Immobilization Of Recombinant E.Coli Containing Nitrile Hydratase

Nitrile hydratase-containing cells are capable of catalyzing the hydration of nitriles to produce amides and have important industrial uses.However,free cells are easily inactivated during the catalysis process,and the disrupted cells affect the separation and purification of the product.Immobilized cell technology can effectively solve these problems.However,it has been generally reported that cells immobilized with nitrile hydratase have low stability or some toxicity during immobilization,which is harmful to cells.In order to solve the above problems,in this study,immobilization of recombinant Escherichia coli containing nitrile hydratase was carried out,and nicotinamide was formed as a model reaction by hydration of 3-cyanopyridine.And the main results are as follows:(1)The immobilization methods such as calcium alginate,polyacrylamide,carrageenan,agar and DA-F127 hydrogel were compared,and the enzyme recovery rate of the immobilized enzyme and the intermittent reaction of the immobilized cells were studied.Two kinds of immobilization methods with better effects were obtained by screening,namely calcium alginate immobilization method and DA-F127 hydrogel immobilization method.The enzymatic activity recovery rate of calcium alginate immobilized cells reached 48.08%,and the enzymatic activity recovery rate of DA-F127 hydrogel immobilized cells reached 61.28%.Both immobilized cells can be reused in more than 3 batches with less loss of enzyme activity.(2)The immobilization conditions were optimized for the calcium alginate method.The optimal conditions for immobilization were as follows:Sodium alginate concentration was 3%,calcium chloride concentration was 3%,cell addition amount was 17.5 mg/g carrier,and the enzyme recovery rate of calcium alginate immobilized cells was optimized to 56.73%.And the optimum pH of the immobilized cells was determined to be 7.0,and the optimum reaction temperature was 18 ?.The reaction process of the production of nicotinamide by immobilized cells was studied,and the optimal conditions were determined.Cells immobilized with calcium alginate under optimal conditions can catalyze the conversion of 150 g/L 3-cyanopyridine to 6 batches.The nicotinamide production per unit mass of cells was increased by 4.91 times compared to the free cell catalyzed process.(3)The optimization conditions for the immobilization conditions of the DA-F127 hydrogel method are as follows:The concentration of DA-F127 was 15%,the ultraviolet irradiation distance was 20 cm,the irradiation time was 6 min,the cell embedding amount was 20 mg/g carrier,and the optimal recovery rate of the immobilized cell enzyme activity was 89.74%.And the optimum pH of the immobilized cells was determined to be 7.0,and the optimum reaction temperature was 18?.Preliminary study on the production of nicotinamide by DA-F127 immobilized cells.Under optimal conditions,DA-F127 immobilized cells can catalyze the conversion of at least 9 batches of 150 g/L 3-cyanopyridine with free cells.The nicotinamide production per unit mass of free cells was increased by a factor of 12 compared to the free cell catalyzed process.