DK2420570T3 - Arabinofuranosidaseenzymer, nucleinsyrer, som koder for dem, og fremgangsmåder til at fremstille og anvende dem - Google Patents

Arabinofuranosidaseenzymer, nucleinsyrer, som koder for dem, og fremgangsmåder til at fremstille og anvende dem Download PDF

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DK2420570T3
DK2420570T3 DK11007477T DK11007477T DK2420570T3 DK 2420570 T3 DK2420570 T3 DK 2420570T3 DK 11007477 T DK11007477 T DK 11007477T DK 11007477 T DK11007477 T DK 11007477T DK 2420570 T3 DK2420570 T3 DK 2420570T3
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nucleic acid
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Kevin A Gray
Lishan Zhao
Michelle H Cayouette
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Bp Corp North America Inc
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Claims (19)

1. Isoleret, syntetisk eller rekombinant nucleinsyre omfattende (a) en nucleinsyresekvens med 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 komplet (100%) sekvensidentitet til SEQ ID NO: 103, hvor nucleinsy-ren, som koder for et polypeptid, har en arabinofuranosidaseaktivitet, og eventuelt er sekvensidentiteterne bestemt ved analyse med en sekvenssammenligningsalgoritme eller ved en visuel inspicering, eller (b) en nucleinsyresekvens, som hybridiserer ved stringente betingelser til en nucleinsyre omfattende SEQ ID NO: 103, hvor nucleinsyren koder for et polypeptid med en arabinofuranosidaseaktivitet, og har mindst 70% identitet med SEQ ID NO: 103, og de stringente betingelser indbefatter et vasketrin omfattende en vask med 0,2 X SSC ved en temperatur på ca. 65°C i ca. 15 minutter, (c) en nucleinsyresekvens, som koder for et polypeptid med mindst 70% identitet med sekvensen i SEQ ID NO: 104, hvor polypeptidet har arabinofuranosidaseaktivitet, (d) nucleinsyren i (a), (b) eller (c), som koder for et polypeptid med en arabinofuranosidaseaktivitet, men mangler en signalsekvens eller et carbohy-dratbindingsmodul, (e) nucleinsyren i (a), (b), (c) eller (d), som koder for et polypeptid med en arabinofuranosidaseaktivitet, men har en heterolog sekvens, hvor den heterologe sekvens eventuelt omfatter en heterolog signalsekvens, et carbohydratbindingsmodul, et katalytisk domæne (CD) eller en kombination heraf, og eventuelt den heterologe signalsekvens, carbohy-dratbindingsmodulet eller det katalytiske domæne (CD) er afledt fra en anden oligomerase, cellulase eller cellulytisk enzym, eller en non-oligomera-se, -cellulase eller -cellulytisk enzym, eller (f) en nucleinsyresekvens fuldstændig komplementær til (a), (b), (c), (d) eller (e).
2. Ekspressionskassette, vektor eller kloningsvehikel omfattende en nuclein-syre omfattende sekvensen i krav 1, hvor eventuelt kloningsvehiklen omfatter en viral vektor, et plasmid, en phag, et phagemid, et cosmid, et fosmid, en bak-teriophag eller et kunstigt kromosom, og hvor eventuelt kloningsvehiklen omfatter en viral vektor omfattende en adenovirusvektor, en retroviral vektor eller en adenoassocieret viral vektor, eller hvor eventuelt kloningsvehiklen omfatter et bakterielt kunstigt kromosom (BAC), et plasmid, en bakteriophag P1-afledt vektor (PAC), et kunstigt gærkromosom (YAC) eller et kunstigt patterdyrskromosom (MAC).
3. Transformeret celle omfattende en nucleinsyre omfattende sekvensen i krav 1, eller en vektor eller kloningsvehikel som anført i krav 2, hvor eventuelt cellen er en bakteriel celle, en pattedyrscelle, en svampecelle, en gærcelle, en insektcelle eller en plantecelle, og hvor den transformerede celle producerer et polypeptid kodet for af nucleinsyren ifølge krav 1, og som har arabinofurano-sidaseaktivitet.
