Preparation Of Metal Chelating Carriers And Application In Immobilized Enzyme

Numerous approaches have been explored for the preparation of immobilized enzymes because they have considerable advantages over enzymes in bulk solution. The advantages of using immobilized enzyme include stability and ease of recovery, both of which are key considerations in practical applications. However, it has been found that, in general, enzymatic activity decreases when enzymes are immobilized. There are many possible effects of immobilization on the kinetic behavior of an immobilized enzyme, which include (a) conformational and steric effects, (b) micro-environmental effects on active site or allosteric sites, and (c) diffusion or mass transfer effect.Based on the technology of protein separation with immobilized metal ion affinity chromatography (IMAC), a method for oriented immobilization of papain has been selected. Papain was successfully immobilized on magnetic agarose carriers. Cu²⁺ with iminodiacetate (IDA) was used as the chelating ligand to be correlated with the histidine on papain (His-81). The optimum immobilization conditions of enzyme were as flows: Cu²+ 0.15×10^-2mol/g carriers, time 4 h, enzyme load 30mg/g carrier ,pH 7.0 ,respectively. The pH and temperature were 8.0 and 70°C for immobilized enzyme. The recovery activity of immobilized enzyme was retained 68.4%. The carriers could be recovered from the spent immobilized enzyme, to be reused. After 5 times, the reimmobilization of papain on the regenerated matrix was 79.71% effective with the retention of maximum enzyme activity.Polyphenol oxidase (PPO) has also been immobilized on magnetic agarose carriers. The optimum immobilization condition of enzyme isCu2+ 6x10 2mol/g carriers. The pH and temperature were 7.0 and 50°C for immobilized enzyme. The carriers could be recovered from the spent immobilized enzyme, to be reused. After 5 times, the reimmobilization of polyphenol oxidase on regenerated matrix was 65.3% effective with the retention of maximum enzyme activity.The cost of carriers used for industrial applications is very important. The regenerability of carriers is therefore, relevant. The mild conditions used for immobilization, the high recovery of immobilized preparations, the stability and the regenerability of the matrix are the main features of the method reported here. All above indicate this method can be applicable and promising in enzyme immobilization field.