Purification and biochemical characterization of the LIP2 lipase from Yarrowia lipolytica.
The LIP2 lipase from the yeast Yarrowia 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 (tributyrin), medium (trioctanoin) and long (olive oil) chain triglyceride substrates at various pH and bile salt concentrations, and compared with those of human gastric and pancreatic lipases. YLLIP2 was not inhibited by bile salts at micellar concentrations with any of the substrates tested, and maximum specific activities were found to be 10,760+/-115 U/mg on tributyrin, 16,920+/-480 U/mg on trioctanoin and 12,260+/-700 U/mg on olive oil at pH 6.0. YLLIP2 was found to be fairly stable and still active on long chain triglycerides (1590+/-430 U/mg) at pH 4.0, in the presence of bile salts. It is therefore a good candidate for use in enzyme replacement therapy as a means of treating pancreatic exocrine insufficiency.
Aloulou
Ahmed
A
Laboratory of Enzymology at Interfaces and Physiology of Lipolysis, CNRS-UPR 9025-IBSM, 31 Chemin Joseph-Aiguier, 13009 Marseille, France.
Rodriguez
Jorge A
JA
Puccinelli
Delphine
D
Mouz
Nicolas
N
Leclaire
Julien
J
Leblond
Yves
Y
Carrière
Frédéric
F
D001647:Bile Acids and Salts / Q000494:pharmacology; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D015966:Gene Expression Regulation, Fungal; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008049:Lipase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D010740:Phospholipases / Q000378:metabolism / Q000502:physiology; D010743:Phospholipids / Q000737:chemistry; D013379:Substrate Specificity; D013997:Time Factors; D014280:Triglycerides / Q000378:metabolism*; D014304:Triolein / Q000378:metabolism; D025062:Yarrowia / Q000201:enzymology*
D001647:Bile Acids and Salts; D005656:Fungal Proteins; D010743:Phospholipids; D014280:Triglycerides; D014304:Triolein; D010740:Phospholipases; D008049:Lipase