Full Article
Purification and Characterization of a Trypsin-Like Protease from Flatfish (Paralichthys olivaceus) Intestine
Misook Kim,
Yoonhwa Jeong,
Misook Kim
Department of Food Science and Nutrition, Dankook University, 126 Jukjeon-Ro, Suji-gu, Yongin, Gyeonggi-Do, 448-701 Korea
Search for more papers by this authorCorresponding Author
Yoonhwa Jeong
Department of Food Science and Nutrition, Dankook University, 126 Jukjeon-Ro, Suji-gu, Yongin, Gyeonggi-Do, 448-701 Korea
Corresponding author. TEL: 82-31-8005-3176; FAX: 82-31-8005-4054; EMAIL: [email protected]Search for more papers by this authorMisook Kim,
Yoonhwa Jeong,
Misook Kim
Department of Food Science and Nutrition, Dankook University, 126 Jukjeon-Ro, Suji-gu, Yongin, Gyeonggi-Do, 448-701 Korea
Search for more papers by this authorCorresponding Author
Yoonhwa Jeong
Department of Food Science and Nutrition, Dankook University, 126 Jukjeon-Ro, Suji-gu, Yongin, Gyeonggi-Do, 448-701 Korea
Corresponding author. TEL: 82-31-8005-3176; FAX: 82-31-8005-4054; EMAIL: [email protected]Search for more papers by this authorAbstract
enA trypsin-like protease was purified from the intestine of flatfish (Paralichthys olivaceus) by gel filtration and anion-exchange chromatography. The molecular weight was estimated to be 29.6 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Flatfish protease had maximal activity at 70C and pH 7.5 using N-α-benzoyl-dl-arginine-ρ-nitroanilide as substrate. It was stable to heat treatment up to 50C and to pH ranges between 7.0 and 10.0. It was activated by calcium ion and completely inhibited by mercury ion and known serine-protease inhibitors, such as phenylmethylsulfonyl fluoride, tosyl lysine chloromethyl ketone and benzamidine.
Practical Applications
frFlatfish trypsin could be an alternative to commercial trypsins in a wide variety of applications in the food, detergent and pharmaceutical industries. In the food industry, trypsin is used to produce protein hydrolysates, extract flavor, tenderize meat and improve oil extractions. It is also used in detergent, bating of leather and in diabetes diagnosis and therapy. Moreover, it can break down tumors and cancer cells in cancer therapy.
References
- Ali, N.E.H., Hmidet, N., Bougatef, A., Nasri, R. and Nasri, M. 2009. A laundry detergent-stable alkaline trypsin from striped seabream (Lithognathus mormyrus) viscera: Purification and characterization. J. Agric. Food Chem. 57, 10943–10950.
- Aranishi, F., Watanabe, T., Osatomi, K., Cao, M., Hara, K. and Ishihara, T. 1998. Purification and characterization of thermostable dipeptidase from carp intestine. J. Mar. Biotechnol. 6, 116–123.
- Asgeirsson, B., Fox, J.W. and Bjarnason, J.B. 1989. Purification and characterization of trypsin from the poikilotherm Gadus morhua . Eur. J. Biochem. 180, 85–94.
- Barkia, A., Bougatef, A., Nasri, R., Fetoui, E., Balti, R. and Nasri, M. 2010. Trypsin from the viscera of bogue (Boop boops): Isolation and characterization. Fish Physiol. Biochem. 36, 893–902.
- Barrett, A.J. 1994. Classification of peptidases. In Methods in Enzymology, ( A.J. Barrett, ed.) pp. 1–59, Academic Press, New York, NY.
- Bezerra, R.S., Santos, J.S., Paiva, P.M.G., Correia, M.T.S., Coelho, L.C.B., Vieira, V.L.A. and Carvalho, L.B., Jr 2001. Partial purification and characterization of a thermostable trypsin from pyloric caeca of Tambaqui (Colossoma macropomum). J. Food Biochem. 25, 199–209.
