Purification and Antimicrobial Activity of Antimicrobial Protein from Brown-spotted Grouper, <I>Epinephelus fario</I>

Abstract

Antimicrobial proteins and peptides had been found from a wide variety of organisms in the last few years. These molecules have attracted much research interest because of their biochemical diversity, broad specificity on anti-viral, anti-bacterial, anti-fungi, anti-protozoan parasites, anti-tumoural, and wound-healing effects. Antimicrobial proteins and peptides play key roles in innate immunity. They interact directly with bacteria and kill them. The brown-spotted grouper, Epinephelus fario, is an important marine fish cultured in southern China. Recently, bacteria and virus have caused high mortality in E. fario cultures, but its endogenous antimicrobial peptides and proteins have not been explored. An antimicrobial component was found from the skin homogenate of E. fario. After the skin homogenate was digested with trypsin, its antimicrobial activity was lost, which showed that the antimicrobial component is a protein. The antimicrobial protein (Efap) was purified from the skin homogenate of E. fario by successive ion-exchange and gel filtration chromatography. Efap was demonstrated to be single protein band by SDS-PAGE, with the apparent molecular weight of 41 kD. Efap exhibited antimicrobial activity both for the Gram-positive bacteria, Staphylococcus aureus, Micrococcus luteus and Bacillus subtilis, and for the Gram-negative bacteria, Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio fluvialis, Pasteurella multocida, Aeromonas hydrophila, Eschrrichiu coli, and Pseudomonas aeruginosa. Except A. hydrophila, P. aeruginosa, and E. coli (MIC>20 mol/L), most of the tested Gram-negative bacteria were sensitive to Efap (MIC<20 mol/L). Interestingly, Efap showed potent antimicrobial activity against Gram-positive bacteria S. aureus (MIC 5-10 mol/L) but comparatively weak antimicrobial activity against M. luteus and B. subtilis. The broad antimicrobial activities of Efap suggest that it contributes to the innate host defence of E. fario.

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