臺灣博碩士論文加值系統

維他命C具有多種生理學及藥理學上的功能，同時也是生物體內重要的抗氧化劑。近年來隨著被證實具有抗癌及抗老的作用，在醫學界形成研究的熱潮。但維他命C非常不穩定，容易因光熱、空氣等影響，而氧化為不具活性的2,3-Diketogulonic Acid。為解決這樣的窘境，研究上採取修飾維他命C結構，達穩定維他命C衍生物之策略。醣化方法又以酵素催化法，反應條件溫和，不生成有害廢棄物而優於化學反應法。
過去的研究指出：轉醣酵素可將維他命C修飾成穩定的衍生物，且普遍存在於生物體內。本實驗採用黑黴菌Aspergillus niger的轉醣酵素，催化維他命C醣苷反應。A. niger可在菌體糾結的Pellet和培養的Broth中獲得大量的轉醣酵素。實驗上考量培養時間的成本效益，以及酵素活性的保存分便性，採用Pellet作為醣化酵素來源，初步測試最大酵素活性約500 U/ g-pellet。
吾人為增加培養基成分被菌體充分利用，經過一連串培養基篩選，由數種成分中選擇農產品殘餘物Rice Bran當作氮源，採用可充分被利用的Maltose作為碳源。經過實驗設計法討論後，可獲得最佳培養基組成：Rice Bran 1.8 %, Starch 4 % 以及鹽類K2HPO4 0.05 %。實驗發現：接菌量控制在1.5 %，經過前培養12小時和主培養24小時後，Pellet的轉醣酵素活性，有效提升到730 U/ g-pellet。
催化反應主要兩種酵素為：Glucosyltransferase 和 Amyloglucosidase。以Maltose 0.55 M和維他命C 0.14 M當做反應基質，溫度控制在30 oC，pH 3及200 rpm轉速下，反應6小時，可獲得89 % - 93 % 的轉化率。
研究觀察Pellet的酵素多數位於細胞膜上。以p-NPG當做反應基質，細胞內的醣化酵素比活性為約為2.3 ~ 5.9 U/ mg-protein；而Pellet細胞膜上的醣化酵素比活性為7.8 ~ 14.9 U/ mg-protein。轉醣酵素的最大反應速率 (Vm) 及Michaelis常數 (Km) 分別是 0.99 U 和0.19 M。實驗求得醣化反應活化能為1.97 cal/ mole與維他命C失活活化能比較，得知醣化反應可在極低的能量狀態下進行反應，因此維他命C在失活前即可有效的轉化成為維他命C醣苷。

Ascorbic acid (AA) plays an important role in physiological reactions and serves as a biological antioxidant. It possesses various functions in immune system modulation, regulation of collagen synthesis, and nitrous acid neutralization. Recently, it is confirmed that AA also plays a beneficial role against skin aging and shows anticancer activity. Therefore, it has the potential for uses in medicine and in cosmetic industry. However, ascorbic acid easily undergoes oxidation to an inactive compound under oxidative conditions such as heat, light, presence of transition metals and enzymes such as oxidases and consequently loses its anti-oxidative activity. Ascorbic acid can be converted into stable derivatives by structurally modifying it. Enzyme-mediated structural modifications are prefered over chemical processes.
Several studies conducted in the past revealed that glycosyltransferase, an enzyme that modifies the structure of ascorbic acid to a more stable ascorbic acid glucoside, can be obtained from several microorganisms. A mold, Aspergillus niger, is used as a biocatalyst in this study for the transglucosylation of ascorbic acid to ascorbic acid glucoside.
Through the design of medium by Response Surface Methodology (RSM), enzyme activity was increased by 230 U/ g-pellet compared to that using the unoptimized medium, using pellets from 2 days culture at 30oC and 200 rpm. It was observed that by using yeast extract, tryptone or soytone as nitrogen sources resulted in compact pellets than by using rice bran or corn steep liquor. Optimal cultivation medium contained: rice bran (1.8 %), starch (4.0 %) and K2HPO4 (0.05 %).
Glucosyltransferase and amyloglucosidase were identified to be the two key enzymes for the biosynthesis of ascorbic acid glucoside. 89%-93% conversion was obtained at 30oC and 200 rpm after 6 h of reaction using maltose (0.55 M) as sugar donor and ascorbic acid (0.14 M). The maximum velocity (Vm) and Michaelis constant (Km) for the transglucosylation were 0.99 U and 0.19 M, respectively. The activation energy (Ea) was 1.97 cal /g-mole

目錄
摘要 I
Abstract III
目錄 IV
第一章 緒論
1.1 前言 1
1.2 抑制黑色素生成機制 3
1.2.1黑色素生成與代謝機制 3
1.2.2抑制黑色素機制 7
1.3維他命C及其衍生物 9
1.3.1維他命C 的分布 9
1.3.2維他命C 的分布 10
1.3.3維他命C的應用 11
1.3.4維他命C衍生物 13
1.4 醣化反應 16
1.4.1化學法醣化反應 16
1.4.2酵素法醣化反應 17
1.5 研究動機 19
第二章 研究材料與方法
2.1 研究材料 21
2.1.1 Aspergillus niger簡介 21
2.1.2實驗材料 24
2.2 研究方法 26
2.2.1 Aspergillus niger的保存及培養方法 26
2.2.2維他命C醣苷反應方法 27
2.2.3 HPLC分析方法 27
2.2.4培養基的篩選 28
2.2.5蛋白質活性分析 28
2.2.6酵素催化反應動力學 30
第三章 實驗結果與討論
3.1 Aspergillus niger的生長 34
3.1.1 Aspergillus nige催化維他命C醣苷反應 35
3.1.2菌體的酵素分布 36
3.2最適培養基篩選 39
3.2.1氮源的篩選 39
3.2.2碳源的篩選 40
3.2.3無機鹽類的影響 40
3.2.4最適培養基設計 46
3.3酵素催化反應條件 53
3.3.1醣化反應基質組成 53
3.3.2醣化反應酵素比率 54
3.3.3溫度與pH效應 54
3.4酵素催化反應動力學 60
3.4.1 Michaels動力學參數 60
3.4.2活化能研究 62
3.4.3 A. niger酵素醣化能力研究 63
第四章 結論與未來展望
4.1結論 65
4.2未來展望 68
Reference 70

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李仰川，化妝品學原理 第二版。新文京開發出版 2003