感溫性共聚體固定化胰蛋白酵素作為胰蛋白酵素抑制劑之親和性分離膠體之研究

The Immobilization of Trypsin by Thermo-Responsive Copolymer for the Affinity Separation of Trypsin Inhibitor

李蓓慈(Pei-tzu Lee)

指導教授 ： 陳志成

大同大學/工程學院/生物工程學系所/碩士(2004年)

摘要

本研究乃利用含N-isopropylacrylamide (NIPAAm)和glycidyl methacrylate (GMA)所合成的感溫性高分子共聚物，作為固定胰蛋白酵素（trypsin）之載體。此共聚物在低於32℃時為可溶狀態而方便與基質反應，當溫度提高至37℃時則成為不溶膠體而與基質分離，且此種相變化反應具有可逆性。控制適當的固定化條件時，胰蛋白酵素的活性回收率可高達70%，而固定後之胰蛋白酵素的比活性保有未固定時之92 ﹪。共聚物之物性影響固定胰蛋白酵素的效率，具有較好效率的共聚物，其分子量較低，並且其極限黏度較類似分子量之共聚物為高。此固定了胰蛋白酵素的感溫性共聚物可作為親和分離之載體，並直接從蕃薯萃取液之硫氨沈澱蛋白質中，分離出胰蛋白酵素的抑制劑sporamin。於pH 8及25℃之條件下，69 ﹪sporamin活性被吸附於固定化酵素之共聚物。

關鍵字

感溫性高分子 ； 固定胰蛋白酵素 ； 胰蛋白酵素抑制劑 ； 親和分離

並列摘要

In this work, the N-isopropylacrylamide (NIPAAm) and glycidyl methacrylate (GMA) copolymer, which is a reversible soluble-insoluble thermo-responsive copolymer, was synthesized and used as a support to immobilize trypsin. The trypsin conjugated NIPAAm-GMA copolymer was soluble below 32℃. When the temperature raised above 37℃, the conjugated trypsin was easily separated from the reaction solution. Under suitable conditions for immobilization of trypsin, the activity recovery could reach 70 ﹪and the specific activity of immobilized trypsin retained 92 ﹪activity of the free enzyme. The copolymer with low molecular weight of 1755 and relative higher intrinsic viscosity shown a good immobilized capacity of trypsin. The trypsin conjugated copolymer could be used as an affinity matrix for sporamin from the sweet potato juice. 69 ﹪sporamin activity was adsorbed to the conjugated trypsin at pH 8 and 25℃.

並列關鍵字

affinity separation ； immobilized trypsin ； reversible soluble-insoluble thermo-responsible ； trypsin inhibitor

參考文獻

1. Arasaratnam V., I. Y. Galaev, and B. Mattiasson. 2000. Reversibly soluble biocatalyst: optimization of trypsin coupling to Eudragit S-100 and biocatalyst activity in soluble and precipitated forms. Enzyme and Microbial Technology 27, 254-263

2. Boulis Y., G. G. March, V. Gomord, H. Adenier, L. Faye, M. A.Vijayalakshmi. 2003. Analysis of sporamin forms expressed in different subcellular compartments of transgenic tobacco plants by IMAC and ESI-MS. Plant Physiology and Biochemistry 41, 215–221

4. Cong L., R. Kaul, U. Dissing, and B.Mattiasson. 1995. A model study on Eudragit and polyethyleneimine as soluble carriers of α-amylase for repeated hydrolysis of starch. Journal of Biotechnology 42, 75-84

5. Chen C. W., B. C. Lei, K. W. Yeh, K. J. Duan. 2003. Recombinant sweet potato sporamin production via glucose/pH control in fed-batch cultures of Saccharomyces cerevisiae. Process Biochemistry 38, 1223-1229

6. Chen J. P., M. S. Hsu. 1997. Preparations and properties of temperature-sensitive poly(N-isopropylacrylamide)α-chymotrypsin conjugates. Journal of Molecular Catalysis B: Enzymatic 2, 233-241