Biochemical and structural characterization of non‐glycosylated Yarrowia lipolytica LIP2 lipase

Abstract

AbstractThe LIP2 lipase from the yeast Yarrowia lipolytica (YLLIP2) is assumed to be a good drug candidate for enzyme replacement therapy in patients with pancreatic exocrine insufficiency. Understanding and improving its biochemical properties are essential for its oral administration. YLLIP2 is a highly glycosylated protein, with glycan chains accounting for about 13% of the molecular mass of the native protein. Two potential N‐glycosylation sites (N113IS and N134NT) can be identified from YLLIP2 amino acid sequence. YLLIP2 mutants with single (N113Q or N134Q) or combined (N113Q/N134Q) substitutions of these glycosylation sites were expressed in the yeast Pichia pastoris, purified and characterized. Lipase specific activity and adsorption at the lipid–water interface were found to be decreased in the absence of N‐glycosylation. It was thus shown that the glycosylated enzyme had a better ability to bind and penetrate a DLPC monolayer than the non‐glycosylated N113Q/N134Q mutant. Comparison of wild‐type glycosylated and non‐glycosylated YLLIP2 shows that the N‐glycosylation clearly contributes to the high stability of YLLIP2 in the presence of pepsin in vitro, and to a lower extent in the presence of chymotrypsin. The X‐ray structure of the YLLIP2 N113Q/N134Q double mutant was obtained at 2.6 Å resolution and was found to be identical to that of wild‐type YLLIP2, with the lid in a closed conformation. Glycosylation is therefore not essential for a proper folding of YLLIP2.Practical applications: The LIP2 lipase from the yeast Yarrowia lipolytica is one of the most active lipases identified so far. Among the various applications envisioned for this enzyme, it seems particularly well adapted for enzyme replacement therapy in patients with pancreatic exocrine insufficiency. It is active and stable at low pH values, resistant to bile salts, and its glycosylation allows a high resistance to pepsin. All these properties are important for developing the oral administration of digestive enzymes used as drugs.