4. Transgent ikke-humant dyr, plante, plantedel eller plantefrø omfattende sekvensen i krav 1 eller en vektor eller kloningsvehikel ifølge krav 2, hvor eventuelt det transgene ikke-humane dyr er en mus eller en rotte, og hvor det ikke-humane dyrs, plantes, plantedels eller plantefrøs transformerede celle fremstiller et polypeptid kodet for af nucleinsyren i krav 1, og har arabinofuranosidase-aktivitet.
5. Isoleret, syntetisk eller rekombinant polypeptid (i) med en aminosyresekvens, som 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 til SEQ ID NO: 104, hvor polypepti-det har en arabinofuranosidaseaktivitet, hvor sekvensidentiteterne eventuelt bestemmes ved analyse med en sekvenssammenligningsalgoritme eller ved visuel inspektion, og eventuelt er sekvenssammenligningsalgoritmen en BLAST version 2.2.2-algoritme, hvor en filtreringsindstilling er sat til blastall-p blastp -d, ”nr pataa”-F F, og alle andre valg er sat til default, (ii) med en aminosyresekvens kodet for af en nucleinsyre ifølge krav 1, hvor polypeptidet har en arabinofuranosidaseaktivitet eller har immunogen aktivitet derved, at den er i stand til at generere et antistof, som specifikt binder til et polypeptid med sekvensen i SEQ ID NO: 104, (iii) polypeptidet i (i) eller (ii) med en arabinofuranosidaseaktivitet, men uden en signalsekvens, (iv) polypeptidet fra (i), (ii) eller (iii) med en arabinofuranosidaseaktivitet, men som har en heterolog sekvens, hvor den heterologe sekvens ifølge krav omfatter en heterolog signalsekvens, et carbohydratbindingsmodul, et katalytisk domæne (CD) eller en kombination heraf, og den heterologe signalsekvens, carbohydratbindings-modulet eller det katalytiske domæne (CD) stammer eventuelt fra en anden oligomerase, cellulase eller cellulytisk enzym, eller en non-oligomera-se, -cellulase eller non-cellulytisk enzym, eller (v) polypeptidet fra (i), (ii), (iii) eller (iv), hvor polypeptidet er glycosyleret.
6. Isoleret, syntetisk eller rekombinant polypeptid med en aminosyresekvens fra SEQ ID NO: 104.
7. Polypeptid ifølge krav 6 med arabinofuranosidaseaktivitet, en signalsekvens og et carbohydratbindingsmodul.
8. Fremgangsmåde til fremstilling af et rekombinant polypeptid omfattende trinnene: (a) tilvejebringelse af en nucleinsyre ifølge krav 1, og (b) ekspression af nucleinsyren i trin (a) ved betingelser, som tillader ekspression af polypeptidet, og derved producere et rekombinant polypeptid, hvor eventuelt fremgangsmåden yderligere omfatter at transformere en værtscelle med nucleinsyren fra trin (a) fulgt at ekspression af nucleinsyren fra trin (a) og derved producere et rekombinant polypeptid i en transformeret celle.
9. Fremgangsmåde til at generere en variant af en nucleinsyre, som koder for et polypeptid med en arabinofuranosidaseaktivitet omfattende trinnene: (a) tilvejebringelse af en templatnucleinsyre omfattende sekvensen ifølge krav 1, og (b) modificering, deletering eller addering af en eller flere nucleotider i tem-platsekvensen, eller en kombination heraf for at generere en variant af templatnucleinsyren, hvor fremgangsmåden eventuelt omfatter at udtrykke nucleinsyrevarianten for at generere en oligomerasevariant og/eller cellulasepolypeptidvariant, og eventuelt indføres modifikationerne, additionerne eller deleteringerne ved en fremgangsmåde omfattende fejlbehæftet PCR, shuffling, oligonucleotidrettet mutagenese, samlings-PCR, seksuel PCR-mutagenese, in vivo mutagenese, kassettemutagenese, rekursiv gruppemutagenese, eksponentiel gruppemutage-nese, stedsspecifik mutagenese, gensamling, Gene Site Saturation Mutagenesis (GSSM), syntetisk ligeringsgensamling (SLR), rekombination, rekursiv sekvensrekombination, phosphothiat-modificeret DNA mutagenese, uracilholdig templatmutagenese, gapped dupleks mutagenese, punktfejlparringsreparations-mutagenese, reparationsdeficient værtsstammemutagenese, kemisk mutagenese, radiogenisk mutagenese, deleteringsmutagenese, restriktions/selekterings-mutagenese, restriktionsoprensningsmutagenese, kunstig gensyntese, homo-mutagenese, kimærisk nucleinsyre multimerskabelse og en kombination heraf, og hvor fremgangsmåden eventuelt iterativt gentages indtil en oligomerase og/-eller en cellulase med en ændret eller forskellig aktivitet, en ændret eller forskellig stabilitet, en ændret kodonanvendelse eller en højere eller lavere grad af messageekspressioner sammenlignet med et polypeptid kodet af templatnucleinsyren fremstilles.