- Bezerra, R.S., Lins, E.J.F., Alencar, R.B., Paiva, P.M.G., Chaves, M.E.C., Coelho, L.C.B.B. and Carvalho, L.B., Jr 2005. Alkaline proteinase from intestine of Nile tilapia (Oreochromis niloticus). Proc. Biochem. 40, 1829–1834.
- Bougatef, A., Souissi, N., Fakhfakh, N., Ellouz-Triki, Y. and Nasri, M. 2007. Purification and characterization of trypsin from the viscera of sardine (Sardina pilchardus). Food Chem. 102, 343–350.
- Bougatef, A., Balti, R., NASRI, R., Jellouli, K., Souissi, N. and Nasri, M. 2010. Biochemical properties of anionic trypsin acting at high concentration of NaCl purified from the intestine of a carnivorous fish: Smooth hound (Mustelus mustelus). J. Agric. Food Chem. 58, 5763–5769.
- Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.
- Cao, M., Osatomi, K., Suzuki, M., Hara, K., Tachibana, K. and Ishihara, T. 2000. Purification and characterization of two anionic trypsins from the hepatopancreas of carp. Fish Sci. 66, 1172–1179.
- Castillo-Yanez, F.J., Pacheco-Aguilar, R., Garcia-Carreno, F.L. and Toro, M.A.N. 2005. Isolation and characterization of trypsin from pyloric caeca of Monterey sardine Sardinops sagax caerulea . Comp. Biochem. Physiol. 140B, 91–98.
- Chong, A.S.C., Hashim, R., Chow-Yang, L. and Ali, A.B. 2002. Partial characterization and activities of proteases from the digestive tract of discus fish (Symphysodon aequifasciata). Aquaculture 203, 321–333.
- Erlanger, B.F., Kokowsky, N. and Cohen, W. 1961. The preparation and properties of two new chromogenic substrates of trypsin. Arch. Biochem. Biophys. 95, 271–278.
- Fuchise, T., Kishimura, H., Sekizaki, H., Nonami, Y., Kanno, G., Klomklao, S., Benjakul, S. and Chum, B. 2009. Purification and characteristics of trypsins from cold-zone fish, Pacific cod (Gadus macrocephalus) and saffron cod (Eleginus gracilis). Food Chem. 116, 611–616.
- Godfrey, T. and Reichelt, J.R. 1983. Introduction to industrial enzymology. In Industrial Enzymology – the Application of Enzymes in Industry, ( T. Godfrey and J. Reichelt, eds.) p. 1, The Nature Press, New York, NY.
- Gudmundsdóttir, A. and Pálsdóttir, H.M. 2005. Atlantic cod trypsins: From basic research to practical applications. Mar. Biotechnol. 7, 77–88.
- Guizani, N., Rolle, R.S., Marshall, M.R. and Wei, C.I. 1991. Isolation, purification and characterization of a trypsin from the pyloric caeca of mullet (Mugil cephalus). Comp. Biochem. Physiol. 98B, 517–521.
- Hjelmeland, K. and Raa, J. 1982. Characteristics of two trypsin isozymes isolated from the arctic fish capelin (Mallotus villosus). Comp. Biochem. Physiol. 71B, 557–562.
- Hwang, J.Y., Hirono, I. and Aoki, T. 2007. Cloning and expression of a novel serine protease from Japanese flounder, Paralichthys olivaceus . Dev. Comp. Immunol. 31, 587–595.
- Kanno, G., Yamaguchi, T., Kishimura, H., Yamaha, E. and Saeki, H. 2010. Purification and characteristics of trypsin from masu salmon (Oncorhynchus masou) cultured in fresh-water. Fish Physiol. Biochem. 36, 637–645.
- Keil, B. 1971. Trypsin. In The Enzymes, ( P.D. Boyer, ed.) pp. 250–275, Academic Press, New York, NY.
- Khaled, H.B., Bougatef, A., Balti, R., Triki-Ellouz, Y., Souissi, N. and Nasri, M. 2008. Isolation and characterization of trypsin from sardinelle (Sardinella aurita) viscera. J. Sci. Food Agric. 88, 2654–2662.
- Kim, H.R., Meyer, S.P. and Godber, J.S. 1992. Purification and characterization of anionic trypsins from the hepatopancreas of crayfish, Procambarus clarkii . Comp. Biochem. Physiol. 103B, 391–398.
