Inulin hydrolysis and citric acid production from inulin using the surface-engineered Yarrowia lipolytica displaying inulinase.

The INU1 gene encoding exo-inulinase cloned from Kluyveromyces marxianus CBS 6556 was ligated into the surface display plasmid and expressed in the cells of the marine-derived yeast Yarrowia lipolytica which can produce citric acid. The expressed inulinase was immobilized on the yeast cells. The activity of the immobilized inulinase with 6 x His tag was found to be 22.6 U mg(-1) of cell dry weight after cell growth for 96 h. The optimal pH and temperature of the displayed inulinase were 4.5 and 50 degrees C, respectively and the inulinase was stable in the pH range of 3-8 and in the temperature range of 0-50 degrees C. During the inulin hydrolysis, the optimal inulin concentration was 12.0% and the optimal amount of added inulinase was 181.6 U g(-1) of inulin. Under such conditions, over 77.9% of inulin was hydrolyzed within 10h and the hydrolysate contained main monosaccharides and disaccharides, and minor trisaccharides. During the citric acid production in the flask level, the recombinant yeast could produce 77.9 g L(-1) citric acid and 5.3 g L(-1) iso-citric acid from inulin while 68.9 g L(-1) of citric acid and 4.1 g L(-1) iso-citric acid in the fermented medium were attained within 312 h of the 2-L fermentation, respectively.