利用水溶液兩相系統與親合層析法從甘藷中 純化胰蛋白酶抑制劑

Purification of sporamin from sweet ptotao by aqueous two phase system and affinity chromatography

趙坤山(Kuen-Shan Jaw)

指導教授 ： 段國仁

大同大學/工程學院/生物工程學系所/博士(2006年)

摘要

本研究利用水溶液兩相系統(aqueous two-phase system)及親和層析法(affinity chromatography)兩種方法，從台農57號甘藷中純化胰蛋白酶抑制劑。在水溶液兩相系統上，是利用聚乙二醇(polyethylene glycol, PEG)與磷酸塩(phosphate)形成水溶液兩相系統。在PEG 6000 (11%)-phosphate (16%)-KCl (9%)-pH 6.0條件下，胰蛋白酶抑制劑集中於富含PEG的上相中。純化倍率增加3.7倍及回收率為95%。SDS-PAGE可以看出一條明顯的蛋白質染色，其分子量為27 kDa。在親和層析法上，利用固定胰蛋白酶至戊二醛(glutaraldehyde) 活化之商業化的多孔性幾丁聚糖顆粒(chitosan beads)，形成親和性層析單體。用以純化甘薯塊根的抽出液中的胰蛋白酶抑制劑。最佳的胰蛋白酶固定的條件為幾丁聚糖顆粒經過0.1 % (w/v)戊二醛活化、胰蛋白酶濃度2 mg/ml、pH 8.0~9.0及反應時間6小時為最佳。最大的固定化胰蛋白酶活性為10 unit/g-beads。親和性層析擔體可回收55%台農57號粗抽甘藷汁純化胰蛋白酶抑制劑，SDS-PAGE可以看出7條明顯的蛋白質染色，其分子量都在 20 kDa以上。無論是水溶液兩相萃取系統或者是親和層析法，均可快速純化粗抽甘藷汁中胰蛋白酶抑制劑。

關鍵字

胰蛋白酶抑制劑 ； 水溶液兩相系統 ； 親和層析法

並列摘要

In this study, two methods were used to the purification of Kunitz soybean trypsin inhibitor with aqueous two-phase system and affinity chromatography from sweet potato Tainong 57. In aqueous two-phase, the polyethylene glycol (PEG) and phosphate were used to construct an aqueous two-phase system (ATPS). Trypsin inhibitor was recovered with high yield and high concentration in the top PEG-rich phase when the aqueous two phase system was constructed using PEG 6000 (11%), phosphate (16%), KCl (9%) at pH 6.0. The purity of the trypsin inhibitor was enhanced at 3.7-fold, and the recovery was 95%. The purified trypsin inhibitor showed one visible band, and the molecular weight was 27 kDa by SDS-PAGE. In affinity chromatography, the immobilized trypsin on the glutaraldehyde activated chitosan beads was employed to purify trypsin inhibitor from the tuberous roots of sweet potato by means of affinity chromatography. Maximum trypsin activity (10 unit/g-beads) of immobilized beads was obtained in treating with 0.1 % (w/v) glutaraldehyde, adding trypsin solution (2 mg/mL, pH 8) and shaking at 37 C for 6 hours in this study. Fifty-six percent of the activity of the trypsin inhibitor in the crude extract of sweet potato could be recovered through affinity chromatography. Seven visible bands by SDS-PAGE were suspected to be trypsin inhibitors, and all of the molecular weights were more than 20 kDa. The trypsin inhibitor of sporamin could be rapidly purified by either aqueous two-phase systems or affinity chromatography from sweet potato Tainong 57.

並列關鍵字

aqueous two-phase system ； affinity chromatography ； trypsin inhibitor

參考文獻

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