臺灣博碩士論文加值系統

本研究主要是利用帶有離子官能基與疏水官能基的雙功能基材來幫助大分子的蛋白質，澱粉分解酶，在高濃度下復性。我們探討的內容包括蛋白質疏水程度，帶雙硫鍵與否對復性的影響，並研究如何以調整疏水鏈長及疏水官能基密度來提高復性成效。在研究中比較了三種α-amylase的復性，Bacillus amyloliquefaciens α-amylase（BAA）、較疏水的Bacillus licheniformis α-amylase（BLA）及較親水但帶有分子內雙硫鍵的porcine pancreatic α-amylase（PAA）。結果發現，當以直接稀釋方式來進行復性時，將Bacillus amyloliquefaciens α-amylase稀釋到1μg/mL的最終蛋白質濃度時，可獲得93%的相對比活性。而疏水的Bacillus licheniformis α-amylase較Bacillus amylolique- faciens α-amylase容易聚集，將其稀釋到1μg/mL的最終蛋白質濃度時，只獲得了45%的相對比活性。而較親水但帶有分子內雙硫鍵的porcine pancreatic α-amylase則更易聚集，即使稀釋至1μg/mL的極低濃度下，也只有9 %的相對比活性。
而以批次吸脫附方式利用雙功能吸附基材幫助澱粉分解酶進行復性時，發現短鏈的propyl-CM-sepharose因吸附效果不佳所以導致活性回收率不高，而長鏈的octyl-CM-sepharose則因疏水性太強，阻礙了澱粉分解酶的再摺疊。反而是四個碳鏈長的butyl-CM-sepharose發揮了最好的復性效果。在疏水基密度方面，太高的疏水基密度也同樣會造成阻礙的復性效果，只有在適合的疏水基密度下，才能有最好的復性效果。所以當使用結合密度30.62μmol/mL的butyl-CM- sepharose幫助Bacillus amyloliquefaciens α-amylase復性時，可以在最終蛋白質濃度130μg/mL下得到66%的相對比活性。
而在親疏水性不同的蛋白質復性上，整體疏水性強的蛋白質，需使用疏水性較弱的短鏈基材來復性，且疏水鏈間的距離不宜過近。所以利用較低結合密度12.04μmol/mL的butyl-CM-sepharose幫助Bacillus licheniformis α-amylase復性時，可以在最終蛋白質濃度130μg/mL下得到69%的相對比活性。相對地，對於較親水的蛋白質，則需選取疏水性較強的長鏈基材來復性。但如果蛋白鏈內存在雙硫鍵因而易聚集的蛋白質，則基材上的疏水鏈間距離也不宜過近。因此使用較低結合密度18.03μmol/mL的octyl-CM-sepharose幫助porcine pancreaticα-amylase復性時，可以在最終蛋白質濃度87μg/mL下得到60%的相對比活性。由此可知，根據蛋白質的特性，來選取適當的雙功能基材，是有可能在較高的蛋白質濃度下獲得不錯的復性效果。
最後再以層析方式幫助Bacillus amyloliquefaciens α-amylase進行復性，結果發現最終蛋白質濃度提升到146μg/mL，略比批次吸脫附實驗的結果好，與直接稀釋法相比更是有明顯的提升。在活性回收與相對比活性方面的結果，以層析方式進行復性可以得到55%的活性回收與85%的相對比活性，都比批次吸脫附實驗的復性效果更好些。這也再一次的証明了利用雙功能吸附基材能夠成功幫助蛋白質進行復性！

Three α-amylases from different origins are refolded by the assistance of a novel dual-functional adsorbent containing both ionic carboxylic and hydrophobic alkyl groups. Both batch adsorption/ desorption and chromatographic operations were performed and achieved the similar level of refolding yield. The effects of alkyl density, alkyl chain length, adsorption condition and protein hydrophobicity on refolding yield were under investigation.
The three α-amylases used in this study are: Bacillus amyloliquen- faciens α-amylase (BAA), Bacillus licheniformis α-amylase (BLA) and porcine pancreatic α-amylase (PAA). Among these proteins, BLA is the most hydrophobic. PAA is the least hydrophobic but is the only one containing intra-molecular disulfide bonds. It was found that 93% of guanidine hydrochloride denatured BAA can be renatured by direct dilution at a final concentration of 1μg/mL. However, only 45% of BLA and 9% of PAA can be refolded at the same dilution level.
