Biochemical characterization of lysozyme extracted from Caspian kutum, Rutilus kutum, Kamensky 1901

Document Type: Research Paper


1 Department of Biology, Faculty of Science, University of Guilan, Rasht, Iran

2 Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran

3 Department of Physics, Faculty of Science, Payame Noor University, Theran, Iran


This paper presents the findings of a basic study on biochemical characteristics and activity of lysozyme extracted from a commercially important fish, Caspian kutum, Rutilus kutum, Kamensky 1901. The enzyme was purified by the ammonium sulphate precipitation method and cationic exchange chromatography. Molecular weight of lysozyme was estimated as 15.8kDa by SDS-PAGE. The optimum pH and temperature were 6.0 and 45.0°C, respectively,while the Km and Vmax values were 0.00007g mL-1 and 200units min-1, respectively. Thermo-stability of the enzyme was low at the temperatures higher than 50°C. The enzyme activity increased in the presence of NaCl (1-50mM), KCl (1-50mM) and CrCl2 (1-5mM), then decreased at the higher concentrations. Furthermore, the enzyme activity significantly decreased in the presence of FeCl2 and CuCl2 (1-100mM), but not stable in the presence of MgCl2. SDS inhibited the activity, but the enzyme exhibited a good degree of resistance to urea. Fluorescence quenching was observed in the presence of FeCl2 and CuCl2. Emission intensities in the presence of NaCl, KCl, CrCl2 and SDS were elevated, while was negligible for the urea. Our results exhibitedthat NaCl and KCl are well-established salts for elevating the enzyme activity.However, the purified lysozyme did not display a moderate stability against others salts. Besides, the purified lysozyme from R. kutum was not heat stable. Therefore, some phenomena such as water hardness and warming can exert negative effects on the innate immunity of the fish. So, the enzyme structure can be made more stable by medium engineering through changing the salt composition of aqueous solution.


Buonocore, F, Randelli, E, Trisolino, P, Facchiano, A,  Pascale, D&Scapigliati, G 2014, Molecular characterization, gene structure and antibacterial activity of a g-type lysozyme from the European sea bass (Dicentrarchus labrax L). Molecular Immunology, 62:10-18.

Di Stasio, E, Bizzarri, P, Misiti, F, Pavoni, E&Brancaccio, A 2004, A fast and accurate procedure to collect and analyze unfolding fluorescence signal: the case of dystroglycan domains. Biophysical Chemistry, 107:197–211.

Fu, GH, Bai, ZY, Xia, JH, Liu, F, Liu, P&Yue, GH 2013, Analysis of two lysozyme genes and antimicrobial functions of their recombinant proteins in Asian seabass. Public Library of Science One 8:1-12.

Grinde, B, Jollès, J, Jollès, P 1988, Purification and characterization of two lysozymes from rainbow trout (Salmo gairdneri), European Journal of Biochemistry, 173: 269–273.

Hikima, JI, Minagawa, S, Hirono, I&Aoki, T 2001, Molecular cloning, expression and evolution of the japanese flounder goose-type lysozyme gene, and the lytic activity of its recombinant protein. Biochimica et Biophysica Acta 1520(1):35–44.

Hikima, S, Hikima, J, Rojtinnakorn, J, Hirono, I&Aoki T 2003, Characterization and function of kuruma shrimp lysozyme possessing lytic activity against Vibrio species. Gene, 316:187-195.

Kim, M, Park, M&Jeong, Y 2012, Purification and characterization of lysozyme from filipino venus, Ruditapes philippinarum. Food Science and Biotechnology, 21:1463–1468.

 Klomklao, S, Benjakul, S, Visessanguan, W, Kishimura, H, Simpson, BK& Saeki, H 2006, Trypsins from yellowfin tuna (Thunnus albacores) spleen: Purification and characterization. Comparative BiochemistryPhysiology, 144: 47-56.

Larsen,AN, Solstad, T, Svineng, G, Seppola, M&Jørgensen, T 2009, Molecular characterisation of a goose-type lysozyme gene in Atlantic cod (Gadus morhua L.); Fish and ShellfishImmunology, 26 122–132.

Lie, Ø, Evensen Ø, Sørensen, A, Frøysadal&E 1989, Study on lysozyme activity in some fish species. Diseases of Aquatic Organisms, 6:1–5.

Lockey, TD&Ourth, DD 1996, Purification and characterization of lysozyme from hemolymph of Heliothis virescens larvae. Biochemical and Biophysical ReserchCommunications, 220: 502-508.

Mai, W& Hu, C 2009, cDNA cloning, expression and antibacterial activity of lysozyme C in the blue shrimp (Litopenaeus stylirostris). Progress in Natural Science, 19:837–844.

Nilsen, IW, Overbo, K, Sandsdalen, E, Sandaker, E, Sletten, K&Bjornar, M 1999, Protein purification and gene isolation of chlamysin, a cold active lysozyme-like enzyme with antibacterial activity. Federation of European Biochemical Societies Letters, 464:153-158.

Purich D 2010, Enzyme Kinetics: Catalysis & Control: A Reference of Theory and Best-practice Methods, Elsevier Science, eBook ISBN: 9780123809254.

Saurabh, S & Sahoo, PK 2008, Lysozyme: an important defence molecule of fish innate immune system. Aqua culture Research, 39: 223–239.Shugar, D 1952, The measurement of lysozyme activity and the ultra-violet inactivation of lysozyme. Biochimica et Biophysica Acta, 8(3):302-309.

Callwaert L & Michiels CW 2010, Lysozymes in the animal kingdom. Journal of Biosciences, 35(1):127-60.

Uribe, C, Folch, H, Enriquez, R& Moran, G 2011, Innate and adaptive immunity in teleost fish: a review. journal of Veterinary Medicine,56:486–503.

Walter, M, Eyring, N&Eyring, H 1972, Hydrostatic Pressure and Ionic Strength Effects on the Kinetics of Lysozyme, Proceedings of the National Academy of Sciences ofthe Uuited States of America, 69:2417-2419.

Xue, QG, Schey, KL, Volety, AK, Chu, FL, La Peyre, JF 2004, Purification and characterization of lysozyme from plasma of the eastern oyster (Crassostrea virginica). Comparative Biochemistry Physiology, 139: 11-25.

Yazawa, R, Hirono, I&Aoki, T 2006, Transgenic zebrafi sh expressing chicken lysozyme show resistance against bacterial diseases; Transgenic Research, 15 385–391.

Ye, X, Zhang, L&Tian 2010, Identification and expression analysis of the g-type and c-type lysozymes in grass carp Ctenopharyngodon idellus. Development and Comparative Immunology, 34:501–509.

Yu, L, ping, Sun, B, guang, Li J&Sun, L 2013, Characterization of a c-type lysozyme of Scophthalmus maximus: Expression, activity, and antibacterial effect. Fish and ShellfishImmunology, 34:46–54.

Zheng, W, Tian, C&Chen, X 2007, Molecular characterization of goose-type lysozyme homologue of large yellow croaker and its involvement in immune response induced by trivalent bacterial vaccine as an acute-phase protein. Immunology Letters, 113:107–116.