2.4. Kaempferol Conversion Using BmTy at Various pHs

In 1 mL reaction volume, 0.2 mM kaempferol, 1% (w/v) PVP 40k, 1% (v/v) DMSO, 3 mM L-ascorbic acid, 2 μM CuSO₄ and 50 mU/mL BmTy (427, 216, 133 and 181 nM enzyme at pH 5, 6, 7 and 8, respectively, Table 1) were added into 50 mM buffer (pH 5.0~7.0: citrate-phosphate buffer, pH 8.0: Tris-HCl buffer) as final concentrations. Aliquots of 100 μL were vortexed with 400 μL EA at specific sampling points in time, and 200 μL of EA parts were retrieved and dried. The residues were dissolved with 100 μL MeOH and analyzed with HPLC.