Method for preparing rebaudioside A from stevioside
The present invention relates to an in-situ method for preparing rebaudioside A from sucrose and stevioside by sucrose synthase and glycosyltransferase.
1. A method for preparing rebaudioside A from stevioside, comprising performing in a single reaction system the following two reactions:
(1) reacting sucrose and nucleotide diphosphate in the presence of a sucrose synthase to prepare nucleotide diphosphate to which glucose is bonded, and
(2) reacting the nucleotide diphosphate to which glucose is bonded with stevioside in the presence of a glycosyltransferase to prepare rebaudioside A,
wherein the sucrose synthase comprises the sequence of SEQ ID NO:3,
wherein the glycosyltransferase comprises the sequence of SEQ ID NO:4, and
wherein the reaction system pH is in the range of pH 5.5 to 11.0.
2. The method for preparing rebaudioside A from stevioside according to claim 1 , wherein the reaction system temperature is 20° C. to 60° C., and the reaction system pH is in the range of pH 7 o pH 8.
3. The method for preparing rebaudioside A from stevioside according to claim 1 , wherein the sucrose synthase and the glycosyltransferase are independently produced from recombinant Escherichia coli, Bacillus , yeast, Corynebacterium or Agrobacterium.
4. The method for preparing rebaudioside A from stevioside according to claim 3 , wherein the sucrose synthase and the glycosyltransferase are purified sucrose synthase and purified glycosyltransferase, respectively.