Studies On Fermentation, Purification Of RhNDPK-α And Its Physicochemical Properties

OBJECTIVE.To establish the pilot fermentation process and the purification process of bio-active NDPK-A METHODS:Ampicillin resistance test, plasmid extracting, double cleavage test, expression test was performed to study the genetic stability of the recombinant strain (DH5a-pBVnm23-Hl). The effect of different media ingredient and different ingredient concentration was compared. The establishment of the optimal fermentation process was based primarily on the flask-shaking test. And the efficiency of different purification pathway was compared. RESULTS:Through the experiments mentioned above, we found that after 50 generations of successive culture, the genetic stability of the DH5a-pBVnm23-Hl was not affected. Use the selected media and fermented under established condition, we could achieve more than 25g wet bacteria per liter culture, and the NDPK-A derivative was more than 28% of total protein. Employ the optimal purification process, target protein can reach a purity of more than 99% while enzyme's specific activity is above 800U/mgin the ATP-UDP reaction system.CONCLUSION:The fermentation process and purification pathway of rhNDPK- a was established.