大腸桿菌中亞精胺合成酵素與氧化壓力相關性的研究

Studies on the relationships between spermidine synthase and oxidative stress in Escherichia coli

夏萬順(Wan-shun Hsia)

指導教授 ： 黃海美

國立清華大學/生命科學院/生物科技研究所/碩士(2012年)

摘要

亞精胺合成酶(spermidine synthase)在大腸桿菌合成亞精胺是相當重要的酵素，其可以催化亞精胺的生合成。在缺乏多胺酸的大腸桿菌中，在VB培養基中加入亞精胺(spermidine)和腐胺(putrescine)可以保護大腸桿菌免於受到雙氧水和氧氣的傷害。此外，近年的報告指出，若在阿拉伯芥中，使亞精胺合成酶過量表現能夠增加阿拉伯芥克���多種環境壓力的傷害，其中，也包含了氧化壓力。根據這些研究，這個研究方向將主要用來觀察亞精胺合成酶和氧化壓力間的關係。
為了研究氧化壓力和亞精胺合成酶的關係，不同濃度雙氧水引起的活性氧化物和大腸桿菌內亞精胺合成酶的多寡，被用來當作觀察這兩者是否相關的一個指標。將大腸桿菌分別以1、 2、或4 mM濃度的雙氧水處理一個小時後，在吸光值的觀測上，可以分別對大腸桿菌產生80.2、 64.7、 和 45.5 %的生長抑制。從連續稀釋方法所得到的菌落生長單位，和控制組比起來，細胞的存活率則降到70.4、 46.7、 17.7 %。而且在亞精胺合成酶含量的觀察上，與控制組相比，大約為控制組的83、 67、 和 50 %。
這個研究也探討了亞精胺合成酶對另一種氧化壓力的關係，活性氮物，不同濃度的亞硝基鐵氰化鈉(SNP)所以起的引起的活氮壓力和大腸桿菌內亞精胺合成酶的多寡，如同先前提過的，在將大腸桿菌分別以2、4、或8 mM濃度的亞硝基鐵氰化鈉處理一個小時後，在吸光值的觀測上，可以分別對大腸桿菌產生69.3、 65.6、 和 58.3 %的生長抑制。從連續稀釋方法所得到的菌落生長單位，和控制組比起來，可以分別對大腸桿菌產 75、 71、 和 31 %的生長抑制。而在亞精胺合成酶含量的觀察上，與控制組相比，大約減少了88、 102、和 59 %。
此外，這個實驗也測試了大腸桿菌在分別以8、12、16 mM濃度的亞精胺合成酶活性抑制劑，二環己胺(dicyclohexylamine)，處理一小時候的反應。在將大腸桿菌以8、12、 或16 mM濃度的二環己胺處理一個小時後，在吸光值的觀測上，可以分別對大腸桿菌產生82.9、 58.7、 和 41.7 %的生長抑制。從連續稀釋方法所得到的菌落生長單位，和控制組比起來，和控制組比起來，細胞的存活率則降到72.7、 18.7、 和 4.9 %。在DCHA處理的大腸桿菌中，與控制組相比，在將大腸桿菌處理8 mM 濃度左右的DCHA時，大腸桿菌中亞精胺合成酶含量減少了大約15%。，在12 ~16 mM濃度時，似乎能刺激亞精胺合成酶在大腸桿菌中含量大約增加了60%。在不同生長期的大腸桿菌中，也發現了的亞精胺合成酶含量的改變。主要會隨著在不同培養時間，如培養1、 2 和4 小時，亞精胺合成酶的含量都有顯著的減少。
另外加入4 mM濃度的二環己胺，與2 mM雙氧水處理的大腸桿菌一起培養一個小時，與只有加2 mM雙氧水的控制組相比，則減少了約20%的存活率。這些實驗指出大腸桿菌在雙氧水中的存活率，與亞精胺合成酶的活性在細胞中的含量是有關係的。

關鍵字

亞精胺合成酶 ； 二環己胺 ； 亞精胺 ； 氧化壓力

並列摘要

Spermidine synthase is an enzyme which catalyzed the biosynthesis of spermidine from putrescine. In the presence of putrescine and spermidine, polyamine deficient mutant E. coli was protected from hydrogen peroxide (H2O2) or oxygen in Vogel-Bonner medium (Chattopadhyay et al., 2003). In addition, recent reports showed that Arabidopsis thaliana with over-expressed spermidine synthase enhance the tolerance to multiple environmental stresses including ROS stress (Kasukabe et al., 2004). According to those report, this study is focused on investigating the relationship between spermidine synthase and oxidative stress.
Various concentrations of H2O2 induced ROS and the intracellular contents of spermidine synthase in E. coli were analyzed. After 1 hour treatment with 1, 2, 4 mM H2O2, the growth inhibition based on optical density at 600 nm was down to 80.2, 64.7, and 45.5 % of untreated control, respectively. The survivability based on colony forming unit (CFU) assay, the percentages of viable E. coli colony were detected decreased to 70.4, 46.7, 17.7 % of untreated control, respectively. And the amount of spermidine synthase decreased to 83, 67, and 50 % of untreated control sample.
The relationship between spermidine synthase and reactive nitrogen species (RNS) was also investigated in this study. Based on optical density at 600 nm, E. coli after 1 hour treatment with 2, 4, 8 mM sodium nitroprusside (SNP) resulted in growth inhibition to 69.3, 65.6, and 58.3 % of that from untreated control, respectively. Based on CFU assay, the percentages of viable E. coli were reduced to 75, 71, 31 % compared to untreated control, respectively. And the amount of spermidine synthase decreased to 88, 102, and 59 % of untreated control.
The response of E. coli after 1 hour treatment with 8, 12, 16 mM of spermidine synthase enzyme inhibitor, dicyclohexylamine (DCHA), was also determined. Based on optical density at 600 nm, 8, 12, 16 mM DCHA treatment resulted in growth inhibition to 82.9, 58.7, and 41.7 % of that from untreated control, respectively. Based on CFU assay, the percentages of viable E. coli were decreased to 72.7, 18.7, 4.9 % compared to untreated control, respectively. And the amount of spermidine synthase was approximately 15% decreased with 8 mM DCHA treatment. However, the amount of spermidine synthase was 60 % increased with 12~16 mM DCHA treatment. Further study investigated that intracellular amount of spermidine synthase at 1, 2, 4 hour incubating time which corresponded to log, late-log and stationary phase was decreased in E. coli culture.
Furthermore, 4 mM non-toxic DCHA co-incubated with 2 mM H2O2 treated E. coli for 1 hour, bacteria growth based in O.D.600 reading from DCHA plus H2O2 co-cultured samples reduced approximately 18% of that from H2O2 treated samples. The results suggest the survivability of E. coli co-incubated with H2O2 was highly correlated to the activity and intracellular amount of spermidine synthase.

並列關鍵字

spermidine synthase ； dicyclohexylamine ； spermidine ； oxiative stress

參考文獻

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