- Kishimura, H. and Hayashi, K. 2002. Isolation and characteristics of carboxypeptidase B from the pyloric ceca of the starfish Asterias amurensis . Comp. Biochem. Physiol. 133B, 183–189.
- Kishimura, H., Hayashi, K., Miyashita, Y. and Nonami, Y. 2005. Characteristics of two trypsin isozymes from the viscera of Japanese anchovy (Engraulis japonica). J. Food Biochem. 29, 459–469.
- Kishimura, H., Hayashi, K., Miyashita, Y. and Nonami, Y. 2006a. Characteristics of trypsins from the viscera of true sardine (Sardinops melanostictus) and the pylotic ceca of arabesque greenling (Pleurogrammus azonus). Food Chem. 97, 65–70.
- Kishimura, H., Tokuda, Y., Klomklao, S., Benjakul, S. and Ando, S. 2006b. Comparative study of enzymatic characteristics of trypsins from the pyloric ceca of yellow tail (Seriola quinqueradiata) and brown hakeling (Physiculus japonicus). J. Food Biochem. 30, 521–534.
- Kishimura, H., Tokuda, Y., Klomklao, S., Benjakul, S. and Ando, S. 2006c. Enzymatic characteristics of trypsin from pyloric ceca of spotted mackerel (Scomber australasicus). J. Food Biochem. 30, 466–477.
- Kishimura, H., Klomklao, S., Benjakul, S. and Chun, B. 2008. Characteristics of trypsin from the pyloric ceca of walleye pollock (Theragra chalcogramma). Food Chem. 106, 194–199.
- Kishimura, H., Klomklao, S., Nalinanon, S., Benjakul, S., Chun, B. and Adachi, K. 2010. Comparative study of thermal stability of trypsin from the pyloric ceca of threadfin hakeling (Laemonema longipes). J. Food Biochem. 34, 50–65.
- Klomklao, S. 2008. Digestive proteinases from marine organisms and their applications. Songklanakarin J. Sci. Technol. 30, 37–46.
- Klomklao, S., Benjakul, S., Visessanguan, W., Kishimura, H. and Simpson, B.K. 2006. Purification and characterization of trypsin from the spleen of tongol tuna (Thunnus tonggol). J. Agric. Food Chem. 54, 5617–5622.
- Klomklao, S., Benjakul, S., Visessanguan, W., Kishimura, H. and Simpson, B.K. 2007a. Trypsin from the pyloric caeca of bluefish (Pomatomus saltatrix). Comp. Biochem. Physiol. 148B, 382–389.
- Klomklao, S., Benjakul, S., Visessanguan, W., Kishimura, H. and Simpson, B.K. 2007b. Purification and characterization of trypsins from the spleen of skipjack tuna (Katsuwonus pelamis). Food Chem. 100, 1580–1589.
- Klomklao, S., Kishimura, H., Benjakul, S., Simpson, B.K. and Visessanguan, W. 2010. Cationic trypsin: A predominant proteinase in Pacific saury (Cololabis saira) pyloric ceca. J. Food Biochem. 34, 1105–1123.
- Korea National Statistics Office (KNSO). 2011. Statistics Korea. Fishery Statistics. Korea.
- Kristjansson, M.M. 1991. Purification and characterization of trypsin from the pyloric caeca of rainbow trout (Oncorhynchus mykiss). J. Agric. Food Chem. 39, 1738–1742.
- Kristjansson, M.M. and Nielsen, H.H. 1992. Purification and characterization of two chymotrypsin-like proteases from the pyloric caeca of rainbow trout (Oncorhynchus mykiss). Comp. Biochem. Physiol. B 101, 247–253.
- Laemmli, U.K. 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680–685.
- Martinez, A., Olsen, R.L. and Serra, J.L. 1988. Purification and characterization of two trypsin-like enzymes from the digestive tract of anchovy Engraulis encrasicolus . Comp. Biochem. Physiol. 91B, 677–684.
