DK2327765T3 - Nitrilaser - Google Patents

Nitrilaser Download PDF

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Publication number
DK2327765T3
DK2327765T3 DK10189100.0T DK10189100T DK2327765T3 DK 2327765 T3 DK2327765 T3 DK 2327765T3 DK 10189100 T DK10189100 T DK 10189100T DK 2327765 T3 DK2327765 T3 DK 2327765T3
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Denmark
Prior art keywords
acid
sequence
polypeptide
sequences
nucleic acid
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DK10189100.0T
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English (en)
Inventor
Jennifer Ann Chaplin
David Paul Weiner
Aileen Milan
Ellen Chi
Jay M Short
Mark Madden
Mark Burk
Dan Robertson
Grace Desantis
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Basf Enzymes Llc
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    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/002—Nitriles (-CN)
    • C12P13/004—Cyanohydrins
    • 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)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • 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
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Processing Of Solid Wastes (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Treatment Of Sludge (AREA)

Claims (17)

1. Isoleret, syntetisk eller rekombinant nukleinsyre indeholdende (a) en nukleinsyresekvens, der har mindst 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%, 99% eller mere, eller 100% sekvensidentitet med SEQ ID NO: 103 og kodende for et polypeptid med nitrilaseaktivitet, hvor nukleinsyren koder for mindst ét polypeptid med en nitrilaseaktivitet; eller (b) en nukleinsyresekvens, der er fuldstændigt komplementær til (a).
2. Ekspressionskassette, vektor eller kloningsmedium indeholdende en nukleinsyre omfattende en sekvens som angivet i krav 1.
3. Transformeret celle omfattende (i) en nukleinsyre med en sekvens som angivet i krav 1 eller en ekspressionskassette, en vektor eller et kloningsmedium som angivet i krav 2; (ii) den transformerede celle af (i), hvor cellen er (a) en gramnegativ bakterie, en grampositiv bakterie eller en eukaryotisk monocellulær organisme; (b) en Escherichia coli; (c) en Streptomyces diversa, Lactobacillus gasseri, Lactococcus lactis, Lacto-coccus cremoris eller en Bacillus subtilis; eller (d) en Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha eller en Aspergillus niger.
4. Transgen, ikke-human organisme indeholdende en sekvens som angivet i krav 1 eller en ekspressionskassette, en vektor eller et kloningsmedium som angivet i krav 2.
5. Isoleret, syntetisk eller rekombinant polypeptid indeholdende: (a) en sekvens, der har mindst 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%, 99% eller mere, eller 100% sekvensidentitet med SEQ ID NO:104, hvor polypeptidet har en nitrilaseaktivitet; eller (b) en aminosyre kodet af nukleinsyren ifølge krav 1 (a).
6. Isoleret, syntetisk eller rekombinant polypeptid ifølge krav 5, hvor nitrilaseaktiviteten omfatter (a) hydrolyse af et nitril til en carboxylsyre; (b) hydrolyse af en cyanhydrindel eller en aminonitrildel af et molekyle, hvor molekylet eventuelt indeholder (S)-2-amino-4-phenyl-butansyre, eller molekylet indeholder en L-aminosyre, eller molekylet indeholder et næringsmiddeladditiv eller et farmaceutisk lægemiddel; eller (c) en enantio-selektiv nitrilaseaktivitet, hvor den enantio-selektive aktivitet omfatter dannelse af et chiralt alfa-hydroxysyremolekyle eller et chiralt aminosyre-molekyle ved katalyse af hydrolysen af et molekyle med en cyanhydrindel eller en aminonitrildel, hvor det dannede chirale molekyle eventuelt er en (fl)-enantiomer eller en (S)-enantiomer.
7. Isoleret, syntetisk eller rekombinant polypeptid ifølge krav 5 eller krav 6, hvor nitrilaseaktiviteten omfatter hydrolyse af et hydroxyglutarylnitril til dannelse af en (fl)-ethyl 4-cyano-3-hydroxysmørsyre, hvor polypeptidet eventuelt enantio-selektivt hydrolyserer hydroxyglutarylnitrilet til dannelse af en (F?)-enantiomer (F?)-ethyl 4-cyano-3-hydroxysmørsyre, hvor polypeptidet eventuelt danner et produkt med en enantiomerrenhed omfattende en ee på mindst 95%, 96%, 97%, 98% eller 99%, og hvor hydroxyglutarylnitrilet eventuelt indeholder 1,3-di-cyano-2-hydroxy-propan eller et hydroxyglutaronitil, eller hvor hydroxyglutaronitrilet eventuelt omfatter et 3-hydroxyglutaronitril.
