Purification,characterization and antimicrobial activities of the Lectin in the skin mucus of African catfish(clarias gariepinus,Burchell, 1822)

Odekanyin, Oludele Olayemi (2012)

iv,166

Thesis

The study purified a galactose-specific lectin from the skin mucus of African catfish (Clarias gariepinus) and determined the physicochemical properties and antimicrobial activities of the lectin. This was with a view to examining the involvement of the protein in the host defense mechanism and the adaptability of the fish to diverse environmental conditions. African catfish (Clarias gariepinus) was obtained from the Osin Fish Farm, Yakoyo via Ile-Ife. The skin mucus was scraped and homogenized in 10 mM phosphate buffer, pH 7.2 containing 50 mM NaCl to obtain the crude extract. The blood and sugar specificities of the lectin in the extract were determined. The lectin was purified to homogeneity by a two-step purification procedure - gel filtration on Sephadex G-150 and affinity chromatography on Lactose-Sepharose 4B column. The effects of pH, temperature, Ethylenediamine tetraacetic acid (EDTA) and divalent cations such as Ba2+, Ca2+ , Mn2+ , Mg2+, Fe2+ and Sn2+ on the haemagglutinating activity of the lectin was investigated using standard methods. The molecular weight of the native protein and the subunit was estimated by gel-filtration on Sephadex G-100 and Sodium Dodecyl Sulphate – Polyacrylamide gel electrophoresis (SDS-PAGE) respectively. The amino acid composition of the protein was determined using standard methods. The presence of covalently-bound carbohydrate in the protein was investigated with Periodic Acid-Schiff’s reagent (PAS Staining). Ouchterlony double diffusion technique in agar gel was carried out to examine the interaction of the lectin with carbohydrates. The antimicrobial activities of the skin mucus lectin of catfish were investigated by identifying and characterizing different isolates of pathogens in the fish environment. The ability of the lectin to agglutinate and suppress bacteria growth was tested and the inhibitory effect of the lectin on the mycelia growth of the fungi isolates was investigated. Phosphate buffered saline extract of the skin mucus of African catfish specifically agglutinated erythrocytes of rabbit and human blood group B, but did not agglutinate bat, rat, hen and human blood A and O erythrocytes. The haemagglutinating activity of the lectin was completely inhibited by lactose and slightly inhibited by galactose and melibiose and was calcium-independent. The purified lectin has a native and subunit molecular weight of 63, 000 daltons and 20, 000 daltons respectively suggesting a trimeric structure for the protein. The protein contained 126 amino acids residues per subunit. This was characterized by large amount of polar amino acids that constituted about 60 % of the total amino acids. The lectin showed maximum activity over the pH range 6 – 9 and was heat stable up to 50 oC. EDTA had no inhibitory effect on its haemagglutinating activity. PAS staining showed that the lectin was not a glycoprotein. Chemical modifications of serine and arginine residues of the protein did not affect its haemagglutination activity while modifications of cysteine, tryptophan and histidine residues led to total loss of its activity. The microbial load of the fish pond water was high, with highest total bacterial count of 7.1 x 108 cfu/ml and fungal count of 6.7 x 103 cfu/ml. Twelve bacterial species and 14 fungi species were isolated and identified from the pond water. The lectin specifically agglutinated different Gram-negative bacteria such as Aeromonas hydrophila, Alcaligenes faecalis, Klebsiella edwardsii and Vibrio metschnikovii, but had no effect on Gram-positive bacteria. It inhibited strongly the growth of A. hydrophila, A. faecalis, Bacillus cereus, B. polymyxa, K. pneumoniae, K. edwardsii, Pseudomonas aeruginosa and V. metschnikovii. In the same manner, the lectin inhibited the mycelia growth of yeast, Kluyveromyces marxianus and a phytopathogenic fungus, Fusarium oxysporum. The study concluded that African catfish skin mucus lectin exhibited similar physicochemical properties with lectins from other fish skin mucus. The lectin was involved in the innate host defense mechanism of the fish.

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