Schulze B, Klibanov A M
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Biotechnol Bioeng. 1991 Nov;38(9):1001-6. doi: 10.1002/bit.260380907.
The stability of the serine proteases from Bacillus amyloliquefaciens (subtillisin BPN') and Bacillus licheniformis (subtilisin Carlsberg) was investigated in various anhydrous solvents at 45 degrees C. The half-life of subtilisin BPN' in dimethyl-formamide dramatically depends on the pH of the aqueous solutions from which the enzyme was lyophilized, increasing from 48 min to 20 h when the pH is raised from 6.0 to 7.9. Both subtilisins exhibited substantial inactivation during multihour incubations in tert-amyl alcohol and acetonitrile when enzymatic activities were also measured in these solvents; however, when the enzymes were assayed in water instead, hardly any loss of activity was detected. This surprising difference appears to stem from the partitioning of the bound water essential for catalytic activity from the enzymes into the solvents. When assayed in organic solvents, this time-dependent stripping of water results in decay of enzymatic activity; however, when assayed in water, where the dehydrated subtilisins can undergo rehydration thereby recovering catalytic activity, little inactivation is observed. In agreement with this hypothesis, the addition of small quantities of water tert-amyl alcohol stabilized the subtilisins in it even when enzymatic activity was measured in the nonaqueous solvent. Ester substrates (vinyl butyrate and trichloroethyl butyrate) greatly enhanced the stability of both subtilisins in organic solvents possibly because of the formation of the acyl-enzymes.
研究了来自解淀粉芽孢杆菌（枯草杆菌蛋白酶BPN'）和地衣芽孢杆菌（枯草杆菌蛋白酶Carlsberg）的丝氨酸蛋白酶在45℃的各种无水溶剂中的稳定性。枯草杆菌蛋白酶BPN'在二甲基甲酰胺中的半衰期极大地取决于冻干该酶所用的水溶液的pH值，当pH值从6.0提高到7.9时，半衰期从48分钟增加到20小时。当在叔戊醇和乙腈中进行数小时孵育并同时在这些溶剂中测量酶活性时，两种枯草杆菌蛋白酶均表现出显著失活；然而，当改为在水中测定酶活性时，几乎未检测到活性损失。这种惊人的差异似乎源于对催化活性至关重要的结合水从酶中分配到溶剂中。在有机溶剂中测定时，这种随时间的水的剥离导致酶活性衰减；然而，在水中测定时，脱水的枯草杆菌蛋白酶可以重新水合从而恢复催化活性，几乎观察不到失活。与该假设一致，即使在非水溶剂中测量酶活性，向叔戊醇中添加少量水也能稳定其中的枯草杆菌蛋白酶。酯底物（丁酸乙烯酯和丁酸三氯乙酯）极大地增强了两种枯草杆菌蛋白酶在有机溶剂中的稳定性，这可能是由于形成了酰基酶。
