Expedited Award for Novel Research: Enzyme Biocatalysis in Reverse Micelles: Thermodynamic and Kinetic Studies

酶在有机合成中的广泛应用，由于其高度的区域选择性和立体选择性难以用传统催化剂实现，严重阻碍了许多感兴趣的底物的低水溶性。将酶包封在反胶束中，为酶的稳定提供了一种新的方法，是一种在有机溶剂中进行生物合成或有机化合物转化的新方法。该项目涉及开发使用这种封装酶的新型生物反应器的基础研究。它包括对多巴胺β -单加氧酶（一种非特异性酶，可使环取代苯乙胺、烯烃、醛、酰胺和硫化物羟基化）的包封效果的研究。利用ESR和动力学测量研究了胶束结构对酶构象的影响。还建立了一个分子热力学模型，将水相的性质与当时的条件联系起来。利用反胶束保护酶不受有机溶剂的变性影响是一种稳定酶的新方法。目前的研究主要集中在利用反胶束进行蛋白质纯化。反向胶束的内部水池可以溶解大的蛋白质分子，胶束为反应物和底物进出酶的运输提供了大的表面积。人们对酶在这种胶束中的行为或它们的动力学性质与溶液中的酶有何不同知之甚少。研究人员使用非极性表面活性剂创建胶束，因此极性有机底物的运输可以通过在有机相中添加载体分子（如季烷基胺）来增强。从相平衡热力学的观点描述水在反胶束中的行为是目前的兴趣。更重要的是开发一种分子热力学模型，将水相的性质与外部条件联系起来，从而可以将预测模型用于生物反应器的设计。

The wider use of enzymes in organic syntheses, where their high degree of regio- and stereo-selectivity is difficult to achieve with conventional catalysts, is severely hampered by the low aqueous solubility of many substrates of interest. The encapsulation of enzymes in reverse micelles, which provides a method for stabilization of the enzyme, is a new and particularly promising approach for biological synthesis or transformation of organic compounds in organic solvents. This project concerns fundamental studies for the development of new bioreactors employing such encapsulated enzymes. It includes a study of the effect of encapsulation of dopamine beta- monooxygenase, a non-specific enzyme which hydroxylates ring- substituted phenethylamines, alkenes, aldehydes, amides and sulphides. The effect of micelle structure on the conformation of the enzyme is studied by use of ESR and kinetic measurements. A molecular- thermodynamic model, relating the properties of the aqueous miniphase to prevailing conditions, is also developed. The use of reverse micelles to protect the enzyme from the often denaturing influence of organic solvents is a new approach to enzyme stabilization. Current research has focussed on the use of reverse micelles for protein purification. The inner water pool of a reverse micelle can solubilize large protein molecules and the micelle provides a large surface area for transport of reactant and substrate to and from the enzyme. Little is understood of the behavior of enzymes within such micelles or of how their kinetic properties differ from enzymes in solution. The investigators create micelles using non-polar surfactants, so that transport of polar organic substrates can be enhanced by the addition of carrier molecules in the organic phase, such as quaternary alkyl amines. There is current interest in describing the behavior of water in reverse micelles from a phase equilibrium thermodynamic viewpoint. Of even greater importance is the development of a molecular-thermodynamic model to relate the properties of the aqueous miniphase to the external conditions, so that a predictive model can be employed for bioreactor design.