Expression and characterization of surface-displayed sucrose:sucrose 1-fructosyltransferase on Yarrowia lipolytica cells

Abstract:

[Objective] Sucrose:sucrose 1-fructosyltransferase (1-SST) catalyzes the transfer of a fructosyl residue from one sucrose to another sucrose, forming 1-kestose and glucose. 1-Kestose has the highest prebiotic activity among fructooligosaccharides. In this study, 1-SST displayed on the cell-surface of Yarrowia lipolytica was used to prepare 1-kestose. [Methods] 1-SST gene from Lactuca sativa was cloned into the surface-display vector and expressed in the cells of Y. lipolytica. Biochemical characteristics of the displayed 1-SST were investigated with sucrose as its substrate. [Results] Immunofluorescence microscopy assay and high performance liquid chromatography (HPLC) indicated that the expressed protein was displayed on the cell-surface of Y. lipolytica and possessed 1-SST activity. The displayed 1-SST showed the highest activity at 45 °C and pH 7.5. The activity of the displayed 1-SST was inhibited by Zn2+ and Cu2+, while stimulated by Ca2+. The enzyme activity reduced 50% of initial activity after the displayed 1-SST was repeatedly used for seven times. The highest content of 1-kestose reached 20.8 mmol/L after the reaction mixture containing the displayed 1-SST and 3% sucrose was incubated at 40 °C for 30 min. [Conclusion] 1-SST was successfully expressed and displayed on the cells of Y. lipolytica, and the fructosyltransferase activity was detected. The surface-displayed 1-SST as a whole-cell catalyst can be applied to the 1-kestose preparation.