Purification and Characterization of a Milk-Clotting Aspartic Proteinase from Globe Artichoke (Cynara scolymus L.)

Abstract

The study of proteinase expression in crude extracts from different organs of the globe artichoke (Cynara scolymus L.) disclosed that enzymes with proteolytic and milk-clotting activity are mainly located in mature flowers. Maximum proteolytic activity was recorded at pH 5.0, and inhibition studies showed that only pepstatin, specific for aspartic proteinases, presented a significant inhibitory effect. Such properties, in addition to easy enzyme inactivation by moderate heating, make this crude protease extract potentially useful for cheese production. Adsorption with activated carbon, together with anion exchange and affinity chromatography, led to the isolation of a heterodimeric milk-clotting proteinase consisting of 30- and 15-kDa subunits. MALDI-TOF MS of the 15-kDa chain determined a 15.358-Da mass, and the terminal amino sequence presented 96% homology with the smaller cardosin A subunit. The amino terminal sequence of the 30-kDa chain proved to be identical to the larger cardosin A subunit. Electrophoresis evidenced proteinase self-processing that was confirmed by immunoblots presenting 62-, 30-, and 15-kDa bands.

Caseinolytic and milk-clotting activities from Moringa oleifera flowers

“…Cysteine proteases from B. hieronymi fruits with milk-clotting ability were also described (Bruno et al, 2010). Chymosin and milk-clotting enzymes from C. cardunculus flowers and Strebus aspler twigs are aspartic proteases (Heimgartner et al, 1990;Llorente, Brutti, & Caffini, 2004;Senthilkumar, Ramasamy, & Subramanian, 2006).…”

Comparative evaluation of Bacillus licheniformis 5A5 and Aloe variegata milk-clotting enzymes

“…This situation encourages the search for other sources of rennet substitutes from microbial or plant sources to meet the needs of the expanding cheese industry. Coagulation of milk can be achieved by a number of proteolytic enzymes from various sources, such as animals (e.g., pig, bovine, and chicken pepsins), microorganisms (Rhizomucor miehei, Rhizomucor pusillus, and Cryphonectria parasitica), and plants (e.g., pineapple, papaya, and sodom apple (Llorente et al, 2004;Yu and Chou, 2005;Low et al, 2006 andEl-Bendary et al, 2007). The present investigation aims at comparing the properties of microbial milk-clotting enzyme with those of plant origin for selecting the appropriate one as a substitute of calf rennet.…”

A two-stage oxygen supply control strategy for enhancing milk-clotting enzyme production by Bacillus amyloliquefaciens

“…However, this method is ethically arguable and has low productivity. Thus, research has focused on the identification of alternative milk-clotting enzymes (MCEs) among animals, plants and microorganisms as calf rennet substitutes [2][3][4][5][6][7][8].…”