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

Protein Refolding by Dual-Functional Adsorbents : The Choice of Hydrophobic Alkyl Chain Length and Density.

柯玲潔(Ling-Chieh Ko)

指導教授 ： 劉英麟 ； 阮若屈

中原大學/工學院/化學工程學系/碩士(2006年)

https://doi.org/10.6840/cycu200600172

摘要

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！

關鍵字

雙功能吸附基材 ； 蛋白質復性 ； 澱粉分解?

並列摘要

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.

並列關鍵字

dual-functional adsorbent ； protein refolding ； alpha-amylase

參考文獻

參考文獻

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