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

Abstract

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.

Keywords

• characterization
• lysozyme
• rutilus kutum