Purification and Characterization of Milk-clotting Enzyme from Solanum dubium Seeds

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A. Mohamed, Isam,
E. Babiker, Efadil,
Mori, N.
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This research was carried out to purify and characterize a milk-clotting enzyme from Solanum dubium seeds and to investigate the efficiency of the purified enzyme as a rennet substitute in cheese making. A novel milk-clotting serine protease was purified to 6.7 folds and 35% recovery from S. dubium seeds by combination of (NH4)2SO4 precipitation, cation-exchange and gel filtration chromatographies. The molecular mass of the enzyme was 66 kDa as estimated by gel filtration and SDS-PAGE. The purified enzyme was a chymotrypsin-like serine protease with pI value of 9.3, acts optimally at pH 11.0 and stable over a wide range of pH (4.0-11.0). The purified enzyme was also thermostable protease with stability of up to 60°C for one hour and acts optimally at 70°C for 30 min. When compared with other plant enzymes, S. dubium enzyme had higher ratio of milk-clotting activity /proteolytic activity. The most striking property of S. dubium milk-clotting serine protease, among all reported plant serine proteases, was its high stability under several conditions (pH, temperature, organic solvents and denaturants). The enzyme exhibited broad specificity toward bovine whole casein and its components (α-, β- and κ-caseins). Unlike the other milk clotting enzymes, S. dubium milk-clotting protease degraded β-casein faster than α- casein. The high stability of the enzyme under various conditions, in accordance with its milk-clotting ability, could therefore, pave the way for its uses in cheese industry and biotechnology.
Casein hydrolysis; enzyme activity; milk-clotting, Solanum dubium