Department of Chemistry, Massachusetts Institute of Technology, cambridge, Massachusetts 02139, USA.
Biotechnol Bioeng. 1991 Apr 15;37(9):843-53. doi: 10.1002/bit.260370908.
Three unrelated enzymes (ribonuclease, chymotrypsin, and lysozyme) display markedly enhanced thermostability in anhydrous organic solvents compared to that in aqueous solution. At 110-145 degrees C in nonaqueous media all three enzymes inactivate due to heat-induced protein aggregation, as determined by gel filtration chromatography. Using bovine pancreatic ribonuclease A as a model, it has been established that enzymes are much more thermostable in hydrophobic solvents (shown to be essentially inert with respect to their interaction with the protein) than in hydrophilic ones (shown to strip water from the enzyme). The heat-induced aggregates of ribonuclease were characterized as both physically associated and chemically crosslinked protein agglomerates, with the latter being in part due to transamidation and intermolecular disulfide interchange reactions. The thermal denaturation of ribonuclease in neat organic solvents has been examined by means of differential scanning calorimetry. In hydrophobic solvents, the enzyme exhibits greatly enhanced thermal denaturation temperatures (T(m) values as high as 124 degrees C) compared to aqueous solution. The thermostability of ribonuclease towards heat-induced denaturation and aggregation decreases as the water content of the protein powder increases. The experimental data obtained suggest that enzymes are extremely thermostable in anhydrous organic solvents due to their conformational rigidity in the dehydrated state and their resistance to nearly all the covalent reactions causing irreversible thermoinactivation of enzymes in aqueous solution.
三种互不相关的酶（核糖核酸酶、胰凝乳蛋白酶和溶菌酶）在无水有机溶剂中的热稳定性明显高于在水溶液中的热稳定性。在非水介质中 110-145°C 下，由于热诱导的蛋白质聚集，所有三种酶都会失活，这可以通过凝胶过滤色谱法来确定。使用牛胰核糖核酸酶 A 作为模型，已经确定酶在疏水性溶剂（被证明对其与蛋白质的相互作用基本惰性）中比在亲水性溶剂（被证明会从酶中除去水）中更稳定。核糖核酸酶的热诱导聚集物被表征为物理上相关和化学上交联的蛋白质聚集体，其中后者部分归因于转酰胺化和分子间二硫键交换反应。通过差示扫描量热法研究了核糖核酸酶在纯有机溶剂中的热变性。在疏水性溶剂中，与水溶液相比，酶表现出大大增强的热变性温度（高达 124°C 的 T(m) 值）。核糖核酸酶对热诱导变性和聚集的热稳定性随着蛋白粉中水含量的增加而降低。获得的实验数据表明，由于在脱水状态下的构象刚性以及对导致酶在水溶液中不可逆热失活的几乎所有共价反应的抵抗力，酶在无水有机溶剂中非常稳定。
