Yarrowia lipolytica Lipase 2 Is Stable and Highly Active in Test Meals and Increases Fat Absorption in an Animal Model of Pancreatic Exocrine Insufficiency.
OBJECTIVE
Pancreatic exocrine insufficiency (PEI) reduces pancreatic secretion of digestive enzymes, including lipases. Oral pancreatic enzyme replacement therapy (PERT) with pancreatin produces unsatisfactory results. The lipase 2 produced by the yeast Yarrowia lipolytica (YLLIP2; GenBank: AJ012632) might be used in PERT. We investigated its ability to digest triglycerides in a test meal and its efficacy in reducing fecal fat in an animal model of PEI.


METHODS
YLLIP2 was produced by genetically engineered Y lipolytica and purified from culture media. YLLIP2 or other gastric (LIPF) and pancreatic (PNLIPD) lipases were added to a meal paste containing dietary triglycerides, at a range of pH values (pH 2-7), with and without pepsin or human bile and incubated at 37°C. We collected samples at various time points and measured lipase activities and stabilities. To create an animal model of PEI, steatorrhea was induced by embolization of the exocrine pancreas gland and pancreatic duct ligation in minipigs. The animals were given YLLIP2 (1, 4, 8, 40, or 80 mg/d) or pancreatin (100,000 US Pharmacopeia lipase units/d, controls) for 9 days. We then collected stool samples, measured fat levels, and calculated coefficient of fat absorption (CFA) values.


RESULTS
YLLIP2 was highly stable and poorly degraded by pepsin, and had the highest activity of all lipases tested on meal triglyceride at pH 4-7 (pH 6 with bile: 94 ± 34 U/mg; pH 4 without bile: 43 ± 13 U/mg). Only gastric lipase was active and stable at pH 3, whereas YLLIP2 was sensitive to pepsin hydrolysis after pH inactivation. From in vitro test meal experiments, the lipase activity of YLLIP2 (10 mg) was estimated to be equivalent to that of pancreatin (1200 mg; 100,000 US Pharmacopeia units) at pH 6. In PEI minipigs, CFA values increased from 60.1% ± 9.3% before surgery to 90.5% ± 3.2% after administration of 1200 mg pancreatin (P < .05); CFA values increased to a range of 84.6% ± 3.0% to 90.0% ± 3.8% after administration of 4-80 mg YLLIP2 (P < .05).


CONCLUSIONS
The yeast lipase YLLIP2 is stable and has high levels of activity against test meal triglycerides in a large pH range, with and without bile. Oral administration of milligram amounts of YLLIP2 significantly increased CFA values, similar to that of 1.2 g pancreatin, in a minipig model of PEI.
CNRS-Aix-Marseille Université-UMR7282 Enzymology at Interfaces and Physiology of Lipolysis, Marseille, France; Laboratoires Mayoly Spindler SAS, Chatou, France.
Ahmed
A
Aloulou
CNRS-Aix-Marseille Université-UMR7282 Enzymology at Interfaces and Physiology of Lipolysis, Marseille, France; Proteabio Europe SAS, Langlade, France.
Mathieu
M
Schué
Laboratoires Mayoly Spindler SAS, Chatou, France.
Delphine
D
Puccinelli
Wil Research Europe-Lyon, Saint-Germain Nuelle, France.
Stéphane
S
Milano
Laboratoires Mayoly Spindler SAS, Chatou, France.
Chantal
C
Delchambre
Laboratoires Mayoly Spindler SAS, Chatou, France.
Yves
Y
Leblond
CNRS-Aix-Marseille Université-UMR7282 Enzymology at Interfaces and Physiology of Lipolysis, Marseille, France; AP-HM, Gastroenterology Department, La Timone Hospital, 13005 Marseille, France.
René
R
Laugier
CNRS-Aix-Marseille Université-UMR7282 Enzymology at Interfaces and Physiology of Lipolysis, Marseille, France. Electronic address: carriere@imm.cnrs.fr.
Frédéric
F
Carrière
D000284:Administration, Oral; D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D004195:Disease Models, Animal; D004285:Dogs; D056947:Enzyme Replacement Therapy; D004795:Enzyme Stability; D010188:Exocrine Pancreatic Insufficiency / Q000188:drug therapy* / Q000201:enzymology; D005243:Feces / Q000737:chemistry; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007408:Intestinal Absorption / Q000187:drug effects*; D008049:Lipase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D008066:Lipolysis / Q000187:drug effects*; D010194:Pancreatin / Q000494:pharmacology; D010434:Pepsin A / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D013552:Swine; D013556:Swine, Miniature; D013997:Time Factors; D014280:Triglycerides / Q000008:administration & dosage / Q000378:metabolism*; D025062:Yarrowia / Q000201:enzymology* / Q000235:genetics
D005656:Fungal Proteins; D011994:Recombinant Proteins; D014280:Triglycerides; D010194:Pancreatin; D002265:Carboxylic Ester Hydrolases; D008049:Lipase; C000606101:YLLIP2 protein, Yarrowia lipolytica; D010434:Pepsin A
Chronic Pancreatitis; Enzyme Therapy; Fat Digestion; Lipolysis