Characterization of trypsin immobilized on oxirane-acrylic beads for obtaining phosphopeptides from casein.

The aim of the study was to characterize the proteolytic properties of immobilized trypsin for obtaining phosphopeptide-rich fractions from casein. Trypsin was covalently bound to oxirane-acrylic beads. After incubation for 48 h immobilization degrees of about 85% were achieved. 20% of the immobilized enzyme exhibited no activity towards the substrate N-benzoyl-L-arginine ethyl ester. Compared with homogeneous catalysis with the soluble enzyme a 25% lower degree of proteolysis was calculated and a modified peptide pattern of the resulting proteolysates established. A caseinophosphopeptide (CPP) from alpha s1-CN (59-79) was not detectable after incubation with the carrier-bound enzyme. At a substrate concentration (S) of 15% (w/w) substrate saturation of the enzyme (E) was achieved. Increasing the substrate concentration to 20% (w/w) decreased the conversion rates (content of soluble amino-N) and the liberation of CPPs. Proteolysis of small (1% w/w) and partly also large (20% w/w) substrate concentrations (E/S = 1/100) is subject to changed kinetic conditions. The same was true for small and large enzyme concentrations (S = 5% w/w). Compared with enzyme saturation (E/S = 1/50), lack of enzyme (E/S = 1/800) led to a disproportional decrease in the proteolysis rate and to a markedly decreased content of hydrophobic peptides in the resulting proteolysates. Increasing the pH from 7.8 to 8.8 and the temperature from 37 degrees to 47 degrees C caused only a slight increase in conversion rates, but an overproportional liberation of CPPs (pH 8.8 = + 47%, 47 degrees C = +89%), in particular from beta-casein. Repeated use of immobilized trypsin resulted after nine runs in a loss in proteolytic activity and in CPP yields of approximately 25%, while the peptide pattern of the proteolysates remained qualitatively unchanged. Light microscopy shows that the oxirane-acrylic beads disintegrate to a large extent after nine repetitions.