10. Fremgangsmåde til at modificere kodoner i en nucleinsyrer, som koder for et arabinofuranosidasepolypeptid, hvilken fremgangsmåde omfatter følgende trin: (a) tilvejebringelse af en nucleinsyre, som koder for et polypeptid ifølge krav 5, og (b) identificering af en kodon i nucleinsyren i trin (a) og erstatning af den med en forskellig kodon, som koder for den samme aminosyre som den erstattede kodon og derved modificerer kodoner i en nucleinsyre, som koder for en cellulase.
11. Kimærisk polypeptid omfattende et første domæne og mindst et andet domæne, hvor det første domæne omfatter et enzym som anført i krav 5, og det andet domæne omfatter et heterologt eller modificeret carbohydratbindingsdo-mæne eller et heterologt eller modificeret dockerin-domæne, og carbohydratbin-dingsdomænet eventuelt er et cellulosebindende modul (CBM), eller et lignin-bindende domæne, og det andet domæne eventuelt vedhæftet tæt ved enzymets katalytiske domæne, og det andet domæne er eventuelt hæftet omtrent ved C-terminalen af enzymets katalytiske domæne.
12. Sammensætning eller fremstillingsprodukt omfattende (a) en blanding (eller ’’cocktail”) af hemicellulose- og cellulose-hydrolyse-rende enzymer, hvor de cellulosehydrolyserende enzymer omfatter mindst et af hver af en endoglucanase, cellobiohydrolase I (CBHI), cellobiohydro-lase 11 (CBH11) og β-glucosidase, og de hemicellulosehydrolyserende enzymer omfatter mindst et af hver af en xylanase, β- xylosidase og arabino-furanosidase, hvor mindst en arabinofuranosidase er et polypeptid ifølge krav 5 eller et polypeptid kodet for af en nucleinsyre ifølge krav 1, (b) en blanding (eller ’’cocktail”) af hemicellulose- og cellulosehydrolyserende enzymer omfattende mindst et af hver af en endoglucanase, oligomera-se, cellobiohydrolase I (CBHI), cellobiohydrolase II (CBHII), arabinofuranosidase og xylanase, hvor oligomerasen eventuelt er en oligomerase-1 eller β-g lu cos id ase eller oligomerasen eventuelt er en oligomerase-2 eller β-xy-losidase, og hvor mindst en arabinofuranosidase er et polypeptid ifølge krav 5 eller et polypeptid kodet for af en nucleinsyre ifølge krav 1, (c) en blanding (eller ’’cocktail”) af hemicellulose- og cellulosehydrolyse-rende enzymer omfattende mindst en af hver af endoglucanase, en cello-biohydrolase I (CBHI), en cellobiohydrolase II (CBHII), en arabinofuranosidase, en xylanase, en oligomerase-1 (en β-glucosidase), og en oligomerase-2 eller β-xylosidase, og hvor mindst en arabinofuranosidase er et polypeptid ifølge krav 5 eller et polypeptid kodet for af en nucleinsyre ifølge krav 1, (d) et polypeptid med cellobiohydrolase l-aktivitet og et polypeptid med ara-binofuranosidaseaktivitet, hvor mindst et polypeptid med cellobiohydrolase l-aktivitet er SEQ ID NO: 34 eller SEQ ID NO: 46, og hvor mindst en arabinofuranosidase er et polypeptid ifølge krav 5 eller et polypeptid kodet for af en nucleinsyre ifølge krav 1, hvor eventuelt (i) endoglucanasen omfatter SEQ ID NO: 106, cellobiohydrolase I omfatter SEQ ID NO: 34 eller SEQ ID NO: 