Efficient enzymatic production of rebaudioside A from stevioside

Figures & data

Fig. 1. Concept of the two-enzyme system for efficient synthesis of rebaudioside A from stevioside.

Notes: UGT76G1, UDP-glucosyltransferase from Stevia rebaudiana; AtSUS1, sucrose synthase from Arabidopsis thaliana.

Fig. 2. pEUGT-SUS. The modified genes encoding UGT76G1 and AtSUS1 were subcloned into the expression vector pET-28a (+), resulting in pEUGT-SUS.

Fig. 3. HPLC showing the peaks of stevioside and rebaudioside A detected in the glucosyltransferase activity assay.

Notes: The samples were taken immediately after reaction (a) or for 2 h (b).

Fig. 4. HPLC-MS analysis of rebaudioside A standard (a) and the sample prepared for the glucosyltransferase activity assay (b).

Fig. 5. Sucrose effects in the two-enzyme (UGT76G1 and AtSUS1) coupling reaction system.

Notes: About 2 mU/mL enzymes from the crude extract were incubated with 0.6 mM stevioside, 0.12 mM UDP-glucose, and 0.6 mM sucrose or without sucrose.

Fig. 6. Effect of the initial UDP-glucose concentrations on enzymatic production of rebaudioside A.

Notes: About 2 mU/mL enzymes from the crude extract were incubated with 0.6 mM stevioside, 0.6 mM sucrose and UDP-glucose.

Fig. 7. Effect of the initial UDP concentrations on enzymatic production of rebaudioside A.

Notes: About 2 mU/mL enzymes from the crude extract were incubated with 0.6 mM stevioside, 0.6 mM sucrose and UDP.

Fig. 8. Effect of sucrose concentrations on enzymatic production of rebaudioside A.

Notes: 19.8 mU/mL enzymes from the crude extract were incubated with 0.6 mM stevioside, 0.006 mM UDP and sucrose. The mole ratios of sucrose to stevioside varied from 1:1 to 4:1.

Fig. 9. Effect of stevioside concentrations on enzymatic production of rebaudioside A.

Notes: About 15 mU/mL enzymes from the crude extract were incubated with stevioside, UDP (0.006 mM) and sucrose. The mole ratios of sucrose to stevioside were fixed at 3:1.