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Purification and Biochemical Characterization of Digestive Lipase in Whiteleg Shrimp

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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 Km, app of 3.22 mM and kcat, app/Km, app of 0.303 × 103 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.

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References

  • Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    Article  PubMed  CAS  Google Scholar 

  • Arreguín-Espinosa R, Arreguín B, González C (2000) Purification and properties of a lipase from Cephaloleia presignis (Coleoptera, Chrysomelidae). Biotechnol Appl Biochem 31:239–244

    Article  PubMed  Google Scholar 

  • Arrese EL, Wells MA (1994) Purification and properties of a phosphorylatable triacylglycerol lipase from the fat body of an insect Manduca sexta. J Lipid Res 35:1652–1660

    PubMed  CAS  Google Scholar 

  • Bhardwaj K, Raju A, Rajasekharan R (2001) Identification, purification, and characterization of a thermally stable lipase from rice Bran. A new member of the (Phospho) Lipase Family. J Plant Physiol 127:1728–1738

    Article  CAS  Google Scholar 

  • Borgstrom B, Donner J (1976) Interactions of pancreatic lipase with bile salts and dodecyl sulfate. J Lipid Res 17:491–497

    PubMed  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Buscá R, Pujana MA, Pognonec P, Auwerx J, Deeb SS, Reina M, Vilaró S (1995) Absence of N-glycosylation at asparagine 43 in human lipoprotein lipase induces its accumulation in the rough endoplasmatic reticulum and alters this cellular compartment. J Lipid Res 36:939–951

    PubMed  Google Scholar 

  • Dall W, Moriarty DJW (1983) Nutrition and digestion. In: Mantle LH (ed) The biology of Crustacea. Academic, New York and London

    Google Scholar 

  • De Caro A, Figarella C, Amic J, Michel R, Guy O (1977) Human pancreatic lipase: a glycoprotein. Biochim Biophys Acta 490:411–419

    PubMed  Google Scholar 

  • Deb C, Daniel J, Sirakova TD, Abomoelak B, Dubey VS, Kolattukudy PE (2006) A novel lipase belonging to the hormone-sensitive lipase family induced under starvation to utilize stored triacylglycerol in Mycobacterium tuberculosis. J Biol Chem 281:3866–3875

    Article  PubMed  CAS  Google Scholar 

  • Del Monte A, Nolasco H, Forrellat A, Aragón C, García A, Díaz J, Carrillo O (2002) Evidencias de la presencia de lipasas en el hepatopáncreas de Litopenaeus schmitti. Memorias del Congreso Iberoamericano Virtual de Acuicultura CIVA. Zaragoza, España

    Google Scholar 

  • Dugi KA, Dichek HL, Santamarina-Fojo S (1995) Human hepatic and liporpotein lipase: the loop covering the catalytic site mediates lipase substrate specificity. J Biol Chem 270:25396–25401

    Article  PubMed  CAS  Google Scholar 

  • Forrellat Barrios A, Del Monte A, Estévez T, Boburg B, Nolasco H, Carrillo Farnés O (2004) Caracterización de lipasas en tres especies de camarones peneidos. Su importancia en la digestión. memorias del Congreso Iberoamericano Virtual de Acuicultura CIVA. Zaragoza, España

    Google Scholar 

  • Fullbrook PD (1996) Practical applied kinetics. Stockton Press, New York

    Google Scholar 

  • Gargouri Y, Julien R, Pieroni G, Verger R, Sarda L (1984) Studies on the inhibition of pancreatic and microbial lipases by soybean proteins. J Lipid Res 25:1214–1221

    PubMed  CAS  Google Scholar 

  • Gargouri Y, Chahinian H, Moreau H, Ransac S, Verger R (1991) Inactivation of pancreatic and gastric lipases by THL C12:0-TNB: a kinetic study with emulsified tributyrin. Biochim Biophys Acta 1085:322–328

    PubMed  CAS  Google Scholar 

  • Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel RD, Bairoch A (2003) ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res 31:3784–3788

    Article  PubMed  CAS  Google Scholar 

  • Gonzalez-Baró MdR, Pollero RJ (1998) Fatty acid metabolism of Macrobrachium borellii: dietary origin of arachidonic and eicosapentaenoic acids. Comp Biochem Physiol 119A:747–752

