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標題: 應用陶瓷薄膜高細胞密度培養基因重組大腸桿菌
Application of ceramic membrane in high cell-density cultivation of recombinant Escherichia coli
作者: 吳信府
Wu, ShinFu
關鍵字: 陶瓷薄膜;Ceramic membrane;高細胞密度培養;基因重組大腸桿菌;盤尼西林醯胺酵素;High-cell-density cultivation;Recombinant E. coli;Penicillin G Acylase
出版社: 化學工程學系
The objective of this study was to cultivate a recombinant E. coli strain that carries plasmid pGL-5 for Penicillin G acylase production through the ceramic membrane filtration. Penicillin G acylase (PAC) hydrolyzes penicillin G to 6-aminopenicillanic acid (6-APA), which is used in the semi-synthetic antibiotics. Recombinant E. coli is preferentially used for production of PAC enzyme in industry, owing to its well-known molecular genetics and physiology. In order to enhance the PAC productivity, high cell density cultivation is required. In this study, high cell density cultivation of rE. coli was investigated in fermentation process. The concentration of acetic acid in the fermentor was reduced by ceramic membrane micro-filtration. When cultivating rE. coli in the fermentor without ceramic membrane, the result revealed that accumulation of acetic acid concentration would reached 37.5 g/L with the cell concentration of 32.5 g-DCW/L, and the cell growth yield of 0.163 g-DCW/g-glucose. When the fermentation conditions were set as the fed-batch culture with oxygen enrichment, and removing the metabolic by-product through the ceramic membrane filtration, the acetic acid concentration reduced to only 12 g/L and the cell concentration could reach 86.098 g-DCW/L with the cell growth yield of 0.431 g-DCW/g-glucose. It was proven that ceramic membrane filtration could effective remove the inhibitive metabolite-acetic acid. In addition, in order to speed the filtration rate and improve the efficacy, regularly back flushing is applied, with the help of the membrane to continuous remove acetic acid, the cell concentration could increase to 113.96 g-DCW/L with the cell yield of 0.57 g-DCW/g-glucose.

本研究主要目的是利用一個陶瓷膜微過濾裝置,串聯一基因重組大腸桿菌反應器,以生產帶有pGL-5質體可產盤尼西林醯胺酵素。以大腸桿菌生產PAC酵素為工業上常用之方式,由於PAC酵素係E.coli之胞內酵素,為了達到PAC酵素的高產率,本研究構想於醱酵過程中,進行高密度細胞醱酵培養。利用陶瓷薄膜孔隙度大小產生的過濾作用,藉此降低醋酸在醱酵槽中累積的濃度。研究結果顯示,未使用濾膜時,醋酸濃度高達37.5 g/L,菌體濃度為32.5 g-DCW/L,而其細胞產率為0.163 g-DCW/g-glucose。在高密度細胞醱酵培養過程中,足夠的溶氧量和適當的饋料模式,加入陶瓷薄膜的過濾作用後,醋酸累積濃度僅12 g/L,菌體濃度為86.098 g-DCW/L,其細胞產率為0.431 g-DCW/g-glucose。證實陶瓷薄膜過濾可有效的移除抑制性代謝物-醋酸。另外使用薄膜過濾法中,為了提高濾速,增進過濾效果,採用定期性的逆洗以除去阻塞物,持續移除醋酸,菌體濃度可提升至113.96 g-DCW/L,其細胞產率為0.57g-DCW/g-glucose。
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