Production of Thermostable D-Hydantoinase from Recombinant Escherichia coli

• Main Authors: Chia-Hua Hsu, 許嘉華
• Other Authors: Yuan-Chi Su, Dr. Agr
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/69299189201114376492

碩士 === 國立臺灣大學 === 農業化學研究所 === 87 === D-Hydantoinase (Dht) is an industrially useful enzyme in the production of D-amino acids for the synthesis of semi-synthetic antibiotics, peptide hormones, pyrethroids, and pesticides. A DNA fragment containing the dht gene was cloned from Bacillus caldolyticus and expressed in E. coli under the control of the lac promoter. The effects of culture variables on Dht production by the recombinant E.coli NovaBlue BCDHT-1 were investigated by using an automatically controlled fed-batch fermentation system, to improve the productivity through high cell density cultivation. The compositions of semi-synthetic production (SSP) medium were 1% glucose, 1% yeast extract, 0.7% K2HPO4, 0.3% KH2PO4. About 100 U/mL Dht activity could be achieved in a 500 mL Hinton’s flask. The plasmid harboring dht gene was found to be stable maintained in E. coli for 200 generations. In a 5-liter jar fermentor, the compositions of both batch medium and feeding solution were 1% glucose, 0.75% yeast extract, 0.25% (NH4)2SO4, 0.2% KH2PO4 and 70% glucose, 35% yeast extract, 14% ammonia water, respectively. Glucose concentration was limited in the culture broth, and the pH was maintained at 7.0. Following a 15 h cultivation, 0.01 mM IPTG was added to induce dht gene expression and the temperature was shifted from 32 to 27℃ to avoid the formation of inclusion bodies. Under optimal conditions, a cell density of about 25 g DCW/L and a high volumetric yield of 400 U/mL could be achieved after 55 h cultivation at agitation speed and aeration rates of 1000 rpm and 1 vvm, respectively. The enzyme had a half-life of 16 days and retained 10% of its activity for 31 days at 50℃.