橙黃壺菌BL10 (Aurantiochytrium mangrovei strain BL10) 能利用特殊的多元不飽和脂肪酸合成酶 (Aurantiochytrium mangrovei polyunsaturated fatty acid synthase, AmPFA) 合成22碳6烯酸 (docosahexaenoic acid, DHA)。本研究的目的，在於建立AmPFA的純化平台，利於後續的活性及結構分析，以了解AmPFA是如何參與在DHA的生合成。首先確認能讓BL10穩定生產AmPFA的培養條件，接著優化AmPFA的萃取方法，隨後利用硫酸銨沉澱法，搭配陰離子交換樹脂或膠濾層析法進行純化。在以50 mL離心管搭配27oC及GYSS 培養基進行搖晃培養所獲得之BL10藻體可穩定的生產AmPFA。以液態氮進行反覆凍溶BL10的作法，可破壞BL10細胞，大幅提高AmPFA的萃取效率。在利用30%和40%飽和度硫酸銨進行處理時，可以將粗萃液中的AmPFA沉澱下來，並移除絕大多數小分子量的雜蛋白，然而在進一步的進行離子交換層析時，AmPFA3有明顯降解的情況。在改用膠濾層析管柱進行純化時，雖然沒有出現前述AmPFA3降解的問題，但依其滯留的時間研判，其未與其他兩個次單元: AmPFA1及2形成聚合物。進一步以Blue native PAGE分析經硫酸銨粗分的樣品，發現AmPFA3出現在分子量370 kDa，與預測的完整聚合物 (730 kDa) 大小不符，證實在經過硫酸銨沉澱處理後AmPFA聚合物有分離的現象。

Aurantiochytrium mangrovei strain BL10 is a microalga which is rich in docosahexaenoic acid (DHA). BL10 is different from other creatures by using polyunsaturated fatty acid synthase (PFA) to synthase DHA. Under the condition of nitrogen deficiency, three gene of AmPFA subunits have increased distinguished and accompanied by a great yield of DHA. How DHA synthase by AmPFA and if there are others proteins involved in the biosynthesis of DHA?
The aim of this study is to establish the AmPFA purification process to investigate the how PFA complex produce DHA through proteomic. We used thaw-frozen method to extract the proteins from BL10 and then we used ammonium sulfate precipitation (ASP), ion exchange chromatography and size exclusion chromatography to purify AmPFA complex. We had found that, 30% and 40% of ammonium sulfate precipitate can be detected the signal of AmPFA3 by western blotting. Both anion exchange chromatography and size exclusion chromatography showed that AmPFA complex were departed and degraded during purification. Blue native PAGE was used to study whether AmPFA complex was already depart during the process, and it was found that during ASP, the complex is departed. Last, we used gel filtration chromatography to purify crude extraction protein. But AmPFA complex still departed during purification.

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