A Novel Purification Process of Sardine Lipases Using Protein Ultrafiltration and Dye Ligand Affinity Chromatography

Protein purification is often performed for various applications. However, enzyme purification processes typically involve multiple steps that reduce yield and increase production costs. To overcome these challenges, we developed a novel three-step process to purify a lipase from whole sardine viscera (WSV), leveraging protein properties and the structural affinity of lipases for dye ligands. A crude extract of the viscera (CEV) was obtained by grinding the whole viscera in 50 mM phosphate buffer (pH 7.0, Solution B) followed by centrifugation (6000× g; 30 min, 0 °C). Lipolytic activity (3.3 U/mg) was recorded only in the supernatant. The purification process began with ammonium sulfate fractionation (30–50% saturation), increasing lipolytic activity in the precipitate (PF30-50) to 32.9 U/mg. PF30-50 was then ultrafiltered using a 30 KDa MWCO membrane, where 5% of semi-purified lipases (SPLSV) was retained with an activity of 156.5 U/mg (UF30). Finally, the SPLSV was injected into a column packed with dye ligand affinity adsorbent, pre-equilibrated with 1.0 M ammonium sulfate in buffer A. The WSV lipase was eluted using a step gradient to progressively reduce salt concentration. SDS-PAGE analysis revealed a single band of purified lipase from sardine viscera (PLSV) corresponding to a molecular weight of 123.4 kDa, with a specific activity of 266.4 U/mg. The combination of ammonium sulfate precipitation, ultrafiltration, and dye-ligand affinity chromatography provides a scalable and reproducible approach with potential industrial relevance, particularly in biocatalysis and waste valorization contexts.