Characterization of "lettucine", a serine-like protease from Lactuca sativa leaves, as a novel enzyme for milk clotting.
In this work we focused on the characterization of a novel plant rennet purified from lettuce leaves (Lactuca sativa L. cv Romana). The lettuce protease, lettucine, showed trypsin-like, SV8-like, and caseinolytic activities. Although the enzyme did not recognize peptides having hydrophobic amino acid residues in the P(1) position of the target bond, it did show milk-clotting activity, suggesting that different bonds rather than the Phe(105)-Met(106) of the kappa-casein might be cleaved, still inducing milk-clotting. The enzyme exhibited proteolytic activity toward alpha-casein, beta-casein, kappa-casein, and milks with different fat contents, with the highest activity observed with partially skimmed milk, total casein, and alpha- and kappa-casein. SDS-PAGE studies showed that lettucine cleaved alpha-casein, beta-casein, and kappa-casein. In particular, we showed that alpha-casein breakdown occurred even though total casein or milks were supplied, suggesting that the lettuce enzyme is able to operate a significant disorganization of the casein's micellar structure. Moreover, the proteolytic activity of the enzyme analyzed under various technological parameters, such as temperature and pH, indicated that the lettuce enzyme is highly consistent with the milk-clotting process.
Lo Piero
Angela R
AR
Dipartimento di Scienze Agronomiche, Agrochimiche e delle Produzioni Animali, Università di Catania, via S. Sofia no. 98, 95123 Catania, Italy.
Puglisi
Ivana
I
Petrone
Goffredo
G
D000595:Amino Acid Sequence; D000818:Animals; D002122:Calcium Chloride / Q000494:pharmacology; D002364:Caseins / Q000378:metabolism; D012085:Chymosin; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D018545:Lactuca / Q000201:enzymology*; D008892:Milk / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D002364:Caseins; C025350:rennet; D012697:Serine Endopeptidases; D012085:Chymosin; D002122:Calcium Chloride