Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Biotechnol Bioeng. 1993 Mar 5;41(5):566-71. doi: 10.1002/bit.260410509.
When seven different hydrolytic enzymes (four proteases and three lipases) were lyophilized from aqueous solution containing a ligand, N-Ac-L-Phe-NH(2), their catalytic activity in anhydrous solvents was far greater (one to two orders of magnitude) than that of the enzymes lyophilized without the ligand. This ligand-induced activation was expressed regardless of whether the substrate employed in organic solvents structurally resembled the ligand. Furthermore, nonligand lyoprotectants [sorbitol, other sugars, and poly(ethylene glycol)] also dramatically enhanced enzymatic activity in anhydrous solvents when present in enzyme aqueous solution prior to lyophilization. The effects of the ligand and of the lyoprotectants were nonadditive, suggesting the same mechanism of action. Excipient activated and nonactivated enzymes exhibited identical activities in water. Also, addition of the excipients directly to suspensions of nonactivated enzymes in organic solvents had no appreciable effect on catalytic activity. These observations indicate that the mechanism of the excipient-induced activation is based on the ability of the excipients to alleviate reversible denaturation of enzymes upon lyophilization. Activity enhancement induced by the excipients is displayed even after their removal by washing enzymes with anhydrous solvents. Subtilisin Carlsberg, lyophilized with sorbitol, was found to be a much more efficient practical catalyst than its "regular" counterpart.
当七种不同的水解酶（四种蛋白酶和三种脂肪酶）从含有配体 N-Ac-L-Phe-NH(2) 的水溶液中冻干时，它们在无水溶剂中的催化活性（比没有配体冻干的酶高一个到两个数量级）要高得多。这种配体诱导的激活与在有机溶剂中使用的底物是否在结构上与配体相似无关。此外，非配体保护剂[山梨醇、其他糖和聚乙二醇]在冻干前存在于酶水溶液中时，也能显著提高无水溶剂中的酶活性。配体和保护剂的作用不是加性的，这表明它们的作用机制相同。赋形剂激活和非激活的酶在水中表现出相同的活性。此外，赋形剂直接添加到非激活酶在有机溶剂中的悬浮液中，对催化活性没有明显影响。这些观察结果表明，赋形剂诱导激活的机制基于赋形剂缓解酶在冻干过程中可逆变性的能力。即使通过用无水溶剂洗涤酶去除赋形剂，赋形剂诱导的活性增强仍会显示出来。与“常规”对应物相比，用山梨醇冻干的枯草杆菌蛋白酶 Carlberg 被发现是一种更高效的实用催化剂。