The alkyl chain length and ligand density were found to have tremendous influence on protein refolding. High levels of adsorption and elution were key factors of high refolding yield. The adsorbents owned longer alkyl chains had stronger adsorption toward denatured, but it was difficult to elute the partially refolded protein. Similarily, high ligand density resulted in high adsorption but low desorption. But low ligand density resulted in low adsorption. The levels of adsorption and desorption were also affected by the hydrophobicities of proteins. Therefore, the highest relative specific activity, 66%, obtained from BLA refolding was facilitated by the butyl-CM-sepharose resin of ligand density at 12.04 μmol/mL. The highest relative specific activity, 60%, obtained from BAA refolding was facilitated by the butyl-CM-sepharose resin of ligand density at 30.62 μmol/mL. And the best resin for BAA refolding was the octyl-CM-sepharose resin of ligand density at 18.03 μmol/mL.
Finally, the refolding of denatured Bacillus amyloliquenfaciens α-amylase was operated chromatographically through a butyl-CM-sepharose column. The activity recovery and relative specific activity were 55% and 85%, respectively. A high concentration of refolded BAA, 0.146 mg/mL, could be obtained.

總 目 錄
中文摘要．．．．．．．．．．．．．．．．．．．．．．．．．I
英文摘要．．．．．．．．．．．．．．．．．．．．．．．．．IV
總目錄．．．．．．．．．．．．．．．．．．．．．．．．．VI
圖目錄．．．．．．．．．．．．．．．．．．．．．．．．．XI
表目錄．．．．．．．．．．．．．．．．．．．．．．．．．XIV

第一章 緒論．．．．．．．．．．．．．．．．．．．．．．．．1
第二章 文獻回顧．．．．．．．．．．．．．．．．．．．．．．6
2.1 蛋白質內聚體與糾結體之形成．．．．．．．．．．．．．．6
2.2 蛋白質變性．．．．．．．．．．．．．．．．．．．．．．7
2.2.1 常見化學變性劑．．．．．．．．．．．．．．．．．．8
2.2.2 穩定蛋白質各級結構的作用力．．．．．．．．．．．10
2.3 蛋白質復性．．．．．．．．．．．．．．．．．．．．．13
2.3.1以分子大小排斥層析方式進行復性．．．．．．．．．．15
2.3.2以離子交換層析方式進行復性．．．．．．．．．．．．18
2.3.3以固定金屬離子親和性層析方式進行復性．．．．．．．21
2.3.4以疏水作用層析方式進行復性．．．．．．．．．．．．24
2.4 澱粉分解酶的簡介與差異性比較．．．．．．．．．．．．26
2.4.1 澱粉分解酶的功能與用途．．．．．．．．．．．．．．26
2.4.2 三種不同來源之澱粉分解酶差異比較．．．．．．．．．28
2.4.2.1 胺基酸組成．．．．．．．．．．．．．．．．．．．28
2.4.2.2 熱穩定性．．．．．．．．．．．．．．．．．．．．30
2.4.2.3 親疏水性．．．．．．．．．．．．．．．．．．．．32
2.4.2.4 立體結構上的差異．．．．．．．．．．．．．．．．34

第三章 實驗藥品、設備與方法．．．．．．．．．．．．．．37
3.1 實驗藥品．．．．．．．．．．．．．．．．．．．．．．37
3.1.1 目標蛋白．．．．．．．．．．．．．．．．．．．．37
