Determination of the Influence of Substrate Concentration on Enzyme Selectivity Using Whey Protein Isolate and Bacillus licheniformis Protease

Abstract

Increasing substrate concentration during enzymatic protein hydrolysis results in a decrease in hydrolysis rate. To test if changes in the mechanism of hydrolysis also occur, the enzyme selectivity was determined. The selectivity is defined quantitatively as the relative rate of hydrolysis of each cleavage site in the protein. It was determined from the identification and quantification of the peptides present in the hydrolysates. Solutions of 0.1-10% (w/v) whey protein isolate (WPI) were hydrolyzed by Bacillus licheniformis protease at constant enzyme-to-substrate ratio. The cleavage sites were divided into five groups, from very high (>10%) to very low selectivity (<0.1%). The selectivity toward cleavage sites after Glu 62 and 134 was 2 times higher at 10% (w/v) WPI than at the lower protein concentrations. This finding shows that both the rate of hydrolysis and the enzyme selectivity were influenced by the substrate concentration.