Fish Physiology and Biochemistry

, Volume 36, Issue 4, pp 1041–1060 | Cite as

Purification and properties of digestive lipases from Chinook salmon (Oncorhynchus tshawytscha) and New Zealand hoki (Macruronus novaezelandiae)

  • Ivan Kurtovic
  • Susan N. Marshall
  • Xin Zhao
  • Benjamin K. Simpson


Lipases were purified from delipidated pyloric ceca powder of two New Zealand-sourced fish, Chinook salmon (Oncorhynchus tshawytscha) and hoki (Macruronus novaezelandiae), by fractional precipitation with polyethylene glycol 1000, followed by affinity chromatography using cholate-Affi-Gel 102, and gel filtration on Sephacryl S-300 HR. For the first time, in-polyacrylamide gel activity of purified fish lipases against 4-methylumbelliferyl butyrate has been demonstrated. Calcium ions and sodium cholate were absolutely necessary both for lipase stability in the gel and for optimum activity against caprate and palmitate esters of p-nitrophenol. A single protein band was present in native polyacrylamide gels for both salmon and hoki final enzyme preparations. Under denaturing conditions, electrophoretic analysis revealed two bands of 79.6 and 54.9 kDa for salmon lipase. It is proposed that these bands correspond to an uncleaved and a final form of the enzyme. One band of 44.6 kDa was seen for hoki lipase. pI values of 5.8 ± 0.1 and 5.7 ± 0.1 were obtained for the two salmon lipase forms. The hoki lipase had a pI of 5.8 ± 0.1. Both lipases had the highest activity at 35°C, were thermally labile, had a pH optimum of 8–8.5, and were more acid stable compared to other fish lipases studied to date. Both enzymes were inhibited by the organophosphate paraoxon. Chinook salmon and hoki lipases showed good stability in several water-immiscible solvents. The enzymes had very similar amino acid composition to mammalian carboxyl ester lipases and one other fish digestive lipase. The salmon enzyme was an overall better catalyst based on its higher turnover number (3.7 ± 0.3 vs. 0.71 ± 0.05 s−1 for the hoki enzyme) and lower activation energy (2.0 ± 0.4 vs. 7.6 ± 0.8 kcal/mol for the hoki enzyme) for the hydrolysis of p-nitrophenyl caprate. The salmon and hoki enzymes are homologous with mammalian carboxyl ester lipases.


Bile salt Calcium Chinook salmon Enzyme inhibition Hoki Lipase Organic solvents Zymography 



This study was supported by funds from the New Zealand Foundation for Research, Science & Technology (FRST) under contracts C02X0301 and C02X0806. We thank The New Zealand King Salmon Co Ltd. for providing salmon tissue samples and Sealord Ltd. for providing hoki tissue samples. This research was facilitated using infrastructure provided by the Australian Government through the National Collaborative Research Infrastructure Strategy (NCRIS), and we thank Australian Proteome Analysis Facility, Macquarie University for amino acid composition and N-terminal analyses.


