由雙功能吸附基材在幫助澱粉分解酶-Bacillus amyloliquefaciens α-amylase復性的過程中，我們發現到在高鹽濃度下，蛋白彼此之間會因鹽離子所造成的靜電遮蔽效應較強，使蛋白在尚未吸附在基材之前而形成部分聚集，再以此種較少疏水區域暴露在外的構形吸附在基材表面，且因為部分聚集的團聚物可和基材配體作用的疏水區域不多或吸附力不強，所以只需很低的靜電排斥力便可將其從基材上脫附下來，。
　在蛋白在較低鹽濃度進行吸附時，我們發現需要較高的靜電排斥力才能將吸附在基材上的蛋白脫附下來，可見蛋白與基材之間吸附頗強，蛋白的吸附型態是以單一伸展型的蛋白不同數目的疏水基進行吸附，才使得蛋白與基材之間的吸附力強到需要較高的靜電排斥力與高濃度的變性劑才能使蛋白脫附下來。我們發現蛋白與單一個疏水基的吸附型態，其蛋白的再摺疊過程中也較不會收到干擾，比較容易形成正確的構形，我們在兩種低鹽濃度下([(NH4)2SO4]=0.05 M 、0.1 M)其以此種吸附型態吸附的脫附蛋白比活性測試分別得為1.5與4.86Unit/mg；螢光測試中，兩者脫附蛋白的三級結構再摺疊也顯示出蛋白確實有摺疊回部份正確的結構。
而我們也發現從脫附蛋白的相對比活性中，經由層析復性的蛋白比活性仍太低，可見蛋白和基材之間的疏水性吸附過強，使蛋白在基材表面進行再摺疊時遭遇到疏水性區域無法再摺疊回正確的構形內，才會造成蛋白復性效果不如預期。

The main purpose of this research is to study the relation between the absorbed state of Bacillus amyloliquefaciens α-amylase adsorbed on a dual-functional absorbent and different conditions of refolding. Further, we also study the effect of different absorbed states on the activity recovery of eluted protein.
From our results, we find that denatured proteins will partially aggregate before proteins absorbed on the adsorbent under high salt concentration. During refolding process and the hydrophobic area of the partial-aggregate structure isn’t sufficient to maintain the interaction, the proteins are easily eluted by increasing pH value. Because the structure of proteins are mis-folded during absorbed on the adsorbent, the eluted proteins without native-tertiary structure measured by fluorescence spectrum.
We also find proteins be eluted under higher pH value at the refolding condition of low salt concentration. It is obvious to realize that the hydrophobic interaction is quite strong. So the absorbed states of proteins could be single denatured protein adsorbed on a few ligands or more protein’s hydrophobic area adsorbed on plenty of ligands. And the absorbed proteins aren’t interrupted during refolding step and form a correct structure compared with proteins adsorbed on adsorbents under high salt concentration . From the amylase activity test and fluorescence spectrum of proteins of tertiary structure，it showed that relative activity of eluted proteins are 1.5 and 4.86 Unit/mg under salt concentration of 0.1 M and 0.05 M; each refolding ratio of tertiary structure is about 70%. It points out indeed that eluted proteins have been refolded to partial native structure. According to relative activity of eluted proteins, we find that the activity of proteins refolded with hydrophobic interaction chromatography is too low.
It demonstrates that the hydrophobic interaction between proteins and substrate is still too high to make hydrophobic area refold to native structure. This affect refolded activities of proteins. If we lower the hydrophobicity of substrate by decreasing the number of ligand or ligand density, it may could improve this problem.

1. Agarwal R.P. and Henkin R.I.，“Metal binding characteristics of human salivary and porcine pancreatic amylase.”，The Journal of Biological Chemistry 1987；262：2568-257
2.Altamirano M.，Golbik R.，Zahn R.，Buckle A.M. and Fersht A.R.，“Refolding chromatography with immobilized mini-chaperones.”，Processing of the National Academy of Sciences of the United Structure of America 1997；94：3576-3578
3.Batas B. and Chaudhuri J.B.，“Protein refolding at high concentration using size-exclusion chromatography.”，Biotechnology and Bioengineering 1996；50：16-23
4.Bertriz M.，Claudia Pazos，Laura Franco-Fraguas and Francisco Batista-Viera，“Chromatographic methods for amylases.”，Journal of Chromatography A 1996；684：217-237
5.Boyer R.F.，“Biochemistry”，Brook/Cole Publishing Company，1999
6.Brems D.N.，Plaisted S.M.，Kauffman E.W. and Havel H.A.，“Characterization of an associated equilibrium folding intermediate of bovine growth-hormone.”，Biochemistry 1986；25：6539-6543
7.Campbell M.K.，“Biochemistry”，Saunders College Publishing，1995
8.Copeland R.A.，“Method for Protein Analysis：A practical guide to laboratory protocols.”，New York，Chapman and Hall，1994
9.Creighton T.E.，“Proteins structure and molecular properties.”，W.H. Freeman，New York；1984；9：49-58
10. Diamant S.， Azem A.，Weoss C.，and Golubinoff, P.，“Increased
efficiency of GroE-assisted protein folding by Manganese ions.”，The Journal of Biological Chemistry 1995；270：28378-28391.
