Development of fed-batch culture and recombinant proteins expression system in Bacillus subtilis DB430

• Main Authors: Shih-Yang Wu, 吳世揚
• Other Authors: Ching-Ping Tseng
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/81076043157997203816

碩士 === 國立交通大學 === 生物科技研究所 === 87 === Bacillus subtilis has many advantages for biotechnology that including nonpathogenic, easy to cultivate, store and secrete protein. Therefore, it has been used to product the recombinant DNA proteins. The goal of this study is to improve the biomass as well as recombinant proteins production ability in Bacillus subtilis. In order to obtain the high cell density, the optimization of fed-batch culture was studied. The experiment was carried out by using glucose as carbon source and yeast extract as nitrogen source. The parameters of control fermentation have been examined which includes how to manipulate Carbon and nitrogen sources feeding, agitation and aerate, as well as trace elements supply. The results showed that the optimal glucose concentration is 0.5% ( w/v ), and the consumption tare of yeast extract is 1%( w/v ) when O.D.600 value increased 8. When Bacillus subtilis DB430 grew in 2.5L fermenter at 37℃ and 700 rpm of stir rate and used 50% of oxygen, we can got the highest cell density ( O.D.600 value is 154 ) and the cell dry weight ( biomass is 54 g/l ). The highest expression of recombinant VP3 protein was1.4 g/l in fed-batch culture at 37℃, 700 rpm of stir rate. To protect the recombinant protein, a recE mutant was also construct in Bacillus subtilis DB430.