The function of the milk-clotting enzymes bovine and camel chymosin studied by a fluorescence resonance energy transfer assay.
Enzymatic coagulation of bovine milk can be divided in 2 steps: an enzymatic step, in which the Phe105-Met106 bond of the milk protein bovine κ-casein is cleaved, and an aggregation step. The aspartic peptidases bovine and camel chymosin (EC 3.4.23.4) are typically used to catalyze the enzymatic step. The most commonly used method to study chymosin activity is the relative milk-clotting activity test that measures the end point of the enzymatic and aggregation step. This method showed that camel chymosin has a 2-fold higher milk-clotting activity toward bovine milk than bovine chymosin. To enable a study of the enzymatic step independent of the aggregation step, a fluorescence resonance energy transfer assay has been developed using a peptide substrate derived from the 98-108 sequence of bovine κ-casein. This assay and Michaelis-Menten kinetics were employed to determine the enzymatic activity of camel and bovine chymosin under milk clotting-like conditions (pH 6.65, ionic strength 80 mM). The results obtained show that the catalytic efficiency of camel chymosin is 3-fold higher than bovine chymosin. The substrate affinity and catalytic activity of bovine and camel chymosin increase at lower pH (6.00 and 5.50). The glycosylation of bovine and camel chymosin did not affect binding of the fluorescence resonance energy transfer substrate, but doubly glycosylated camel chymosin seems to have slightly higher catalytic efficiency. In the characterization of the enzymes, the developed assay is easier and faster to use than the traditionally used relative milk-clotting activity test method.
Department of Chemistry, University of Copenhagen, DK-2100 Copenhagen, Denmark; Chr. Hansen a/s, Bøge allé 10-12, DK-2970 Hørsholm, Denmark.
Jesper Langholm
JL
Jensen
Chr. Hansen a/s, Bøge allé 10-12, DK-2970 Hørsholm, Denmark.
Jonas
J
Jacobsen
BioZyme Inc., Apex 27523, NC.
Marcia L
ML
Moss
BioZyme Inc., Apex 27523, NC.
Fred
F
Rasmussen
Chr. Hansen a/s, Bøge allé 10-12, DK-2970 Hørsholm, Denmark.
Karsten Bruun
KB
Qvist
Department of Chemistry, University of Copenhagen, DK-2100 Copenhagen, Denmark.
Sine
S
Larsen
Chr. Hansen a/s, Bøge allé 10-12, DK-2970 Hørsholm, Denmark. Electronic address: dkhvb@chr-hansen.com.
Johannes M
JM
van den Brink
D000818:Animals; D002162:Camelus; D002364:Caseins / Q000378:metabolism*; D002417:Cattle; D012085:Chymosin / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods / Q000662:veterinary*; D006031:Glycosylation; D007700:Kinetics; D008892:Milk / Q000201:enzymology*
D002364:Caseins; D012085:Chymosin
Michaelis-Menten; coagulant; fluorescence resonance energy transfer assay; milk clotting