Purification and Characterization of Lipase from the Liver of Carp, Cyprinus carpio L. (1758), Living in Lake Tödürge (Sivas, Türkiye)

Bu çalışmada, Tödürge Gölü’nde yaşayan Cyprinus carpio Linnaeus (1758)’nun karaciğerinden 90.38 μmol/dk.mg protein spesifik aktivite ve 75,50 katlık saflaştırma parametreleri ile bir lipaz saflaştırılmıştır. Saflaştırma prosedürü homojenatın hazırlanması, poletilen glikol-6.000 (PEG-6000) ile çöktürme ve Q sefaroz, sefakril S 200 HR, fenil sefaroz CL- 4B’yi kapsayan kromatografik tekniklerden oluşmuştur. Saflaştırılmış lipaz, sodyum dodesil sülfat-poliakrilamit jel elektroforezi (SDS-PAGE) ile yaklaşık 74 kDa’luk bir molekül ağırlığı ile tek bant göstermiştir. Optimum pH ve sıcaklık, substrat olarak p-nitrofenil butirat (p-NPB) kullanarak sırasıyla 8,0 ve 37°C olarak belirlenmiştir. Km ve Vmax değerleri, sırasıyla, 0,17 mM p-NPB ve 2,6 μmol/ml.dk olarak bulunmuştur. Sodyum dodesil sülfat (SDS), Triton X-100 ve Nataurokolat gibi yüzey aktif maddelerin lipaz aktivitesi üzerinde inhibitor olarak etki ettikleri gözlenmiştir.

Anahtar Kelimeler:

SDS-PAGE, sazan balığı, Türkiye

Tödürge Gölü (Sivas, Türkiye)’nde yaşayan Cyprinus carpio Linnaeus (1758)’nun karaciğerindeki lipaz’ın saflaştırılması ve karakterizasyonu

In this study, a lipase from the liver of Cyprinus carpio Linnaeus (1758) living in Tödürge Lake (Sivas) was purified with purification parameters of 90.38 μmol/dk.mg protein specific activity and 75.50 fold purity. The purification procedure consisted of preparation of homogenate, precipitation with polyethylene glycol-6000 (PEG-6000) and chromatographic techniques including in Q sepharose, sephacryl S 200 HR and phenyl sepharose CL-4B, respectively. The purified lipase showed a single band with approximately a molecular weight of 74 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). pH and temperature optimums for the purified enzyme were determined as 8.0 and 37°C using p-nitrophenyl butirate (p-NPB) as substrate. Km and Vmax values were found as 0.17 mM p-NPB and 2.6 μmol/ml.dk, respectively. It was observed that surface active agents, such as sodium dodecyl sulfate (SDS), Triton X-100 and Nataurocholate act as inhibitor on lipase activity.

Keywords:

SDS-PAGE, carp, Turkey,

PDF

___

Amara, S., Fendri, A., Ben Salem, N., Gargouri, Y. and Miled, N. 2010. Snail hepatopancreatic lipase: A new member of invertebrates lipases’ group. Applied Biochemistry and Biotechnology, 162: 942-952. doi: 10.1007/s12010-009-8837-z
Arrese, E.L. and Wells, M.A. 1994. Purification and properties of a phosphorylatable triacylglycerol lipase from the fat body of an insect, Manduca sexta. Journal of Lipid Research, 35: 1652-1660.
Aryee, A.N.A., Simpson, B.K. and Villalonga, R. 2007. Lipase fraction from viscera of grey mullet (Mugil cephalus). Isolation, partial purification and some biochemical characteristics. Enzyme and Microbial Technology, 40: 394-402. doi: 10.1016/j.enzmictec.2006.07.009
Beisson, F., Tiss, A., Riviere, C. and Verger, R. 2000. Methods for lipase detection and assay: a critical review. European Journal of Lipid Science and Technology, 133-153. doi: 10.1002/(SICI)1438-9312(200002)102:2_____133::AID-EJLT133>3.0.CO;2-X
Bollag, D.M., Rozycki, M.D. and Edelstein, S.J. 1996. Protein methods, A John Wıley and Sons, Inc., Publication, 57-82.
Bülow, L. and Mosbach, K. 1987. The expression in E. coli of a polymeric gene coding for an esterase mimic catalyzing the hydrolysis of p-nitrophenyl esters. FEB, 210(2): 147-152.
Cherif, S., Fendri, A., Miled, N., Trabelsi, H., Mejdoub, H. and Gargouri, Y. 2007. Crab digestive lipase acting at high temperature: Purification and biochemical characterization. Biochimie, 89: 1012-1018. doi: 10.1016/j.biochi.2007.02.005
Diogo, M.M., Silva, S., Cabral, J.M.S. and Queiroz, J.A. 1999. Hyrophobic interaction chromatography of Chromobacterium viscosum lipase on polypropylene glycol immobilized on sepharose. Journal of Chromatography, 849(A): 413-419. doi: 10.1016/S0021-9673(99)00619-6
Ferrer, M., Plou, F.J., Nuero, O.M., Reyes, F. and Ballesteros, A. 2000. Purification and properties of a lipase from Penicillium chrysogenum isolated from industrial wastes. Journal of Chemical Technology and Biotechnology, 75: 569-576. doi: 10.1002/1097-4660(200007)75:7_____569::AID-JCTB258>3.0.CO;2-S
Gilham, D. and Lehner, R. 2005. Techniques to measure lipase and esterase activity in vitro. Methods, 36: 139-147. doi: 10.1016/j.ymeth.2004.11.003 Gjellesvik, D.R. 1991. Enzymatic lipid digestion in teleosts: bile salt-dependent lipase as mojor lipolytic enzyme in cod pancreas. PhD thesis, Bergen: Norway, University of Bergen.
Gjellesvik, D.R., Lombardo, D. and Walther, B.T. 1992. Pancreatic bile salt dependent lipase from cod (Gadus morhua): purification and properties. Biochimica Biophysica Acta, 1124(2): 123-134.
Harmon, J.S., Michelsen, K.G. and Sheridan, M.A. 1991. Purification and characterization of hepatic triacylglycerol ipase isolated from rainbow trout, Oncorhynchus mykiss. Fish Physiology and Biochemistry, 9 (4): 361-368. doi: 10.1007/BF02265156
Hoehne-Reitan, K., jorsvik, E. and Gjellesvik, D.R. 2001 a. Development of bile salt-activated lipase in larval turbot. Journal of Fish Biology, 58: 737-745 doi: 10.1111/j.1095-8649.2001.tb00526.x
Hoehne-Reitan, K., Kjorsvik, E. and Reitan, K.I. 2001b. Bile salt-dependent lipase in larval turbot, as influenced by density and lipid content of fed prey. Journal of Fish Biology, 58: 746-754. doi: 10.1111/j.1095-8649.2001.tb00527.x
Hoehne-Reitan, K., Kjorsvik, E. and Reitan, K.I. 2003. Lipolytic activities in developing turbot larvae as influenced by diet. Aquaculture International, 11: 477-489. doi: 10.1023/B:AQUI.0000004192.36467.0d
Iijima, N., Tanaka, S. and Ota, Y. 1998. Purification and characterization of bile salt-activated lipase from the hepatopancreas of red sea bream, Pagrus major. Fish Physiology and Biochemistry, 18: 59-69. doi: 10.1023/A:1007725513389