3.1.2 變性劑．．．．．．．．．．．．．．．．．．．．．．37
3.1.3 復性劑．．．．．．．．．．．．．．．．．．．．．．37
3.1.4 緩衝溶液．．．．．．．．．．．．．．．．．．．．．38
3.1.5 活性分析試劑．．．．．．．．．．．．．．．．．．．39
3.1.6 雙功能吸附基材合成．．．．．．．．．．．．．．．．39
3.1.7 其他藥品．．．．．．．．．．．．．．．．．．．．．40
3.2 實驗設備．．．．．．．．．．．．．．．．．．．．．．41
3.3 實驗方法．．．．．．．．．．．．．．．．．．．．．．43
3.3.1 雙功能吸附基材合成．．．．．．．．．．．．．．．．43
3.3.1.1 CM-Sepharose 膠體的清洗．．．．．．．．．．．．．43
3.3.1.2 胺基烷溶液的配製．．．．．．．．．．．．．．．．44
3.3.1.3 疏水官能基合成反應．．．．．．．．．．．．．．．45
3.3.1.4 反應完膠體的清洗．．．．．．．．．．．．．．．．45
3.3.1.5 不同結合密度的基材之合成．．．．．．．．．．．．47
3.3.2 雙功能吸附基材疏水基結合密度之測定．．．．．．．．48
3.3.3 澱粉分解酶濃度測定．．．．．．．．．．．．．．．．49
3.3.4 澱粉分解酶活性測定．．．．．．．．．．．．．．．49
3.3.5 澱粉分解酶的變性．．．．．．．．．．．．．．．．．51
3.3.6 直接稀釋法復性澱粉分解酶．．．．．．．．．．．．52
3.3.7 利用批次吸脫附方式以雙功能吸附基材復性Bacillus amyloliquefaciens a-amylase．．．．．．．．．．．．53
3.3.8 利用批次吸脫附方式以雙功能吸附基材復性Bacillus lich- eniformis a-amylase．．．．．．．．．．．．．．55
3.3.9 利用批次吸脫附方式以雙功能吸附基材復性porcine pancreas a-amylase．．．．．．．．．．．．．．．．．．．．．57
3.3.10 利用層析方式以雙功能吸附基材復性澱粉分解酶．．．59
第四章 結果與討論．．．．．．．．．．．．．．．．．．．．61
4.1 利用直接稀釋法幫助澱粉分解酶復性．．．．．．．．．．61
4.1.1 Bacillus amyloliquefaciens α-amylase直接稀釋法復性結果．61
4.1.2 Bacillus licheniformis α-amylase直接稀釋法復性結果．．63
4.1.3 Porcine pancreatic α-amylase直接稀釋法復性結果．．．．64
4.2 利用雙功能吸附基材進行批次吸脫附以幫助α-amylase復性．．．．．．．．．．．．．．．．．．．．．．．．．66
4.2.1 Bacillus amyloliquefaciens α-amylase之復性．．．．．．．66
4.2.1.1 吸附條件之探討．．．．．．．．．．．．．．．．．66
4.2.1.2 不同碳鏈長度之雙功能吸附基材對Bacillus amylolique- faciens α-amylase復性之影響．．．．．．．．．．69
4.2.1.3 不同結合密度之雙功能吸附基材對Bacillus amylolique- faciens α-amylase復性之影響．．．．．．．．．．73
4.2.2 Bacillus licheniformis α-amylase復性．．．．．．．．．77
4.2.2.1 吸附條件之探討．．．．．．．．．．．．．．．．．77
4.2.2.2 雙功能吸附基材對Bacillus licheniformis α-amylase復性的影響．．．．．．．．．．．．．．．．．．．．．．80
4.2.3 Porcine pancreatic α-amylase復性．．．．．．．．．．．84
4.2.3.1 吸附條件之探討．．．．．．．．．．．．．．．．．84
4.2.3.2 雙功能吸附基材對porcine pancreatic α-amylase復性的影響．．．．．．．．．．．．．．．．．．．．．．．87
4.3 利用管柱層析的方式充填雙功能吸附基材幫助α-amylase進行復性之結果．．．．．．．．．．．．．．．．．．．93
第五章 結論與建議．．．．．．．．．．．．．．．．．．．．102
5.1 結論．．．．．．．．．．．．．．．．．．．．．．．．102
5.2 建議．．．．．．．．．．．．．．．．．．．．．．．．105
參考文獻．．．．．．．．．．．．．．．．．．．．．．．．．106
附 錄．．．．．．．．．．．．．．．．．．．．．．．．．．115
附錄A Bacillus amyloliquefaciens α-amylase之濃度檢量線．．．115
附錄B Bacillus licheniformis α-amylase之濃度檢量線．．．．．116
附錄C Porcine pancreatic α-amylase之濃度檢量線．．．．．．117
