Immobilization Of Trypsin And Its Application In Preparation Of Casein Phosphopeptide

In this paper, the method for immobilization of trypsin and the properties of immobilized trypsin were studied. Casein phosphopeptides (CPP) was separated from the resulted casein hydrolysates, which was prepared with immobilized trypsin as catalyst, the properties of CPP in enhancing the absorption of calcium and the molecular mass of CPP were studied as well.From the study, following conclusions were obtained.Through experiments, trypsin was immobilized on anion exchange resin D380 and the optimum conditions were: glutaraldehyde concentration 0.3%, crosslinked temperature 20℃, crosslinked time 10 hours, the pH value of solvent trypsin 8.0, the volume of solvent trypsin 2ml (Img/ml), immobilized time 1 hour, immobilized temperature 20℃.Under above conditions, activity retention value was over 18.86%.Optimum operation temperature of immobilized trypsin was 55℃, that of native trypsin was 50℃. Optimum pH value of immobilized trypsin was 8.0, the same as the native trypsin. Thermo-stability was improved in comparison to that of native trypsin. The experiments of operation stability showed that the immobilized trypsin was stable for casein hydrolysis. By using the immobilized trypsin, the cost of preparation of CPP would be lower than using native trypsin.The production of CPP included two steps, the immobilized enzyme hydrolysis of casein and the separation of CPP from the resulted casein hydrolysates. We chose N/P molar ratio and the yield of CPP as the indexes to evaluate the conditions of CPP production technology. The optimum conditions for the hydrolysis of casein were: casein concentration 4%, enzyme dosage 900units/g substrate, temperature 40 ℃, hydrolysis time 60 minutes. The isolating conditions of the CPP were: pH value of hydrolysate 6.5 and precipitation time 2-4 hours.The effect of CPP on retarding the formation of calcium phosphate was monitored. 0.2g/L and 0.5g/L of CPP prevented the precipitation of calcium phosphate during one hour of reaction. 0.1 g/L CPP can retard the formation of calcium phosphate 20 minutes. With SDS-PAGE, molecular mass of CPP were measured: 3300 and 4600, respectively.