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Proteolytic activity of proteinases on macropeptide isolated from kappa-casein
K M Shammet1, R J Brown, D J McMahon
1Department of Nutrition and Food Sciences, Utah State University, Logan 84322-8700.
Abstract:
Proteolytic activities of chymosin, bovine pepsin, Mucor miehei rennet, Cryphonectria parasitica (formerly Endothia parasitica) rennet, trypsin, and chymotrypsin on kappa-casein macropeptide were measured. Macropeptide solutions (10 mg/ml of .05 M, pH 6.6 phosphate buffer) were incubated with the enzymes at 37 degrees C for various times, and their reactions were stopped by adding .025 ml of pepstatin (1 mg/ml of methanol). Peptides released from kappa-casein macropeptide were then fractionated using reverse-phase HPLC. At the pH of milk (pH 6.6), kappa-casein macropeptide was resistant to enzymic action by chymosin, bovine pepsin, and M. miehei and C. parasitica rennets. Bovine pepsin hydrolyzed kappa-casein macropeptide at pH 3. kappa-Casein macropeptide was readily hydrolyzed at pH 6.6 by trypsin and chymotrypsin. Possible physiological functions of the kappa-casein macropeptide are discussed in light of these findings.
Insights
Kappa-casein macropeptide showed resistance to common milk-clotting enzymes at milk
Area of Science:
- Dairy science and enzymology.
- Biochemistry of milk proteins.
Background:
- Kappa-casein macropeptide is a key component of casein micelles.
- Understanding its enzymatic hydrolysis is crucial for dairy processing and milk's physiological roles.
Purpose of the Study:
- To investigate the proteolytic activity of various enzymes on kappa-casein macropeptide.
- To determine the optimal conditions for kappa-casein macropeptide hydrolysis.
Main Methods:
- Incubation of kappa-casein macropeptide with chymosin, bovine pepsin, microbial rennets, trypsin, and chymotrypsin.
- Enzymatic reactions were stopped using pepstatin.
- Hydrolysis products were analyzed using reverse-phase High-Performance Liquid Chromatography (HPLC).
Main Results:
- Kappa-casein macropeptide was resistant to chymosin, bovine pepsin, and rennets at pH 6.6 (milk's natural pH).
- Bovine pepsin hydrolyzed kappa-casein macropeptide effectively at a lower pH of 3.
- Trypsin and chymotrypsin readily hydrolyzed kappa-casein macropeptide at pH 6.6.
Conclusions:
- Standard milk-clotting enzymes do not significantly degrade kappa-casein macropeptide under typical milk conditions.
- Specific proteases like trypsin and chymotrypsin can effectively hydrolyze kappa-casein macropeptide.
- Findings suggest potential physiological roles for kappa-casein macropeptide beyond its structural function in micelles.