Date of Award:
Doctor of Philosophy (PhD)
Nutrition, Dietetics, and Food Sciences
Department name when degree awarded
Nutrition and Food Sciences
Rodney J. Brown
This work reviews studies of bovine K-casein and specifically K-casein macropeptide. Properties of K-casein, its structure and heterogeneity, proteolytic activity of some milk clotting enzymes on K-casein, and K-casein sensitive bonds are discussed. Macropeptides of other species are also presented. The carbohydrate moieties of bovine macropeptide together with their biological and physiological functions are reviewed.
Macropeptides were produced by enzymic hydrolysis from whole casein solution using crystalline chymosin (EC 22.214.171.124). Trichloroacetic acid (final concentrations 2, 8 and 12%) was added after 5, 30 and 60 min of incubation to precipitate protein and inactivate the enzyme. The filtrate was then exhaustively dialyzed against distilled water to remove trichloroacetic acid and small molecules. The dialyzate was lyophilized and stored at -20deg;c until required for analysis. These macropeptides were then compared using RP-HPLC with macropeptides obtained from purified K-casein isolates by the same method (15 min incubation).
Proteolytic activity of some milk-clotting enzymes (chymosin, Mucor miehei rennet and Endothia parasitica rennet) and some proteinases (trypsin and chymotrypsin) on K-casein and macropeptide isolated from K-casein was followed by RP-HPLC. The milk-clotting enzymes were standardized to the same clotting activity using a Formagraph.
Each enzyme was incubated with .5 mix-casein and macropeptide solutions (10 mg in 1 ml .05 MpH 6.6 phosphate buffer) at 37°C for various incubation times. Reactions were stopped by addition .5 ml of 8 Murea containing 10-5 Mpepstatin or .025 ml pepstatin (1 mg pepstatin in 1 ml methanol). These reaction mixtures were separated into fractions using RP-HPLC and chromatograms of the different enzymes compared.
Shammet, Khalid M., "Proteolytic Activity of Some Milk-Clotting Enzymes on K-casein and K-casein Macropeptide" (1989). All Graduate Theses and Dissertations. 5371.
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