Acetate → acryloyl-CoA

HRID 2354518Notizen

Reaktionsgleichung

Reaktionsgleichung von HRID 2354518
[C:1]([S:4][CH2:5][CH2:6][NH:7][C:8](=[O:51])[CH2:9][CH2:10][NH:11][C:12](=[O:50])[C@H:13]([OH:49])[C:14]([CH3:48])([CH3:47])[CH2:15][O:16][P:17]([OH:46])(=[O:45])[O:18][P:19]([OH:44])(=[O:43])[O:20][CH2:21][C@H:22]1[O:26][C@@H:25]([N:27]2[C:36]3[N:35]=[CH:34][N:33]=[C:31]([NH2:32])[C:30]=3[N:29]=[CH:28]2)[C@H:24]([OH:37])[C@@H:23]1[O:38][P:39]([OH:42])([OH:41])=[O:40])(=[O:3])[CH3:2].[C:52]([O-])(=O)C>>[C:1]([S:4][CH2:5][CH2:6][NH:7][C:8](=[O:51])[CH2:9][CH2:10][NH:11][C:12](=[O:50])[C@H:13]([OH:49])[C:14]([CH3:47])([CH3:48])[CH2:15][O:16][P:17]([OH:46])(=[O:45])[O:18][P:19]([OH:44])(=[O:43])[O:20][CH2:21][C@H:22]1[O:26][C@@H:25]([N:27]2[C:36]3[N:35]=[CH:34][N:33]=[C:31]([NH2:32])[C:30]=3[N:29]=[CH:28]2)[C@H:24]([OH:37])[C@@H:23]1[O:38][P:39]([OH:42])([OH:41])=[O:40])(=[O:3])[CH:2]=[CH2:52]

Komponenten

3

Edukt 2

HCID 175AcetateHCID 444493acetyl CoA

Produkt 1

HCID 9543026acryloyl-CoA

Versuchsvorschrift

S-acetyl CoA synthetase (acetate thiokinase from bakers yeast, Sigma, Saint Louis, Mo., USA; catalogue No. 1765) (1.9 mg/ml) is added. Using the fed-batch approach meaning step-wise addition (feeding) of H-CoA, 15 μl aliquots of H-CoA are added after 5, 10, 15 and 20 minutes, raising the H-CoA concentration by 1 mM each time, and a further 5 mM ATP is added after 25 minutes. Samples are analysed for H-CoA by HPLC (Instrumentation: Agilent Technologies HP1090L with HP1100 Variable Wavelength UV Detector and Chemstation (rev. A.06.01) Data System; column: Luna C18 from Phenomenex, 25 cm×4.6 mm inner diameter, bead diameter 5 μm (No. 92); mobile phase: A: 25 mM ammonium formate (pH 7.0), B: methanol; gradient 5% B for 0 min, then to 30% B in 20 min; flow 1 ml/min (approximately 130 bar), oven temperature 40° C., Detector: UV at 210 nm; injection volume 5 μl, Run time 28 min; peaks are found at the following retention times: ATP approximately 3.710 min, Coenzyme A at approximately 10.554 min, acetyl CoA at approximately 14.457 min, acrylyl CoA at approximately 17.500 min, bisadduct at approximately 15.993 min). When no H-CoA is detected, further 15 μl aliquots of H-CoA are added after 80, 95, 110 and 120 minutes, again raising the H-CoA concentration by 1 mM each time. Further 15 μl aliquots of H-CoA are added after 145 and 155 minutes and sufficient ATP to raise the concentration by 5 mM is added after 150 minutes.

Aufarbeitung

  1. addition1765) (1.9 mg/ml) is added
  2. additionare added after 5, 10, 15 and 20 minutes
  3. temperatureraising
  4. concentrationthe H-CoA concentration by 1 mM each time
  5. additiona further 5 mM ATP is added after 25 minutes
  6. waitto 30% B in 20 min
  7. customflow 1 ml/min (approximately 130 bar), oven temperature 40° C., Detector
  8. customtime 28 min
  9. waitATP approximately 3.710 min, Coenzyme A at approximately 10.554 min
  10. waitacetyl CoA at approximately 14.457 min
  11. waitacrylyl CoA at approximately 17.500 min
  12. waitbisadduct at approximately 15.993 min)
  13. additionfurther 15 μl aliquots of H-CoA are added after 80, 95, 110 and 120 minutes
  14. temperatureagain raising
  15. concentrationthe H-CoA concentration by 1 mM each time
  16. additionFurther 15 μl aliquots of H-CoA are added after 145 and 155 minutes and sufficient ATP
  17. temperatureto raise
  18. concentrationthe concentration by 5 mM
  19. additionis added after 150 minutes

Quellen und Nachweise

ORD-Eintrag
ord-c4e60a15534d41d5931aeb64a309d73b
Quelldatensatz
uspto-grants-2010_07
Patentnummer
US07754455B2

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