    Google Scholar 

  • González-Félix ML, Pérez-Velazquez M (2002) Current status of lipid nutrition of pacific white shrimp, Litopenaeus vannamei. Avances en Nutrición AcuícolaVI Memorias del VI Simposium Internacional de Nutrición Acuícola. Cancún, Quintana Roo, México: Cruz-Suárez, L.E., Ricque-Marie, D., Tapia-Salazar, M., Gaxiola-Cortés, M.G., Simoe, N

  • Grillo LAM, Majerowickz D, Gondim KC (2007) Lipid metabolism in Rhodnius prolixus (Hemiptera:Reduviidae): role of a midgut triacylglycerol-lipase. Insect Biochem Mol Biol 37:579–588

    Article  PubMed  CAS  Google Scholar 

  • Hermoso J, Pignol D, Kerfelec B, Crenon I, Chapus C, Fontecilla Campos JC (1996) Lipase activation by nonionic detergents. J Biol Chem 271:18007–18016

    Article  PubMed  CAS  Google Scholar 

  • Hide WA, Chan L, Hsiung LW (1992) Structure and evolution of the lipase superfamily. J Lipid Res 33:168–178

    Google Scholar 

  • Holm C, Osterlund T, Laurell H, Contreras JA (2000) Molecular mechanism regulating hormone-sensitive lipase and lipolysis. Annu Rev Nutr 20:365–393

    Article  PubMed  CAS  Google Scholar 

  • Jaeger K-E, Dijkstra BW, Colson C, Mv H, Misset O (1994) Bacterial lipases. FEMS Microbiol Rev 15:29–63

    Article  PubMed  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • Lim C, Ako H, Brown CL, Hahn K (1997) Growth response and fatty acids composition of juvenile Litopenaeus vannamei fed different source of dietary lipids. Aquaculture 151:143–153

    Article  CAS  Google Scholar 

  • Loizzi RF, Peterson DR (1971) Lipolytic sites in crayfish hepatopancreas and correlation with the fine structure. Comp Biochem Physiol Part B 39:227–236

    Article  CAS  Google Scholar 

  • López-López S, Nolasco H, Vega Villasante F (2003) Characterization of digestive gland esterase lipase activity of juvenile redclaw crayfish Cherax quadricarinatus. Comp Biochem Physiol B 135:337–347

    Article  PubMed  Google Scholar 

  • Lowe ME (1997) Molecular mechanism of rat and human pancreatic triglyceride lipases. J Nutr 127:549–557

    PubMed  CAS  Google Scholar 

  • Marcus PI, Talalay P (1956) Induction and purification of alpha and beta-hydroxysteroid dehydrogenases. J Biol Chem 218:661–674

    PubMed  CAS  Google Scholar 

  • Matsumura S, Matsuo M, Nishizuka Y (1976) Partial purification and characterization of a triacylglyceride lipase from pig adipose tissue. J Biol Chem 251:6267–6273

    PubMed  CAS  Google Scholar 

  • Merril CR, Washart KM (1998) Gel electrophoresis of proteins: a practical approach. Oxford University Press, New York

    Google Scholar 

  • Miled N, Canaan S, Dupuis L, Roussel A, Rivière M, Carrière F, de Caro A, Cambillau C, Verger R (2000) Digestive lipases: from three-dimensional structure to physiology. Biochimie 82:973–986

    Article  PubMed  CAS  Google Scholar 

  • Moreau H, Abergel C, Carrière F, Ferrato F, Fontecilla-Camps JC, Cambillau C, Verger R (1992) Isoform purification of gastric lipases: towards crystallization. J Mol Biol 225:147–153

    Article  PubMed  CAS  Google Scholar 

  • Mrdakovic M, Lazarevic J, Peric-Mataruga V, Ilijin L, Vlahovic M (2008) Partial characterization of a lipase from gypsy moth (Lymantria dispar L.) larval midgut. Folia Biol (Cracow) 56:103–110

    Article  CAS  Google Scholar 

  • Nayak J, Nair PGV, Mathew S, Ammu K (2004) A study on the intestinal lipase of Indian major carp Labeo rohita. Asian Fisch Sci 17:333–340

