DK2622069T3 - Beta-glucosidasevarianter og polynukleotider, der koder for dem - Google Patents

Beta-glucosidasevarianter og polynukleotider, der koder for dem Download PDF

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DK2622069T3
DK2622069T3 DK11767880.5T DK11767880T DK2622069T3 DK 2622069 T3 DK2622069 T3 DK 2622069T3 DK 11767880 T DK11767880 T DK 11767880T DK 2622069 T3 DK2622069 T3 DK 2622069T3
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seq
variant
mature polypeptide
substitutions
beta
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Mark Wogulis
Paul Harris
David Osborn
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Novozymes Inc
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Claims (23)

1. Beta-glucosidasevariant, som omfatter eller består af en substitution i positioner svarende til position 89, 91, 140 og 186 i det modne polypeptid ifølge SEQ ID NO: 2, hvor varianten har beta-glucosidaseaktivitet, og hvor varianten udviser mindst 80%, men mindre end 100%, sekvensidentitet med det modne polypeptid ifølge SEQ ID NO: 2.
2. Variant ifølge krav 1, hvor varianten udviser mindst 90% sekvensidentitet med det modne polypeptid ifølge SEQ ID NO: 2.
3. Variant ifølge krav 1, hvor varianten udviser mindst 95% sekvensidentitet med det modne polypeptid ifølge SEQ ID NO: 2.
4. Variant ifølge et hvilket som helst af de foregående krav, som er en variant af en stam-beta-glucosidase valgt fra gruppen bestående af: (a) et polypeptid, som udviser mindst 80% sekvensidentitet med det modne polypeptid ifølge SEQ ID NO: 2; (b) et polypeptid, der kodes for af et polynukleotid, som under betingelser med høj stringens eller betingelser med meget høj stringens hybridiserer med (i) det modne polypeptids kodningssekvens ifølge SEQ ID NO: 1, (ii) cDNA-sekvensen deraf eller (iii) komplementet til (i) eller (ii) i fuld længde; (c) et polypeptid, der kodes for af et polynukleotid, som udviser mindst 80% sekvensidentitet med det modne polypeptids kodningssekvens ifølge SEQ ID NO: 1 eller cDNA-sekvensen deraf; og (d) et fragment af det modne polypeptid ifølge SEQ ID NO: 2, som har beta-glucosidaseaktivitet.
5. Variant ifølge krav 4, som udviser mindst 80%, men mindre end 100%, sekvensidentitet med stam-beta-glucosidasens aminosyresekvens.
6. Variant ifølge et hvilket som helst af de foregående krav, som omfatter en eller flere substitutioner valgt fra gruppen bestående af L89M, G91L, F100D, I140V, I186V, S283G, N456E og F512Y.
7. Variant ifølge et hvilket som helst af kravene 1-6, hvor varianten omfatter eller består af substitutionerne i positioner svarende til position 89, 91, 100, 140, 186, 283, 456 og 512 i det modne polypeptid ifølge SEQ ID NO: 2.
8. Variant ifølge et hvilket som helst af kravene 1-5 og 7, som omfatter eller består af substitutionerne L89M, G91L, F100D, I140V, I186V, S283G, N456E og F512Y.
9. Variant ifølge et hvilket som helst af kravene 1-6, hvor varianten omfatter eller består af substitutionerne L89M + G91L + I140V + I186V i det modne polypeptid ifølge SEQ ID NO: 2.
10. Isoleret polynukleotid, der koder for varianten ifølge et hvilket som helst af kravene 1 -9.
11. Værtscelle, som omfatter polynukleotidet ifølge krav 10.
12. Fremgangsmåde til fremstilling af en variant, hvilken fremgangsmåde omfatter: (a) dyrkning af værtscellen ifølge krav 11 under betingelser, som er egnede til ekspression af varianten; og (b) indvinding af varianten.
13. Fremgangsmåde til opnåelse af en variant, hvilken fremgangsmåde omfatter indføring i en stam-beta-glucosidase af en substitution i positioner svarende til position 89, 91,140 og 186 i det modne polypeptid ifølge SEQ ID NO: 2, hvor varianten har beta-glucosidaseaktivitet; og indvinding af varianten.
14. Fremgangsmåde til nedbrydning eller omdannelse af et celluloseholdigt materiale, hvilken fremgangsmåde omfatter: behandling af det celluloseholdige materiale med en enzymsammensætning i nærværelse af varianten ifølge et hvilket som helst af kravene 1-9.
15. Fremgangsmåde ifølge krav 14, som endvidere omfatter indvinding af det nedbrudte celluloseholdige materiale.
16. Fremgangsmåde ifølge krav 14 eller 15, hvor enzymsammensætningen omfatter et eller flere enzymer valgt fra gruppen bestående af en cellulase, et polypeptid med cellulolytisk forbedrende aktivitet, en hemicellulase, et expansin, en esterase, en laccase, et ligninolytisk enzym, en pectinase, en peroxidase, en protease og et swollenin.
17. Fremgangsmåde til fremstilling af et fermenteringsprodukt, hvilken fremgangsmåde omfatter: (a) forsukring af et celluloseholdigt materiale med en enzymsammensætning i nærværelse af varianten ifølge et hvilket som helst af kravene 1-9; (b) fermentering af det forsukrede celluloseholdige materiale med en eller flere fermenterende mikroorganismer for at fremstille fermenteringsproduktet; og (c) indvinding af fermenteringsproduktet fra fermenteringen.
18. Fremgangsmåde ifølge krav 17, hvor enzymsammensætningen omfatter et eller flere enzymer valgt fra gruppen bestående af en cellulase, et polypeptid med cellulolytisk forbedrende aktivitet, en hemicellulase, et expansin, en esterase, en laccase, et ligninolytisk enzym, en pectinase, en peroxidase, en protease og et swollenin.
19. Fremgangsmåde til fermentering af et celluloseholdigt materiale, hvilken fremgangsmåde omfatter: fermentering af det celluloseholdige materiale med en eller flere fermenterende mikroorganismer, hvor det celluloseholdige materiale er forsukret med en enzymsammensætning i nærværelse af varianten ifølge et hvilket som helst af kravene 1-9.
20. Fremgangsmåde ifølge krav 18, hvor enzymsammensætningen omfatter et eller flere enzymer valgt fra gruppen bestående af en cellulase, et polypeptid med cellulolytisk forbedrende aktivitet, en hemicellulase, et expansin, en esterase, en laccase, et ligninolytisk enzym, en pectinase, en peroxidase, en protease og et swollenin.
21. Fremgangsmåde ifølge krav 19 eller 20, hvor fermenteringen af det celluloseholdige materiale frembringer et fermenteringsprodukt valgt fra gruppen bestående af brændstof, drikkeligt ethanol og platformskemikalier.
22. Fremgangsmåde ifølge krav 21, som endvidere omfatter indvinding af fermenteringsproduktet fra fermenteringen.
23. Totalmedieformulering (whole broth formulation) eller cellekultursammensætning (cell culture composition), som omfatter varianten ifølge et hvilket som helst af kravene 1 -9.
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