2.6 Coupling reactions
To explore the application of Mc SuSy, we established SuSy-GT reactions to catalyze PPD to produce Rh2. The reaction mixtures (5 mL) contained 6 or 8 mM PPD, 200 mM sucrose, potassium phosphate buffer (50 mM, pH 7.0), and 8 mg/mL of total protein from the crude extract prepared from E. coliBL21 (pRSF-UGT51m-Mc SuSym) or E. coli BL21 (pRSF-UGT51m-At SuSy1). Expressions of two recombinant enzymes were under the same conditions as SuSys, except for the induction for 36 h. The reaction was incubated at 37 °C and 200 rpm for 3 h. The GT activity of UGT51m was measured at 37 °C in 0.5 mL reaction mixture containing 0.1 mg of total protein from crude extract, 2 mM PPD, 2 mM UDPG, 2% Tween 80 (v/v), 10% DMSO and 50 mM potassium phosphate buffer (pH7.0). Reactions were terminated by heating for 10 min at 95°C and diluted with methanol. One unit (U) of GT activity was defined as the amount of enzyme that produced 1 μmol of Rh2 from PPD. The concentrations of PPD and Rh2 were determined by UltiMate 3000 using an Agilent C18 column (250x4.6 mm) at UV 203 nm. The flow rate was 1.0 mL/min, and the column temperature was set at 30°C. The mobile phase consisted of water with 0.1% phosphoric acid (A) and acetonitrile (B), and a gradient program of 70–95% B in 0–25 min was applied. The crude enzyme activities of SuSys were measured as described in Supporting Information.