反应详情

(R)-3-hydroxyoctanoyl-CoA was synthesized according to Rehm B H A, Kruger N, Steinbuchel A (1998) Journal of Biological Chemistry 273 pp 24044-24051 with certain changes. Acyl-CoA synthetase (Sigma Co.) was dissolved in trishydrochloric acid buffer (50 mM, pH 7.5) containing 2 mM ATP, 5 mM MgCl2, 2 mM coenzyme A and 2 mM (R)-3-hydroxyoctanoate to a concentration of 0.1 mU/μL. It was retained in a bath of 37° C. and was sampled suitably and the proceeding of reaction was analyzed by HPLC. After the enzyme reaction was terminated by adding sulfuric acid to 0.02 N to the sampled reaction liquid, the unreacted substrate (R)-3-hydroxyoctganoate was removed by extraction with n-heptane. The HPLC analysis employed RP18 column (nucleosil C18, 7 μm, Knauser), and elusion was made under a linear concentration slope of acetonitrile, utilizing 25 mM phosphoric acid buffer (pH 5.3) as the moving phase, and the thioester compound generated by the enzymatic reaction was detected by monitoring the absorption spectrum of 200 to 500 nm with a diode array detector. (R)-3-hydroxy-5-phenylvaryl CoA and (R)-3-hydroxy-5-(4-fluorophenyl)valeryl CoA were also synthesized in a similar manner.

1. customIt was retained in a bath of 37° C.
2. aliquotwas sampled suitably
3. customthe proceeding of reaction
4. customAfter the enzyme reaction
5. customwas terminated
6. additionby adding sulfuric acid to 0.02 N
7. customto the sampled reaction liquid
8. customthe unreacted substrate (R)-3-hydroxyoctganoate was removed by extraction with n-heptane
9. concentrationwas made under a linear concentration slope of acetonitrile
10. customthe thioester compound generated by the enzymatic reaction