Purification and stability analysis of antimicrobial proteins from Varuna litterata

Ten marine species, including different crabs, bivalve molluscs, and fish intestines were selected to screen the natural antimicrobial protein or peptide as they are enriched with various microorganisms. The crude extract from Varuna litterata, a marine crab which is used as a raw material in the preparation of pickled crabs in Chaoshan area of China, was proved to have a potent bacteriostatic effect against gram-negative bacterium (Escherichia coli) and gram-positive bacterium(Staphylococcus aureus) compared with other marine species. The crude proteins of Varunalitterata were salted-out for preliminary purification and further purified by gel filtration (Sephadex G-150) or anion exchange (DEAE-cellulose 52) chromatographic column. An increase in the antimicrobial activity was noted with the increase in the purity level of the protein. A relatively pure protein was eventually obtained, which was determined to be belonging to the hemocyanin family based on the mass spectrometric data analysis. The purified proteins solution (1 mg/ml) from Varuna litterata exhibited similar antimicrobial activity to that of gentamycin sulfate (0.2 mg/ml), which were relatively stable in a certain pH or temperature range. A structure-activity relationship of the purified hemocyanin was determined based on the interaction of hemocyanin and different chromatographic medium, which revealed that the integrated hexamers played a remarkable role in its bacteriostatic activity. Moreover, the phenoloxidase activity of hemocyanin from Varuna litterata was found as the underlying cause of its antimicrobial potential.