반응 #348330
ord-deb97bd49c524110a64ea3ae885506b7
반응 방정식
반응물
반응 조건
후처리
- 1농축Concentrated enzyme (0.5 ml)
- 2기타was incubated at 28°
- 3추출The supernatant was extracted with ethylacetate (2.5 ml)
- 4기타The organic phase was collected
- 5기타evaporated to dryness
- 6기타The cephalosporins it contained were then separated by hplc [100 mm×5 diam. column Partisil 5 eluted at 1500 psi with chloroform:methanol:90% formic acid (25:2:1 v/v) saturated with water]
- 7기타were detected by their absorption at 270 nm
실험 절차
Concentrated enzyme (0.5 ml) was incubated at 28° in a final volume of 1.0 ml water containing (6R,7R)-3-hydroxymethyl-7-[2-methoxyimino-2-(fur-2-yl) acetamido]ceph-3-em-4-carboxylic acid (syn-isomer) (0,1 μmole), carbamoyl phosphate (3 μmoles), ATP (1 μmole), magnesium chloride (1 μmole), manganese chloride (1 μmole) & pipes buffer (50 μmoles) adjusted to pH 6.0 with potassium hydroxide. After 2 hr the reaction was stopped by the addition of acetic acid (20 μl). The pH was then adjusted to 2.0 with M hydrochloric acid and the precipitated protein was centrifuged off. The supernatant was extracted with ethylacetate (2.5 ml). The organic phase was collected and evaporated to dryness. The cephalosporins it contained were then separated by hplc [100 mm×5 diam. column Partisil 5 eluted at 1500 psi with chloroform:methanol:90% formic acid (25:2:1 v/v) saturated with water] and were detected by their absorption at 270 nm. Their retention time was compared with standard samples. The size of the U.V. absorbing peaks showed that 20% of the cephalosporin starting material had been converted to title compound.