title: Purification and biochemical characterization of digestive lipase in whiteleg shrimp.
abstract: Penaeus vannamei lipase was purified from midgut gland of whiteleg shrimp. Pure lipase (E.C. 3.1.1.3) was obtained after Superdex 200 gel filtration and Resource Q anionic exchange. The pure lipase, which is a glycosylated molecule, is a monomer having a molecular mass of about 44.8 kDa, as determined by SDS-PAGE analysis. The lipase hydrolyses short and long-chain triacylglycerols and naphthol derivates at comparable rates. A specific activity of 1787 U mg(-1) and 475 U mg(-1) was measured with triolein and tributyrin as substrates, respectively, at pH 8.0 and 30°C in the absence of colipase. The lipase showed a K (m, app) of 3.22 mM and k (cat, app)/K (m, app) of 0.303 × 10(3) mM(-1) s(-1) using triolein as substrate. Natural detergents, such as sodium deoxycholate, act as potent inhibitors of the lipase. This inhibition can be reversed by adding fresh oil emulsion. Result with tetrahydrolipstatin, an irreversible inhibitor, suggests that the lipase is a serine enzyme. Peptide sequences of the lipase were determined and compared with the full-length sequence of lipase which was obtained by the rapid amplification of cDNA ends method. The full cDNA of the pvl was 1,186 bp, with a deduced protein of 362 amino acids that includes a consensus sequence (GXSXG) of the lipase superfamily of α/β-hydrolase. The gene exhibits features of conserved catalytic residues and high homology with various mammalian and insect lipase genes. A potential lid sequence is suggested for pvl.
authors.affiliation: Centro de Investigaciones Biologicas del Noroeste (CIBNOR), Apdo. Postal 128, La Paz, B.C.S. 23000, Mexico.
authors.forename: Crisalejandra
authors.initials: C
authors.lastname: Rivera-Pérez
authors.forename: Fernando L
authors.initials: FL
authors.lastname: García-Carreño
authors.forename: Reinhard
authors.initials: R
authors.lastname: Saborowski
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001647:Bile Acids and Salts / Q000378:metabolism; D003089:Colipases / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004063:Digestion / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D033561:Penaeidae / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism
chemical_list: D001647:Bile Acids and Salts; D003089:Colipases; D018076:DNA, Complementary; D008049:Lipase
references.citation: Biochimie. 2007 Aug;89(8):1012-8
references.pmid: 17395356
references.citation: J Dairy Sci. 1976 Mar;59(3):369-75
references.pmid: 4478
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: Biotechnol Appl Biochem. 2000 Jun;31(3):239-44
references.pmid: 10814595
references.citation: J Biol Chem. 1956 Feb;218(2):661-74
references.pmid: 13295221
references.citation: J Nutr. 1997 Apr;127(4):549-57
references.pmid: 9109604
references.citation: J Lipid Res. 1992 Feb;33(2):167-78
references.pmid: 1569370
references.citation: Nucleic Acids Res. 2003 Jul 1;31(13):3784-8
references.pmid: 12824418
references.citation: J Biol Chem. 1996 Jul 26;271(30):18007-16
references.pmid: 8663362
references.citation: J Biol Chem. 1995 Oct 27;270(43):25396-401
references.pmid: 7592706
references.citation: Biochimie. 2000 Nov;82(11):973-86
references.pmid: 11099794
references.citation: Biochim Biophys Acta. 1977 Feb 22;490(2):411-9
references.pmid: 402159
references.citation: Plant Physiol. 2001 Dec;127(4):1728-38
references.pmid: 11743116
references.citation: J Biol Chem. 1976 Oct 25;251(20):6267-73
references.pmid: 10295
references.citation: Protein Eng. 1992 Apr;5(3):197-211
references.pmid: 1409539
references.citation: J Lipid Res. 1976 Sep;17(5):491-7
references.pmid: 965841
references.citation: Anal Biochem. 1976 May 7;72:248-54
references.pmid: 942051
references.citation: J Lipid Res. 1994 Sep;35(9):1652-60
references.pmid: 7806979
references.citation: Biochim Biophys Acta. 1991 Oct 1;1085(3):322-8
references.pmid: 1911866
references.citation: Insect Biochem Mol Biol. 2007 Jun;37(6):579-88
references.pmid: 17517335
references.citation: BMB Rep. 2008 Mar 31;41(3):254-8
references.pmid: 18377731
references.citation: J Lipid Res. 1995 May;36(5):939-51
references.pmid: 7658166
references.citation: Comp Biochem Physiol B Biochem Mol Biol. 2003 Jun;135(2):337-47
references.pmid: 12798943
references.citation: J Lipid Res. 1984 Nov;25(11):1214-21
references.pmid: 6542933
references.citation: Annu Rev Nutr. 2000;20:365-93
references.pmid: 10940339
references.citation: Folia Biol (Krakow). 2008;56(1-2):103-10
references.pmid: 19055033
references.citation: J Mol Biol. 1992 May 5;225(1):147-53
references.pmid: 1583687
references.citation: J Biol Chem. 2006 Feb 17;281(7):3866-75
references.pmid: 16354661
references.citation: Nucleic Acids Res. 1997 Sep 1;25(17):3389-402
references.pmid: 9254694
references.citation: Biochimie. 1981 Mar;63(3):227-34
references.pmid: 7225466
references.citation: Nucleic Acids Res. 1994 Nov 11;22(22):4673-80
references.pmid: 7984417
references.citation: J Biol Chem. 1999 Jun 11;274(24):16995-7002
references.pmid: 10358049
references.citation: FEMS Microbiol Rev. 1994 Sep;15(1):29-63
references.pmid: 7946464
references.citation: Biochim Biophys Acta. 2005 Oct 30;1726(1):67-74
references.pmid: 16154275