Catalase is an important antioxidant enzyme that virtually exists in all oxygen respiring organisms. It protects the organism against various oxidative stress by eliminating excessive hydrogen peroxide (H2O2) generated in the cell and thereby maintaining the correct redox balance in the cells. Two types of catalases have been identified from organisms as classical Fe heme enzymes and catalase -peroxidases which are engaging in different mechanisms. Despite the main function of ROS scavenging ability, several evidences are available for the intervention of catalases in host immune responses according to previous studies. Amphiprion clarkii is a so-called anemone fish and in the wild it will always try to find an anemone that can protect it from predators. This fish is one of the most commonly kept anemone fishes among saltwater aquarists . In this study we have characterized catalase from Amphiprion clarkii (ClCat) and determined the antioxidant activity of the purified recombinant protein against H2O2.
The deduced amino acid sequence of ClCat was identified and it consisted with an open reading frame (ORF) of 1581 bp which codes a protein with 527 amino acids. The molecular weight of ClCat was predicted as 60 kDa with a 7.25 theoretical isoelectric point (pI ) followed by an instability index of 26.37. Following the in-silico predictions, ClCat consisted with highly conserved proximal active site signature (64-80) and proximal heme-ligand signature (354-362) which resemble the typical catalase characteristic domain signatures.
The recombinant protein of ClCat (rClCat) was purified using pMAL protein fusion and purification system. Following the functional assays, increased peroxidase activity was obtained with the increment of the rClCat concentration. The potential protective ability of rClCat against oxidative DNA damage was determined by the thiol mixed-function oxidation (MFO) assay. The relative activity of the rClCat was evaluated using a range of temperature and pH conditions in order to determine the favorable reaction conditions of its enzyme catalytic activity. According to the plotted graphs, the highest residual activity for rClCat was obtained within 30 ℃ to 50 ℃ of temperature range while optimum pH was obtained at pH 7. These results indicate that the rClCat has detectable peroxidase activity against H2O2 and protective activity against DNA damage while exhibiting significant activity towards range of temperature and pH conditions.