利用黑黴菌轉醣基酵素修飾維他命C之研究

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。

關鍵字

轉醣酵素；維他命C ；維他命C醣苷；醣化反應

並列摘要

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

並列關鍵字

無資料

參考文獻

Austria, R. A. Semenzato and A. Bettero, “Stability of Vitamin C Derivatives in Solution and Topical Formulations,” J. of Pharmaceutical and Biomedical Analysis, 15, 795(1997).

Bae, H. K., S. B. Lee and C. S. Park, “Modification of Ascorbic Acid Using Transglycosylation Activity of Bacillus Stearothermophilus Maltogenic Amylase to Enhance Its Oxidative Stability,” J. of Agricultural and Food Chemistry, 50, 3309(2002).

Box, G. E. P. and K. B. Wilson, “On The Experimental Attainment of Optimum Conditions,” J. of the Royal Statistical Society, B 13, 1(1951).

Bradford M. M. A., “A Rapid and Sensitive Method for The Quantitation of Microgram Quantities of Protein Utilizing The Principle of Protein-dye Binding,” Anal Biochem, 72, 248(1976).

Braun, S. and S. E. Vecht-Lifshitz, “Mycelial Morphology and Metabolite Production,” Trends in Biotechnology, 9, 63(1991).

國際替代計量

利用黑黴菌轉醣基酵素修飾維他命C之研究

全文下載

主題瀏覽