DK2287317T3 - Fremgangsmåde til fremstilling af en proteinester eller en ester af en proteinunderenhed - Google Patents

Fremgangsmåde til fremstilling af en proteinester eller en ester af en proteinunderenhed Download PDF

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DK2287317T3
DK2287317T3 DK10178368.6T DK10178368T DK2287317T3 DK 2287317 T3 DK2287317 T3 DK 2287317T3 DK 10178368 T DK10178368 T DK 10178368T DK 2287317 T3 DK2287317 T3 DK 2287317T3
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Kreij Arno De
Susan Mampusti Madrid
Jørn Dalgaard Mikkelsen
Jørn Borch Søe
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Dupont Nutrition Biosci Aps
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Priority claimed from GBGB0301117.8A external-priority patent/GB0301117D0/en
Priority claimed from GBGB0301122.8A external-priority patent/GB0301122D0/en
Priority claimed from GB0301118A external-priority patent/GB0301118D0/en
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Claims (20)

1. Fremgangsmåde til fremstilling af en proteinester og/eller en ester af en proteinunderenhed, hvilken fremgangsmåde omfatter samblanding af en acyldonor, en acylacceptor og vand til fremstilling af et vandrigt miljø, der omfatter 5-98 % vand, hvor acyldonoren er et lipidsubstrat, der er udvalgt fra én eller flere af gruppen bestående af et phospholipid, et lysophospholipid, et triacylglycerid, et diglycerid, et glycolipid eller et lysoglycolipid, og acylacceptoren er et protein og/eller en proteinunderenhed; og etablering af kontakt mellem samblandingen og en lipid-acyltransferase, således at lipid-acyltransferasen katalyserer én eller begge af følgende reaktionsblandinger: alkoholyse eller transesterificering, hvor lipid-acyltransferasen, når den testes ved anvendelse af Transferase-assay i Buffersubstrat, har mindst 2 % acyltransferaseaktivitet; hvilket transferase-assay omfatter trinnene med: i) opløsning af 450 mg phosphatidylcholin og 50 mg cholesterol i chloroform, inddampning til tørhed under vakuum; ii) overføring af 300 mg cholesterol/phosphatidylcholinblanding til et Wheaton-glas, tilsætning af 15 ml 50 mM HEPES-buffer pH 7 og dispergering af væsken i bufferen under omrøring; iii) opvarmning af substratet til 35 °C under omrøring med en magnetisk omrører og tilsætning af 0,25 ml enzymopløsning; iv) udtagning afprøver på 2 ml ved 0, 5, 10, 15, 25, 40 og 60 minutters reaktionstid og umiddelbar standsning af enzymreaktionen ved tilsætning af 25 μΐ 4M HC1 til forsuring af den frie fedtsyre; v) tilsætning af 3 ml chloroform og kraftig rystning i 30 sekunder, centrifugering og isolering af 2 ml af chloroformfasen, filtrering gennem et 0,45 μηι filter i et 10 ml tareret Dram-glas; vi) inddampning af chloroformen under en strøm af nitrogen ved 60 °C og vejning af prøverne; vi i) analyse af det ekstraherede lipid ved hjælp af GLC.
2. Fremgangsmåde ifølge krav 1, hvor lipid-acyltransferasen er immobiliseret.
3. Fremgangsmåde ifølge krav 1 eller krav 2, hvor fremgangsmåden omfatter oprensning af proteinesteren og/eller esteren af proteinunderenheden.
4. Fremgangsmåde ifølge et hvilket som helst af kravene 1-3, hvor lipid-acyltransferasen er kendetegnet ved at være et enzym, der har acyltransferase-aktivitet, og som omfatter aminosyresekvensmotivet GDSX, hvor X er én eller flere af følgende aminosyrerester L, A, V, I, F, Y, H, Q, T, N, M eller S.
