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SUMMARY BackgroundVarious pancreatic enzyme preparations are used for the treatment of pancreatic insufficiency but their bioequivalence is often unknown.

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The 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. enzyme replacement therapy; RML, Rhizomucor miehei lipase; TAG, triglycerides; TLL, Thermomyces lanuginosus lipase; YLLIP2, LIP2 lipase from the yeast Yarrowia lipolytica; YPD, yeast-peptone-glucose culture medium Eur.

The LIP2 lipase from the yeast Y. lipolytica (YLLIP2) was obtained from two genetically modified strains with multi‐copies of the lip2 gene and further purified using gel filtration and cation exchange chromatography. Four YLLIP2 isoforms were identified and subjected to N‐terminal amino‐acid sequencing and mass spectrometry analysis. These isoforms differed in their glycosylation patterns and their molecular masses ranged from 36,874 to 38,481 Da, whereas the polypeptide mass was 33,385 Da. YLLIP2 substrate specificity was investigated using short, medium and long chain triglyceride (TG) at various pH and bile salt (BS) concentrations, and compared with those of human digestive lipases. YLLIP2 was not inhibited by bile salts with any of the substrate tested, and maximum specific activities were found to be 10,760 IU/mg on tributyrin, 16,920 IU/mg on trioctanoin and 12,260 IU/mg on olive oil at pH 6.0. YLLIP2 possesses the same pattern of regioselectivity as human pancreatic lipase, generating 2‐MG and free fatty acids, the lipolysis products absorbed at the intestinal level. YLLIP2 was found to be fairly stable and still active on long chain TG (1590 IU/mg) at pH 4.0, in the presence of BS. YLLIP2 is therefore tailored to act optimally under the physiological conditions pertaining in the gastro‐intestinal tract, and might be an ideal candidate for enzyme replacement therapy for pancreatic exocrine insufficiency.

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