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Volume 37, Number 6, December 2004

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   

 

Corresponding author:

Dr. Jan-Hsiung Huang, Institute of Microbiology and Biochemistry, College of Life Science, NationalTaiwan University, Taipei, Taiwan 10617, ROC.E-mail: jhhuang@ntu.edu.tw This e-mail address is being protected from spam bots, you need JavaScript enabled to view it

 



 

Methods:

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.

 



 

Key words:

Escherichia coli, hydantoinase, inclusion bodies, plasmids, recombinant proteins



 



 

J Microbiol Immunol Infect 2004;37:313-321.