8. Isoleret, syntetisk eller rekombinant polypeptid ifølge krav 5 eller krav 6, hvor nitrilaseaktiviteten omfatter hydrolyse af et hydroxyglutarylnitril til dannelse af en ethyl 4-cyano-3-hydroxysmørsyre, hvor polypeptidet eventuelt enantio-selektivt hydrolyserer hydroxyglutarylnitrilet til dannelse af en (S)-enantiomer (S)-ethyl-4-cyano-3-hydroxysmørsyre, og hvor polypeptidet eventuelt danner et produkt med en enantiomerrenhed omfattende en ee på 95%, 96%, 97%, 98% eller 99%.
9. Isoleret, syntetisk eller rekombinant polypeptid ifølge krav 5 eller krav 6, hvor nitrilaseaktiviteten omfatter hydrolyse af et mandelonitril til dannelse af en mandelsyre eller et mandelsyrederivat, hvor mandelsyren eventuelt omfatter (f?)-2-chlormandelsyre, hvor mandelsyren eventuelt omfatter en aromatisk ringsubstitution i ortho-, me-ta- eller para-stillingerne; et 1-naphthylderivat af (F?)-mandelsyre, et pyridylderi-vat af (F?)-mandelsyre eller thienylderivat af (F?)-mandelsyre eller en kombination deraf, hvor mandelsyren eventuelt omfatter (S)-methyl-benzylcyanid, og mandelo-nitrilet omfatter (S)-methoxy-benzylcyanid, eller hvor mandelsyren eventuelt omfatter en aromatisk ringsubstitution i ortho-, me-ta- eller para-stillingerne; et 1-naphthylderivat af (S)-mandelsyre, et pyridylderi-vat af (S)-mandelsyre eller thienylderivat af (S)-mandelsyre eller en kombination deraf.
10. Isoleret, syntetisk eller rekombinant polypeptid ifølge krav 5 eller krav 6, hvor nitrilaseaktiviteten omfatter hydrolyse af et phenyllactocyanonitril til dannelse af et (S)-phenyl-mælkesyrederivat eller et (F?)-phenyl-mælkesyrederivat.
11. Enzympræparat indeholdende et polypeptid som angivet i ethvert af kravene 5 til 10, hvor præparatet er et flydende eller tørt præparat.
12. Industriel fremgangsmåde omfattende anvendelse af det isolerede, syntetiske eller rekombinante polypeptid som angivet i ethvert af kravene 5 til 10 ved hydrolysen af et substrat med en cyanhydrindel eller en aminonitrildel til dannelse af en sammensætning indeholdende en alfa-hydroxysyre eller en aminosyre.
13. Industriel fremgangsmåde ifølge krav 12, hvor den industrielle fremgangsmåde yderligere omfatter: (i) fremstillingen af en farmaceutisk sammensætning indeholdende den dannede alfa-hydroxysyre eller aminosyre, (ii) fremstillingen af en kemisk forbindelse indeholdende den dannede alfa-hydroxysyre eller aminosyre, (iii) katalyse af nedbrydningen af et affaldsprodukt, hvor nedbrydningen omfatter hydrolyse af et substrat med en cyanhydrindel eller en aminonitrildel, (iv) fremstillingen af et lægemiddelmellemprodukt, hvor lægemiddelmellemproduktet omfatter den dannede alfa-hydroxysyre eller aminosyre, og hvor po-lypeptidet eventuelt anvendes til at hydrolysere et hydroxyglutarylnitril-substrat, (v) fremstillingen af et detergent, eller (vi) fremstillingen af et næringsmiddelprodukt.
14. Isoleret, syntetisk eller rekombinant polypeptid ifølge krav 5 kodet af en nukleinsyre udtrykt i en værtscelle, hvor værtscellen omfatter: (a) en gramnegativ bakterie, en grampositiv bakterie eller en eukaryotisk monocellulær organisme; (b) en Escherichia coli; (c) en Streptomyces diversa, Lactobacillus gasseri, Lactococcus lactis, Lacto-coccus cremoris eller en Bacillus subtilis; eller (d) en Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha eller en Aspergillus niger.
15. Fremgangsmåde til fremstilling af en carboxylsyre, omfattende hydrolyse af et nitril under anvendelse af det isolerede, syntetiske eller rekombinante polypeptid ifølge krav 5 eller krav 6 under vandige betingelser.
16. Polypeptid ifølge krav 5, men som mangler en signalsekvens.
17. Polypeptid ifølge krav 5, der yderligere omfatter en heterolog sekvens.
DK10189100.0T 2001-06-21 2002-05-15 Nitrilaser DK2327765T3 (da)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US30018901P 2001-06-21 2001-06-21
US30900601P 2001-07-30 2001-07-30
US35133602P 2002-01-22 2002-01-22
EP08075753A EP2039762A3 (en) 2001-06-21 2002-05-15 Nitralases

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DK2327765T3 true DK2327765T3 (da) 2015-07-13

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EP (6) EP1578910A4 (da)
JP (2) JP4405258B2 (da)
AU (2) AU2006252258B2 (da)
CA (2) CA2445046C (da)
DK (3) DK2327765T3 (da)
WO (1) WO2003000840A2 (da)

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