46, cellobiohydrolase II omfatter SEQ ID NO: 98, β-glucosidasen omfatter SEQ ID NO: 94, xylanasen omfatter SEQ ID NO: 100, SEQ ID NO: 102 eller SEQ ID NO: 524, β-xylosodasen omfatter SEQ ID NO: 96, en yderligere arabinofuranosidase, såfremt den er til stede, omfatter SEQ ID NO: 92 eller en vilkårlig kombination heraf, hvor SEQ ID NO: 106 eventuelt omfatter et yderligere carbohydratbindingsdo-mæne, (ii) blandingen omfatter en endoglucanase omfattende SEQ ID NO: 106, en oligomerase-1 (en β-glucosidase) omfattende SEQ ID NO: 522, en cellobiohydrolase I (CBHI) omfattende SEQ ID NO: 34 eller SEQ ID NO: 46, en cellobiohydrolase II (CBHII) omfattende SEQ ID NO: 98, en yderligere eventuel arabinofuranosidase, hvis til stede, omfattende SEQ ID NO: 92, en oligomerase-2 (en β-xylosidase) omfattende SEQ ID NO: 520, og en xy- lanase omfattende SEQ ID NO: 102, SEQ ID NO: 524 og/eller SEQ ID NO: 100, eller (iii) sammensætningen eller fremstillingsproduktet omfattende yderligere et biomassemateriale, hvor biomassematerialet eventuelt omfatter et lignocel-lulosemateriale afledt fra en landbrugsmæssig afgrøde, eller biomassematerialet er et biprodukt af en fødevare- eller en foderstofproduktion, eller biomassematerialet er et lignocellulosespildprodukt, eller biomassematerialet er en planteremanens eller affaldspapir eller et affaldspapirprodukt, eller biomassematerialet omfatter en planteremanens, og planteremanensen eventuelt omfatter stængler, blade, avner, majskolber, majsblade, hø, træ, træflis, træpulp og/eller savsmuld, og papiraffaldet omfatter eventuelt bortkastet og brugt fotokopieringspapir, computerprinterpapir, notesblokpapir, notesbogpapir, maskinskrivningspapir, aviser, magasiner, pap- og papirbaserede emballagematerialer.
13. Sammensætning eller fremstillingsprodukt omfattende en blanding af enzymer omfattende: (a) SEQ ID NO: 106, en cellobiohydrolase I (CBHI), en cellobiohydrolase II (CBHII) og en arabinofuranosidase i SEQ ID NO: 104, (b) blandingen fra (a), hvor CBHI er SEQ ID NO: 46 eller SEQ ID NO: 34, (c) blandingen fra (a) eller (b), hvor CBHII er SEQ ID NO: 98, (d) blandingen fra (a), (b) eller (c) omfattende en yderligere arabinofuranosidase i SEQ ID NO: 92, (e) blandingen fra (a), (b), (c) eller (d) omfattende yderligere en xylanase, (f) blandingen fra (e), hvor xylanasen er SEQ ID NO: 100, SEQ ID NO: 102 eller SEQ ID NO: 524 eller en kombination heraf, (g) blandingen fra (a), (b), (c), (d), (e) eller (f) omfattende yderligere en oligomerase, (h) blandingen fra (g), hvor oligomerasen er SEQ ID NO: 520 eller SEQ ID NO: 522 eller en kombination heraf, (i) blandingen fra (a), (b), (c), (d), (e), (f), (g) eller (h) omfattende yderligere mindst en af SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 264, SEQ ID NO: 440 eller SEQ ID NO: 442 eller en kombination heraf, eller (j) blandingen fra (a), (b), (c), (d), (e), (f), (g), (h) eller (i) yderligere omfattende en endoglucanase, hvor endoglucanasen eventuelt omfatter SEQ ID NO: 108, SEQ ID NO: 112, SEQ ID NO: 114 eller SEQ ID NO: 116.
