Controlled in vitro proteolysis of casein using immobilized trypsin. Influence of variation of the enzyme‐substrate ratio and the re‐use of the immobilized enzyme on the preparation of phosphopeptide‐rich fractions

Abstract

AbstractStudies 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.