CONTROLLED IN-VITRO PROTEOLYSIS OF CASEIN USING IMMOBILIZED TRYPSIN - INFLUENCE OF VARIATION OF THE ENZYME-SUBSTRATE RATIO AND THE REUSE OF THE IMMOBILIZED ENZYME ON THE PREPARATION OF PHOSPHOPEPTIDE-RICH FRACTIONS

Studies on in vitro proteolysis of casein using dissolved trypsin or covalently bound to oxirane beads have shown that immobilization leads to a change in the peptide pattern of the resulting proteolysate. Experiments on the variation of the enzyme-substrate ratio (E/S = 1/50, 1/100, 1/200, 1/400, 1/800) revealed that, compared with saturation (E/S = 1/50), lack of enzyme (E/S = 1/800) results not only in a disproportional decrease in the proteolysis rate, but leads additionally to a qualitatively differing peptide composition of the proteolysates. This variation, which appears, on the one hand, not acceptable in regard to a reproducible preparation of biologically active peptides is, on the other hand, a means of controlling proteolysis via the enzyme-substrate ratio. Re-use of the immobilized trypsin caused, after 9 repetitions, a loss in proteolytic activity and in phosphopeptide yields of approximately 25%, whilst the peptide pattern of the proteolysate remained qualitatively unchanged.