Cell Surface Display of Yarrowia lipolytica Lipase Lip2p Using the Cell Wall Protein YlPir1p, Its Characterization, and Application as a Whole-Cell Biocatalyst.

Yuzbasheva, Evgeniya; Yuzbashev, Tigran; Perkovskaya, Natalia; Mostova, Elizaveta; Vybornaya, Tatiana; Sukhozhenko, Aleksei; Toropygin, Ilya; Sineoky, Sergey

The Yarrowia lipolytica lipase Lip2p was displayed on the yeast cell surface via N-terminal fusion variant using cell wall protein YlPir1p. The hydrolytic activity of the lipase displayed on Y. lipolytica cells reached 11,900 U/g of dry weight. However, leakage of enzyme from the cell wall was observed. The calculated number of recombinant enzyme displayed on the cell surface corresponds to approximately 6 × 10 molecules per cell, which is close to the theoretical maximum (2 × 10 molecules/cell). Furthermore, the leaking enzyme was presented as three N-glycosylated proteins, one of which corresponds to the whole hybrid protein. Thus, we attribute the enzyme leakage to the limited space available on the cell surface. Nevertheless, the surface-displayed lipase exhibited greater stability to short-term and long-term temperature treatment than the native enzyme. Cell-bound lipase retained 74 % of its original activity at 60 °C for 5 min of incubation, and 83 % of original activity after incubation at 50 °C during 5 h. Cell-bound lipase had also higher stability in organic solvents and detergents. The developed whole-cell biocatalyst was used for recycling biodiesel synthesis. Two repeated cycles of methanolysis yielded 84.1 and 71.0 % methyl esters after 33- and 45-h reactions, respectively.

LIPASES; YEAST; FUNGAL cell walls; ENZYMES; PROTEINS; ORGANIC solvents; DETERGENTS

Applied Biochemistry & Biotechnology, 2015, Vol 175, Issue 8, p3888

0273-2289

Academic Journal

10.1007/s12010-015-1557-7