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A proteolytic susceptibility index was proposed to evaluate the reaction performance of different food by-products with subtilisin. Whey, salmon muscle and feather keratine were hydrolyzed at the same peptide bond concentration at 50 °C and pH 8.0 with different subtilisin concentrations. The logarithmic equation P = 1/b ln(abt+1) was fitted to estimate the kinetic constants a and b. The a/b ratio was proposed as a proteolytic susceptibility index, which was correlated with the chemical structure and conformation of protein sources in increasing order of feather keratin, salmon muscle and whey proteins with corresponding a/b values of 0.1, 8.2 and 14.4 mM2/min, respectively. The methodology proposed in this work can be used to evaluate the proteolytic susceptibility of different protein by-products to different proteases and to evaluate the changes in proteolytic susceptibility after treatment.

A novel methodology for the modeling and characterization of the enzymatic hydrolysis of proteins is proposed. The hydrolysis time course can be predicted at different operating conditions for protein concentration, enzyme concentration and temperature. The hydrolysis kinetics of salmon muscle proteins and whey protein isolate by Alcalase were studied using a central composite design. A combination of the logarithmic equation P = 1/bln (abt + 1) to model the hydrolysis time course with the response surface methodology to correlate the kinetic constants a and b with the operating conditions: protein concentration, enzyme concentration and temperature were achieved. The logarithmic equation was a very good fit with the hydrolysis time courses, with both substrates achieving R2 > 0.995. The kinetic constants a and b were significantly affected by the operating conditions. Empirical models were obtained for a and b as functions of operating conditions. The kinetic constant values were predicted, and a strong correlation between predicted and experimental values was obtained for a (R2 = 0.949) and b (R2 = 0.945). The predicted and experimental time courses resulted in good correlation for both salmon muscle proteins (R2 > 0.987) and whey protein isolate (R2 > 0.978). The model allowed calculation of the hydrolysis time course of proteins from a given set of operating conditions with good predictability. This methodology can be used with different sources of proteins and enzymes to test the susceptibility of proteins to hydrolysis as well as the catalytic efficiency of proteases. Additionally, the combined model can be used as a design and optimization function.

Through decades of study, regardless the protein source, the enzymatic hydrolysis of proteins has always resulted in a hydrolysis curve with a typical shape: an initial rapid hydrolysis phase followed by a rapid decrease in the reaction rate. Several hypotheses have been proposed, such as substrate exhaustion, product inhibition and enzyme inactivation. In current study the effect of substrate, product and thermal inactivation were systematically studied using the commercial enzyme Alcalase and salmon muscle protein as the substrate. The low Km value of 4.39 mM and the stabilization of Alcalase in the presence of substrate and hydrolysis products could not explain the progressive decrease in the reaction rate. On the other hand, a strong product inhibition was observed (Ki = 2.31 mM; K ′ i = 7.12 mM ). These results along with computer simulations revealed that neither substrate exhaustion nor enzyme inactivation generated the typical hydrolysis curve; however, inhibition by hydrolysis products resulted in a remarkable and progressive decrease in the reaction rate, which satisfactorily explained the shape of the hydrolysis curve.