Partial purification of intestinal triglyceride lipase from Cyprinion acrostomus Heckel, 1843 and effect of pH on enzyme activity

Cyprinion macrostomus incebağırsak Lipaz’ının% 30 polietilen glikol (PEG) ile çöktürülmesinden sonra, hidrofobik etkileşim kromatografisi ile kısmi saflaştırılması ve pH’nın enzimatik aktivite üzerine etkisi titrasyon yöntemiyle araştırılmıştır. incebağırsak doku homojenatı %30 PEG-8000 ile çöktürülmüş ve fenil sefaroz ile hidrofobik etkileşim kromatografisine uygulanmıştır. Matriks ile bağ kurmuş olan ve bu şekilde kısmi olarak saflaştırılan lipaz aktivitesi taurokolik asit ve triton X-100 çözeltileri ile geri alınmıştır. Saflaştırılan enzim deneysel koşullar altında doğal zeytin yağı substrat’ını etkili şekilde hidrolizleşmiştir. incebağırsak lipaz aktivitesi için optimum pH’nın 7.50 olduğu saptanmıştır. %94.37’lik bir verim ve 71.54-kat saflaştırma parametreleri saptanmıştır. Poliakrilamid disc-continuos jel elektroforezi (Disk-PAGE) ile denatöre edici etken içermeyen koşullar altında bu Lipaz’ın molekül kütlesi 51 kDa. olarak tespit edilmiştir. Bu balığın incebağırsak doku lipaz’ının alkali karakterde olduğu sonucuna varılmıştır.

Cyprinion macrostomus Heckel, 1843 incebağırsak lipaz'ının kısmi saflaştırılması ve pH'nın enzim aktivitesi üzerine etkisi

: Intestinal triglyceride lipase (TG) of Cyprinion macrostomus after precipitation with 30% polyethylene glycol (PEG), was partially purified by hydrophobic interaction chromatography on phenyl sepharose CL-4B, and the effect of pH on this enzyme activity was determined titrimetrically. Intestinal tissue homogenates were precipitated using 30% PEG-8000, and then applied onto a phenyl sepharose CL-4B column for hy-drophobic interaction chromatography. The lipase that bound to this hydrophobic resin and was partially purified by this single step application was then eluted from this resin with taurocholic acid and Triton X-100 elution. This purified enzyme effectively hydrolysed natural olive oil substrate under the experimental conditions. An optimal pH of 7.50, and 94.37% yield, and 71.54-fold purification parameters were estimated for this lipase. The molecular mass of this enzyme was determined to be 51 kDa under non-denaturing conditions by polyacrylamide disc-gel electrophoresis (Disc-PAGE). It was concluded that the intestinal lipase of this fish is alkaline in character.

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1. Vadehra, D.V., Harmon, L.G. Characterisation of purified Staphylococcal lipase. App. Microbiology. 15; 3, 480-483, 1967.
2. Hassing, G.S. Partial purification and some properties of a lipase from Cornebacte-rium acnes. Biochimica et Biophysica Acta. 242; 381-394, 1971.
3. Tyski, S., Hryniewicz, W., Jeljaszewicz, J. Purification and Some Properties of the Staphylococcal Extracellular Lipase. Biochimica et Biophysica Acta. 749; 312-317, 1983.
4. Veerraragavan, K.A., Colpitts, T., Gibbs, B.F. Purification and characterisation of two distinct lipases from Geotrichum candidum. Biochimica et Biophysica Acta. 1044; 26 - 33, 1990.
5. Hedrich, C.H., Spener, F., Menge, U., et al. LargeÐscale purification, enzymic characterization, and crystallisation of the lipase from Geotrichum candidum. Enzyme Microb. Tecnol. 13; 840 - 847, 1990.
6. Kordel, M., Hofmann, B., Schomburg, D., Schmid, R. Extracellular lipase of Pseudomonas sp. Strain ATCC 21808: Purification, characterization, crystallization, and preliminary x-ray diffraction data. Journal of Bacteriology. 173; 4836 - 4841, 1991.
7. Sztajer, H., Lansdorf, H., Erdmann, H., Menge, U., Schmid, R. Purification and properties of lipase from Pennicillium simplicissimum. Biochimica et Biophysica Acta. 1124, 253 - 261, 1992.
8. Sugihara, A., Ueshima, M., Shimada, Y., Tsunasawa, S., Tominaga, Y. Purification and characterization of a novel thermostable lipase from Pseudomonas cepacia. J. Biochem. 112; 598-603, 1992.
9. Haas, J.M., Cichowicz, J.D., Bailey, G.D. Purification and characterisation of an extracellular lipase from the fungus, Rhizopus delemar. Lipids. 27; 8, 571-576, 1992.
