Chloroperoxidase Immobilization And Its Application

Chloroperoxidase(CPO) is recognized as the most versatile enzyme in the group of peroxidases because of its unique active site structure,it is noticeable particularly that it has the function of chiral recognition for substrates,so it has very captivating development and application in asymmetric organic synthesis,biotransformation,chiral pharmaceuticals,detoxication of environmental pollutants and etc.However,due to its inactivation in extreme conditions,including high-temperature,strong acids and base,the industrial application of native CPO has been severely limited.In this paper,immobilized CPO improved thermal stability and catalytic activity of enzyme, and increased the adaptive ability to the synthesis environment.Meanwhile,it will provide a basis for immobilized enzyme ha the practical application.Immobilization technology of enzyme is an effective measure to achieve repeatly using of enzyme and improve stability.In the article,two new materials were selected for the carriers of immobilized CPO,including chitosan membranes/beads and magnetic microspheres.Chitosan is a very ideal immobilized carrier because of its rich resource,good biocompatibility and susceptibility to chemical modification.Meanwhile,magnetic microspheres have super paramagnetism,so the immobilized enzyme by magnetic microspheres is easer to be separate and recycle than other carriers, and it can reach a higher-load.The results of optimized conditions of immobilization of CPO, differences between native CPO and immobilized CPO had be investigated by MCD assay,and application of immobilized CPO in catalytic polymerization of pyrogaUol had be showed as follows:1.CPO was covalently immobilized on chitosan membranes(beads) under mild condition with glutararldehyde as a crosslinking agent.The results indicated that the optimum concentration of glutaraldehyde and pH were determined to be 0.8%and 3.5,respectively.Compared with the native CPO,immobilized CPO has excellent thermal stability,and the stability of immobilized CPO against urea and hydrogen peroxide exposure was improved.The immobilized CPO can retain about 40%of its activity after 1h incubation at 50℃,whereas native CPO could maintain only about 0.02%of its activity under the same conditions.Furthermore,immobilized CPO had unchanged residual activity (about 100%),but the 32%loss of activity for native CPO in the presence of 1.5 M urea.When incubated with 300μM H₂O₂,the immobilized CPO retained 58%after 20 h whereas the residual activity of native CPO only retained 42%.2.Thiocyanate groups containing magnetic micropheres were used in covalent immobilization of the enzyme "CPO".The results showed the optimal conditions of immobilization:the optimal pH was 5.5,and the optimal reaction time was 10 min.The maximum amount of immobilization CPO on the magnetic micropheres was 25.59 mg/g support.Compared with the native CPO,the affinity of immobilized CPO decreased toward substrate and its stability was considerably improved. Immobilized CPO is incubated in the presence of 300μM H₂O₂ in different times,the immobilized CPO still remain about 50%of its activity after 96 h,whereas native CPO only remained around 9%. This result suggested that the covalently immobilized CPO was considerably stable on the magnetic micropheres surface and the immobilized CPO retained more than 85%of its initial activity after 16 cycles of usage.3.So the catalyzed polymerization of pyrogallol using immobilized CPO was investigated.The result indicates that the immobilized enzyme was used for transforming pyrogallol into purpurogallin in aqueous phase.