Study Of Biological Catalysis Of Rebaudioside A To Produce Rebaudioside D

Steviol glycosides?SGs?are natural sweetening agents found in the leaves of S.rebaudiana and can be widely used in food and pharmaceutical industries due to their low-calorie and high sweetness.SGs are a group of highly sweet diterpene glycosides.More than 100 species of SGs have been discovered in the leaves of S.rebaudiana.Taking into account the sweetness and aftertaste,rebaudioside D?RD?exhibits the best properties.Base on the current research progress,we aim to cloning and expression of the glycosyltransferase gene in host strains,to realize the biological production of RD from rebaudioside A?RA?by adding a glucosyl residue in the position of C₁₉in rebaudioside A.First,a specific glycosyltransferase gene was selected and synthesized with codon optimized sequence.It was ligased to the pESC and pVTU plasmids,respectively and the recombinant expression vectors pESC-LEU-EUGT11 and pVTU26-EUGT11 were then transformed into Saccharomyces cerevisiae W303-1A and Saccharomyces cerevisiae BY4741 host strains.The proteins were successfully expressed in the yeast strains.The EUGT11 gene was later inserted into the plasmid of pET-28a?+?and the recombinant expression plasmid pET-28a?+?-EUGT11 was transformed into E.coli BL21?DE3?Codon Plus-RIL competent cell.The gene was successfully expressed and an expressed protein with 51 kDa size was obtained.The enzymatic catalytic conditions were studied:the optimum working pH was 7.0,the optimum reaction temperature was 35?,and the optimum proportion of substrate concentration is UDPG:RA=2:1?c/c?.The enzyme activity was inhibitied by adding metal ions and EDTA.With above optimized condition,the yield of RA to RD reached 87.36%in5 hours reaction with 60 g/L cells and 0.25 g/L RA as substrate and the EUGT11 activity reached 62.7 U/g...