重組芋頭半乳糖水解酵素之表現與定性

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Title:	重組芋頭半乳糖水解酵素之表現與定性
Other Titles:	Expression and characterization of recombinant α-galactosidase from taro
Authors:	陳逸書;Chen, I-Su
Contributors:	淡江大學化學學系碩士班簡素芳;Chien, Su-Fang
Keywords:	芋頭;α-半乳糖水解酵素;基因表現;酵素純化;紅血球轉型;Taro;α-galactosidase;gene expression;enzyme purification;blood conversion
Date:	2013
Issue Date:	2014-01-23 13:46:42 (UTC+8)
Abstract:	α-半乳糖水解酵素（α-galactosidase）是一種可以將非還原端半乳糖水解的酵素，在各種生物體中都存在，並且擁有許多不同的生物功能，例如在植物中α-半乳糖水解酵素參與醣類的代謝和運輸。在應用方面，α-半乳糖水解酵素可以使B型紅血球表面抗原上的半乳糖水解而變成O型紅血球，因此藉由基因工程的方法取得基因並利用 Pichia pastoris 的蛋白質表現系統來產生大量的芋頭α-半乳糖水解酵素以進行血型轉換的測試和應用。本研究將已轉殖入α-半乳糖水解酵素基因的酵母菌大量培養，並進行誘導一天產生蛋白質，細胞內酵素活性單位最高達到3.6 units/mL。利用水解酵母菌細胞壁的方式，將酵母菌打破取出α-半乳糖水解酵素。大量的萃取液經過濃縮之後，分別進行四步純化步驟，依序是分子篩 SephadexTM G-100管柱、陰離子交換樹脂Q SepharoseTM Fast flow管柱、疏水性吸附 HiTrap Phenyl FF (high sub), 1mL管柱，分子篩SepharoseTM 6 10/300 GL。純化後的酵素樣品經過十二烷基硫酸鈉-聚丙烯醯胺凝膠電泳與基質輔助雷射脫附離子化－飛行時間質譜儀的分析，確認是α-半乳糖水解酵素。進行一系列酵素的定性分析，包含紅血球轉型實驗，2 units的純化酵素可於2小時將實驗中總體積為120 μL的B型紅血球懸浮液轉型為O型紅血球，其轉型百分比約為80％。 α-Galactosidase is capable of hydrolyzing terminal non-reducing galactosyl residues, and it is distributed in most organisms with many different biological functions such as metabolism and transportation of photoassimilates in plants. In application, α-galactosidase can be used to hydrolyze the galactosyl group of type B antigen on the red blood cell surface, and covert the type B red blood cell into type O. Thus, the Pichia patoris expression system was used to fastly produce large amount of taro α-galactosidase to proceed the seroconversion of erythrocyte. In this work, the Pichia patoris cells harboring the taro α-galactosidase gene on the expression vector were grown to large scale. After the recombinant gene had been induced for one day, the highest intracellular enzyme activities were 3.6 units/mL. Lysis of yeasts was achieved by using Lyticase to extract the crude taro α-galactosidase. From the crude extractsα-galactosidase was then purified to homogeneity by using four different types of column chromatography, include SephadexTM G-100, Q SepharoseTM Fast flow, HiTrap Phenyl FF (high sub), and SepharoseTM 6 10/300 GL. The purified enzyme showed single band on SDS-PAGE, and then identified by by MALDI-TOF MS analysis. The purified α-galactosidase was characterized in terms of blood conversion, and a conversion rate of 80％ type B red blood cell into type O with use 2 units of purified α-galactosidase in 2h was achieved.
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