Study On The Immobilization Of A Nitrilase By Magnetic Materials

In this paper, an innovative technique of immobilizing E. coli BL21 (DE3) PET21-NIT whole cells using Fe3O4-alginate as carrier was developed. It can catalyze the hydrolysis of 1-cyano-cyclohexyl-acetonitrile to 1-cyano-cyclohexyl-acetic acid, which is an important synthetic intermediate of Gabapentin. Firstly, the immobilization conditions was studied and the optimized conditions includes:50 g/L E. coli cells,3.0% (M/V) sodium alginate,50 g/L Fe3O4,0.2 M CaCl2 solution and 1.5 mm bead diameter. Secondly, the magnetic immobilized cell was characterized. They possess magnetic feature that could be separated quickly (within several seconds) under the external magnetic field. And the mechanical strength of bead was stronger than calcium alginate bead without Fe3O4. Finally, compared with free cell, Fe3O4-alginate beads showed better pH stability when the amount of alkaline was increased, and resistance to high temperature was enhanced. Enzyme activity could remain more than 60% after 30 days storage while free cells remained only 40%. Moreover, after 40 batches the yield of 1-cyano-cyclohexyl-acetic acid catalyzed by Fe3O4-alginate beads still remained 84%.The immobilization of nitrilase was studied using beaver beads as carriers. The optimal immobilization conditions included:15 mM imidazole of Buffer A,6 g/L nitrilase,30 min time for immmobilization. The optimal concentrations of beaver beads for 3-cyanopyridine and 1-cyano-cyclohexyl-acetonitrile was 3 g/L and 4 g/L, respectively, and the optimal reaction pH value was 7.0. The results of the free enzyme and immobilized enzyme showed that the immobilized nitrilase had better pH stability than the free enzyme. The reaction batches catalyzed by immobilized nitrilase can reach 8 times, and the residual activity was still 68%.