Of Cyanide Hydrolysis Enzyme Preparation, Immobilization And Its Application Research

Nitrilase is such an enzyme that can convert nitriles into carboxylic acid or ammonium. Compared with the harsh condition of chemical synthesis, such as high pressure, high temperature, poisonous heavy metal catalysis and alkali, catalysis through enzyme brings about high value and wide application under mild condition in organic synthesis and industrial production. In this paper, extraction, purification, immobilization and application of nitrilase were studied.Phenylglycinonitrile were prepared by chemical synthesis. Recombinant nitrilase was purified by Ni-NTA affinity chromatography, and displayed a single band in SDS-PAGE, which indicates the purification of nitrilase. Then, the properties of recombinant nitrilase were studied, including optimum reaction temperature, pH and the tolerance to organic reagent. Considering the application of methanol as cosolvent for improving dissolution of subtracts, we also researched the tolerance of recombinant nitrilase to methanol.Recombinant nitrilase was separately immobilized on both amino-support and epoxy-support. We optimized the immobilized conditions, such as immobilized buffer pH, immobilized temperature and immobilized time, and so on. Under optimized conditions, we successfully prepared the immobilized nitrilase, and then studied the optimum reaction temperature, pH and stability of temperature and pH compared with purified recombinant nitrilase. The temperature and pH stability of nitrilase were enhanced after immobilization on amino-support or epoxy-support.