Related Experiment Videos
Characterization of trypsin immobilized on oxirane-acrylic beads for obtaining phosphopeptides from casein
1Bundesanstalt für Milchforschung Institut für Chemie und Physik, Kiel.
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.
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.