Enzyme Reaction Conditions Optimization Of A Nitrile Hydratase Producing Strain And Immobilization Of Cells

Acrylamide is a fine chemical product which was widely used. The acrylamide production by microbial method has prominent advantages such as mild reacting conditons, high convertion rate of acrylonitrile, low cost, therefore, it becomes one of the researching hotspots in recent years. The nitrile hydratase is the catalyst of the microbial production of acrylamide, so the high efficient expression of nitrile hydratase is the key to improve the production efficiency of acrylamide.The detecting method of acrylaimide, the reaction conditions and kinetics of nitrile hydratase, separation technique of nitrile hydratase and the immobilization of cells were studied with the strain Rhodococcus sp. HUST-1 which was isolated and breeded from activated sludge of wastewater treatment plant in the first period research. The main results are as follows:1.The optimum acrylamide detecting conditions of bromating method, GC and RP HPLC were determined, and by comparing with three detecting methods, the plan of detecting the acrylamide was determined: the RP HPLC was used as qualitative analysis and the bromating method was used to determine the acrylamide content.2.The reaction conditions were optimized, the maximum specific enzyme activity was 119U/(mL dry cells) in conditions as follows: the acrylonitrile concentration was 40 g/L, the reaction temperature was 28℃, pH was 7.0. The highest amount of cells put in was 15mL, the enzyme activity was 1218U. The km of nitrile hydratase of strain HUST-1 was 6.124g/L, the observed activation energy Ea was 47.8kJ/mol. The epuation of de-active dynamics of nitrile hydratase was that: lnk1=0.9391 0.0548-(1334.3 38.7)/T, and the observed activation energy Ea of the de-active reaction was 85.9kJ/mol.3 . Fragmentated method of strain HUST-1 by some kinds of non-mechanical means such as toluene, acetone, Triton X-100, lysozyme and autolysis was investigated. The results showed that the lysozyme had better fragmenting effect. The highest enzyme activity was 1308U, which was 29.2 percentage points higher than before treatment. The enzyme activity was got under these conditions as follows: The concentration was 7mg lysozyme in 1mL cells and the fragmentating time was 10 hous.4.Three immobilized methods of strain HUST-1 were studied, which were calcium alginate entrapping, polyvinyl alcohol crosslinking, and united entrapping with calcium alginate and polyvinyl alcohol. The results showed that the united entrapping with calcium alginate, polyvinyl alcohol and activated carbon had best effect. The relatively enzyme activity of the immobilized cells was 115.7%, the united entrapping method had evidently improved the biomass in per unit volume, the relative enzyme activity was 15.7 percentage points higher than the corresponding period in per unit volume, and the immobilized cells had higher stability.