以限制葡萄糖濃度饋料醱酵培養重組大腸桿菌生產Colicin E7 Nuclease 之研究

The Study of Colicin E7 Nuclease Production by Recombinant Escherichia coli in Glucose-limited Fed-batch Cultivation

雷博欽(Bo-Chin Lei)

指導教授 ： 陳志成

大同大學/工程學院/生物工程學系所/博士(2006年)

摘要

於本研究中利用饋料批次醱酵培養重組Escherichia coli DH5 以生產含有核酸水解酶(nuclease domain)與免疫蛋白(immunity protein, Im7)之coliciny Im7 complex。由於核酸水解酶為細胞毒素，所以當IPTG進行誘導表現時，必須藉由coliciny Im7 complex 的生成以降低細胞毒素的毒性。利用Luria-Bertani (LB) medium 進行批次醱酵培養，所獲得的coliciny Im7 complex比活性(specific biomass activity)、單位體積活性(volumetric nuclease activity) 與產率活性(productivity of nuclease activity)分別為763 U/g cell、4223 U/l 與384 U/l/h。
於研究中利用2III3-1 部分因子實驗設計獲得之semi-synthetic medium (SS medium) 可以獲得最大的細胞產量。採用SS medium 進行批次醱酵培養可以增進coliciny Im7 complex 之產量。因此，於SS medium中進行限制葡萄糖濃度(1 g/l glucose)之控制策略，並且於饋料過程添加酵母抽出物(yeast extract)可以coliciny Im7 complex比活性(specific biomass activity)、單位體積活性(volumetric nuclease activity) 與產率活性(productivity of nuclease activity)分別為24852 U/g cell、618187 U/l 與41212 U/l/h。

關鍵字

重組大腸桿菌 ； 饋料醱酵培養

並列摘要

In this work, we developed a fed-batch culture to enhance the production of the coliciny Im7 complex, which consisted of nuclease domain and immunity protein (Im7) of colicin E7 using a recombinant Escherichia coli DH5. Since the nuclease activity is cytotoxic, the cell biomass may degrade after isopropyl--D-thiogalacto- pyranoside
(IPTG) induction for gene expression. Therefore, it is beneficial to reduce the cytotoxicity of nuclease domain with coliciny Im7 complex. When Luria-Bertani (LB) medium is added to the batch culture, the specific biomass, volumetric and productivity of nuclease activity of coliciny Im7 complex were measured to be 763 U/g cell, 4223 U/l and 384 U/l/h.
We successfully developed a semi-synthetic medium (SS medium) to obtain maximum biomass by 2III3-1 fractional factorial design. SS medium could be utilized to increase coliciny Im7 complex production. By using a SS medium, along with the glucose-limited control strategy (at 1 g/l of glucose concentration), and adding yeast extract (YE) at post-induction stage, increased the specific biomass, volumetric and productivity of nuclease activity were increased to be 24852 U/g cell, 618187 U/l and 41212 U/l/h, respectively.

並列關鍵字

Fed-batch cultivation ； Recombinant E.coli

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