附錄D Bacillus amyloliquefaciens α-amylase之活性檢量線．．．118
附錄E Bacillus licheniformis α-amylase之活性檢量線．．．．．119
附錄F Porcine pancreatic α-amylase之活性檢量線．．．．．．120
附錄G Bacillus amyloliquefaciens α-amylase在基材上復性之質量回收率與相對比活性間的關係．．．．．．．．．．．．121
附錄H. Bacillus licheniformis α-amylase在基材上復性之質量回收率與相對比活性間的關係．．．．．．．．．．．．．122
附錄I. Porcine pancreatic α-amylase在基材上復性之質量回收率與相對比活性間的關係．．．．．．．．．．．．．．．123

圖 目 錄

圖2.1 常見變性劑之分子結構．．．．．．．．．．．．．．．．9
圖2.2 利用在大小排次層析管柱前端先進行變性劑梯度以避免蛋白聚集而阻塞管柱的復性方法裝置圖．．．．．．．．．．．16
圖2.3 利用大小排次層析幫助血小板衍生因子進行復性之層析圖譜
．．．．．．．．．．．．．．．．．．．．．．．．．17
圖2.4 利用大小排次層析幫助溶菌酶進行復性之層析圖譜．．．．17
圖2.5 利用離子交換層析幫助牛血清蛋白進行復性之結果．．．．20
圖2.6 二種金屬螯合層析最常使用的基材．．．．．．．．．．．21
圖2.7 利用固定金屬離子親和性層析幫助蛋白進行復性之示意圖．．
．．．．．．．．．．．．．．．．．．．．．．．．．22
圖2.8 利用雙功能吸附基材幫助蛋白進行復性之示意圖．．．．．25
圖2.9 三種不同澱粉分解酶之胺基酸序列．．．．．．．．．．．29
圖2.10 六種澱粉分解酶在不同溫度下的之螢光比值與Cp值．．．31
圖2.11 Bacillus licheniformis α-amylase之立體構型．．．．．．．36
圖2.12 Porcine pancreatic ��-amylase之立體構型．．．．．．．．36
圖4.1 利用直接稀釋法幫助不同澱粉分解酶復性之結果．．．．．62
圖4.2 硫酸銨濃度與胍鹽酸濃度對去摺疊態的Bacillus amyloliquefa- ciens α-amylase溶解度之影響．．．．．．．．．．．．68
圖4.3 Bacillus amyloliquefaciens α-amylase在不同碳鏈長度的雙功能吸附基材上之吸脫附效果．．．．．．．．．．．．．．72
圖4.4 Bacillus amyloliquefaciens α-amylase在不同碳鏈長度的雙功能吸附基材上復性之復性結果．．．．．．．．．．．．．72
圖4.5 Bacillus amyloliquefaciens α-amylase在不同結合密度的雙功能吸附基材上之吸脫附效果．．．．．．．．．．．．．．76
圖4.6 Bacillus amyloliquefaciens α-amylase在不同結合密度的雙功能吸附基材上復性之復性結果．．．．．．．．．．．．．76
圖4.7 硫酸銨濃度與胍鹽酸濃度對去摺疊態的Bacillus licheniformis α-amylase溶解度之影響．．．．．．．．．．．．．．．79
圖4.8 Bacillus licheniformis α-amylase在雙功能吸附基材上之吸脫附效果．．．．．．．．．．．．．．．．．．．．．．83
圖4.9 Bacillus licheniformis α-amylase在雙功能吸附基材上復性之復性結果．．．．．．．．．．．．．．．．．．．．．．83
圖4.10 硫酸銨濃度與胍鹽酸濃度對去摺疊態porcine pancreatic alpha- amylase溶解度的影響．．．．．．．．．．．．．．．86
圖4.11 Porcine pancreatic α-amylase在不同雙功能吸附基材上之吸脫附效果．．．．．．．．．．．．．．．．．．．．．92
圖4.12 Porcine pancreatic α-amylase在不同雙功能吸附基材上之復性結果．．．．．．．．．．．．．．．．．．．．．．92
圖4.13 利用層析方式幫助Bacillus amyloliquefaciens α-amylase復性之結果．．．．．．．．．．．．．．．．．．．．．．95
圖4.14 在0.2M硫酸銨濃度下，利用層析方式幫助Bacillus amylo- liquefaciens α-amylase復性之結果．．．．．．．．．．98
圖4.15利用層析方式幫助較大量的Bacillus amyloliquefaciens α-amylase復性之結果．．．．．．．．．．．．．．100
圖A Bacillus amyloliquefaciens α-amylase之濃度檢量線．．．．115
圖B Bacillus licheniformis α-amylase之濃度檢量線．．．．．．116
