Rich Joseph O, Mozhaev Vadim V, Dordick Jonathan S, Clark Douglas S, Khmelnitsky Yuri L
Biocatalysis Division, Albany Molecular Research Inc., 601 E. Kensington Road, Mt. Prospect, Illinois 60056, USA.
J Am Chem Soc. 2002 May 15;124(19):5254-5. doi: 10.1021/ja012219z.
Attaining higher levels of catalytic activity of enzymes in organic solvents is one of the major challenges in nonaqueous enzymology. One of the most successful strategies for enhancing enzyme activity in organic solvents involves tuning the enzyme active site by molecular imprinting with substrates or their analogues. Unfortunately, numerous imprinters of potential importance are poorly soluble in water, which significantly limits the utility of this method. In the present study, we have developed strategies that overcome this limitation of the molecular-imprinting technique and that thus expand its applicability beyond water-soluble ligands. The solubility problem can be addressed either by converting the ligands into a water-soluble form or by adding relatively high concentrations of organic cosolvents, such as tert-butyl alcohol and 1,4-dioxane, to increase their solubility in the lyophilization medium. We have succeeded in applying both of these strategies to produce imprinted thermolysin, subtilisin, and lipase TL possessing up to 26-fold higher catalytic activity in the acylation of paclitaxel and 17beta-estradiol compared to nonimprinted enzymes. Furthermore, we have demonstrated for the first time that molecular imprinting and salt activation, applied in combination, produce a strong additive activation effect (up to 110-fold), suggesting different mechanisms of action involved in these enzyme activation techniques.
在有机溶剂中实现酶的更高催化活性水平是非水酶学中的主要挑战之一。提高酶在有机溶剂中活性的最成功策略之一是通过用底物或其类似物进行分子印迹来调节酶的活性位点。不幸的是，许多具有潜在重要性的印迹剂在水中溶解度很差，这极大地限制了该方法的实用性。在本研究中，我们开发了克服分子印迹技术这一局限性的策略，从而将其适用性扩展到水溶性配体之外。溶解度问题可以通过将配体转化为水溶性形式或通过添加相对高浓度的有机共溶剂（如叔丁醇和1,4 - 二氧六环）来解决，以增加它们在冻干介质中的溶解度。我们成功地应用这两种策略制备了印迹嗜热菌蛋白酶、枯草杆菌蛋白酶和脂肪酶TL，与非印迹酶相比，它们在紫杉醇和17β - 雌二醇酰化反应中的催化活性提高了26倍。此外，我们首次证明，分子印迹和盐激活相结合会产生强烈的加和激活效应（高达110倍），这表明这些酶激活技术涉及不同的作用机制。