10. Bernback, S., Hernell, O., Blackberg, L. Purification and molecular characteri-sation of bovine pregastric lipase. Eur. J. Biochem., 148; 223-238, 1985.
11. Kuusi, T., Kinnunen, K.J.P., Ehnholm, C., Nikkila, E.A. A simple purification procedure for rat hepatic lipase. Febs Letter. 98; 2, 314-318, 1979.
12. Charbonnier, M., Arnaud, J., Boyer, J. Partial purification and characterization of a fatty acyl monoester lipase from human adipose tissue. Biochimica et Biophysica Acta. 296: 471 - 480, 1973.
13. Momsen, W., Brockman, H. Purification and characterisation of cholesterol esterase from porcine pancreas. Biochimica et Biophysica Acta. 486: 103-113, 1977.
14. Warner, T.G., Tennant, L.L., Veath, M.L., O'Brien, J.S. An improved method for the isolation and assay of the acid lipase from human liver. Biochimica et Biophysica Acta. 572: 201-210, 1979.
15. Becht, I., Schrecker, O., Klose, G., Greten, H. Purification of human lipoprotein lipase. Biochimica et Biophysica Acta. 620: 583-591, 1980.
16. Imanaka, T., Muto, K., Ohkuma, S., Takano, T. Purification and properties of rabbit liver (4 Ð methylumbelliferyl oleate hydrolase). Biochimica et Biophysica Acta. 665: 322-330, 1981.
17. Jensen, G.L., Bensadoun, A. Purification, stabilisation, and characterisation of rat hepatic triglyceride lipase. Analytical Biochemistry. 113: 246-252, 1981.
18. Gargouri, Y., Julien, R., Bois, A.G., Verger, R., Sarda, L. Studies on the detergent inhibition of pancreatic lipase activity. Journal of Lipid Research. 24: 1336- 1342, 1983.
19. Albro, W.P., Hall, R.D., Corbett, J.T., Schroeder, J. Activation of non-specific lipase (EC 3.1.1.-) by bile salts. Biochimica et Biophysica Acta. 835: 477-490, 1985.
20. Gargouri, Y., Pieroni, G., Riviere C., et al. Importance of human gastric lipase for intestinal lipolysis. An in vitro study. Biochimica et Biophysica Acta. 879: 419-423, 1986.
21. Walde, P., Sunamoto, J., O'Connor, C.J. Activity of bile-saltstimulated human milk lipase in the presence of liposomes and mixed taurocholate- phosphadylcholine micelles. Biochimica et Biophysica Acta. 905: 39-47, 1987.
22. Moreau, H., Gargouri, Y., Lecat, D., Junien, J.-L., Verger, R. Purification, char-acterisation and kinetic properties of the rabbit gastric lipase. Biochimica et Biophysica Acta. 960. 286-283, 1988.
23. Aboukil, N., Rogalska, E., Bonicel, J., Lombardo, D. Purification of pancreatic carboxylic-ester hydrolyse by immunoaffinity and its application to human bile-salt-simulated lipase. Biochimica et Biophysica Acta. 961: 299-308, 1988.
24. Aboukil, N., Lombardo, D. Inhibition of human milk bile-saltdependent lipase by boronic acids. Implication to the bile salts activator effect. Biochimica et Biophysica Acta. 1004: 215-220, 1989.
25. Camulli, D.E., Linke, M.J., Brockman, H.L., Hui, D.Y. Identity of a cytosolic neutral cholesterol esterase in rat liver with the bile salt stimulated cholesterol esterase in pancreas. Biochimica et Biophysica Acta. 1005: 177-182, 1989.
26. Lombardo, D., Chapus, C., Bourne, Y., Cambillau, C. Crystallisation and pre-liminary x-ray study of horse pancreatic lipase. J. Mol. Biol., 205: 259-261, 1989.
27. Cox, D.G., Leung, T.K.C., Kyger, E.M., Spilburg, C.A., Lange, G.L. Pancreatic cholesterol esterase induction during maturation. Biochemistry. 29: 3842-3848, 1990.
28. Rao, R.H., Mansbach, C.M. Purification and partial characterisation of intestinal lipase. Biochimica et Biophysica Acta. 1046: 19-26, 1990.
29. Carriere, F., Moreau, H., Raphel, V., Laugier, R., et al. Purification and biochemical characterisation of dog gastric lipase. Eur. J. Biochem., 202: 75-83, 1991.
30. Ghosh, S., Grogan, W.M. Rapid three-step purification of a hepatic neutral cholesteryl ester hydrolyse which is not the pancreatic enzyme. Lipids. 26: 793-798, 1991.
