On the activity loss of hydrolases in organic solvents: II. a mechanistic study of subtilisin Carlsberg.
On the activity loss of hydrolases in organic solvents: II. a mechanistic study of subtilisin Carlsberg.
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DOI：
10.1186/1472-6750-6-51
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发表时间：
2006-12-22
影响因子：
3.5
通讯作者：
Barletta G
中科院分区：
文献类型：
--
作者：
Castillo B;Bansal V;Ganesan A;Halling P;Secundo F;Ferrer A;Griebenow K;Barletta G
酶在有机溶剂中被广泛用于催化具有生物和工业意义的各种转化。人们普遍认为，在干燥的非质子有机溶剂中，由于酶展开所需的高能屏障，酶在动力学上被困在它们的构象中，这表明在这种介质中它们应该长时间保持催化活性。然而，最近对各种酶的研究表明，它们最初的高活性在暴露于有机溶剂几个小时后严重降低。据推测，这可能是由于结构扰动，酶的pH记忆的变化，酶聚集，或脱水，由于水的溶剂去除。在此，我们系统地研究了在1，4-二氧六环中这种不希望的活性损失的可能原因。以枯草杆菌蛋白酶Carlsberg为模型酶，采用冷冻干燥法和低温冷冻干燥法制备枯草杆菌蛋白酶，并添加甲基-β-环糊精（MβCD）。我们的研究结果排除了一种机制，涉及的酶的电离状态的变化，因为酶的活性显示出类似的pH值的依赖性之前和之后孵育5天，在1，4-二氧六环。未检测到因长期暴露于该溶剂中而导致的明显二级或三级结构扰动。此外，活性位点滴定显示，在孵育过程中，活性位点的数量保持恒定。此外，酶的水合水平似乎不影响其稳定性。电子顺磁共振光谱研究表明，在纯1，4-二氧六环中孵育期间，当使用BaBr 2水合盐使水活度保持恒定时，结合到活性位点（自旋标记）的顺磁氮氧自由基抑制剂的旋转自由度没有实质性增加。孵育还伴随着Vmax/KM的大幅降低。这些结果排除了在1，4-二氧六环中观察到的低酶储存稳定性的一些最明显的原因，主要是结构、动力学和电离状态的变化。最可能的解释是酶内的水分子可能发生重排，从而影响其介电环境。然而，此时不能排除其他机制，例如活性位点周围的小扭曲或反离子的重排。
Enzymes have been extensively used in organic solvents to catalyze a variety of transformations of biological and industrial significance. It has been generally accepted that in dry aprotic organic solvents, enzymes are kinetically trapped in their conformation due to the high-energy barrier needed for them to unfold, suggesting that in such media they should remain catalytically active for long periods. However, recent studies on a variety of enzymes demonstrate that their initial high activity is severely reduced after exposure to organic solvents for several hours. It was speculated that this could be due to structural perturbations, changes of the enzyme's pH memory, enzyme aggregation, or dehydration due to water removal by the solvents. Herein, we systematically study the possible causes for this undesirable activity loss in 1,4-dioxane. As model enzyme, we employed the protease subtilisin Carlsberg, prepared by lyophilization and colyophilization with the additive methyl-β-cyclodextrin (MβCD). Our results exclude a mechanism involving a change in ionization state of the enzyme, since the enzyme activity shows a similar pH dependence before and after incubation for 5 days in 1,4-dioxane. No apparent secondary or tertiary structural perturbations resulting from prolonged exposure in this solvent were detected. Furthermore, active site titration revealed that the number of active sites remained constant during incubation. Additionally, the hydration level of the enzyme does not seem to affect its stability. Electron paramagnetic resonance spectroscopy studies revealed no substantial increase in the rotational freedom of a paramagnetic nitroxide inhibitor bound to the active site (a spin-label) during incubation in neat 1,4-dioxane, when the water activity was kept constant using BaBr2 hydrated salts. Incubation was also accompanied by a substantial decrease in Vmax/KM. These results exclude some of the most obvious causes for the observed low enzyme storage stability in 1,4-dioxane, mainly structural, dynamics and ionization state changes. The most likely explanation is possible rearrangement of water molecules within the enzyme that could affect its dielectric environment. However, other mechanisms, such as small distortions around the active site or rearrangement of counter ions, cannot be excluded at this time.
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DOI：
10.1016/j.bbapap.2006.03.016
发表时间：
2006-06-01
影响因子：
3.2
作者：
Ganesan, Ashok;Price, Nicholas C.;Halling, Peter J.
通讯作者：
Halling, Peter J.
影响因子：
15
作者：
Lee, T;Jones, JB
通讯作者：
Jones, JB
影响因子：
--
作者：
Castillo, B;Pacheco, Y;Barletta, G
通讯作者：
Barletta, G
影响因子：
2.9
作者：
Fernandes, JFA;Halling, PJ
通讯作者：
Halling, PJ
影响因子：
15
作者：
Griebenow, K;Laureano, YD;Barletta, G
通讯作者：
Barletta, G