Purification and biochemical characterization of the LIP2 lipase from Yarrowia lipolytica

被引:83
作者
Aloulou, Ahmed
Rodriguez, Jorge A.
Puccinelli, Delphine
Mouz, Nicolas
Leclaire, Julien
Leblond, Yves
Carriere, Frederic
机构
[1] IBSM, CNRS, UPR 9025, Lab Enzymol Interfaces & Physiol Lipolysis, F-13009 Marseille, France
[2] Mayoly Spindler SA, F-78401 Chatou, France
[3] ProteineXpert SA, Zone Minatec Enterprises, F-38040 Grenoble, France
[4] Ecole Cent Marseille, CNRS, UMR 6180, F-13397 Marseille, France
来源
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR AND CELL BIOLOGY OF LIPIDS | 2007年 / 1771卷 / 02期
关键词
microbial lipase; lipolysis; pH-dependency; bile salts; yeast; triglycerides;
D O I
10.1016/j.bbalip.2006.12.006
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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. (c) 2006 Elsevier B.V. All rights reserved.
引用
收藏
页码:228 / 237
页数:10
相关论文
共 42 条
[1]   LINGUAL LIPASE IN CYSTIC-FIBROSIS - QUANTITATION OF ENZYME-ACTIVITY IN THE UPPER SMALL-INTESTINE OF PATIENTS WITH EXOCRINE PANCREATIC INSUFFICIENCY [J].
ABRAMS, CK ;
HAMOSH, M ;
HUBBARD, VS ;
DUTTA, SK ;
HAMOSH, P .
JOURNAL OF CLINICAL INVESTIGATION, 1984, 73 (02) :374-382
[2]   Exploring the specific features of interfacial enzymology based on lipase studies [J].
Aloulou, Ahmed ;
Rodriguez, Jorge A. ;
Fernandez, Sylvie ;
van Oosterhout, Dirk ;
Puccinelli, Delphine ;
Carriere, Frederic .
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR AND CELL BIOLOGY OF LIPIDS, 2006, 1761 (09) :995-1013
[3]   Directed evolution of mesophilic enzymes into their thermophilic counterparts [J].
Arnold, FH ;
Giver, L ;
Gershenson, A ;
Zhao, HM ;
Miyazaki, K .
MOLECULAR STRATEGIES IN BIOLOGICAL EVOLUTION, 1999, 870 :400-403
[4]   Physiology and genetics of the dimorphic fungus Yarrowia lipolytica [J].
Barth, G ;
Gaillardin, C .
FEMS MICROBIOLOGY REVIEWS, 1997, 19 (04) :219-237
[5]   Human pancreatic lipase:: Colipase dependence and interfacial binding of lid domain mutants [J].
Bezzine, S ;
Ferrato, F ;
Ivanova, MG ;
Lopez, V ;
Verger, R ;
Carrière, F .
BIOCHEMISTRY, 1999, 38 (17) :5499-5510
[6]  
BONINI P, 1992, EUR J CLIN CHEM CLIN, V30, P881
[7]   PANCREATIC LIPASE AND CO-LIPASE - INTERACTIONS AND EFFECTS OF BILE-SALTS AND OTHER DETERGENTS [J].
BORGSTRO.B ;
ERLANSON, C .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1973, 37 (01) :60-68
[8]  
Bornscheuer UT, 2006, HYDROLASES IN ORGANIC SYNTHESIS: REGIO- AND STEREOSELECTIVE BIOTRANSFORMATIONS, 2ND EDITION, P1
[9]  
BROCKERHOFF H, 1971, J BIOL CHEM, V246, P5828
[10]  
Brockman H., 2001, INTESTINAL LIPID MET, P61, DOI DOI 10.1007/978-1-4615-1195-3_4