  1. Abouakil N, Rogalska E, Bonicel J, Lombardo D (1988) Purification of pancreatic carboxylic-ester hydrolase by immunoaffinity and its application to the human bile-salt-stimulated lipase. Biochim Biophys Acta Lipids Lipid Metab 961:299–308CrossRefGoogle Scholar
  2. Abouakil N, Rogalska E, Lombardo D (1989) Human milk bile-salt stimulated lipase: further investigations on the amino-acids residues involved in the catalytic site. Biochim Biophys Acta 1002:225–230PubMedGoogle Scholar
  3. Albro PW, Hall RD, Corbett JT, Schroeder J (1985) Activation of nonspecific lipase (EC 3.1.1.-) by bile salts. Biochim Biophys Acta Lipids Lipid Metab 835:477–490CrossRefGoogle Scholar
  4. Anonymous (2008) Information Centre, New Zealand Seafood Industry Council (SeaFIC), Wellington New Zealand. Accessed 16 Jan 2009
  5. Anthonsen HW, Baptista A, Drablos F, Martel P, Petersen SB, Sebastiao M, Vaz L (1995) Lipases and esterases: a review of their sequences, structure and evolution. In: El-Gewely MR (ed) Biotechnology annual review. Elsevier, Amsterdam, pp 315–371CrossRefGoogle Scholar
  6. Aryee ANA, Simpson BK, Villalonga R (2007) Lipase fraction from the viscera of grey mullet (Mugil cephalus): isolation, partial purification and some biochemical characteristics. Enzyme Microb Technol 40:394–402CrossRefGoogle Scholar
  7. Bier M (1955) Lipases. In: Colowick SP, Kaplan NO (eds) Methods in enzymology. Academic Press, New York, pp 627–642CrossRefGoogle Scholar
  8. Blackberg L, Hernell O (1993) Bile salt-stimulated lipase in human milk: evidence that bile salt induces lipid binding and activation via binding to different sites. FEBS Lett 323:207–210CrossRefPubMedGoogle Scholar
  9. Bollag DM, Rozycki MD, Edelstein SJ (1996) Gel electrophoresis under nondenaturing conditions. In: Protein methods, 2nd edn. Wiley-Liss, New York, pp 155–172Google Scholar
  10. Cherif S, Fendri A, Miled N, Trabelsi H, Mejdoub H, Gargouri Y (2007) Crab digestive lipase acting at high temperature: purification and biochemical characterization. Biochimie 89:1012–1018CrossRefPubMedGoogle Scholar
  11. Derewenda U, Swenson L, Wei YY, Green R, Kobos PM, Joerger R, Haas MJ, Derewenda ZS (1994) Conformational lability of lipases observed in the absence of an oil-water interface: crystallographic studies of enzymes from the fungi Humicola lanuginosa and Rhizopus delemar. J Lipid Res 35:524–534PubMedGoogle Scholar
  12. Diaz P, Prim N, Javier Pastor FI (1999) Direct fluorescence-based lipase activity assay. Biotechniques 27:696–700PubMedGoogle Scholar
  13. Georlette D, Blaise V, Collins T, D’Amico S, Gratia E, Hoyoux A, Marx J-C, Sonan G, Feller G, Gerday C (2004) Some like it cold: biocatalysis at low temperatures. FEMS Microbiol Rev 28:25–42CrossRefPubMedGoogle Scholar
  14. German AB, Neklyudov AD, Ivankin AN, Berdutina AV (2002) The kinetics of hydrolysis of animal fat by pancreatic lipase. Appl Biochem Microbiol 38:517–520CrossRefGoogle Scholar
  15. Gillard M (2008) The NZ Salmon Farmers Association website, Accessed 16 Jan 2009
  16. Gjellesvik DR (1991) Fatty acid specificity of bile salt-dependent lipase: enzyme recognition and super-substrate effects. Biochim Biophys Acta 1086:167–172PubMedGoogle Scholar
  17. Gjellesvik DR, Lombardo D, Walther BT (1992) Pancreatic bile salt dependent lipase from cod (Gadus morhua): purification and properties. Biochim Biophys Acta 1124:123–134PubMedGoogle Scholar
  18. Gjellesvik DR, Lorens JB, Male R (1994) Pancreatic carboxylester lipase from Atlantic salmon (Salmo salar). cDNA sequence and computer-assisted modelling of tertiary structure. Eur J Biochem 226:603–612CrossRefPubMedGoogle Scholar
  19. Gomori G (1955) Preparation of buffers for use in enzyme studies. In: Colowick SP, Kaplan NO (eds) Methods in enzymology. Academic Press, New York, pp 138–146CrossRefGoogle Scholar
