利用金屬螯合層析法從甘藷中純化胰蛋白酶抑制劑

本研究利用幾丁質(chitosan)顆粒與銅離子交聯(cross-linked)作用，將銅離子固定在幾丁質顆粒上，形成固定化金屬層析(immobilized metal affinity chromatography, IMAC)擔體。本研究針對CuCl2的濃度、固定化時間、固定化時pH值等條件，找尋幾丁質顆粒與銅離子交聯作用的最適條件及銅離子的最大固定量。結果顯示最適固定銅離子的濃度40 mM、時間48小時和pH 8.0之環境下，幾丁質顆粒銅離子的最大吸附量為190 mM/g-bead。經過銅離子交聯固定化的幾丁質顆粒，填入層析管柱形成金屬層析管柱，並探討金屬層析擔體對台農57號甘藷中胰蛋白酶抑制劑sporamin吸附能力。我們改變pH 與反應時間等因子，找尋金屬螯合擔體對吸附粗抽蛋白質的最適吸附條件及最大吸附能力。結果顯示，固定化金屬層析對胰蛋白酶抑制劑最適吸附的pH 與時間分別9 .0與4小時，每克幾丁質最大吸附能力可達到3.7 mg 和5.2 IU的胰蛋白酶抑制劑。利用此固定化金屬層析系統對台農57 號甘藷中胰蛋白酶抑制劑sporamin進行純化，其純化倍率為1.6倍，回收率為49.6％。吸附在管柱上的胰蛋白酶抑制劑經冲堤後，經SDS-PAGE分析可得部分純化的胰蛋白酶抑制劑sporamin，印證此系統可以從甘藷粗抽蛋白質中快速進行胰蛋白酶抑制劑之層析分離及部分純化。

關鍵字

胰蛋白酶抑制劑；固定化金屬層析；幾丁質

並列摘要

An immobilized metal affinity chromatography (IMAC) absorbent was prepared by coordinating Cu2+ with cross-linked chitosan bead and successfully used for extracting trypsin inhibitor (TI) from sweet potato Tainong 57. Results showed that the optimal immobilized copper ion concentration on Chitosan beads ( Cu-CSTB) was 40 mM, and the maximum Cu2+ adsorption capacity per gram was 190 mM. In pH 9.0, and 4 hours, the maximum adsorption capacity for TI on the Cu-CTSB could reach 5.2 IU/g-beads. Based on small-scale purification, the purity of TI was enhanced 1.6-fold. Fifty percent activity of TIs in crude extract from sweet potato could be recovered by IMAC. There was one visible band of the purified TI, and the molecular weight was 27 kDa by SDS-PAGE.

並列關鍵字

sweet potato ； chitosan bead ； Immobilized metal affinity chromatography ； trypsin immobilization ； trypsin inhibitor

參考文獻

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利用金屬螯合層析法從甘藷中純化胰蛋白酶抑制劑

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