Enzyme water exclusion

Before we could take up this study in general, we had to solve one of the more bothersome aspects of cyclodextrin chemistry. It was believed strongly that cyclodextrin would bind substrates only in pure water solution, and this was a serious defect. First of all, it severely restricted the range of substrates that could be examined, since many interesting molecules have low water solubility. As a second point, it made it difficult to examine another feature of enzyme-catalyzed reactions. One of the roles that can be ascribed to the large protein mass, which contains the functional groups of an enzyme, is the function of water exclusion. That is, enzyme reactions can be considered to be operating in a nonaqueous or only partially aqueous medium. 

Much like temperature, many industrial applications of enzymes require activity in the presence of organic solvents. Interestingly, Burton et al.118 have pointed out that thermostability and tolerance of organic solvents are characteristics that are often linked sequence and structural modifications that improve activity to one denaturing influence often also improve tolerance to others. Unfortunately, enzymes have generally evolved to function in aqueous media solvents are believed to denature and inactivate enzymes by exclusion of water from critical structural or functional components. Thus, many enzymes cannot tolerate common bioprocess conditions and techniques such as directed evolution must be used to improve their stability. 

In order to be exploitable for extraction and purification of proteins/enzymes, RMs should exhibit two characteristic features. First, they should be capable of solubilizing proteins selectively. This protein uptake is referred to as forward extraction. Second, they should be able to release these proteins into aqueous phase so that a quantitative recovery of the purified protein can be obtained, which is referred to as back extraction. A schematic representation of protein solubilization in RMs from aqueous phase is shown in Fig. 2. In a number of recent publications, extraction and purification of proteins (both forward and back extraction) has been demonstrated using various reverse micellar systems [44,46-48]. In Table 2, exclusively various enzymes/proteins that are extracted using RMs as well as the stability and conformational studies of various enzymes in RMs are summarized. The studies revealed that the extraction process is generally controlled by various factors such as concentration and type of surfactant, pH and ionic strength of the aqueous phase, concentration and type of CO-surfactants, salts, charge of the protein, temperature, water content, size and shape of reverse micelles, etc. By manipulating these parameters selective sepa-... 

I would like to remind the audience that Mildred Cohn had shown that in some phosphotransferases the binding of ligands to the active site of the enzyme leads to an exclusion of water molecules in the neighborhood of manganese as part of the active site function. 

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