Comparison of the hydrolysis of bovine κ-casein by camel and bovine chymosin: a kinetic and specificity study

Journal of Agricultural and Food Chemistry
Kirsten Kastberg MøllerYlva Ardö

Abstract

Bovine chymosin constitutes a traditional ingredient for enzymatic milk coagulation in cheese making, providing a strong clotting capacity and low general proteolytic activity. Recently, these properties were surpassed by camel chymosin, but the mechanistic difference behind their action is not yet clear. We used capillary electrophoresis and reversed-phase liquid chromatography-mass spectrometry to compare the first site of hydrolysis of camel and bovine chymosin on bovine κ-casein (CN) and to determine the kinetic parameters of this reaction (pH 6.5; 32 °C). The enzymes showed identical specificities, cleaving the Phe105-Met106 bond of κ-CN to produce para-κ-CN and caseinomacropeptide. Initial formation rates of both products validated Michaelis-Menten modeling of the kinetic properties of both enzymes. Camel chymosin bound κ-CN with ∼30% lower affinity (K(M)) and exhibited a 60% higher turnover rate (k(cat)), resulting in ∼15% higher catalytic efficiency (k(cat)/K(M)) as compared to bovine chymosin. A local, less dense negatively charged cluster on the surface of camel chymosin may weaken electrostatic binding to the His-Pro cluster of κ-CN to simultaneously impart reduced substrate affinity and accelerated enzyme-substrate ...Continue Reading

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Citations

Nov 1, 2012·Journal of Agricultural and Food Chemistry·Kirsten Kastberg MøllerYlva Ardö
Jul 10, 2013·Journal of Agricultural and Food Chemistry·Jesper SørensenBirgit Schiøtt
Jan 16, 2016·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Feng HangWei Chen
Dec 9, 2014·Bioengineered·Tessália Diniz LuerceDaniela Santos Pontes
Jan 23, 2016·International Journal of Biological Macromolecules·Feng HangWei Chen
Jan 21, 2020·Analytical Sciences : the International Journal of the Japan Society for Analytical Chemistry·Toshio TakayanagiHitoshi Mizuguchi
May 21, 2017·Protein Expression and Purification·Wen-Guo LiuMing Lu
Mar 3, 2018·Methods : a Companion to Methods in Enzymology·Srikanth GattuLisa A Holland

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