31. Iizuka, K., Higurashi, H., Fujimato, J., et al. Purification of human gastric lipase and the influence of bicarbonate on lipase activity. Ann. Clin. Biochem. 28; 373-378. 1991.
32. Wang, C. S. Acyl-chain specificity of human milk bile-salt-activated lipase. Bio-chem. J. 279: 297-302, 1991.
33. Holm, C., Davis, R.C., Fredrikson, G., Belfrage, P., Schottz, M.C. Expression of biologically active hormone-sensitive lipase in mammalian (COS) cells. FEBS. 1: 139 - 144, 1991.
34. Bernadac, A., Moreau, H., Verger, R. Gastric lipase and pepsinogen during the ontogenesis of rabbit gastric glands. E. J. of Cell Biol. 55: 149 - 157, 1991.
35. Waite, M., Thuren, T.Y., Wilcox, R.W., et al. Purification and substrate specificity of rat hepatic lipase. Methods in Enzymology. 197: 331- 339, 1991.
36. Verhoeven, A.J.M., Jansen, H. Secretion-coupled increase in the catalytic activity of rat hepatic lipase. Biochimica et Biophysica Acta. 1086: 49 - 56, 1991.
37. Kresse, H., Löffler, B.-M., Kunze, H. Improved procedure for the purification of hepatic lipase from rat liver homogenate. Biochemistry International. 23: 885 - 893, 1991.
38. Moreau, H., Abergel, C., Carriere, F., Ferrato, F., et. al. Isoform purification of gas-tric lipases. Towards crystallisation. J. Mol. Biol. 225: 147 - 153, 1992.
39. Hills, M. J., Mukherjee, K. D. Triacylglycerol lipase from rape (Brassica napus L.). Suitable for biotechnological purposes. Applied Biochemistry and Biotechnology. 22: 1 - 9, 1990.
40. Arrese, E.L., Wells, M.A. Purification and properties of a phosphorylatable triacylglycerol lipase from the fat body of an insect, Manduca sexta. Journal of Lipid Research. 35. 1652- 1659, 1992.
41. Simpson, B.K., Haard, N.F. Purification and characterisation of trypsin from the greenland cod (Gadus ogac). 1. Kinetic and thermodynamic characteristics. Can. J. Bio-chem. Cell Biol. 62: 894 - 900, 1984.
42. Raae, A.J., Walther, B.T. Purification and characterisation of chymotrypsin, trypsin and elastase like proteinases from cod (Gadus morhua L.). Comp. Biochem. Physiol. 93B: 317 - 324, 1989.
43. Asgeirsson, B., Fox, J.W., Bjarnason, J.B. Purification and characterisation of trypsin from the poikilotherm Gadus morhua. Euro. J. Biochem. 180: 85 - 94, 1989.
44. Neas, N.P., Hazel, J.R. Temperature-dependent deacylation of molecular species of phosphatidylcholine by microsomal phospholipase A2 of thermally acclimated rainbow trout, Oncorhynchus mykiss. Lipids. 19: No.4, 258- 262, 1984.
45. Sheridan, M.A., Allen, W.V. Partial purification of a triacylglycerol lipase isolated from steelhead trout (Salmo gairdneri) adipose tissue. Lipids. 19: No. 5, 347 - 352, 1984.
46. Neas, N.P., Hazel, J.R. Phospholipase A2 from liver microsomal membranes of thermally acclimated rainbow trout. The Journal of Experimental Zoology. 233: 51-60, 1985.
47. Neas, N.P., Hazel, J.R. Partial purification and kinetic characterisation of the microsomal phospholipase A2 from thermally acclimated rainbow trout (Oncorhynchus mykiss). J. Comp. Physiol B. 155: 461-469, 1985.
48. Gjellesvik, D.R., Lambordo, D., Walther, B.T. Partial purification and characterisation of a triglyceride lipase from cod (Gadus morhua). Aquaculture. 79: 177-184, 1989.
49. Brahimi-Horn, M-C., Guglielmino, M.L., Sparrow, L.G., et al. Lipolytic en-zymes of the digestive organs of the crown-of-thorns starfish (Acanthaster planci): Comparison of the stomach and pyloric caeca. Comp. Biochem. Physiol. 92B: No. 4, 637-643, 1989.
50. Campell, C.M., Duncan, D., Price, N.C., Stevens, L. The secretion of amylase, phospholipase and protease from Aeromonas salmonicida, and the correlation with membrane-associated ribosome. Journal of Fish Diseases. 13: 463-474, 1990.
51. Harmon, S.J., Michelsen, G.K., Sheridan, A.M. Purification and characterisation of hepatic triacylglycerol lipase isolated from rainbow trout, Oncorhynchus mykiss. Fish Phy. Biochem. 9; 4, 360-368, 1991.