14. Enzymgruppe eller ’’cocktail” til depolymerisering af cellulose- og hæmicel-lulosepolymerer til metaboliseringsdygtige carbongrupper omfattende et po-lypeptid som anført i krav 5 eller et polypeptid kodet for af en nucleinsyre som anført i krav 1.
15. Sammensætning omfattende et polypeptid som anført i krav 5 eller et polypeptid kodet for af en nucleinsyre som anført i krav 1 eller en sammensætning eller et fremstillingsprodukt som anført i et vilkårligt af kravene 12 eller 13 eller en enzymgruppe ifølge krav 14, hvor sammensætningen eventuelt er en farmaceutisk sammensætning, en detergentsammensætning, en kontaktlinseopløsning, en spildbehandlingssammensætning, et desinficerende middel, et bioforsvarsmiddel eller et biodetyficeringsmiddel, eller hvor sammensætningen eventuelt er et brændstof, hvor sammensætningen eventuelt er en alkohol, hvor alkoholen eventuelt er ethanol, eller hvor sammensætningen eventuelt er en biomasse eller et biomassemateriale, et papir, papiraffald, genanvendt papir, papirpulp, et papirprodukt, træ, træprodukt, træpulp, tekstil, væv, garn eller fibre, eller hvor sammensætningen eventuelt er en cellulose- eller en celluloseafledt sammensætning, eller hvor eventuelt sammensætningen er en drikkevare, en fødevare, et foderstof, et mejeriprodukt eller et ernæringsmæssigt supplement, eller hvor eventuelt fødevaren er en dej, et brød eller et bagt produkt, eller hvor eventuelt drikkevaren, fødevaren, foderet eller det ernæringsmæssige supplement er til et dyr, eller hvor eventuelt biomassematerialet omfatter et lignocellu-losemateriale stammende fra en landbrugsmæssig afgrøde, eller biomassematerialet er et biprodukt af en fødevareproduktion eller en foderstofproduktion, eller biomassematerialet er et lignocelluloseaffaldsprodukt, eller biomassematerialet er en planteremanens eller et affaldspapir eller affaldspapirprodukt, eller biomassematerialet omfatter en planteremanens, og planteremanensen omfatter eventuelt stængler, blade, avner, avner, majskolber, majsblade, hø, træ, træflis, træpulp og/eller savsmuld, og affaldspapiret eventuelt omfatter bortkastet og brugt fotokopieringspapir, computerprinterpapir, notesbogpapir, notes-blokpapir, skrivemaskinepapir, aviser, magasiner, pap- og papirbaserede emballagematerialer.
