Effects of pH and chromatographic column on isolation, purification of antibacterial peptide from Japanese eel liver, and determination of antibacterial activity

In order to quickly isolate and purify antimicrobial peptide,different flow phase pH values(cation-change pH 3.0,4.0,5.0,and reverse-phase liquid chromatography pH 2.0,4.5,7.0),and different chromatographic columns(cation-change and gel chromatography coupled with reverse-phase liquid chromatography respectively)were used to isolate and purify antibacterial peptide from Anguilla japonica liver,and the results were analysed and compared.The merits and drawbacks of agar plate diffusion and microporous liquid culture methods on determination of antibacterial activity were analysed.The results showed that pH 4.0 buffer solution was the best flow phase in three-pH-value buffer solutions,which had 16.43% extraction ratio and isolated two elution peaks;We did not obtain perfect result from reverse-phase liquid chromatography(RPC)in three-pH-value buffer solutions;Sephadex G-25 gel coupled with RPC significantly improved the isolation effect,elution peak amounts were greater,peaks were sharper and narrower,baseline was flatter and low than those of cation exchange coupled with RPC.Agar plate diffuse method to determine antibacterial activity was simple to operate,and experimental result was intuitional.It required much protein,so it should be used to determine antibacterial activity during protein crude extraction period;Microporous liquid culture method to determine antibacterial activity was sensitive.It required little protein,so it should be used to determine antibacterial activity during isolation and purification of protein in chromatography period.