利用菸草細胞培養生產輔酵素Q10之研究

Abstract

中文摘要 由菸草植株誘導癒合組織，將所得細胞以懸浮培養生產輔酵素Q10。選擇菸草（Nicotiana tabacum L. c.v. Wisconsin 38）葉片為培植體，使用MS培養基（加2,4-D 2.0 ppm, Kinetin 0.2 ppm, 蔗糖 3%）誘導癒合組織。建立懸浮培養系統後，使用篩析法（Mesh No.為 10, 18, 45）將細胞分成不同粒徑以達到同步化。進行生長曲線測定，求得最合適的繼代天數。Q10定量方法採用HPLC法，於275 nm下分析。細胞於第八天進入定常期（stationary phase），乾重 10.8 g/L，Q10 總量668 μg/L，Q10含量 61.9 μg/g。 針對培養基碳源、氮源、荷爾蒙不同條件作最適化探討，實驗結果顯示，蔗糖為3%時為最適濃度，甘露糖醇雖能提高Q10含量卻會抑制生質量，與控制組相比約降低五分之四。此外，NH4+/NO3－的比例為20/40時Q10產率最佳，但含量卻於30/30時最高。Q10產率則在2,4-D 2.0 ppm，Kinetin 0.2 ppm時可達 732 μg/L。培養環境方面，初始pH值為5.7、溫度26℃下，細胞生長及Q10的累積都較佳。而前驅物的添加，以L-Tyrosine較具效果，當濃度達1000 ppm時，可提升Q10產量，其餘如4-Hydroxybenzoic acid、p-Coumaric acid，Mevalonic acid的添加，對於Q10的增加並無助益。金屬離子Mg2+的添加則會造成Q10和生質量的減少。

Abstract A callus from explant of tobacco was induced with MS medium (supplemented with 2.0 ppm of 2,4-D, 0.2 ppm of kinetin, 3 w/v% of sucrose). The leaf of Nicotiana tabacum L. cv. Wisconsin 38 were used for the callus development. Coenzyme Q10 was produced by suspension culture of induced callus. After suspension culture system was established, fractionated the cells into different sizes by using stainless steel sieves (mesh no. 10, 18, 45) for synchronizing the cells. The growth curve was obtained and the suitable subculture period was determined. Q10 was determined by HPLC with Nacalai Cosmosil 5C18-AR column by UV detector under 275 nm. Cells entered stationary phase on the eighth day. Dry cell weight, total Q10 and Q10 content reached 10.8 g/L, 668 μg/L, 61.9 μg/g dry wt, respectively. The optimum concentrations of carbon source (sucrose, glucose ,fructose and mannitol), nitrogen source (peptone, yeast extract, casein, casamino acid, (NH4)2SO4 and KNO3) and hormones were found. The results showed that the optimum concentration of sucrose was 3 w/v %. Mannitol (7%) favored the accumulation of Q10 content, but suppressed the cell growth. The NH4+/NO3－ ratio of 20/40 maximized the total Q10 productivity and the ratio of 30/30 maximized the Q10 content. Under pH 5.7 and temperature 26℃, both cell growth and Q10 formation were high. 1000 ppm of L-tyrosine dosed as a precursor enhanced Q10 productivity. The other precursor, 4-hydroxybenzoic acid, p-coumaric acid and mevalonic acid did not enhance the productivity of Q10. It was found that the Mg2+ repressed Q10 formation and cell growth.