Monday, July 27, 2009
P36
Large Production of Renilla Luciferase in a Secreted Form by Bacillus subtilis
Chung-Jen Chiang, Department of Medical Laboratory Science and Biotechnology, China Medical University, Taiwan and Yun-Peng Chao, Chemical Engineering, Feng Chia University, 100 Wenhwa Road, Taichung, Taiwan.
Luciferase (Ruc) from the soft coral Renilla reniformis has been widely used as an excellent bioluminescent reporter. Thus far, secreted production of Ruc has been solely performed in mammalian cells. To make it more efficient, extracellular production of Ruc in Bacillus subtilis was attempted based on the pUB110/R6K-based plasmid. Furthermore, a simple and cost-effective medium was designed for the high-level production of Ruc using the response surface methodology. By the Plackett-Burman design, three critical medium components were identified as glucose, yeast extract, and glutamate. Followed by the Box-Behnken design, the medium was optimally formulated. With the optimum medium, the recombinant B. subtilis strain was grown in a bench fermenter to secret Ruc with the yield of 60 mg/L. Subsequently, the production scheme was carried out in a fed-batch mode. Before the cease of cell growth, the feeding solution was pumped into the culture broth at the pre-determined rate. Consequently, it resulted in a high cell density with a 5-fold increase in the secreted production of Ruc. This result indicates the promise of this proposed approach for the massive production of recombinant Ruc in a simple and effective way.
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