添加正十六烷及全氟碳化合物對盤尼西林生產菌之影響

Abstract

由於盤尼西林醯畫酵素是抗生素工業上將盤尼西林作用成6- 胺基盤 尼西林酸的重要酵素, 所以本實驗係針對於醱酵培養基中添加高溶氧化 合物正十六烷及全氟碳化合物FC-40, 使基因改質過的大腸桿菌ATCC 9637 (pGL5) 的生長速率提高, 並增加菌體生產出的盤尼西林醯化酵素活 性. 根據文獻記載, 正十六烷對氧的溶解度為同體積純水的八倍, 而全氟 碳化合物對氧的溶解度則為同體積純水的十至二十倍之譜. 因改質過的大腸桿菌ATCC 9637 (pGL5) 所生產盤尼西林醯化酵素的活性 會受到碳源的抑制, 所以我們選擇使用山梨糖醇當作碳源, 並將山梨糖醇 的濃度固定在百分之四, 以維持菌體在對數生長期, 使菌體的生長速率增 加. 我們發現在培養基中添加體積百分之五 的正十六烷或體積百 分之七的全氟碳化合物FC-40, 能提高菌體生長濃度為1.2-1.3倍,提高盤 尼西林醯化酵素活性約1.1-1.2倍. 我們也發現在進行大腸桿菌ATCC 9637 (pGL5) 醱酵時, 如在菌體對數生長期, 亦即培養第六至八小時加入體積 百分之五的正十六烷或體積百分之七的全氟碳化合物FC-40, 對菌體的生 長及酵素的活性具有明顯的促進效果. 因為產生盤尼西林醯化酵素之 大腸桿菌ATCC 9637 (pGL5) 為一次代謝產物之菌體, 故我們同時也比較 了正十六烷及全氟碳化合物對二次代謝產物之菌體Penicillium chrysogenum的影響, 發現此類化合物對二次代謝的菌體或產物亦有促進 之功效, 菌體增加約1.08-1.09倍, 而產物則增加至1.5倍左右. 所以我們 相信正十六烷及全氟碳化合物對生物體, 無論是一次代謝或是二次代謝產 物, 應該都具有提高生長速率及促進產物產率的功效. Penicillin acylase is an industrial important enzyme. It has been widelyused in the commercial production of 6-aminopenicillanic acid(6-APA) throughthe deacylation of penicillin G. As 6-APA is the key intermediate for the production of a wide range of semisynthetic penicillins, it warrants study on more effective ways to obtain and utilize penicillin acylase. The purpose of this research is to enhance the productivity of 6-APA by addition of n-hexadecane and perfluorocarbon FC-40. In this thesis, we have studies the kinetics of penicillin acylaseproduction by using recombinant E. coli ATCC 9637 (pGL5) cells as the modelsystem. The particular strain is catabolitically repressed by glucose and not induced by phenylacetic acid(PAA). As such, a fed-batch culture system is the choice in order to reach high cell densities in a short period of time. It has been shown that the oxygen solubility in n-hexadecane is eight times higher than that in pure water; and the oxygen solubility in perfluorocarbon (PFC) FC-40 is 10-20 times that of pure water. It has been demonstrated that, by simply adding 5% n-hexadecane and 7% PFC FC-40, we can increase cell growth by 1.2-1.3 fold and the activity of penicillin acylase by 1.1-1.2 fold. We havefound out that the optinum time for the addition of 5% n-hexadecane and 7% PFCFC-40 would be at the log phase, approximately six to eight hours into the fermentation. Penicillin acylase is primary metabolic product of E. coli ATCC 9637 (pGL5).We have compared the findings with secondary metabolic products using the penicillin producing Penicillium chrysogenum for the study. Again, we have observed that adding 5% n-hexaecane and 7% PFC FC-40 can promote P. chrysogenumgrowth by 1.07-1.08 fold and penicillin production by 1.5-1.6 fold, respectively.It is interesting to note that the addition of n-hexadecane and perfluorocarboncan enhance the growth of cells and their production of primary and secondary metabolites.