  20. Hart GJ, O’Brien RD (1973) Recording spectrophotometric method for determination of dissociation and phosphorylation constants for the inhibition of acetylcholinesterase by organophosphates in the presence of substrate. Biochemistry 12:2940–2945CrossRefPubMedGoogle Scholar
  21. Hosie L, Sutton LD, Quinn DM (1987) p-Nitrophenyl and cholesteryl-N-alkyl carbamates as inhibitors of cholesterol esterase. J Biol Chem 262:260–264PubMedGoogle Scholar
  22. Hui DY, Howles PN (2002) Carboxyl ester lipase: structure-function relationship and physiological role in lipoprotein metabolism and atherosclerosis. J Lipid Res 43:2017–2030CrossRefPubMedGoogle Scholar
  23. Iijima N, Tanaka S, Ota Y (1998) Purification and characterization of bile salt-activated lipase from the hepatopancreas of red sea bream, Pagrus major. Fish Physiol Biochem 18:59–69CrossRefGoogle Scholar
  24. Ingham KC (1990) Precipitation of proteins with polyethylene glycol. In: Deutscher MP (ed) Guide to protein purification. Academic Press, San Diego, pp 301–306CrossRefGoogle Scholar
  25. Klibanov AM (1989) Enzymatic catalysis in anhydrous organic solvents. Trends Biochem Sci 14:141–144CrossRefPubMedGoogle Scholar
  26. Kordel M, Hofmann B, Schomburg D, Schmid RD (1991) Extracellular lipase of Pseudomonas sp. strain ATCC 21808: purification, characterization, crystallization, and preliminary X-ray diffraction data. J Bacteriol 173:4836–4841PubMedGoogle Scholar
  27. Kurtovic I, Marshall SN, Simpson BK (2006) Isolation and characterization of a trypsin fraction from the pyloric ceca of chinook salmon (Oncorhynchus tshawytscha). Comp Biochem Physiol Part B 143:432–440CrossRefGoogle Scholar
  28. Kurtovic I, Marshall SN, Zhao X, Simpson BK (2009) Lipases from mammals and fishes. Rev Fish Sci 17:18–40CrossRefGoogle Scholar
  29. Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  30. Leger C, Bauchart D, Flanzy J (1977) Some properties of pancreatic lipase in Salmo gairdnerii rich.: Km, effects of bile salts and Ca2+, gel filtrations. Comp Biochem Physiol Part B 57:359–363CrossRefGoogle Scholar
  31. Lehrer GM, Barker R (1970) Conformational changes in rabbit muscle aldolase. Kinetic studies. Biochemistry 9:1533–1540CrossRefPubMedGoogle Scholar
  32. Lima VMG, Krieger N, Mitchell DA, Fontana JD (2004) Activity and stability of a crude lipase from Penicillium aurantiogriseum in aqueous media and organic solvents. Biochem Eng J 18:65–71CrossRefGoogle Scholar
  33. Lombardo D, Guy O, Figarella C (1978) Purification and characterization of a carboxyl ester hydrolase from human pancreatic juice. Biochim Biophys Acta Enzymol 527:142–149Google Scholar
  34. Loomes KM (1995) Structural organisation of human bile-salt-activated lipase probed by limited proteolysis and expression of a recombinant truncated variant. Eur J Biochem 230:607–613CrossRefPubMedGoogle Scholar
  35. Lopez-Amaya C, Marangoni AG (2000) Lipases. In: Haard NF, Simpson BK (eds) Seafood enzymes. Utilization and influence on postharvest seafood quality. Marcel Dekker, New York, pp 121–146Google Scholar
  36. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  37. Maurin C, Gal Y (1996) Characterization of a hydrophobic esterase from tuna (Thunnus albacares) pyloric caeca. J Mar Biotechnol 4:87–90Google Scholar
  38. Mukundan MK, Gopakumar K, Nair MR (1985) Purification of a lipase from the hepatopancreas of oil sardine (Sardinella longiceps Linnaeus) and its characteristics and properties. J Sci Food Agric 36:191–203CrossRefGoogle Scholar
  39. Palekar AA, Vasudevan PT, Yan S (2000) Purification of lipase: a review. Biocatal Biotransform 18:177–200CrossRefGoogle Scholar
  40. Patkar S, Bjorkling F (1994) Lipase inhibitors. In: Wooley P, Petersen SB (eds) Lipases—their structure, biochemistry and application. Cambridge University Press, Cambridge, pp 207–224Google Scholar
