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Isolation, Purification and Partial Characterization of a Serine-Like Protease from Solanum dubium Seeds

Fatima Musbah Abbas* and Abubaker Elshiekh Abdelrahman

The aim of this study was to extract, purify and characterize an enzyme from Solanum dubium (S. dubium) seeds and to investigate the seed extractability in milk clotting activity. The extracted were using different buffers of sodium phosphate (pH 7.1), Tris HCl (pH 8.5), Sodium Acetate (pH 5.2) in addition to a distilled water treatment. The results shows that buffer extraction (sodium phosphate pH 7.1) and presoaking produced more extractable protein with better clotting ability. The extracted enzymes were filtered and fractionated on Sephadex G-75, anion exchange, and gel filtration chromatography columns. The purified enzyme is characterized by being thermostable (up to 70ºC) with a pH range of 6-7 (for 1% casein substrate). The enzyme is inhibited by serine protease inhibitors. It is, therefore, concluded that the enzyme is of the chymotrypsinlike serine protease class. Gel filtration and SDS-PAGE methods showed that the purified enzyme consists of three bands with molecular masses of 11KD, 35KD, and 40KD. The enzyme has a Km value of 0.6 mM and a Vm value of 66.7unit/min. mg. Crude extracts obtained from S. dubium seeds were shown to exhibit milk clotting activity.