Islam, M.A., Parveen, F., Hossain, K., Khatun, S., Karim, Md.R., Kim, G.S., Absar, N. and Haque, Md.S. 2009. Purification and biochemical characterization of lipase from the dorsal part of Cirrhinus reba. Thai Journal of Agricultural Science, 42(2): 71-80.
Izquierdo, M.S. and Henderson, R.J. 1998. The determination of lipase and phospholipase activities in gut contents of turbot (Scophthalmus maximus) by fluorescence-based assays. Fish Physiology and Biochemistry, 19: 153-162. doi: 10.1023/A:1007734425931
Kurtovic, I., Marshall, S.N., Zhao, X. and Simpson, B.K. 2009. Lipases from mammals and fishes. Reviews in Fisheries Science, 17(1): 18-40. doi: 10.1080/10641260802031322
Kurtovic, I., Marshall, S.N., Zhao, X. and Simpson, B.K. 2010. Purification and properties of digestive lipases from Chinook salmon (Oncorhynchus tshawytscha) and New Zealand hoki (Macruronus novaezelandiae). Fish Physiology and Biochemistry, 36(4): 1041-1060. doi: 10.1007/s10695-010-9382-y
Laemmli, U.K. 1970. Cleavage of structural proteins during assembly of the head of bacteriopage T4. Nature, 227: 680-685. doi: 10.1038/227680a0
Lima, V.M.G., Krieger, N., Mitchell, D.A. and Fontana, J.D. 2004. Activity and stability of a crude lipase from Penicillium aurantiogriseum in aqueous media and organic solvents. Biochemical Engineering Journal, 18(1): 65-71. doi: 10.1016/S1369-703X(03)00165-7
L’opez-Amaya, C. and Marangoni, A.G. 2001. Lipases: Seafood Enzymes. In: N.F. Haard and B.K Simpson, (Eds.), Utilization and Influence on Postmortem Fish Quality. Marcel Dekker; New York:121-146.
Metin, K. and Akpınar, M.A. 2000. Oncorhynchus mykiss Walbaum, 1792 karaciğer lipaz enziminin bazı kinetik özellikleri. In Turkish. Turkish Journal of Biology, 24: 489-502.
Mogensen, J.E., Sehgal, P. and Otzen, D.E. 2005. Activation, inhibition, and destabilization of Thermomyces lanuginosus lipase by detergents. Biochemistry, 44(5): 1719-1730. doi: 10.1021/bi0479757
Mukundan, M.K., Gopakumar, K. and Nair, M.R. 1985. Purification of a lipase from the hepatopancreas of oil sardine (Sardinella longiceps Linnaeus) and its characteristics and properties. Journal of the Science of Food and Agriculture, 36: 191-203. doi: 10.1002/jsfa.2740360310
Noriega-Rodriguez, J.A., Gamez-Meza, N., Alanis-Villa, A., Medina-Juarez, L.A., Tejeda-Mansir, A., Angulo-Guerrero, O. and Garcia, H.S. 2009. Extraction and fractionation of lipolytic enzyme from viscera of Monterey sardine (Sardinops sagax caerulea). International Journal of Food Science and Technology, 44: 1223-1228. doi: 10.1111/j.1365-2621.2009.01951.x
Palekar, A.A., Vasudevan, P.T. and Yan, S. 2000. Purification of lipase: A review. Biocatalysis and Biotransformation, 18: 177-200. doi: 10.3109/10242420009015244
Reetz, M.T. 2002. Lipases as practical biocatalysts. Current Opinion in Chemical Biology, 6: 145-150. doi: 10.1016/S1367-5931(02)00297-1
Rivera-Perez, C., Garcia-Carreno, F.L. and Saborowski, R. 2010. Purification and biochemical characterization of digestive lipase in Whiteleg Shrimp. Marine Biotechnology, 13(2): 284-295. doi: 10.1007/s10126-010-9298-7
Smichi, N., Fendri, A., Chaabouni, R., Ben Rebah, F., Gargouri, Y. and Miled, N. 2010. Purification and biochemical characterization of and acid-stable lipase from the pyloric caeca of Sardine (Sardinella aurita). Applied Biochemistry and Biothecnology, 162: 1483-1496. doi: 10.1007/s12010-010-8920-5
Unver, B. and Erk’akan, F. 2005. A natural hybrid of Leuciscus cephalus (L.) and Chalcalburnus calcoides (Güldenstad) (Osteichthyes-Cyprinidae) from Lake Tödürge (Sivas, Turkey). Journal of Fish Biology, 66: 899-910. doi: 10.1111/j.0022-1112.2005.00610.x