由台灣南部電鍍工廠廢水中分離出的 Klebsiella oxytoca SYSU-011
具有分解氰化物之能力。本實驗利用二維電泳和 MALDI-TOF MS 分析
K. oxytoca 在四氰化鎳（tetracyanonickelate，TCN）處理下，其蛋白質
表現的變化。結果培養在含有 TCN 環境下的菌體，有明顯被誘導出兩
倍以上差異變化的蛋白質共 91 種，經質譜儀成功鑑定出共有 44 種。
TCN 所誘導出蛋白質如: chaperone、glutathione S-transferase (GST) 及
alkyl hydroperoxide reductase (AHR)，可防止菌體受到氧化性傷害；
fructose-bisphosphate aldolase 、glyceraldehyde-3-phosphate
dehydrogenase、phosphoglucomutase 及 6-phosphogluconolactonase，為
產能代謝反應之酵素，可提供耗能功能性蛋白所需要的能量；nitrogenase
及 glutamine synthetase (GS) 兩種氮源調解蛋白，以提供氮源供細胞使
用。另一方面，由於原核生物細胞膜充滿了許多酵素、運輸蛋白及受體
蛋白，參與物質輸出與輸入、蛋白質間的交互作用及呼吸作用等重要的
生理功能，所以本實驗亦萃取膜蛋白，在二維電泳及 MALDI-TOF MS
分析下，希望能瞭解在 TCN 處理下膜蛋白之消長。結果有明顯出現大
於兩倍的蛋白質共有41種，經質譜儀成功鑑定出的蛋白質共有 26 種。
TCN 處理下所誘導出的膜蛋白如: RND-drive efflux ( RND family,MFP subunit、outer membrane factor A 及 outer membrane factor TolC) 和
cation efflux system，可將高濃度的鎳運輸至細胞外，推測此菌可能是利
用此輸出膜蛋白避免細胞受到鎳毒性傷害。另外，誘導出的 ABC
transporter 可能扮演著氰化錯合物 TCN 及營養物質的運輸。這些結果
可以幫助我們更深入的瞭解 K. oxytoca 在 TCN 環境下可能的反應機
制。

Klebsiella oxytoca SYSU-011 isolated from wastewater of a
metal-plating plant in southern Taiwan had been proven to be able to
degrade cyanide. In this study, we performed proteomic analyses to
understand the mechanism of tetracyanonickelate (TCN) resistance in K.
oxytoca by using two-dimensional polyacrylamide gel electrophoresis (2-D
PAGE) and MALDI-TOF-MS techniques. There were 91 protein spots had
been induced or overexpressed (≥2 fold) by TCN. Among them, 44 protein
spots were successfully identified by MALDI-TOF-MS. The expressed
proteins that had been escalated including chaperone, glutathione
S-transferase (GST) and alkyl hydroperoxide reductase (AHR) were
involved in the TCN detoxification process. Fructose-bisphosphate aldolase,
glyceraldehyde-3-phosphate dehydrogenase, phosphoglucomutase and
6-phosphogluconolactonase were involved in energy-producing process;
nitrogenase and glutamine synthetase (GS) were required to regulate
nitrogen assimilation. We also analyzed the K. oxytoca membrane proteins.
Twenty six proteins spots had been successfully identified by
MALDI-TOF-MS (out of 41 protein) that were induced ≥2 fold by TCN.
These proteins induced RND-drive efflux proteins (RND family, MFP
subunit, outer membrane factor A and outer membrane factor TolC) and cation efflux system. These efflux pumps could transport nickel ion out of
the cells. The induced ATP-binding cassette (ABC) proteins may also play a
role in transportation of metal-cyano complexes TCN and nutrition.
By this study, we had a better understanding on the defense mechanism
of K. oxytoca after exposure to TCN.

摘要………………………………………………………………………I
目錄………………………………………………………………………V
圖目錄……………………………………………………………………VI
表目錄……………………………………………………………………VII
壹、前言…………………………………………………………………1
貳、實驗材料……………………………………………………………24
參、實驗方法……………………………………………………………27
肆、結果與討論…………………………………………………………47
伍、參考文獻……………………………………………………………71
陸、圖……………………………………………………………………96
柒、表……………………………………………………………………117
捌、附錄…………………………………………………………………130

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