagenese, kunstig gen-syntese, ensemble-mutagenese, fremstilling af ki-mærisk nukleinsyre-multimer og en kombination deraf; og fremgangsmåden gentages iterativt, indtil en amylase, som har en ændret eller forskellig aktivitet eller en ændret eller forskellig stabilitet end polypepti-det, der kodes af skabelonnukleinsyren, er fremstillet, og amylase-polypeptid-varianten er termotolerant og beholder noget aktivitet efter at være udsat for en forhøjet temperatur, eller amylase-polypeptid-varianten har forhøjet glyko-sylering i sammenligning med den amylase, som kodes af en skabelonnukle-insyre, eller amylase-polypeptid-varianten har en amylase-aktivitet under en høj temperatur; og fremgangsmåden gentages iterativt, indtil en sekvens, som koder for en amylase, der har en ændret kodonanvendelse end skabelonnukleinsyren, er fremstillet; og fremgangsmåden gentages iterativt, indtil et amylase-gen, som har højere eller lavere niveau af beskedeksprimering eller stabilitet end skabelonnukleinsyren, er fremstillet.
11. Fremgangsmåde til hydrolyse af en polysaccharid, oligosaccharid eller stivelse eller at fjerne eller gøre en polysaccharid, oligosaccharid eller stivelse fra en sammensætning flydende, omfattende de følgende trin: (a) tilvejebringe polypeptidet ifølge krav 5, eller et polypeptid, der kodes af nukleinsyren ifølge krav 1; (b) tilvejebringe en sammensætning omfattende polysaccharid, oligosaccha-rid eller stivelse; og (c) bringe polypeptidet af trin (a) i kontakt med sammensætningen af trin (b) under forhold, hvor polypeptidet hydrolyserer, fjerner eller gør polysacchari-den, oligosacchariden eller stivelsen flydende, eller under betingelser, hvor polypeptidet fjerner eller gør polysacchariden, oligosacchariden eller stivelsen flydende. hvor sammensætningen omfatter en a-1,4-glucosidisk binding eller en a-1,6-glucosidisk binding.
12. Sammensætning omfattende polypeptidet af krav 5, eller et polypeptid, der kodes af nukleinsyren ifølge krav 1, hvor sammensætningen er udvalgt fra gruppen bestående af: en drik, et foder eller et levnedsmiddel, et fodertilskud til et dyr, en spiselig enzymindgi-velsesmatrix, et tekstile, papir eller et papirprodukt, papirmasse, en detergentsammensætning, en sammensætning til forhindre udtørring af brød, en farmaceutisk sammensætning, et mundplejeprodukt, en forsinket frigivelseseller kontrolleret frigivelsessammensætning, en olieboringsfluid og en bioblegningsopløsning.
13. Detergentsammensætning ifølge krav 12, hvor polypeptidet formuleres i en ikke-vandig, flydende sammensætning, et støbt faststof, en granulær form, en partikelform, en komprimeret tablet, en gelform, en pasta eller en opslæmningsform.
14. Fremgangsmåde til anvendelse af polypeptidet ifølge krav 5, eller det polypeptid, der kodes af en nukleinsyre ifølge krav 1; hvor anvendelsen er udvalgt fra gruppen bestående af: (a) forbedring af flydeevnen af en produktionsfluid fra en underjordisk formation; (b) forhindre udtørring af et bagt produkt, hvor det bagte produkt er et brød eller brødprodukt; (c) understøtte vækføringen af boreslam; (d) bio-blegning af et papir eller papirmasse; (e) krympning af tekstiler; (f) afsværtning af papir eller fibre; (g) behandling af lignocellulosiske fibre; (h) fremstilling af en sirup med højt indhold af maltose eller glukose; og (i) fremstilling af et foder eller levnedsmiddel.
15. Fremgangsmåde til fremstilling af ethanol, omfattende de følgende trin: (a) tilvejebringe polypeptidet ifølge krav 5, eller et polypeptid, der kodes af nukleinsyren ifølge krav 1; (b) tilvejebringe en sammensætning omfattende polysaccharid, oligosaccha-rid eller stivelse; og (c) bringe polypeptidet af trin (a) i kontakt med sammensætningen af trin (b) under forhold, hvor polypeptidet hydrolyserer polysacchariden, oligosaccha-riden eller stivelsen; eller: en fremgangsmåde til anvendelse af en amylase i brygning eller fremstilling af alkohol, omfattende de følgende trin: (a) tilvejebringe et polypeptid omfattende polypeptidet af krav 5, eller et polypeptid, der kodes af nukleinsyren ifølge krav 1; (b) tilvejebringe en sammensætning, der anvendes til brygning eller produktion af alkohol, omfattende polysaccharid, oligosaccharid eller stivelse; (c) kombinere polypeptidet af trin (a) med sammensætningen af trin (b) under forhold, hvor polypeptidet kan hydrolysere polysacchariden, oligosacchariden eller stivelsen i sammensætningen, som anvendes til brygning eller produktion af alkohol.
DK09180956.6T 2003-03-06 2004-03-08 Amylaser, nukleinsyrer, som koder for dem, og fremgangsmåder til fremstilling og anvendelse deraf DK2194133T3 (da)

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US10/385,305 US7560126B2 (en) 2001-02-21 2003-03-06 Amylases, nucleic acids encoding them and methods for making and using them
US45901403P 2003-03-28 2003-03-28
EP04718513A EP1727898A4 (en) 2003-03-06 2004-03-08 AMYLASES, NUCLEIC ACIDS ENCODING THESE AMYLASES AND METHODS OF PRODUCTION AND USE OF AMYLASES

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CN112384616B (zh) * 2018-05-29 2024-09-03 巴斯夫欧洲公司 淀粉酶
US20210388406A1 (en) * 2018-10-31 2021-12-16 Amano Enzyme Inc. Maltotriose-generating amylase
KR102070415B1 (ko) * 2019-03-06 2020-01-28 고려대학교산학협력단 신규한 알파-아가레이즈 및 이를 이용한 한천 유래 홀수 개의 올리고당 생산 방법
CN113322295A (zh) * 2020-10-20 2021-08-31 河北肽丰生物科技有限公司 一种富硒黑小麦芽低聚糖肽的制备方法及其制品
CN114606221B (zh) * 2022-05-12 2022-09-02 凯莱英医药集团（天津）股份有限公司 固定化酶、其制备方法及应用
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