5. Fremgangsmåde ifølge krav 1, hvor lipid-acyltransferaseensymet omfatter en aminosyreskevens der er fremstillet ved ekspression af én eller flere af følgende nukleotidsekvenser: (a) nukleotidsekvens ifølge SEQ ID NO: 7 (se figur 9); (b) nukleotidsekvens ifølge SEQ ID NO: 8 (se figur 10); (c) nukleotidsekvens ifølge SEQ ID NO: 9 (se figur 11); (d) nukleotidsekvens ifølge SEQ ID NO: 10 (se figur 12); (e) nukleotidsekvens ifølge SEQ ID NO: 11 (se figur 13); (f) nukleotidsekvens ifølge SEQ ID NO: 13 (se figur 15); (g) nukleotidsekvens ifølge SEQ ID NO: 21 (se figur 17); (h) nukleotidsekvens ifølge SEQ ID NO: 23 (se figur 19); (i) nukleotidsekvens ifølge SEQ ID NO: 25 (se figur 21); (j) nukleotidsekvens ifølge SEQ ID NO: 27 (se figur 23); (k) nukleotidsekvens ifølge SEQ ID NO: 29 (se figur 25); (l) nukleotidsekvens ifølge SEQ ID NO: 31 (se figur 27); (m) nukleotidsekvens ifølge SEQ ID NO: 33 (se figur 29); (n) nukleotidsekvens ifølge SEQ ID NO: 35 (se figur 31); (o) eller en nukleotidsekvens, der er 75 % eller mere identisk med en hvilken som helst af sekvenserne ifølge SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO:
29, SEQ ID NO: 31, SEQ ID NO: 33 eller SEQ ID NO: 35.
6. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvor lipid-acyltransferasen er klassificeret som E.C. 2.3.1.x.
7. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvor lipid-acyltransferasen kan opnås fra en organisme fta én eller flere af følgende arter: Aeromonas, Streptomyces, Saccharomyces, Lactococcus, Mycobacterium, Streptococcus, Lactobacillus, Desulfitobacterium, Bacillus, Campylobacter, Vibrionaceae, Xylella, Sulfolobus, Aspergillus, Schizosaccharomyces, Listeria, Neisseria, Mesorhizobium, Ralstonia, Xanthomonas og Candida.
8. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvor lipid-acyltransferasen omfatter én eller flere af følgende aminosyresekvenser: (i) aminosyresekvensen ifølge SEQ ID NO: 2; (ii) aminosyresekvensen ifølge SEQ ID NO: 3; (iii) aminosyresekvensen ifølge SEQ ID NO: 4; (iv) aminosyresekvensen ifølge SED ID NO: 5; (v) aminosyresekvensen ifølge SEQ ID NO: 6; (vi) aminosyresekvensen ifølge SEQ ID NO: 12, (vii) aminosyresekvensen ifølge SEQ ID NO: 20, (viii) aminosyresekvensen ifølge SEQ ID NO: 22, (ix) aminosyresekvensen ifølge SEQ ID NO: 24, (x) aminosyresekvensen ifølge SEQ ID NO: 26, (xi) aminosyresekvensen ifølge SEQ ID NO: 28, (xii) aminosyresekvensen ifølge SEQ ID NO: 30, (xiii) aminosyresekvensen ifølge SEQ ID NO: 32, (xiv) aminosyresekvensen ifølge SEQ ID NO: 34, eller en aminosyresekvens, der er 75 % eller mere identisk med en hvilken som helst af sekvenserne ifølge SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:
24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 eller SEQ ID NO: 34.
9. Anvendelse af en lipid-acyltransferase til fremstilling af en proteinester og/eller en ester af en proteinunderenhed ved katalyse af én eller begge af alkoholyse eller transesterificering i en samblanding af en acyldonor, en acylacceptor og vand, hvilken samblanding omfatter 5-98 % vand, hvor acyldonoren er et lipidsubstrat udvalgt fra én eller flere af gruppen bestående af et phospholipid, et lysophospholipid, et triacylglycerid, et diglycerid, et glycolipid eller et lysoglycolipid, og acylacceptoren er et protein og/eller en proteinunderenhed, hvor lipid-acyltransferasen, når den testes ved anvendelse af et Transferase-assay i Buffersubstrat, har mindst 2 % acyltransferaseaktivitet; hvilket transferase-assay omfatter trinnene med: i) opløsning af 450 mg phosphatidylcholin og 50 mg cholesterol i chloroform, inddampning til tørhed under vakuum; ii) overføring af 300 mg cholesterol/phosphatidylcholinblanding til et Wheaton-glas, tilsætning af 15 ml 50 mM HEPES-buffer pH 7 og dispergering af væsken i bufferen under omrøring; iii) opvarmning af substratet til 35 °C under omrøring med en magnetisk omrører og tilsætning af 0,25 ml enzymopløsning; iv) udtagning afprøver på 2 ml ved 0, 5, 10, 15, 25, 40 og 60 minutters reaktionstid og umiddelbar standsning af enzymreaktionen ved tilsætning af 25 μΐ 4M HC1 til forsuring af den frie fedtsyre; v) tilsætning af 3 ml chloroform og kraftig rystning i 30 sekunder, centrifugering og isolering af 2 ml af chloroformfasen, filtrering gennem et 0,45 μηι filter i et 10 ml tareret Dram-glas; vi) inddampning af chloroformen under en strøm af nitrogen ved 60 °C, og vejning af prøverne; vii) analyse af det ekstraherede lipid ved hjælp af GLC.