16. Fremgangsmåde til at behandle eller modificere en sammensætning omfattende at bringe sammensætningen i kontakt med et polypeptid som anført i krav 5, eller et polypeptid kodet for af en nucleinsyre som anført i krav 1, eller en sammensætning eller fremstillingsprodukt som anført i et vilkårligt af kravene 12 eller 13, eller en enzymgruppering ifølge krav 14, hvor eventuelt sammensætningen er en farmaceutisk sammensætning, en detergentsammensætning, en kontaktlinseopløsning, en affaldssammensætning, et desinficerende middel, et bioforsvarsmiddel eller et biodetoxificeringsmiddel, eller hvor eventuelt sammensætningen er et brændstof, hvor eventuelt sammensætningen er en alkohol, hvor eventuelt alkoholen er ethanol, eller hvor eventuelt sammensætningen er en biomasse eller et biomassemateriale, et papir, papiraffald, genanvendt papirprodukt, papirpulp, papirprodukt, træ, træprodukt, træpulp, tekstil, væv, garn eller fibre, eller hvor eventuelt sammensætningen er en cellulose- eller cel-lulosederivatsammensætning, eller hvor eventuelt sammensætningen er en drikkevare, en fødevare, et foder, et mejeriprodukt eller et ernæringsmæssigt supplement, eller hvor eventuelt fødevaren er en dej, et brød eller et bagt produkt, eller hvor eventuelt drikkevaren, fødevaren, foderstoffet eller det ernæringsmæssige supplement er til dyr, eller hvor eventuelt biomassematerialet omfatter et lignocellulosemateriale stammende fra en landbrugsmæssig afgrøde, eller biomassematerialet er et biprodukt fra en fødevareproduktion eller en foderproduktion eller biomassematerialet er et lignocelluloseaffaldsprodukt eller biomassematerialet er en planteremanens eller et affaldspapir eller affaldspapirprodukt eller biomassematerialet omfatter en planteremanens og planteremanensen omfatter eventuelt stængler, blade, avner, avner, majskolber, majsblade, løv, træ, træflis, træpulp og/eller savsmuld og eventuelt omfatter papiraffaldet bortkastet og brugt fotokopieringspapir, computerprinterpapir, notesbog- papir, notesblokpapir, skrivemaskinepapir, aviser, magasiner, pap- og papirbaserede emballeringsmaterialer.
17. Fremgangsmåde til at hydrolysere, opbryde eller afbryde en arabinoxylan-oligomer omfattende sammensætning omfattende de følgende trin: (a) tilvejebringelse af et polypeptid med en arabinofuranosidaseaktivitet som anført i krav 5 eller et polypeptid kodet for af en nucleinsyre som anført i krav 1, eller en sammensætning eller fremstillingsprodukt som anført i et vilkårligt af kravene 12 eller 13, eller en enzymsamling ifølge krav 14, (b) tilvejebringelse af en sammensætning omfattende en arabinoxylanoli-gomer, og (c) anbringelse af polypeptidet fra trin (a) i kontakt med sammensætningen fra trin (b) ved betingelser, hvor cellulasen hydrolyserer, opbryder eller afbryder den arabinoxylanoligomeromfattende sammensætning.
18. Fremgangsmåde til at fremstille et brændstof omfattende at bringe en sammensætning i kontakt omfattende en cellulose eller en fermenteringsdygtig sukker med et polypeptid som anført i krav 5 eller et polypeptid kodet for af en nucleinsyre som anført i krav 1 eller en sammensætning eller et fremstillet produkt som anført i et vilkårligt af kravene 12 eller 13, eller en enzymgruppe ifølge krav 14, hvor eventuelt sammensætningen omfattende en cellulose eller en fermenteringsdygtig sukker omfatter en plante, planteprodukt eller plantederivat, og planten eller planteproduktet eventuelt omfatter sukkerrørsplanter eller planteprodukter, roer eller sukkerroer, hvede, majs, sojabønner, kartofler, ris eller byg, og eventuelt omfatter brændstoffet en bioethanol eller en blanding af benzin og ethanol.
19. Fremgangsmåde til forarbejdning af et biomassemateriale omfattende at bringe biomassematerialet i kontakt med et polypeptid som anført i krav 5, eller et polypeptid kodet for af en nucleinsyre som anført i krav 1, eller en sammensætning eller et fremstillet produkt som anført i et vilkårligt af kravene 12 eller 13 eller en enzymgruppering ifølge krav 14, hvor biomassematerialet eventuelt stammer fra en landbrugsmæssig afgrøde, er et biprodukt af en fødevare- eller foderproduktion, er et lignocelluloseaffalds-produkt eller er en planteremanens eller et affaldspapir eller et affaldspapirprodukt, og planteremanensen eventuelt omfatter stængler, blade, avner, avner, majskolber, majsblade, hø, træ, træflis, træpulp og savsmuld, og papiraffaldet omfatter bortkastet eller brugt fotokopieringspapir, computerprinterpapir, notes-bogpapir, notesblokpapir, skrivemaskinepapir, aviser, magasiner, pap- og papirbaserede emballeringsmaterialer og eventuelt genererer forarbejdningen af biomassematerialet en bioethanol.
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