    Google Scholar 

  • Ollis DL, Cheah E, Cygler M, Dijkstra B, Frolow F, Franken SM, Harel M, Remington SJ, Silman I, Schrag J, Sussman J (1992) The α/β hydrolase fold. Protein Eng 5:197–211

    Article  PubMed  CAS  Google Scholar 

  • Park J, Soon-Yeong C, Suk-Jung C (2008) Purification and characterization of hepatic lipase from Todarodes pacificus. BMB Rep 41:254–258

    PubMed  CAS  Google Scholar 

  • Prim N, Sánchez M, Ruiz C, Pastor FIJ, Diaz P (2003) Use of methyllumbeliferyl-derivate substrates for lipase activity characterization. J Mol Catal B Enzym 22:339–346

    Article  CAS  Google Scholar 

  • Rathelot J, Julien R, Bosc-Bierne I, Gargouri Y, Canioni PLS (1981) Horse pancreatic lipase interaction with colipase from various species. Biochimie 63:227–234

    Article  PubMed  CAS  Google Scholar 

  • Roussel A, Canaan S, Egloff M-P, Riviére M, Dupuis L, Verger R, Cambillau C (1999) Crystal structure of human gastric lipase and model of lysosomal acid lipase, two lipolytic enzymes of medical interest. J Biol Chem 274:16995–17002

    Article  PubMed  CAS  Google Scholar 

  • Shahani KM, Khan IM, Chandan RC (1976) Bovine pancreatic lipase. I. Isolation, homogenity, and characterization. J Dairy Sci 59:369–375

    Article  PubMed  CAS  Google Scholar 

  • Slim C, Ahmed F, Nabil M, Heykel T, Hafedh M, Youssef G (2007) Crab digestive lipase acting at high temperature: purification and biochemical characterization. Biochimie 89:1012–1018

    Article  Google Scholar 

  • Stauffer C (1989) Enzyme assays for food scientist. New York.

  • Sukarno TK, Hatano M, Sakurai Y (1996) Lipase from neon flying squid hepatopancreas: purification and properties. Food Chem 57:515–521

    Article  CAS  Google Scholar 

  • Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weigh matrix choice. Nucleic Acids Res 22:4673–4680

    Article  PubMed  CAS  Google Scholar 

  • Thornton DJ, Carlstedt I, Sheehan JK (1994) Identification of glycoproteins on nitrocellulose membranes and gels. In: Walker JM (ed) Methods mol biol. Humana Press, Totowa, NJ

    Google Scholar 

  • Versaw WK, Cuppert SL, Winter DD, Williams LE (1989) An improved colorimetric assay for bacterial lipase in nonfat dry milk. J Food Sci 54:1557–1558

    Article  CAS  Google Scholar 

  • Zouari N, Miled N, Cherif S, Mejdoub H, Gargouri Y (2005) Purification and characterization of a novel lipase from the digestive glands of a primitive animal: the scorpion. Biochim Biophys Acta 1726:67–74

    PubMed  CAS  Google Scholar 

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Acknowledgments

C.R.P. is the recipient of a doctoral fellowship from Consejo Nacional de Ciencia y Tecnologia of Mexico.

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Authors and Affiliations

  1. Centro de Investigaciones Biologicas del Noroeste (CIBNOR), Apdo. Postal 128, La Paz, B.C.S. 23000, Mexico

    Crisalejandra Rivera-Pérez & Fernando L. García-Carreño

  2. Alfred-Wegener-Institute für Polar- und Meeresforschung (AWI), Biologische Anstalt Helgoland, P.O. Box 180, 27483, Helgoland, Germany

    Reinhard Saborowski

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  1. Crisalejandra Rivera-Pérez
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  2. Fernando L. García-Carreño
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  3. Reinhard Saborowski
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Correspondence to Fernando L. García-Carreño.

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Rivera-Pérez, C., García-Carreño, F.L. & Saborowski, R. Purification and Biochemical Characterization of Digestive Lipase in Whiteleg Shrimp. Mar Biotechnol 13, 284–295 (2011). https://doi.org/10.1007/s10126-010-9298-7

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