huagongshe.com
HRID2354518

反应详情

EQUATION

反应方程式

HRID 2354518 的结构方程式

REACTANTS

反应物

2
结构：CC(=O)[O-]
HCID175

Acetate

C2H3O2-

结构：CC(=O)SCCNC(=O)CCNC(=O)[C@H](O)C(C)(C)COP(=O)(O)OP(=O)(O)OC[C@H]1O[C@@H](n2cnc3c(N)ncnc32)[C@H](O)[C@@H]1OP(=O)(O)O
HCID444493

Acetyl CoA

C23H38N7O17P3S

PRODUCTS

生成物

1
结构：C=CC(=O)SCCNC(=O)CCNC(=O)[C@H](O)C(C)(C)COP(=O)(O)OP(=O)(O)OC[C@H]1O[C@@H](n2cnc3c(N)ncnc32)[C@H](O)[C@@H]1OP(=O)(O)O
HCID9543026

未命名化合物

PROCEDURE

实验过程

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.

WORKUP

后处理

  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