-
Murakami, K. and Noda, M. 1981. Studies on proteinases from the digestive organs of sardine. I. Purification and characterization of three alkaline proteinases from the pyloric caeca. Biochim. Biophys. Acta 65B, 17–26.
10.1016/0005-2744(81)90245-X Google Scholar
- Palmer, T. 1995. The investigation of active site structure. Understanding Enzymes, 4th Ed., ( T. Palmer, ed.) pp. 176–179, Horwood, London.
- Pavlisko, A., Rial, A., De Vecchi, S. and Coppes, Z. 1997. Properties of pepsin and trypsin isolated from the digestive tract of Parona signata “Palometa.” J. Food Biochem. 21, 289–308.
- Pollock, M.R. 1965. Purification and properties of penicillinases from two stains of Bacillus licheniformis: A chemical, physico-chemical and physiological comparison. Biochem. J. 94, 666–675.
- Raa, A.J. and Walther, W.T. 1989. Purification and characterization of chymotrypsin, trypsin and elastase like proteinases from cod (Gadus morhua). Comp. Biochem. Physiol. B 93, 317–324.
- Rypniewski, W., Perrakis, A., Vorgias, C.E. and Wilson, K. 1994. Evolutionary divergence and conservation of trypsin. Protein Eng. 7, 57–64.
- Sekizaki, H., Itoh, K., Murakami, M., Toyota, E. and Tanizawa, K. 2000. Anionic trypsin from chum salmon: Activity with p-amidinophenyl ester and comparison with bovine and Streptomyces griseus trypsins. Comp. Biochem. Physiol. 127B, 337–346.
- Silva, J.F., Esposito, T.S., Marcuschi, M., Ribeiro, K., Cavalli, R.O., Oliveira, V. and Bezerra, R.S. 2011. Purification and partial characterization of a trypsin from the processing waste of the silver mojarra (Diapterus rhombeus). Food Chem. 129, 777–782.
- Simpson, B.K. 2000. Digestive proteinases from marine animals. In Seafood Enzymes: Utilization and Influence on Postharvest Seafood Quality, ( N.F. Haard and B.K. Simpson, eds.) pp. 119–124, Marcel Dekker, New York, NY.
- Simpson, B.K. and Haard, N.F. 1984. Purification and characterization of trypsin from the Greenland cod (Gadus ogac). I. Kinetic and thermodynamic characteristics. Can. J. Biochem. Cell Biol. 62, 894–900.
- Simpson, B.K. and Haard, N.F. 1985. Characterization of trypsin fraction from cunner (Tautogolabrus adspersus). Comp. Biochem. Physiol. 80B, 475–480.
- Smalas, A.O., Heimstad, E.S., Hordvik, A., Willassen, N.P. and Male, R. 1994. Cold adaptation of enzymes: Structural comparison between salmon and bovine trypsins. Proteins 20, 149–166.
- Souza, A.A.G., Amaral, I.P.G., Santo, A.R.E., Carvalho, L.B., Jr and Bezerra, R.S. 2007. Trypsin-like enzyme from intestine and pyloric caeca of spotted goatfish (Pseudupeneus maculatus). Food Chem. 100, 1429–1434.
- Wang, Q., Gao, Z., Zhang, N., Shi, Y., Xie, X. and Chen, Q. 2010. Purification and characterization of trypsin from the intestine of hybrid tilapia (Oreochromis niloticus x O. aureus). J. Agric. Food Chem. 58, 655–659.
- Wasserman, B.P. 1984. Thermostable enzyme production. Food Technol. 38, 78–88.
- Weber, K. and Osborn, M. 1969. The reliability of molecular weight determinations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406–4412.
- Whitaker, J.R. 1994. Introduction. In Principles of Enzymology for the Food Sciences, 2nd Ed., ( J.R. Whitaker ed.) pp. 1–25, Marcel Dekker, New York, NY.
- Zhou, L., Ruan, M., Cai, Q., Liu, G., Sun, L., Su, W. and Cao, M. 2012. Purification, characterization and cDNA cloning of a trypsin from the hepatopancreas of snakehead (Channa argus). Comp. Biochem. Physiol. 161, 247–254.
