Effects of culturing conditions on production of D-hydantoinase from recombinant Escherichia coli

Jan-Hsiung Huang¹, Wen-Hwei Hsu², Chun-Wei Chen^1
1Institute of Microbiology and Biochemistry, National Taiwan University, Taipei; and ²Institute of Molecular Biology, National Chung-Hsing University, Taichung, Taiwan, ROC

Received: February 9, 2004 Revised: March 12, 2004 Accepted: April 26, 2004

Corresponding author: Dr. Jan-Hsiung Huang, Institute of Microbiology and Biochemistry, College of Life Science, NationalTaiwan University, Taipei, Taiwan 10617, ROC.E-mail:

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--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.