title: 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.
authors.affiliation: Food Engineering, CULTEV, Department of Basic Sciences, National University of Luján, Luján, Argentina. llorente@mail.unlu.edu.ar
authors.forename: Berta E
authors.initials: BE
authors.lastname: Llorente
authors.forename: Cristina B
authors.initials: CB
authors.lastname: Brutti
authors.forename: Néstor O
authors.initials: NO
authors.lastname: Caffini
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D031943:Cynara scolymus / Q000201:enzymology*; D035264:Flowers / Q000201:enzymology; D013058:Mass Spectrometry; D008892:Milk / Q000378:metabolism*; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D016282:Aspartic Acid Endopeptidases