AmPFA是 Aurantiochytrium sp. strain BL10 (簡稱BL10) 用以合成兩種高度不飽和脂肪酸：C22:6n-3 (DHA) 及C22:5n-6 (n-6DPA) 的聚酮合成酶。為了進行立體結構的分析來了解 AmPFA 的催化機制，乃至於是否有其他蛋白參與 PFA 的活化與調控，前人也曾嘗試進行 AmPFA protein complex 的純化，然而發現其在純化過程中極不穩定。本研究的目的，在於建立能穩定萃取及純化 AmPFA complex 的方法。首先改善前人培養方式以2 L 三角錐形瓶進行擴大培養，發現 16 小時當中 BL10 可穩定地產出 AmPFA。隨之進行 BL10破細胞平台的優化;不同於前人，本研究以超音波均質機取得大量 AmPFA 的粗萃。相較於前人使用硫酸銨沉澱法再經過離子交換樹脂後 AmPFA 有 dissociation 的現象，本研究想透過一次性純化-尺寸移除法，來了解AmPFA的分佈。於適當鹽類濃度的緩衝溶液下，以手動進樣搭配新鮮萃取的樣本，並利用膠體過濾層析管柱Superdex 200能夠有效的得到AmPFA complex進而利用HIC可以提升AmPFA的純度。因此本研究證實 AmPFA可以透過HIC進行初步純化後，搭配Superdex 200以及高鹽緩衝溶液，得到完整 AmPFA complex，有效解 決前人對於AmPFA complex的純化問題。

Analysis and purification of polyunsaturated fatty acid synthase in Aurantiochytrium strain BL10

Ying-Hua Ting
Yi-Min Chen
Department of Biotechnology and Bioindustry Sciences
College of Bioscience and Biotechnology

SUMMARY

Aurantiochytrium sp. strain BL10 is a heterotrophic organism rich in docosahexaenoic acid (DHA). Moreover, a few strains can utilize an alternative pathway to synthesize DHA and n-6 DPA by using polyunsaturated fatty acid synthase PFA, which was first found in marine microorganisms such as Schizochytrium, Ukenia, and Auranthchytrium. An analysis of amino acid sequences in BL10 and Schizochytrium ATCC 20888 identified the existence of PFA in BL10, which was then named AmPFA. Furthermore, purification of AmPFA is difficult, and the structure of the active site of AmPFA as well as the mechanism for the synthesis of DHA are still unknown.
In this study, we have optimized an AmPFA purification method in order to explore by proteomic analysis the ways in which the PFA complex synthesizes DHA. First, sonication was used to extract proteins from BL10, which were then subjected to size exclusion chromatography and hydrophobic interaction chromatography for the purification of the AmPFA complex. During size exclusion chromatography, increasing the buffer concentration of NaCl resulted in the clustering of the AmPFA and elution in fraction F2. Finally, the subsequent hydrophobic interaction chromatography step enhanced the purification of AmPFA by removing the presence of non-specific proteins.

Key words: Aurantiochytrium mangrovei, BL10, DHA, PUFA

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