  41. Pencreac’h G, Baratti JC (1996) Hydrolysis of p-nitrophenyl palmitate in n-heptane by the Pseudomonas cepacia lipase: a simple test for the determination of lipase activity in organic media. Enzyme Microb Technol 18:417–422CrossRefGoogle Scholar
  42. Robertson EF, Dannelly HK, Malloy PJ, Reeves HC (1987) Rapid isoelectric focusing in a vertical polyacrylamide minigel system. Anal Biochem 167:290–294CrossRefPubMedGoogle Scholar
  43. Rudd EA, Mizuno NK, Brockman HL (1987) Isolation of two forms of carboxylester lipase (cholesterol esterase) from porcine pancreas. Biochim Biophys Acta 918:106–114PubMedGoogle Scholar
  44. Shi CY, Marshall SN, Simpson BK (2007) Purification and characterization of trypsin from the pyloric ceca of the New Zealand hoki fish (Macruronus novaezelandiae). J Food Biochem 31:772–796CrossRefGoogle Scholar
  45. Simpson BK, Haard NF (1984) Purification and characterization of trypsin from the Greenland cod (Gadus ogac). 1. Kinetic and thermodynamic characteristics. Can J Biochem Cell Biol 62:894–900CrossRefGoogle Scholar
  46. Sun SY, Xu Y, Wang D (2009) Purification and biochemical characterization of an intracellular lipase by Rhizopus chinensis under solid-state fermentation and its potential application in the production of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). J Chem Technol Biotechnol 84:435–441CrossRefGoogle Scholar
  47. Sztajer H, Lunsdorf H, Erdmann H, Menge U, Schmid R (1992) Purification and properites of lipase from Penicillium simplicissimum. Biochim Biophys Acta 1124:253–261PubMedGoogle Scholar
  48. Taniguchi A, Takano K, Kamoi I (2001) Purification and properties of lipase from tilapia intestine: digestive enzyme of Tilapia VI. Bull Jpn Soc Sci Fish 67:78–84Google Scholar
  49. van den Bosch H, Aarsman AJ, De Jong JGN, Van Deenen LLM (1973) Studies on lysophospholipases: I. Purification and some properties of a lysophospholipase from beef pancreas. Biochim Biophys Acta Lipids Lipid Metab 296:94–104CrossRefGoogle Scholar
  50. Wang CS (1980) Purification of human milk bile salt-activated lipase by cholic acid-coupled Sepharose 4B affinity chromatography. Anal Biochem 105:398–402CrossRefPubMedGoogle Scholar
  51. Wang C-S, Hartsuck JA (1993) Bile salt-activated lipase. A multiple function lipolytic enzyme. Biochim Biophys Acta 1166:1–19PubMedGoogle Scholar
  52. Wang CS, Johnson K (1983) Purification of human milk bile salt-activated lipase. Anal Biochem 133:457–461CrossRefPubMedGoogle Scholar
  53. Wang X, Wang C-S, Tang J, Dyda F, Zhang XC (1997) The crystal structure of bovine bile salt activated lipase: insights into the bile salt activation mechanism. Structure 5:1209–1218CrossRefPubMedGoogle Scholar
  54. Whitaker JR (1994) Effect of temperature on rates of enzyme catalyzed reactions. In: Whitaker JR (ed) Principles of enzymology for the food sciences. Marcel Dekker, Inc., New York, pp 301–328Google Scholar
  55. Winkler FK, Gubernator K (1994) Structure and mechanism of human pancreatic lipase. In: Wooley P, Petersen SB (eds) Lipases—their structure, biochemistry and application. Cambridge University Press, Cambridge, pp 139–157Google Scholar
  56. Winkler UK, Stuckmann M (1979) Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens. J Bacteriol 138:663–670PubMedGoogle Scholar
  57. Zaks A, Klibanov A (1988) Enzymatic catalysis in nonaqueous solvents. J Biol Chem 263:3194–3201PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Ivan Kurtovic
    • 1
    • 2
  • Susan N. Marshall
    • 2
  • Xin Zhao
    • 1
  • Benjamin K. Simpson
    • 3
  1. 1.Department of Animal ScienceMcGill University (Macdonald Campus)Ste. Anne de BellevueCanada
  2. 2.The New Zealand Institute for Plant & Food Research LimitedNelsonNew Zealand
  3. 3.Department of Food Science and Agricultural ChemistryMcGill University (Macdonald Campus)Ste. Anne de BellevueCanada

Personalised recommendations