Immobilization Of Chloroperoxidase By Mesoporous Carrier And Its Application In Degradation Of Dye

Chloroperoxidase (CPO) has versitile catalytic activities, including both ones of peroxidase and cytochrome P-450, and wide substrate spectrum due to its unique active site structure. It can be used in many types of reactions, for example, chlorination of saturated hydrocarbons, epoxidation of olefins, hydroxylation, sulfoxidation and so on. It gets more and more applications during catalysis, organic synthesis, drug preparation and biodegradation of environmental toxicants and other areas in recent years as it does not require coenzyme and regeneration. Our group have reported the outstanding performance of CPO in degradation of the water-soluble dye, this research are expected to be applied in the wastewater treatment industry. However, there are many difficulties in practice, such as temperature, pH of wastewater and use of organic solvents. The enzyme is easy to lose its activity in extreme conditions. At the same time, the free CPO can not be isolated from the reaction system, therefore it can’t be reused.Immobilization technology of enzyme is an effective method to achieve repeatability of enzyme and improve stability. At present, immobilized technology are often accompanied by aggregation of the enzyme and reducing the enzyme activity. To overcome this shortcoming, this paper attempts to control the pore size of porous materials, namely choosing a mesoporous material as immobilized carrier which just match the size of enzyme molecule, avoiding aggregation of enzyme molecules possible. The main results are as follows:1. Three different range of pore sizes of the porous material was prepared, namely inorganic mesoporous silica material FDU-12-140,3D-SBA-15and composite hybrid material CaCO3/Lentinan. The pore size of FDU-12-140,3D-SBA-15and CaCO3/Lentinan was12～17nm,8nm,30nm, respectively. Evaluating the immobilization effect depending on the amount of CPO immobilized into the carrier and use the degradation of Crocein Orange G to represent the activity of CPO.Mesoporous silica materials FDU-12-140for CPO immobilization was best, up to7.0mg/g. And material3D-SBA-15the biggest loading for the CPO was6.0mg/g. Due to the pore size of CaCO3/lentinan was too large relative to the CPO, so its loading of CPO was little. The michaelis constant Km of FDU-12-140-CPO was0.5times of free CPO and the Km of3D-SBA-15-CPO was the same with free CPO. It illustrated the affinity of CPO was enhanced to the substrate easily compared with free enzyme. In addition, the maximum reaction rate Vm of two immobilized enzyme FDU-12-140-CPO and3D-SBA-15-CPO was5.0and2.5times of free enzyme, respectively. Especially for substrate specificity, FDU-12-140-CPO improved17times compared with free CPO and3D-SBA-15-CPO increased to5times also, namely, improved substrate selectivity.2. Compared with the free CPO, immobilized CPO has good thermal stability, and the stability of immobilized CPO against organic solvent was improved. The immobilized CPO can maintain80%of its original activity after1h incubation at50℃, however, the free CPO was only about20%of its activity under the same condition. In addition, the degradation of dye by FDU-12-140-CPO can reach71%, while free CPO was only15%in the presence of1%methanol. In the acetonitrile solution, the degradation of dye by FDU-12-140-CPO was53%, while free CPO was only5%. When incubated with most hydrophilic1%N, N-dimethylformamide, free CPO has no activity whereas the immobilized CPO kept30%. After repeated use of22times, FDU-12-140-CPO still maintain80%of the original activity, and even in the1%methanol solution, FDU-12-140-CPO can be reused12times.3. The Composite materials CaCO3/lentinan as the carrier and the adsorption of dye Crystal Violet under the irradiation of a xenon lamp or mercury lamp was good. The results showed that compared with no carrier, dye bleaching rated up to100%when added the carrier within five hours, but in the case of no carrier material, it was only30%close to five hours under the irradiation of a xenon lamp. There were two main reasons for this phenomenon, fistly, electron of dye transitions to the3d or4s orbit of Ca2+to achieve the result of oxidative degradation of dye; secondly, due to the adsorption of the dye molecules. This method was simple in operation and the effect was significant, furthermore it provided evidence for subsequent enzymatic photocatalysis bifunctional bioreactors.