摘要或動機

在嗜甲烷菌中，甲烷與甲醇間的轉化是由甲烷單氧化酵素來進行。目前已知有兩種型態\r
的甲烷單氧化酵素，一種是溶解型甲烷單氧化酵素，存在於較低銅離子濃度之水溶液環境中﹔\r
另外一種為微粒體甲烷單氧化酵素，鑲嵌在細胞內質膜上，表現於較高的銅離子濃度環境下。\r
除了本身的天然基質－甲烷之外，其他種類之簡單烷烯類化合物，甚至芳香族化合物，均可\r
作為此酵素催化的基質。其中，甲烷單氧化酵素將丙烯轉化成環氧丙烯與甲烷轉化成甲醇的\r
催化活性非常接近，因此丙烯普遍被用來作為酵素活性測量的基質。為了直接測量它們的活\r
性，我們設計出一種方法，可以讓我們直接利用氣相色層分析儀，來偵測細胞的催化反應過\r
程。基於異丁烷在甲烷單氧化酵素幾乎不存在任何活性，故我們將其作為內標準氣體，並藉\r
由丙烯在氣相色層分析儀中吸收訊號的遞減來偵測細胞的催化活性。在多樣性的動力學實驗\r
中，我們發現以sMMO 為催化酵素時，丙烯的轉化是依據一級動力學反應趨勢而減少。相對\r
的，以pMMO 為催化酵素時，丙烯的減少趨勢則是依據零級動力學反應模式進行。在比較完\r
Pipes 緩衝液、上清液蛋白質及內質膜蛋白質溶液之丙烯吸附量測試結果後，我們發現內質膜\r
蛋白可吸附的丙烯分子相對於其它兩種溶液是最多的。依據Michaelis-Menten 動力學理論，\r
可得到以下結論﹕丙烯的轉化在sMMO 中是以基質受限的催化形式進行，而在pMMO 中則\r
已達到最佳的催化速率。\r
\r
In methanotrophs, the oxidation of methane to methanol is catalyzed by methane\r
monooxygenase. There are two distinct forms of the enzyme associated with different gene\r
products. One is the soluble methane monooxygenase (sMMO) expressed in the cytosolic portion\r
of the cell and grown under copper-limiting growth conditions. The other enzyme is the\r
particulate methane monooxygenase (pMMO), a membrane-associated protein that is expressed\r
under high copper-to-biomass ratios. In addition to the natural substrates of methane gas,\r
simple aliphatic alkanes, alkenes, or even aromatic compounds could be used as the substrates of\r
the methane monooxygenase. In those gaseous simple alkenes and alkanes, propylene converted\r
to propylene oxide by methane monooxygenase has been considered as popularly use for enzymatic\r
activity determination because of its comparable activity to the methane gas. To measure the\r
catalytic behavior of the methanotroph directly, we design a method to choose isobutane as the\r
internal standard because of the negligible activity in the methane monooxygenase. The catalytic\r
activity can be simply inferred from the decrease of the gaseous propylene signals in the GC\r
chromatograms by generating the liquefied epoxides mediated by MMO within the methanotrophic\r
bacteria. Under various kinetics measurements, when we incubate the methanotroph grown under\r
copper-limiting concentrations, we observed the diminishment of propylene follow a first-order\r
kinetic behavior with the over-expression of soluble methane monooxygenase. However, the\r
growth of bacteria under 40 　M presents the zero-order kinetic trend with the bulk expression of\r
pMMO. After the quantification of the dissolved propylene in the deionized water, soluble\r
proteins solution as well as membrane proteins solution, we observe the membrane proteins could\r
adsorb more propylene molecules in comparison with the other solution mixtures. By considering\r
Michaelis-Menten kinetics, we conclude the propylene conversion in sMMO is under substrate\r
limiting catalysis whereas the pMMO has attended the optimized velocity of propylene conversion.