- 學位論文
自Rhodobacter sphaeroides 分離與純化輔酵素-Q10之研究
Separation and Purification of Coenzyme Q10 from Rhodobacter sphaeroides
摘要
輔酵素Q10 (Coenzyme Q10)是人體電子傳遞鏈中生成能量adenosine triphosphate (ATP)的必須成分，為一種抗氧化物，具有防止脂質過氧化和清除體內過氧化物的功能，如自由基之清除。 本論文研究目的主要探討如何將光合菌之ㄧ---球形紅桿菌(Rhodobacter sphaeroides BCRC 13100)菌株生成之輔酵素Q10從醱酵液中分離並且純化。分成三部分探討。(1)前處理破菌方法，如酵素法、冷熱法、化學法和高壓破菌法，結果顯示，以酵素法而言，其產量最高可達2.85 mg/g-DCW，但其成本花費較高。其次是高壓破菌法效果最好，其產量也可達2.11 mg/g-DCW。另外則使用乙醇破菌，並且直接萃取，立即可省掉破菌的成本，其產量也可達2.01 mg/g-DCW；(2)分離方法分成兩種，破菌後，利用一些有機溶劑從細胞中萃取出輔酵素Q10、另一方法即利用乙醇在40℃水浴下經過30分鐘，直接破菌並且兩次萃取，結果顯示產量可達到2.46 mg/g-DCW，其萃取率可達到酵素法的0.86倍；(3)純化方法分成兩種方法進行，利用矽膠管柱層析使粗萃取物達到純化的效果，另一方法即使用正己烷做液-液萃取，再經過結晶後，結果顯示，純度可達到96.9%。
並列摘要
Coenzyme Q10 acts as an essential component of adenosine triphosphate (ATP) generation in the oxidative phosphorylation chain and as an antioxidant preventing lipid peroxidation and scavenging superoxide. The purpose of this study will focus on separation from the fermentation broth and purification of CoQ10 that produced by Rhodobacter sphaeroides BCRC 13100. Three topics were discussed as follows：(1) Disruption methods：the cells were disrupted by using enzyme, freezing and heating, chemicals or high press homogenizer. The results showed that the product yield could achieve 2.85 mg/g-DCW for enzyme method but its cost was higher. Secondly, the method of high press homogenizer was better that the product yield can achieve 2.11 mg/g-DCW. On the other hand, the ethanol could be used to disrupt and extract directly. The product yield could also achieve 2.01 mg/g-DCW. (2) Separation methods：CoQ10 was quantitatively extracted with various organic solvents. Using ethanol twice extraction could separate the products to release into the surrounding solution.CoQ10 was extracted by ethanol at 40℃ for 30min. The product yield could also achieve 2.46 mg/g-DCW. The product yield of ethanol disrupted method is 0.86 fold of the product yield using enzyme method. (3) Purification method：The crude extract was purified by silica gel column chromatography. Another method was liquid-liquid extraction with hexane. After crystallization, the purity determined by means HPLC could achieve 96.9%.