本研究以基因重組菌Escherichia coli JM109/pSCR05醱酵生產肌酸酵素（Creatinase）。在批次醱酵中發現，於第2小時添加誘導劑0.5 mM IPTG進行誘導時，肌酸酵素的產量能夠於8小時達到約3.0 U/mL，菌體濃度達到約2.0 g/L。然而在每4小時置換60%培養液（內含IPTG）的Fill-and-Draw醱酵中發現，雖然菌體濃度約可回昇到1.5 g/L，但卻無法繼續生產肌酸酵素，其原因為質體已經脫落。研究發現，添加IPTG誘導細胞生產外來蛋白質時，質體會非常不穩定，且當外在選擇性壓力Ampicillin存在時，會導致細胞分泌出較多β-Lactamase以分解Ampicillin。增加Ampicillin的量，對質體穩定性並沒有幫助，只會造成細胞分泌出更多的β-Lactamase至胞外。額外添加Methicillin於培養液中以作為β-Lactamase之抑制劑，雖可短暫提昇質體穩定性，但卻會使細胞分泌更多的β-Lactamase。另外，若降低操作溫度以降低細胞的生長速率，則對質體穩定性的提升有很大的幫助。由此可見以大腸桿菌做為宿主細胞時，質體穩定性與細胞的生長速率有很大的關係。

A recombinant Escherichia coli, JM109/pSCR05, was used for creatinase production. In a batch culture, where 0.5 mM IPTG was added at 2 h, the creatinase yield was 3.0 U/mL at 8 h, and the cell density was 2.0 g/L. During fill-and-draw culture, where 60% of the medium (containing IPTG) was replaced every 4 hours, although the cells grew to 1.5 g/L, creatinase was no longer being produced because of plasmid instability. It was found that production of the foreign protein by IPTG induction resulted in serious plasmid instability, and the selective pressure, ampicillin, made the cells to excrete β-lactamase. Supplementing the medium with more ampicillin could not improve the plasmid stability, but resulted in more β-lactamase excreted. Extraneous addition of methicillin as an inhibitor of β-lactamase could temporarily improve the plasmid stability; nevertheless, it caused even more β-lactamase excreted. Furthermore, lowering culture temperature to reduce growth rate had a great improvement on plasmid stability. It therefore suggests that for E. coli as the host cell, the plasmid stability is highly dependent on the growth rate.

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