2.4 In vitro enzymatic reactions
The yeast strains CPR-CYP5139G1 and CPR-EV (Table S3) were cultivated
for microsome isolation. Microsomal isolation was performed as
previously described (Wang et al., 2018). The enzymatic assay was
conducted in a total volume of 500 μL of 90 mM Tris-HCl (pH 7.5)
containing 500 μg microsomal protein and 2 mM NADPH; and 100 µM of a
substrate - 3-hydroxy-lanosta-8,24-dien-26-ol (HLDO),
3-hydroxy-lanosta-8,24-dien-26-al (HLDA), or HLDOA was added,
respectively. After incubation at 30 °C for 4 h, the product was
extracted by ethyl acetate. The ethyl acetate layer was collected,
evaporated, and redissolved in methanol for LC-MS analysis.