Study On Fermentation Process Of Nitrile Hydratase Production By Nocardia Sp. And Enzymatic Properties

The nitrile hydratase(NHase, EC4.2.1.84) from Microbial, which catalyzes the hydration of nitriles to amides, is an important biological catalyst in the industrial production of acrylamide(AM). It has many advantages. Such as mild reaction conditions, high yields, fewer by-products. With the growing demand for AM, Chinese and foreign schloars have a widely research in NHase producing strain. They did many studies in the microbial strain screen, optimization of fermentation conditions and enzymatic properties, so achieves favourable results.The optimization conditions in the shaking flasks was determined. Under the condition of initial pH7.5, glucose concentration was 20g per liter, urea concentration was 7g per liter, sodium glutamate concertration was 1g per liter, the promoter was 1 milliliter per liter(Co²⁺ terminal concertration was 0.00248g per liter), the NHase enzyme activity reached up to 967.14U/mL.The effect of cell growth, pH and glucose consumption on the production of NHase was studied. It was shown that the production of NHase had positive correlation with the cell growth in the culture. A glucose fed-batch fermentation for production of high activity NHase in a 2L fermentor was established. In the glucose fed-batch fermentation that maintaind the pH at 6.5 and the glucose concentration in 1.5g/L～6g/L, the NHase activity was increased to 2814.3U/mL which was 8.7-fold compared to that in the batch fermentation.The cell ultrasonication condition was optimized aiming at the high specific acttivity of NHase in the cell free extract. Under the conditions of 160W, 5sec of sonication followed by 5sec cooling, the total time was 30min, the NHase specific activity reached up to maximum. The enzyme from cell free extract was preliminary purified by ammonium sulfate fractionation, the purification was 1.25 folds and the activity yield was 48.1%. The optimum temperature and pH of the purified enzyme was 30℃and 7.0.