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Determination of kinetic parameters for casein hydrolysis by chymotrypsin using two ranges of substrate concentration

Vorob'ev, Mikhail M., Kochetkov, Konstantin A.
International dairy journal 2016 v.61 pp. 76-84
casein, chymotrypsin, hydrolysis, models, proteolysis
Hydrolysis of casein by chymotrypsin was performed at low and high substrate concentrations. Hydrolysis kinetics were analysed in the framework of a two-step model, with consecutive demasking and hydrolysis steps. This model took into consideration the limited accessibility of peptide bonds for the enzyme (masking effect). Dependence of the logarithm of the hydrolysis rate via degree of hydrolysis was non-linear at concentrations of 0.04 and 10 g L−1. In the intermediate range of substrate concentrations (0.07–0.72), this dependence was found to be linear, corresponding to the exponential model of proteolysis. Taking into consideration the multiplicity of peptide bonds, coupled to peptide bond demasking, allowed determination of two important parameters: the ratio of the rate constants of demasking and hydrolysis (kd/kh), and the fraction of initially masked bonds (m). An analytical procedure was suggested for the calculation of the apparent Michaelis constants at various degrees of hydrolysis.