圖C Porcine pancreatic α-amylase之濃度檢量線．．．．．．．117
圖D Bacillus amyloliquefaciens α-amylase之活性檢量線．．．．118
圖E Bacillus licheniformis α-amylase之活性檢量線．．．．．．119
圖F Porcine pancreatic α-amylase之活性檢量線．．．．．．．．120
圖G 利用批次吸脫附方式以不同雙功能基材幫助Bacillus amyloli- quefaciens α-amylase復性之質量回收率與相對比活性間的關係．．．．．．．．．．．．．．．．．．．．．．．．121
圖H 利用批次吸脫附方式以不同雙功能基材幫助Bacillus lichenifo- rmis α-amylase復性之質量回收率與相對比活性間的關係．．
．．．．．．．．．．．．．．．．．．．．．．．．．122
圖I 利用批次吸脫附方式以不同雙功能基材幫助porcine pancreatic α-amylase復性之質量回收率與相對比活性間的關係
．．．．．．．．．．．．．．．．．．．．．．．．．123


表 目 錄

表2.1 穩定蛋白質各級結構的作用力．．．．．．．．．．．．．11
表2.2 三種澱粉分解酶之胺基酸組成．．．．．．．．．．．．．33
表2.3 三種澱粉分解酶之胺基酸疏水性計算．．．．．．．．．．33
表2.4 三種澱粉分解酶之基本性質．．．．．．．．．．．．．．35
表4.1 利用不同碳鏈長度的雙功能吸附基材幫助Bacillus amylolique- faciens α-amylase復性之結果．．．．．．．．．．．．．71
表4.2 利用不同結合密度的雙功能吸附基材幫助Bacillus amylolique- faciens α-amylase復性之結果．．．．．．．．．．．．．75
表4.3 利用雙功能吸附基材幫助Bacillus licheniformis α-amylase復性之結果．．．．．．．．．．．．．．．．．．．．．．82
表4.4 利用雙功能吸附基材幫助porcine pancreatic α-amylase復性之結果．．．．．．．．．．．．．．．．．．．．．．．91

參考文獻

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: 2568-2575
2.Altamirano, M., Golbik, R., Zahn, R., Buckle, A.M. and Fersht, A. R., “Refolding chromatography with immobilized mini-chaperones. ”Proceedings of the National Academy of Sciences of the United States of America 1997; 94: 3576-3578
3.Anfinsen, C.B., “The Molecular Basis of Evolution” John Wiley & Sons, 1959.
4.Anfinsen, C. B., “Principles that govern the folding of protein chains. ”Science 1973; 181: 223-230
5.Batas, B. and Chaudhuri, J. B., “Protein refolding at hight conctration using size-exclusion chromatography. ” Biotechnology and Bioengineering 1996; 50: 16-23
6.Bernfeld, P.,“Enzymes of starch degradation and synthesis. ” Advances in Enzymology and Related Subjects 1951 ; 12: 379-428.
7.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.
8.Bret, A., “Protein stability and folding : Theory and Practice:” Humana Press, 1995.
9.Brzozowski, A.M.; Lawson, D.M.;and Turkenburg, J.P.,“Structural Analysis of a Chimeric Bacterial alpha-Amylase. High-Resolution Analysis of Native and Ligand Complexes.” Biochemistry 2000; 39: 9099-9107.
10.Boyer, R.F., “Biochemistry”Brook/Cole Publishing Company, 1999.