52. Gjellesvik, D.R., Lambordo, D., Walther, B.T. Pancreatic bile saltdependent lipase from Cod, Gadus morhua: Purification and properties. Biochimica et Biophysica Acta. 1124; 123 - 134, 1992.
53. Gjellesvik, D.R. Fatty acid specificity of bile salt-dependent lipase: enzyme recog-nition and super substrate effects. Biochimica et Biophysica Acta.1086: 167-172, 1991.
54. Leger, C., Bauchart, D., Flanzy, J. Some properties of pancreatic lipase in Oncorhynchus mykiss: Km, effects of bile salts and Ca++, gel filtration. Comp. Biochem. Physiol. 57B: 359-363, 1997.
55. Munkundan, M.K., Gopakumar, K., Nair, M.R. Purification of a lipase from the hepatopancreas of oil sardine (Sardinella longiceps Linnaeus) and its characteristics and properties. J. Sci. Food Agric. 36: 191-203, 1985.
56. Polson, A., Potgieter, G.M., Largier, J.M., Mears, G.E.F., Joubert, F.J. The fractionation of protein mixtures by linear polymers of high molecular weight. Biochimica et Biophysica Acta. 82: 463- 475. 1964.
57. Değerli, N., Akpınar, M.A. Cyprinion macrostomus Heckel'den karaciğer li-pazğnğn saflaştırılması ve pH'nın enzim aktivitesi üzerine etkisi. S.D.Ü. Eğirdir Su ürünleri Fak. Der. 4: 1 - 10, 1995.
58. Peled, N., Krenz, M. A new assay of microbial lipases with emulsified trioleoyl glycerol. Analytical Biochemistry. 112: 219- 222, 1981.
59. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randal, J. Protein measurement with the folin-phenol reagent. J. Biol. Chem. 193: 265-275, 1951.
60. Parish, S.H., Marchalonis, J.J. Polyacrylamide disc gel electrophoresis. Anal. Biochem. 34: 436. 1970.
61. SPSS Inc, SPSS for Windows release 6.0 444 North Michigan Avenue, Chicago, IL 606111, 1993.
62. Scopes, K.R. Protein purification. Principles and practice, New York, 1982. Springer-Verlag, New York Inc.
63. Roe, S. Protein Purification Methods, A practical approach; Separation based on structure. Harris, E.L.V., Angal, S. Oxford, 1995, IRL Press at Oxford University Press, Chapter 4, 175-242.
64. Taipa, M.A., AiresÐBarros, M.R., Capral, J.M. Purification of Lipases. Biochimica et Biophysica Acta. 1124: 111 - 142, 1992.
65. Değerli, N., Hsiung, K.P. Purification of lipase from fish liver for biotechnologi-cal purpose. IECDF, UCL Biotechnology Workshop Report. IECDF, UCL Biotechnol-ogy Laboratory, Hsinchu, Taiwan R.O.C. 1992. Unpublished.
66. Takahashi, K., Saito, Y., Inada, Y. Lipase made active in hydrophobic media, Jaocs. 65: no. 6, 911- 915. 1988.
67. Roe, S. Separation based on structure: Protein purification methods, A practical approach. Harris, E.L.V., Angal, S. IRL Press at Oxford University Press. 1995. Chapter 4, 175-244.
68. Berglund, L., Khoo, J.C., Jensen, D., Steinberg, D. Resolution of hormone-sensitive triglyceride/diglyceride lipase from monoglyceride lipase of chicken adipose tissue. Biochimica et Biophysica Acta. 255: 5420-5428. 1980.
69. Belfrage, P., Jergil, B., Stralfors, P., Tornqvist, H. Hormonesensitive lipase of rat adipose tissue; identification and some properties of the enzyme protein. Febs lett. 75: 259-264. 1977.
70. Khoo, J.C., Berglund, L., Jensen, D., Steinberg, D. Hormonesensitive lipase of rat adipose tissue: correlation of activity with a protein of molecular weight 84 000. Biochimica et Biophysica Acta. 619: 440-444. 1980.
71. Fredrikson, G., Stralfors, P., Nilsson, N. O., Belfrage, P. Hormonesensitive li-pase of rat adipose tissue: purification and some properties. J. Biol. Chem. 256: 6311-6320. 1981.
72. Khoo, J.C., Steinberg, D., Huang, J.J., Vagelos, P.R. Triglyceride, diglyceride, monoglyceride and cholesterol ester hydrolyses in chicken adipose tissue activated by adenosine 3: 5- monophosphate-dependent protein kinase. J. Biol. Chem. 251: 2882-2890. 1976.