反应详情

KREDs described in Table 5 of Example 1 are screened using NADH and NADPH as co-factors and glucose dehydrogenase/glucose or isopropylalcohol (“IPA”) as co-factor regeneration system. 100 μl of cell lysate was added to a deep well plate (Costar #3960) containing 25 μl 5 mg/ml Na-NADP (Oriental Yeast) and 2 mM MgSO4 in 100 mM triethanolamine(chloride) (pH7.0), and 125 μl isopropyl alcohol containing 2 g/L (S)-1-(4-Fluoro-phenyl)-5-(2-oxo-4-phenyl-oxazolidin-3-yl)-pentane-1,5-dione. After sealing the plates with aluminum/polypropylene laminate heat seal tape (Velocity 11 (Menlo Park, Calif.), Cat#06643-001), reactions were run for at least 16 hrs at ambient temperature. At the end of the reaction 1 ml acetonitrile (for reversed phase HPLC) or MTBE (for normal phase HPLC) was added per well. Plates were resealed, shaken for 20 minutes, and centrifuged (4000 rpm, 10 min, 4° C.). 200 ul of the organic layer was transferred into a new shallow-well microtiter plate for analysis.

1. addition100 μl of cell lysate was added to a deep well plate (Costar #3960)
2. temperatureheat
3. customseal tape (Velocity 11 (Menlo Park, Calif.), Cat#06643-001), reactions
4. waitwere run for at least 16 hrs at ambient temperature
5. additionwas added per well
6. customcentrifuged (4000 rpm, 10 min, 4° C.)