Exploring the Antimicrobial Potential and Biofilm Inhibitory Properties of Hemocyanin from Hemifusus pugilinus (Born, 1778)

The seafood industry plays a huge role in the blue economy, exploiting the advantage of the enriched protein content of marine organisms such as shrimps and molluscs, which are cultured in aquafarms. Diseases greatly affect these aquatic organisms in culture and, hence, there is need to study, in detail, their innate immune mechanisms. Hemocyanin is a non-specific innate defense molecule present in the blood cells of several invertebrates, especially molluscs, arthropods, and annelids. It is concerned with oxygen transport, blood clotting, and immune enhancement. In the present study, this macromolecular metalloprotein was isolated from the hemolymph of the marine snail Hemifusus pugilinus (Born, 1778) using Sephadex G-100 gel filtration column chromatography. It occurred as a single band (MW 80 kDa) on SDS-PAGE. High-performance liquid chromatography (HPLC) of the purified hemocyanin showed a single peak with a retention time of 4.3 min. The secondary structure and stability of the protein were detected using circular dichroism (CD), and the spectra demonstrated negative ellipticity bands close to 208 nm and 225 nm, indicating β-sheets. Further exploration of the purified hemocyanin revealed remarkable antimicrobial and antibiofilm activities against Gram-positive (Enterococcus faecalis and Staphylococcus aureus) and Gram-negative bacteria (Pseudomonas aeruginosa and Proteus vulgaris) at a concentration of 1–5 μg/mL. Spectrophotometric and in situ microscopic analyses (CLSM) unveiled the potential of the purified hemocyanin to inhibit biofilm formation in these bacteria with a minimal inhibitory concentration of 40 μg/mL. Furthermore, H. pugilinus hemocyanin (10 μg/mL concentration) displayed antifungal activity against Aspergillus niger. The purified hemocyanin was also assessed for cytotoxicity against human cancer cells using cell viability assays. Altogether, the present study shows that molluscan hemocyanin is a potential antimicrobial, antibiofilm, antifungal, anticancer, and immunomodulatory agent, with great scope for application in the enhancement of the immune system of molluscs, thereby facilitating their aquaculture.