Effects of culturing conditions on production of D-hydantoinase from recombinant Escherichia coli
Jan-Hsiung Huang, Wen-Hwei Hsu, Chun-Wei Chen
Institute of Microbiology and Biochemistry, National Taiwan University, Taipei; and Institute of Molecular Biology, National Chung-Hsing University, Taichung, Taiwan, ROC
Received: February 9, 2004 Revised: March 12, 2004 Accepted: April 26, 2004
The effects of culturing conditions on D-hydantoinase production by a recombinant Escherichia coli strain were investigated using a controlled fed-batch fermentation system. Glucose concentration and pH of the culture broth were maintained at less than 3.3 g/L and at 7.0, respectively, in a 5 L jar fermentor. The optimal composition of the batch medium was glucose, 0.25%; yeast extract, 0.75%; (NH4)2SO4, 0.25%; KH2PO4, 0.2%. The optimal feeding solution was glucose, 60%; yeast extract, 30%; ammonia water, 9%. Following 25-h cultivation, 0.02 mM isopropyl-ß-thiogalactopyranoside was added to induce dht gene expression and the temperature was shifted from 32 to 27° C to avoid inclusion body formation. The plasmid-harboring dht gene was found to be stably maintained in the E. coli. Under optimal conditions, a cell density of about 25 g dry cell weight/L and a high volumetric productivity of 8300 U/L/h could be achieved after 48 h culture at agitation and aeration rates of 1000 revolutions per minute and 1 vvm, respectively.
Escherichia coli, hydantoinase, inclusion bodies, plasmids, recombinant proteins
J Microbiol Immunol Infect 2004;37:313-321.