11.Campbell, M.K.,“Biochemistry”Saunders College Publishing, 1995.
12.Chance, R.E.; Ellis, R.M.; and Bromer, W.W.,“Porcine proinsulin: characterization and amino acid sequence.” Science 1968; 161:165-167.
13.Chen, H.M.; Lin, K.Y.; and Lu C.H.,“Refolding and activation of recombinant N-carbamoyl-D-amino acid amidohydrolase from Escherichia coli inclusion bodies.” Process Biochemistry 2005; 40: 2135-2141.
14.Cheng, R.Z. and Kawakishi, S.,“Site-specific oxidation of histidine residues in glycated insulin mediated by Cu(II).” European Journal of Biochemistry 1994; 223: 759-764.
15.Choi, W.C.; Kim, M.Y.; Suh, C.W.; and Lee, E.K.,“Solid-phase refolding of inclusion body protein in a packed and expanded bed adsorption chromatography.”Process Biochemistry 2005; 40: 1967-1972.
16.Clark, E.D.B.,“Refolding of recombinant proteins.”Current Opinion in Biotechnology 1998; 9: 157-163.
17.Creighton, T.E., “An empirical-approach to protein conformation stability and flexibility.”Biopolymers 1983; 22: 49-58.
18.Creighton, T.E., “Proteins, structure and molecular properties.”W. H. Freeman, New york 1984; 9: 157-163.
19.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.
20.Feller, G.; dAmico, D.; and Gerday, C.,“Thermodynamic stability of a cold-active alpha-amylase from the Antarctic bacterium Alteromonas haloplanctis.” Biochemistry 1999; 38: 4613-4619.
21.Gaberc-Porekar, V. and Menart, V., “Perspectives of immobilized- metal affinity chromatography.”Journal of Biochemical and Biophysical Methods 2001; 49: 335
22.Goel, A.; Colcher, D.; Koo, J.S.; Booth, B.J.M.; Pavlinkova, G.; and Batra, S.K.,“Relative position of the hexahistidine tag effects binding properties of a tumor-associated single-chain Fv construct.” Biochimica et Biophysica Acta-General Subjects 2000; 1523: 13-20.
23.Goldberg, M.E.; Rudolph, R.; and Jaenicke, R.,“Kinetic Study of the Competition between Renaturation and Aggregation during the Refolding of Denatured-Reduced Egg White Lysozyme.” Biochemistry 1991; 30: 2790-2797.
24.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.
25.Hermanson, G.T.; Mallia, A.K.; and Smith, P.K., “Immobilized affinity ligand techniques.”Academic Press Inc., 1992.
26.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.
27.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.
28.Jungbauer, A.; Kaar, W.; and Schlegl, R.,“Folding and refolding of proteins in chromatographic beds.” Current Opinion in Biotechnology 2004; 15: 1-8.
29.Katoh, S. and Katoh, Y., “Continuous refolding of lysozyme with fed-batch addition of denatured protein solution.”Process Biochemistry 2000; 35:1119-1124.
30.Klibanov, A.M. and Tomazic S.J. “Mechanism of Irreversible thermal inactivation of Bacillus ��-amylases.” The Journal of Biological Chemistry 1988; 263: 3086-3091.
31.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.
32.Lecker, D.N.; and Khan, A.,“Model for inactivation of alpha-amylase in the presence of salts: Theoretical and experimental studies.” Biotechnology Progress 1998; 14: 621-625.
33.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.
34.Machius M.; Declerck N.; Hubert H.; and Wiegand G. “Activaion of Bacillus licheniformis ��-amylase through a disorder/order transition of the substrate-binding site mediated by a calcium- sodium-calcium metal triad.”Structure 1998; 6: 281-292.
35.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.
36.Mamoru, M.; Daisuke, N.; and Tomoya, S., “Difference between guanidinium chloride and urea as denaturants of globular protein : The possibility of application to improve refolding process. ” Chemical and Pharmaceutical Bulletin 1992; 40: 550-552
37.Müller, 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.
