Enzyme-containing micellar solutions

Oparin and coworkers [125,126] have studied the enzymic polymerization of ADP by polynucleotide phosphorylase (PNPase) and Mg ions in coacervates in an attempt to construct primitive forms of precellular structures. Walde et al. [127] have investigated this enzymic ADP polymerization in AOT reversed micellar solutions instead of coacervates. The PNPase-catalyzed synthesis of poly(A) (polyadenylic acid) in the AOT reversed micelles was carried out by mixing two reversed micellar solutions, one containing ADP and the other containing the enzyme. 

Another subject is concerned with biopolymer synthesis utilizing the liquid/solid interface in a reversed micellar system. The enzymic polymerization of ADP in AOT reversed micellar solution containing a Mg ions resulted in the precipitation of poly(A) together with the PNPase. Further polymerization could proceed by the enzyme in the precipitate by feeding ADP through the dynamic AOT monolayer on a glass surface. This is concluded to be a kind of solid polymerization in a reversed micellar solution. This process of polymerization provides a simple isolation of both the product and enzyme the maintenance of the enzyme activity for a long time and a novel solid polymerization on the oil/solid interface. This polymerization at the interfaces in the reversed micellar solution could be applied to other biopolymer syntheses. 

Reverse micellar extraction (RME) has been gaining popularity as an attractive hquid-hquid extraction process [108-111]. This is mainly due to the fact that enzymes can be solubilized in organic solvents with the aid of reverse micellar aggregates [112, 113]. Their inner core contains an aqueous micro-phase, which is able to solubilize polar substances, e.g., hydrophilic enzymes [114]. In many cases not only the enzymes retained their activity in organic environment in some cases they seem to perform even better if they are entrapped into reverse micellar aggregates [111]. One of the remarkable findings that gave this field a major boost is that the solubilization of different proteins into micellar solutions is a selective process [112]. 

How does the enzyme steady state cycle function in reverse micelles Just as in water, enzyme and substrate must encounter, and product must leave the enzyme. In hydrocarbon micellar solutions these events are subordinate to the encounter of enzyme-containing micelles with substrate-containing micelles, whereas the empty micelles may play the important role of incorporating the product (this holds in the case of hydrophilic substrate and products which are soluble only in the water pools and not in the hydrocarbon bulk - otherwise partition coefficients must be included, a problem which for the sake of simplicity we neglect here). In an earlier paper [88], we propose the steady state cycle as shown in Fig. 6. 

Protein solubilized in the reverse micellar solution can be transferred back into an aqueous solution by contacting the micellar solution with an aqueous solution containing a high concentration of salt (e.g., KCl, CaCy, which has the capability to exchange with protein in the micelles (Asenjo and Chaudhuri, 1996). Reverse miceUization has been successfully used to separate a variety of proteins including enzymes (e.g., lysozyme, trypsin and ribonucease). 

Nature uses membrane-based microstructures which organize redox enzymes and reactants to provide living organisms with highly efficient redox processes. Micellar solutions and microemulsions contain surfactant aggregates which can be considered crude models for biological membranes. 

Solutions of surfactant-stabilized nanogels share both the advantage of gels (drastic reduction of molecular diffusion and of internal dynamics of solubilizates entrapped in the micellar aggregates) and of nonviscous liquids (nanogel-containing reversed micelles diffuse and are dispersed in a macroscopicaUy nonviscous medium). Effects on the lifetime of excited species and on the catalytic activity and stability of immobilized enzymes can be expected. 

Rate of protein transfer to or from a reverse micellar phase and factors affecting the rate are important for the practical applications of RME for the extraction and purification of proteins/enzymes and for scale-up. The mechanism of protein exchange between two immiscible phases (Fig. 2) can be divided into three steps [36] the diffusion of protein from bulk aqueous solution to the interface, the formation of a protein-containing micelle at the interface, and the diffusion of a protein-containing micelle in to the organic phase. The reverse steps are applicable for back transfer with the coalescence of protein-filled RM with the interface to release the protein. The overall mass transfer rate during an extraction processes will depend on which of these steps is rate limiting. 

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