The function of the milk-clotting enzymes bovine and camel chymosin studied by a fluorescence resonance energy transfer assay

Abstract

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.

Interaction between sodium chloride and texture in semi-hard Danish cheese as affected by brining time, dl -starter culture, chymosin type and cheese ripening

“…Camel chymosin has a 70% higher clotting activity towards bovine milk compared with bovine chymosin (Bansal et al, 2009;Jensen et al, 2015;Kappeler et al, 2006).…”

Recombinant Camel Chymosin Effectively Acts on Milk Coagulation after Immobilization on Eggshell Membranes

“…Some, such as the relative milk-clotting activity test, traditionally used as the standard test, depend on the visual appearance of flocks of renneted standard milk on the wall of a rotating test, which represents a serious inconvenience of subjectivity . Other tests distinguish the catalytic stage using synthetic fluorescent substrates that allow precise kinetic studies, but are not well suited for routine determinations and appear to be rather expensive. The method described here accounts for the integrated result of the two processes mentioned, but it does not have the disadvantage of subjective assessment and is easy to implement to perform routine measurements at a low cost.…”

Can Recombinant Tree Shrew (Tupaia belangeri chinensis) Chymosin Coagulate Cow (Bos taurus) Milk?

“…The complex of bio-chemical properties of bovine Chn was considered optimal for cheese making until 2006, when camel rChn was obtained and studied (Camelus dromedarius) [7]. Camel rChn is now widely used in cheese making along with cow rChn [3,[8][9][10].…”

“…Obtaining the rChn of the dromedary camel was the beginning of a new stage of research aimed at studying and understanding the mechanisms of the substrate specificity of neonatal gastric proteinases [9][10][11][12][13][14]. Moreover, the results of the study of C. dromedarius rChn continue to stimulate research aimed at the search and study of novel Chn species.…”