A Technology Of One Step Enzyme Purification And Immobilization

Enzyme is the core of biocatalysis.A main form of enzyme participating in biocatalysis is Immobilized enzyme.But traditional method of enzyme Immobilization,purification and immobilization of the enzyme have to be completed by two steps.It takes more cost and more lost of the aim enzyme during the production.Immobilized metal affinity chromatography(IMAC)is a mature technology of protein purification.Isolation of histidine containing proteins to high purity can be efficiently performed using metal-chelate interactions within a single chromatographic step.When the crude extract flows through the pip of the vector,the recombinant protein with His-tag will be pulled out by the ion on the vector.But the chelating power between ion and His-tag is too weak to be used in enzyme immobilization.The enzyme caught by the vector is easy to get off.In order to solve this problem,make purification and immobilization of the enzyme completed with one step,based on the techniques of immobilized metal affinity chromatography(IMAC),a recombinant E.coli.strain was constructed to produce chimeric protein with a His-Arg-Asn-Tyr-Gly-Gly-Cys-Cys-Gly tag(Lab).The carrier of the immobilized enzyme in our work also can be reused by washing it with 0.3M EDTA.The researches of the immobilized enzyme with the new tag show that:The immobilized enzyme had a better thermal and storage stability compared to the free enzyme.It has good catalytic activities at 30℃,pH=8.0.The immobilized enzyme had a better storage stability compared to the free enzyme.The batch-catclytic of the new immobilized enzyme is 100 or more and does not get the half-life of the enzyme.The vector of the immobilized enzyme can be reuse at lest 5 times.The achievement of this research breaks a new path for the immobilized of industrial enzyme.