Identification of the key mechanisms involved in the hydrolysis of fish protein by Alcalase

• Enzymatic hydrolysis of fish protein by Alcalase was studied.
• A mechanistic approach for the protein hydrolysis was assessed.
• Enzyme inhibition by hydrolysis products was the more significant phenomena.
• The product inhibition explained the typical shape of the hydrolysis curve.

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 mMK′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.