3.5. Active Assay of Glycosyltransferase by HPLC

The glycosyltransferase activity of the recombinant UGT76G1 was evaluated by the conversion rate from the substrate to the goal product. The reaction mixture with a total volume of 200 μL contained 40 μL soluble glycosyltransferase samples, 20 μL of rebaudioside A (100 mM) or rebaudioside D (20 mM), 20 μL of UDP-glucose (60 mM), and 120 μL of sodium phosphate buffer (50 mM, pH 7.0). Reactions were performed at 35 °C with 220 rpm shaking for 3–24 h. The reaction was terminated by adding 160 μL of methanol solution (60%, v/v) and 16 μL of 2 N sulfuric acid solution. The mixed solution was then centrifuged at 13,800× g for 10 min and the supernatant was carried out by membrane filtration for product analysis by high performance liquid chromatography (HPLC). The steviol glycosides (SGs) concentration was determined using an Innoval C18 ODS-2 column (250 mm × 4.6 mm, Bonna-Agela Technologies, Tianjin, China) maintained at 40 °C with UV detection at 210 nm in a Waters e2695 HPLC system. The mobile phase was consistent of 68% acetonitrile and 32% water and the flow rate was 0.5 mL/min, the injection volume was 10 µL. St (50%, mixed with 50% RebA), Reb A (50%, mixed with 50% St) and Reb D standard (97.6%) for HPLC was purchased from Shanghai PureOne Biotechnology Co., Ltd. (Shanghai, China). The conversion rate (%) indicated the yield of target products which were calculated as follows:

where A0 (St) and A0 (Reb D) represent the initial concentration of the substrates St and Reb D respectively, and A1 (Reb A) and At (Reb M) respectively represent the increased concentration of terminal products Reb A and Reb M after reaction.