38.Nakajima, R.; Imanaka, T.; and Aiba, S., “Comparison of amino acid sequences of eleven different alpha-amylases.”Applied Microbiology and Biotechnology 1986; 23: 355-360
39.Neurath, H.; Greenstein, J.P.; Putnam, F.W.; and Erickson J.O., “The chemistry of protein denaturation.” Chemical Reviews 1943; 32: 157-265
40.Nozaki, Y. and Tanford C.,“ The Solubility of Amino Acids and Two Glycine Peptides in Aqueous Ethanol and Dioxane Solutions.” The Journal of Biological Chemistry 1971; 246: 2211-2217.
41.Pain, R.H., “Mechanisms of protein folding.”Oxford University press, 1994.
42.Porath J.; Carlsson, J.; Olsson, I.; and Belfrage, G., “Metal chelate affinity chromatography, a new approach to protein fractionation.” Nature 1975; 258: 598
43.Saboury, A.A. and Karbassi, F.,“Thermodynamic studies on the interaction of calcium ions with alpha-amylase.” Thermochimica Acta 2000; 362: 121-129.
44.Sadana, A., “Protein refolding and inactivation during bioseparation : Bioseparation implication.” Biotechnology and Bioengineering 1995; 48: 481-489
45.Saxena, V.P. and Wetlaufer, D.B.,“Formation of three-dimensional sturcture in proteins. I: Rapid Nonenzymic reactivation of reduced lysozyme.” Biochemistry 1970; 9: 5015-5022.
46.Shastry, M.C.R. and Eftink, M.R. “Reversible thermal unfolding of ribonuclease T1 in reverse micelles.” Biochemistry 1996; 35: 4094-4101
47.Shire, S.J., “Purification and immunogenicity of fusion vp1 protein of foot and mouth-disease virus.”Biochemistry 1984; 23: 6474- 6480
48.Smith, V.R. and Walker J.E.,“Purification and folding of recombinant bovine oxoglutarate/malate carrier by immobilized metal-ion affinity chromatography.”Protein Expression and Purification 2003; 29: 209-216.
49.Suzuki, Y.; Ito, N.; Yuuki, T.; Yamagata, H.; and Udaka, S.,“Amino acid residues stabilizing a Bacillus alpha-amylase against irreversible thermoinactivation.”The Journal of Biological Chemistry 1989; 264: 18933-18938.
50.Ueda, E.K.M.; Gout, P.W.; and Morganti, L.,“Current and prospective applications of metal ion–protein binding.” Journal of Chromatography A 2003; 988: 1-23.
51.Vetsch, M.; Sebbel, P.; and Glockshuber, R.,“Chaperone- independent Folding of Type 1 Pilus Domains.” Journal of Molecular Biology 2002; 322: 827-840.
52.Vihinen, M. and Mäntsälä, P. “Microbial amylolytic enzymes” Critical reviews in biochemistry and molecular biology 1989; 24: 329-418
53.Vladka, G.P. and Viktor, M.,“Perspectives of immobilized-metal affinity chromatography.” Journal of Biochemical and Biophysical Methods 2001; 49: 335–360.
54.Voet, J.G.; Pratt, C.W.; and Voet, D., “Fundamentals of Biochemistry: Upgrade Edition” John Wiley & Sons Inc., 2001.
55.Weemaes, C.; DeCordt, S.; Goossens, K.; and Ludikhuyze, L.,“High pressure, thermal, and combined pressure-temperature stabilities of alpha-amylase from Bacillus species.” Biotechnology and Bioengineering 1996; 50: 49-56.
56.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
57.Yuuki, T.; Nomura, T.; and Tezuka, H.,“Complete nucleotide sequence of a gene coding for heat- and pH-stable alpha-amylase of Bacillus licheniformis: Compaison of the amino acid sequences of three bacterial liquefying alpha-amylase deduced from the DNA sequences.” Journal of Biochemistry 1985; 98: 1147-1156.
58.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。
59.張哲魁，“饋料式與間歇式復性法對溶菌酶復性行為之研究”，台灣大學碩士論文，1998。
60.黃世權，“澱粉分解酶摺疊程序之研究”，中原大學碩士論文，2001。
61.楊傑予，“澱粉分解酶之熱穩定性與其復性難易之關係”，中原大學碩士論文，2002。