Investigation of Feeding Strategy in Fed-Batch Culture

• Main Authors: Li-Chun Cheng, 程麗君
• Other Authors: Teh-Liang Chen
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/13780469944694586887

博士 === 國立成功大學 === 化學工程學系碩博士班 === 91 === 　　A simple feeding method for controlling specific growth rate in fed-batch culture was developed. With a concentrate reservoir and two mixing chambers in series, this method can use a constant feed rate to simulate the exponential feeding. Fed-batch cultures with Escherichia coli DH5αshowed that the present feeding method could sustain cell growth at predetermined specific growth rates, where the time length for exponential growth was dependent on the magnitude of the growth rate. The present feeding method is ease to operate, requires no computerized control equipments, and thus could expect an extensive application in fed-batch culture to produce heterologous proteins. 　　Vibrio vulnificus, a halophilic gram-negative bacterium, is associated with serious wound infections and fatal septicemia in human. The nuclease of V. vulnificus was produced in the periplasmic space, which was found to possess both DNase and RNase activities and to be thermally stable. This research use a recombinant strain, E. coli DH5α/pSI026, to produce V. vulnificus nuclease. The effect of specific growth rate m on the nuclease production was investigated in fed-batch cultures. The pseudo-exponential feeding method for controlling m was employed to obtain the desired data. It was found that the nuclease content of cells gP varied with m, with a maximum occurred at m = 0.05 h-1; however, the specific nuclease production rate qp increased with an increase in m. The nuclease production was dependent on m, irrespective of the feeding methods. 　When concerning the compromise between the nuclease yield and its production rate, the linear-gradient feeding method, being simple and adaptable, was adequate for the nuclease production. 　　Although the production of recombinant protein has been extensively investigated, very few studies have discussed strategies of inducer addition. For better understanding of the effect of IPTG inducing strategies on the Interleukin 20 (IL-20) production, a recombinant strain, E. coli BL21(DE3)/pET-43a, was used in this study under controlled specific growth rates to produce IL-20 at the fed-batch mode. The results revealed that different strategies of inducer addition affected cell growth, plasmid stability, IL-20 synthesis, and the formation of inclusion body. It was found that the IL-20 content in cells (gP) varied with when IPTG was added by pulse. In addition, the maximum gP was found to occur at = 0.1 h-1. However, the specific growth rate and recombinant gene expression was not correlated when IPTG was added continuously. The results also demonstrated that continuous IPTG feeding could prevent a decrease in specific growth rate, but could increase IL-20 yield; moreover, it could sustain higher plasmid stability and allow less inclusion body formation.