Purification and biochemical characterization of digestive lipase in whiteleg shrimp.
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.
Rivera-Pérez
Crisalejandra
C
Centro de Investigaciones Biologicas del Noroeste (CIBNOR), Apdo. Postal 128, La Paz, B.C.S. 23000, Mexico.
García-Carreño
Fernando L
FL
Saborowski
Reinhard
R
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
D001647:Bile Acids and Salts; D003089:Colipases; D018076:DNA, Complementary; D008049:Lipase
Biochimie. 2007 Aug;89(8):1012-8
17395356
J Dairy Sci. 1976 Mar;59(3):369-75
4478
Nature. 1970 Aug 15;227(5259):680-5
5432063
Biotechnol Appl Biochem. 2000 Jun;31(3):239-44
10814595
J Biol Chem. 1956 Feb;218(2):661-74
13295221
J Nutr. 1997 Apr;127(4):549-57
9109604
J Lipid Res. 1992 Feb;33(2):167-78
1569370
Nucleic Acids Res. 2003 Jul 1;31(13):3784-8
12824418
J Biol Chem. 1996 Jul 26;271(30):18007-16
8663362
J Biol Chem. 1995 Oct 27;270(43):25396-401
7592706
Biochimie. 2000 Nov;82(11):973-86
11099794
Biochim Biophys Acta. 1977 Feb 22;490(2):411-9
402159
Plant Physiol. 2001 Dec;127(4):1728-38
11743116
J Biol Chem. 1976 Oct 25;251(20):6267-73
10295
Protein Eng. 1992 Apr;5(3):197-211
1409539
J Lipid Res. 1976 Sep;17(5):491-7
965841
Anal Biochem. 1976 May 7;72:248-54
942051
J Lipid Res. 1994 Sep;35(9):1652-60
7806979
Biochim Biophys Acta. 1991 Oct 1;1085(3):322-8
1911866
Insect Biochem Mol Biol. 2007 Jun;37(6):579-88
17517335
BMB Rep. 2008 Mar 31;41(3):254-8
18377731
J Lipid Res. 1995 May;36(5):939-51
7658166
Comp Biochem Physiol B Biochem Mol Biol. 2003 Jun;135(2):337-47
12798943
J Lipid Res. 1984 Nov;25(11):1214-21
6542933
Annu Rev Nutr. 2000;20:365-93
10940339
Folia Biol (Krakow). 2008;56(1-2):103-10
19055033
J Mol Biol. 1992 May 5;225(1):147-53
1583687
J Biol Chem. 2006 Feb 17;281(7):3866-75
16354661
Nucleic Acids Res. 1997 Sep 1;25(17):3389-402
9254694
Biochimie. 1981 Mar;63(3):227-34
7225466
Nucleic Acids Res. 1994 Nov 11;22(22):4673-80
7984417
J Biol Chem. 1999 Jun 11;274(24):16995-7002
10358049
FEMS Microbiol Rev. 1994 Sep;15(1):29-63
7946464
Biochim Biophys Acta. 2005 Oct 30;1726(1):67-74
16154275