Anchorage of cyclodextrin glycosyltransferase on outer membrane of Saccharomyces cerevisiae to produce 2-O-α-D-glucopyranosyl-L-ascorbic acid

Abstract:

Abstract:［Objective］ Displaying cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans 251 on the cell surface of Saccharomyces cerevisiae to improve 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) production.［Methods］CGTase encoding gene cgt was inserted into the 3' terminal of Aga2p of vector pYD1 and the obtained recombinant plasmid pYD1-cgt was then transformed into S.cerevisiae EBY100 to produce surface displayed CGTase and culture conditions (culture medium，inductive temperature and concentration of inducer galactose) were optimized． Moreover，resulted CGTase displayed on the yeast cell surface was used for the AA-2G biosynthesis under the optimized condition.［Results］ CGTase activity on the cell surface of recombinant yeast，S.cerevisiae EBY100-pYD1-cgt，reached 0.5 U/ml in 48 h fermentation using Yeast Peptone Galactose culture medium with 20% galactose as sole carbon source and inducer at 25 ℃．The displayed CGTase exhibited better thermostability and pH stability than that of free CGTase．The concentration of AA-2G produced by the surface displayed CGTase was 37% higher than that produced by free CGTase at its optimal transformation conditions of 30 ℃ and pH4.5．［Conclusion］The cell surface display system based on a-agglutinin is an effective system for displaying CGTase． During AA-2G production by surface displayed CGTase，the by-product glucose might be consumed by yeast cell and thus facilitated AA-2G production． The whole cell EBY100-pYD1-cgt will have better prospects for applications．