反应详情

(R)-3-hydroxyoctanoyl-CoA was synthesized in accordance with the following procedure, based on the method of Rehm BHA, Kruger N, Steinbuchel A (1998) Journal of Biological Chemistry 273 pp 24044–24051, with the method slightly modified. Acyl-CoA synthetase (manufactured by Sigma Co., Ltd.) was dissolved in a tris hydrochloric buffer solution (50 mM, pH 7.5) containing 2 mM ATP, 5 mM MgCl2, 2 mM CoA and 2 mM (R)-3-hydroxyoctanoate so that the concentration was 0.1 milliunit per microliter. The solution was stored in a warm bath at 37° C., and was sampled at appropriate times to analyze the progress of the reaction by HPLC. Sulfuric acid was added in the sampled reaction solution to make a concentration 0.02 N to stop the enzyme reaction, and thereafter (R)-3-hydroxyoctanoate being an unreacted substrate was extracted with n-heptane and removed. For the analysis by HPLC, using a RP18 column (nucleosil C18, 7 μm, Knauser), elution was conducted with the linear concentration gradient of acetonitrile using a 25 mM phosphate buffer solution (pH 5.3) as a mobile phase, and absorption spectra of 200 to 500 nm were monitored by a diode array detector, thereby detecting a thioester compound produced through the enzyme reaction. In a similar way, (R)-3-hydroxy-5-phenylvaleryl CoA, and (R)-3-hydroxy-5-(4-fluorophenyl)valeryl CoA were prepared.

1. concentrationso that the concentration
2. customThe solution was stored in a warm bath at 37° C.
3. aliquotwas sampled at appropriate times
4. customthe progress of the reaction by HPLC
5. customthe enzyme reaction
6. extraction(R)-3-hydroxyoctanoate being an unreacted substrate was extracted with n-heptane
7. customremoved
8. washa RP18 column (nucleosil C18, 7 μm, Knauser), elution
9. concentrationwas conducted with the linear concentration gradient of acetonitrile using a 25 mM phosphate buffer solution (pH 5.3)
10. customas a mobile phase, and absorption spectra of 200 to 500 nm
11. customproduced through the enzyme reaction