澱粉分解脢摺疊程序之研究

A study of alpha-amylase unfolding and refolding processes

黃世權(Shih-Chuan Huang)

指導教授 ： 阮若屈

中原大學/工學院/化學工程研究所/碩士(2001年)

https://doi.org/10.6840/cycu200100193

摘要

摘 要

本研究利用活性變化、螢光光譜、圓極偏光光譜探討兩個胺基酸組成相似的澱粉分解脢Bacillus amyloliquefaciens α-amylase（BAA）及Bacillus licheniformis α-amylase（BLA）摺疊的過程。利用活性變化觀察活性破壞的情形，螢光光譜觀察胺基酸tryptophan暴露情形，圓極偏光光譜觀察二級結構破壞情形。

利用變性劑尿素主要是破壞疏水作用力與氫鍵，變性劑胍鹽酸主要是破壞離子作用力、疏水性作用力與氫鍵，因此我們利用尿素和胍鹽酸當變性劑，探討變性劑濃度對變性程度影響與變性過程隨時間變化，比較BAA與BLA去摺疊情形，以了解兩者穩定結構力量與摺疊機制的不同。

在變性劑濃度對變性程度影響，尿素濃度需高於6M，但胍鹽酸只需0.5M時，BAA呈現完全展開的狀態；而BLA只有在胍鹽酸1~3M才會呈現完全展開的狀態，顯示離子性作用力或鹽橋穩定BAA與BLA結構。

在變性過程隨時間變化，BAA不管利用8 M尿素或6 M胍鹽酸當變性劑，活性破壞的速度一樣，不過在二級結構α-helix破壞的速度與胺基酸tryptophan暴露速度上，以尿素當變性劑會比胍鹽酸來的快；而BLA不管利用8 M尿素或6 M和 1 M胍鹽酸當變性劑，都是二級結構破壞最快，然後是活性破壞，最後是胺基酸tryptophan的暴露。

我們發現利用8 M尿素可以將BAA結構破壞，而無法完全破壞BLA，這表示BLA的離子性作用力或鹽橋作用比BAA強；而利用6 M胍鹽酸可以將BAA結構破壞，卻只能部分破壞BLA，但是在1 M 胍鹽酸卻能將BLA完全破壞，這可能是BLA疏水作用極強，或是其二級結構β-pleated sheet增加其穩定作用。

在復性過程方面，BAA在尿素變性後，最後無法復性；而在胍鹽酸變性後，在初始濃度為1 mg/mL時，活性測定只恢復到相對比活性的35﹪，但是從螢光光譜與圓極偏光光譜卻可以知道，大部分蛋白質已經恢復至接近原來的結構。再以初始濃度為0.3mg/mL進行復性時，80﹪的活性可以恢復，這表示在初始濃度為1 mg/mL時，的確大部分摺疊動作已完成，可能由於蛋白質聚集而無法展現活性。

利用變性程度隨時間變化和復性的結果，可以推論BAA的摺疊機制為： ，N：native state、I：鹽橋作用被破壞之中間狀態、U：unfolded state，BLA的摺疊機制為： ， I1：二級結構被破壞之中間狀態、I2：鹽橋作用被破壞之中間狀態，從復性性結果推論，BAA在復性過程中，在 之間產生聚集，而BLA在復性過程中，在 之間產生聚集。

關鍵字

摺疊 ； 澱粉分解脢 ； 圓極偏光光譜 ； 螢光光譜

並列摘要

Abstract

The unfolding and refolding processes of Bacillus amyloliquefaciens and Bacillus licheniformis α-amylase（BAA and BLA）were under investigation. The tertiary structure change was monitored by both the activity change and tryptophan exposure. The secondary structure change was monitored by circular dicroism.

The denaturants, urea and guanidine hydrochloride, were used to study the unfolding processes. Electrostatic interaction (salt bridge), hydrophobic interaction and hydrogen bonding are the three major forces stabilizing protein structure. Guanidine hydrochloride can disrupt all these three forces but urea lacks the ability to hinder electrostatic force. The major forces that stabilize the structure of the two a-amylases can be partially elucidated by comparing the unfolding processes in these two denaturants.

It was found that BAA unfolded in low concentration of guanidine hydrochloride but it needed much higher concentration of urea to fully unfold the protein. This result indicates that the salt bridge plays an important role in a-amylase’s stability. However, when the unfolding rates were compared at high concentration of denaturants, the rate of tryptophan exposure was found faster in urea than in GdnHCl. This indicates that there exist at least one intermediate that can be more easily unfolded by urea than by GdnHCl. It was also found that the rates of activity loss, a-helix disruption and tryptophan exposure were similar when BAA was exposed in 8 M urea. But the rate of activity loss appeared much faster than those of a-helix disruption and tryptophan exposure when BAA was unfolded in 6 M GdnHCl. This indicates that the intermediate can temporarily hold its secondary and tertiary structure after the salt bridge is broken.

BLA could only be partially deactivated in 8M urea or 6M GdnHCl. The rates of activity loss and a-helix disruption were faster than that of tryptophan exposure. However, 1.0M of GdnHCl was sufficient to deactivate BLA. The rate of a-helix disruption was faster than that of activity loss and the activity loss was faster than the exposure of tryptophan in 1.0 M GdnHCl. It is believed that there exist two intermediates during the unfolding of BLA. One intermediate occurs after the a-helix disruption and the other occurs after the salt bridge was broken.

We were not able to regain the activity of the 1 mg/mL unfolded BAA by 100 fold direct dilution. But the CD and fluorescence spectroscopy showed fully restoration of its a-helix structure and tryptophan location. We therefore tried to recover the activity of GdnHCl denatured BAA by further dilution. Eighty percent activity was recovered when the 1 mg/mL unfolded BAA was diluted by 300 fold.

From the results of unfolding and refolding, we can infer the mechanisms of BAA and BLA unfolding/folding. The process of BAA may be . N denotes the native state and I denotes the intermediate after salt bridge disruption. The mechanism of BLA may be . I1 denotes the intermediate state after a-helix disruption, I2 denotes the intermediate after salt bridge disruption. It is possible that BAA forms aggregates during and BLA easily forms aggregates during .

並列關鍵字

amylase ； folding ； circular dicroism ； fluorescence

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