以重覆批次策略培養 Aspergillus niger 及其在維生素C 醣苷生產之研究

Repeated Batch Cultivation of Aspergillus niger and Its Applications for L-Ascorbyl Glucoside Production

施淑銀(Shu-Yin Shih)

指導教授 ： 吳文騰 ； 朱一民

國立清華大學/工學院/化學工程學系/碩士(2006年)

摘要

維生素C具有良好的抗氧化性，亦有抑制黑色素生成的能力。在抑制黑色素生合成方面，維生素C扮演著還原氧化態的多巴奎寧，使其變為無色的多巴，也可直接抑制酪胺酸酶的活性以阻斷黑色素生合成途徑。由於維生素C對光與熱相當敏感，不適合做為保養品中的添加物，因此發展維生素C衍生物作為美白保養品之添加物為研究重點—維生素C醣苷即為其中一種。
本研究培養 Aspergillus niger 利用其醱酵液中含有的醣化酵素來進行維生素C醣化成維生素C醣苷。研究著重於建立較適化的批次醱酵條件及運用重覆批次培養策略來大量生產具有醣化能力的醱酵液。
本研究結果顯示，利用 A. niger 之醱酵液可進行維生素C醣化修飾，較適化的批次培養條件為培養溫度 30oC、轉速 630 rpm、通氣量 1 vvm，培養基組成為 6.6% yeast extract、4.5% maltose、0.5% K2HPO4，培養時間為 48 小時，其醱酵液的醣化轉化率可達 80% 以上。在重覆批次培養策略以單一麥芽糖溶液及原始培養基的交替使用作為饋料成份，每次取出體積為 3 L，整個重覆批次醱酵至少可達七次，且平均批次培養時間為 24 小時，醱酵液之醣化轉化率均可達 80% 以上。
本研究亦嘗試純化醱酵液中參與醣化維生素C反應之醣化酵素。醱酵液經由硫酸銨沈澱及陽離子交換樹脂層析後，可得較純之單一蛋白。將此蛋白質進行 MALDI-TOF mass spectrometry 分析並與資料庫比對，已成功的分離及鑑定出可醣化維生素C之醣化酵素為 alpha-glucosidase。

關鍵字

黑麴黴菌 ； 重覆批次培養 ； 醣解酵素

並列摘要

L-Ascorbic acid is an excellent anti-oxidant, which is widely used in a number of food and cosmetic products. It plays an important role in reducing dopaquinone into dopa and inhibiting the tyrosinase activity. It can directly break down melanin biosynthesis.
The main disadvantage of L-ascorbic acid for use in cosmetic preparations is its sensitivity to light and heat. In order to overcome this problem, it is important to develop stable derivatives of L-ascorbic acid. Ascorbyl glucoside is one such derivative of L-Ascorbic acid, which is produced by the glucosylation of L-ascorbic acid.
In this study, the use of the culture supernatant of Aspergillus niger for glycosylation of L-ascorbic acid was investigated. The study involved the development of suboptimal fermentation conditions and using repeated batch process for obtaining large amount of culture supernatant for the production of ascorbyl glucoside.
The results showed that the culture supernatant of A. niger could effectively glycosylate L-ascorbic acid to ascorbyl glucoside. The suboptimal batch culture conditions were: temp, 30oC; agitation; 630 rpm, aeration, 1 vvm, and the culture medium composition was: 6.6% yeast extract, 4.5% maltose, 0.5% K2HPO4. The conversion using supernatant of 48 h old batch culture reached to 80 %. The reaction time was 6 h at 30oC. For repeated batch culture, the suboptimal condition was found to be by using medium replacement and maltose addition alternatively in a 10 L fermenter. The temperature was 30oC and agitation was 630 rpm . The repeated volume was 3 L, and the fed-batch fermentation was repeated 7 times. The average repeated batch culture time was 24 h. The conversion of ascorbyl glucoside obtained using culture supernatant from repeated batch culture was above 80%.
Identification of the glycosylation enzyme from culture supernatant by ammonium sulfate fractionation, followed by ion-exchange chromatography was also attempted. The enzyme protein was analysis by SDS-PAGE and MALDI-TOF mass spectrometry. The data from mass spectrometry was aligned with NCBI database for identifying the sequence.

並列關鍵字

Aspergillus niger ； Repeated batch ； alpha-glucosidase

參考文獻

Austria, R., Semenzato, A. & Bettero, A. (1997). Stability of vitamin C derivatives in solution and topical formulations. Journal of Pharmaceutical and Biomedical Analysis 15, 795-801.

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-254.

Breton, C., Mucha, J. & Jeanneau, C. (2001). Structural and functional features of glycosyltransferases. Biochimie 83, 713-718.

Chen, W. C., Lee, S. L. & Tung, P. Y. (1996). Effects of initial maltose concentration, agitation and aeration rates on the production of glucosyltransferase by Aspergillus niger CCRC 31494. Biotechnology Letters 18, 489-494.

Colven, R. M. & Pinnell, S. R. (1996). Topical vitamin C in aging. Clinics in Dermatology 14, 227-234.