11.Georgiou G. and Bermardez-Clark E.D.，“Protein refolding”，Washington DC，American Chemical Society，1991
12. Gu Z.，Su Z. and Janson J.C.，“Urea gradient size-exclusion chromatography enhanced the yield of lysozyme refolding.”， Journal of Chromatography A 2001；918：311-318.
13.Humphreys D.P.，Smith B.J. and King L.M.，“Efficient site specific removal of a C-terminal FLAG fusion from a fab using copper(II) ion catalysed protein cleavage.”，Protein Engineering 1999；12：179-184
14.Jungbauer A.，Kaar W. and Schlegl R.，“Folding and refolding of proteins in chromatographic beds.”，Current Opinion in Biotechnology 2004；15：487-494
15. Ishii Y.， Teshima T.， and Kondo, A.，“Operation conditions of　enzyme refolding by chaperonin and recycle system using ultrafiltration.”，Chemical Engineering Journal 1997；65：151-157.
16.Katoh S. and Katoh Y.，“Continuous refolding of　lysozyme with fed-batch addition of denatured protein solution.”，Process Biotechnology 2000；35：1119-1124
17.Kyriaki Glynou，Penelope C.，Ioannou and Theodore K. Christopoulos，“One-step purification and refolding of recombinant photoprotein aequorin by immobilized metal-ion affinity chromatography.，” Protein Expression and Purification 2003；27：384-390
18.Langenhof M.，Leong S.S.J.，Pattenden L.K. and　Middelberg A.P.J.，“Controlled oxidative protein refolding using an ion-exchange column.”，Journal of Chromatography A 2005；1069：195-201.
19. Li J.J.，Venkataramana M.，Wang A.Q.，and Sanyal S.，“A mild hydrophobic interaction chromatography involving polyethylene glycol immobilized to agarose media refolding recombinant Staphylococcus aureus elongation factor G.”，Protein Expression　and Purification 2005；40：327-335.
20. Machold C.，Schlegl R.，Buchinger W.，and Jungbauer A.，“Matrix assisted refolding of proteins by ion exchange chromatography.”，Journal of Biotechnology 2005；117：83-97.
21.Mckee T.，Mckee J.R.，“Biochemistry”，McGraw-Hill Publishers，New York，1996
22.M.Mar Carrio and Antonio Villaverde，“Protein aggregation as bacterial inclusion bodies is reversible.”，FEBS Letters 2001；489：29-33
23.Muller C. and Rinas U.，“Renaturation of heterodimeric platelet-derived growth factor from inclusion bodies of recombinant Escherichia coli using size-exclusion chromatography.”，Journal of Chromatography A 1999；855：203-213
24.Neurath H.，Greenstein J.P.，Putnam F.W. and Erickson J.O.，“The chemistry of protein denaturation.”，Chemical Reviews 1943；32：157-265
25. Pain R.H.，“Mechanisms of protein folding.”，Oxford University
press，1994.
26. Porth J.，Carlsson J.，Olsson I. and Belfrage G.，“Metal chelate affinity chromatography，a new approach to protein fractionation.”Nature 1975；258：598
27.Saboury A.A. and Karbassi F.，“Thermodynamic studies on the interaction of calcium ions with alpha-amylase.”，Thermochimica Acta 2000；362：121-129.
28.Skoog D.A.，Laeary J.J.，“Principles of instrumental analysis.”，Saders college publishing，U.S.A.，1992
29.Shire S.J.，“Purification and immunogenicity of fusion vp1 protein of foot and mouse-disease virus”. Biochemistry 1984；23：6474-6480
30. Vetsch M.，Sebbel P.and Glockshuber, R.，“Chaperoneindependent Folding of Type 1 Pilus Domains.”，Journal of Molecular Biology 2002；322： 827-840
31.Xindu Geng and Chaozhan Wang，“Protein folding liquid chromatography and its recent developments.”，Journal of Chromatography B 2007；849：69-80
32. Yamaguchi H. and Uchida M.，“A chaperone-like function of intramolecular high-mannose chains in the oxidative refolding of bovine pancreatic RNase B.”，The Journal of Biochemistry 1996；120：474-477
33.柯玲潔，“以雙功能吸附基材進行蛋白質復性—疏水基鏈長與密度之選擇”，中原大學碩士論文，2006
34.林俊良，“Alpha-澱粉分解酶復性方法之探討”，中原大學碩士論文，1999
35.蔡易積，“Bacillus amyloliquefaciens α-amylase 去摺疊程序與其熱力學性質探討”，中原大學碩士論文，2004
36.楊國華，“含陰離子之疏水性吸附劑在疏水性蛋白質置換層析的應用”，中原大學碩士論文，1998