COMPARING STUDIES ON THE PROTEOLYTIC PROPERTIES OF SOLUBLE AND IMMOBILIZED PROTEASES FOR GENERATING PHOSPHOPEPTIDES FROM CASEIN

The aim of the study was to characterize and to compare the proteolyti c properties of soluble as well as immobilized serine and aspartic pro teases for obtaining phosphopeptide-rich fractions (CPP) from casein. The proteases were covalently bound to oxirane-acrylic beads. Comparis on of the proteolytic activities revealed that proteolysis with immobi lized chymosin, pepsin and trypsin accounted for only around 50% of th at caused by homogeneous catalysis. Carrier-bound pancreatin, as a mul tienzyme-system, showed only 30% of the proteolysis with the soluble s ystem. The smaller degrees of hydrolysis with immobilized enzymes are mainly due to diffusional limitations and steric hindrance of catalysi s. In addition, noncovalent bounding of small peptides to the carrier leads to a decrease in the detectable activity values. The formation o f stable enzyme/substrate or enzyme/product complexes may also lead to a hindrance of the enzyme activity. ion exchange chromatography and a mino acid analysis were used to characterize the proteolysis products. Looking over the enzyme systems studied it can be concluded that tryp sin is the most suited protease for in vitro preparation of CPPs as fu nctional ingredients. Regarding the peptide pattern it seems to be adv antageous to use soluble trypsin, because a CPP [alpha(s1)-CN (59-79)] , which is detectable after homogeneous catalysis, is not found after proteolysis with the carrier-bound enzyme. On the other hand, it has t o be mentioned that repeated use of immobilized trypsin resulted after nine repetitions only in a loss of enzyme activity of around 25%. The peptide pattern of the proteo-lysate remained unchanged during the re petitions.