Bacillus amyloliquefaciens α-amylase去摺疊程序與其熱力學性質探討

Changes in Thermodynamic States of Unfolding of Bacillus amyloliquefaciens α-amylase

蔡易積(Yi-Chi Tsai)

指導教授 ： 莊清榮 ； 阮若屈

中原大學/工學院/化學工程研究所/碩士(2004年)

https://doi.org/10.6840/cycu200400451

摘要

摘 要
本研究利用圓極偏光光譜儀、螢光光譜儀及恆溫滴定微卡計探討澱粉分解、acillus amyloliquefaciens α-amylase (BAA)其去摺疊機制，並利用熱力學參數分析。
將BAA於不同濃度之變性劑與不同溫度下進行去摺疊，利用圓極偏光光譜儀觀察波長222nm下橢圓率(θ222)的改變，而知BAA二級結構α-helix破壞的程度；並利用螢光光譜儀觀察其最大散射波長紅移程度，而知BAA三級結構破壞的程度。結果得知BAA二級結構破壞速率與程度皆大於三級結構，藉此推論BAA於GdnHCl下其去摺疊程序中存在一個二級結構已破壞，但仍保有絕大部分三級結構之中間狀態，其去摺疊程序為：NpIpU。此中間狀態之橢圓率及最大散射波長分別為-9 mdeg及342nm。
假設BAA之去摺疊過程中之光譜量測值，為完全摺疊狀態(N)、中間狀態(I)及完全去摺疊狀態(U)之線性加成關係，則我們可進一步求得其各狀態之比例，再藉van’t Hoff equation計算BAA去摺疊之各熱力學參數，我們得知BAA由完全摺疊狀態便成中間狀態的enthalpy變化(榎m,NI)為128kJmol-1，熱含量變化(榰p,NI)為2.66 kJmol-1K-1，而由中間狀態變為完全去摺疊狀態過程中伴隨產生較大之榎m,IU與榰p,IU，其榎m,IU為538kJmol-1，榰p,IU為31.77 kJmol-1K-1，因此得知此中間狀態於BAA去摺疊程序中穩定存在。

關鍵字

蛋白質去摺疊程序 ； 澱粉分解脢 ； 蛋白質去摺疊之熱力學參數

並列摘要

Abstract
The main purpose of this study is to study the changes in thermodynamic states of unfolding of Bacillus amyloliquefaciens α-amylase (BAA).
The unfolding process of BAA was monitored by the loss of its secondary structure and tertiary structure at various GdnHCl level. The change in secondary structure was examined by circular dicroism (CD) spectroscopy at 222 nm and the change in tertiary structure was monitored by the shift in its fluorescence spectrum. Comparing the rates of changes in secondary and tertiary structures, we have found the existence of an intermediate state of which the secondary structure was lost but the tertiary structure was mostly conserved. The unfolding process of Bacillus amyloliquefaciens α-amylase was a three-state transition, N→I→U. The ellipicity and the fluorescence shift of the intermediate state were found to be -9 mdeg and 342 nm.
If the CD and fluorescence spectra were linear combination of the spectra of BAA at its native (N), intermediate (I), and fully unfolding (U) state, the fraction of each state could be calculated. The thermodynamic parameters of BAA unfolding could then be estimated by van’t Hoff equation. It was found that the transition from the native to intermediate states proceeded with significant changes in enthalpy. The estimation value of enthalpy change, 榎m,NI, was 128kJmol-1. The transition from the intermediate to fully unfolding states proceeded with comparatively large enthalpy and heat capacity changes, 榎m,IU and 榰p,IU. The results indicated that the intermediate state was stably existed during the BAA unfolding process.

並列關鍵字

BAA ； Changes in Thermodynamic States of protein unfolding

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