10. Anvendelse ifølge krav 9, hvor lipid-acyltransferasen er immobiliseret.
11. Anvendelse ifølge krav 9, hvor proteinesteren og/eller esteren af proteinunderenheden er oprenset.
12. Anvendelse ifølge et hvilket som helst af kravene 9-11, hvor lipid-acyltransferasen er kendetegnet ved at være et enzym, der har acyltransferase-aktivitet, og som omfatter aminosyresekvensmotivet GDSX, hvor X er én eller flere af følgende aminosyrerester L, A, V, I, F, Y, H, Q, T, N, M eller S.
13. Anvendelse ifølge krav 9, hvor lipid-acyltransferaseenzymet omfatter en aminosyresekvens fremstillet ved ekspression af én eller flere af følgende nukleotidsekvenser: (a) nukleotidsekvens ifølge SEQ ID NO: 7 (se figur 9); (b) nukleotidsekvens ifølge SEQ ID NO: 8 (se figur 10); (c) nukleotidsekvens ifølge SEQ ID NO: 9 (se figur 11); (d) nukleotidsekvens ifølge SEQ ID NO: 10 (se figur 12); (e) nukleotidsekvens ifølge SEQ ID NO: 11 (se figur 13); (f) nukleotidsekvens ifølge SEQ ID NO: 13 (se figur 15); (g) nukleotidsekvens ifølge SEQ ID NO: 21 (se figur 17); (h) nukleotidsekvens ifølge SEQ ID NO: 23 (se figur 19); (i) nukleotidsekvens ifølge SEQ ID NO: 25 (se figur 21); (j) nukleotidsekvens ifølge SEQ ID NO: 27 (se figur 23); (k) nukleotidsekvens ifølge SEQ ID NO: 29 (se figur 25); (l) nukleotidsekvens ifølge SEQ ID NO: 31 (se figur 27); (m) nukleotidsekvens ifølge SEQ ID NO: 33 (se figur 29); (n) nukleotidsekvens ifølge SEQ ID NO: 35 (se figur 31); (o) eller en nukleotidsekvens, der er 75 % eller mere identisk med en hvilken som helst af sekvenserne ifølge SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO:
29, SEQ ID NO: 31, SEQ ID NO: 33 eller SEQ ID NO: 35.
14. Anvendelse ifølge et hvilket som helst af kravene 9 til 13, hvor lipid-acyltransferasen er klassificeret somE.C. 2.3.1.x
15. Anvendelse ifølge et hvilket som helst af kravene 9 til 14, hvor lipid-acyltransferasen kan opnås fra en organisme fra én eller flere af følgende arter: Aeromonas, Streptomyces, Saccharomyces, Lactococcus, Mycobacterium, Streptococcus, Lactobacillus, Desulfatobacterium, Bacillus, Campylobacter, Vibrionaceae, Xylella, Sulfolobus, Aspergillus, Schizosaccharomyces, Listeria, Neisseria, Mesorhizobium, Ralstonia, Xanthomonas og Candida.
16. Anvendelse ifølge et hvilket som helst af kravene 9-15, hvor lipid-acyltransferasen omfatter én eller flere af følgende aminosyresekvenser: (i) aminosyresekvensen ifølge SEQ ID NO: 2; (ii) aminosyresekvensen ifølge SEQ ID NO: 3; (iii) aminosyresekvensen ifølge SEQ ID NO: 4; (iv) aminosyresekvensen ifølge SEQ ID NO: 5; (v) aminosyresekvensen ifølge SEQ ID NO: 6; (vi) aminosyresekvensen ifølge SEQ ID NO: 12, (vii) aminosyresekvensen ifølge SEQ ID NO: 20, (viii) aminosyresekvensen ifølge SEQ ID NO: 22, (ix) aminosyresekvensen ifølge SEQ ID NO: 24, (x) aminosyresekvensen ifølge SEQ ID NO: 26, (xi) aminosyresekvensen ifølge SEQ ID NO: 28, (xii) aminosyresekvensen ifølge SEQ ID NO: 30, (xiii) aminosyresekvensen ifølge SEQ ID NO: 32, (xiv) aminosyresekvensen ifølge SEQ ID NO: 34, eller en aminosyresekvens, der er 75 % eller mere identisk med en hvilken som helst af sekvenserne ifølge SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:
24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 eller SEQ ID NO: 34.
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