Entrapment enzyme aggregates

Entrapment of enzymes and cells has played an important role in developing bioprocesses. Applications of entrapment technology to biosensors and bioanalysis have mainly been focused on udlizadon of cells and, to a smaller extent, on enzymes (24). Combining covalent coupling and entrapment cross-links enzymes and inert protein to form a protein membrane that covers the sensitive part of the electrode dp in bioanalytical applications (25). Entrapping enzyme aggregates is another variadon of this methodology (26). 

First, these systems are diphilic, since both hydrophilic and hydrophobic (water-insoluble) substances may be dissolved (solubilized) therein. Second, surfactant aggregates, which are organized in such systems, are particles and may serve as matrix reactors of molecular size by entrapping enzyme molecules and required reagents. This allows one to carry out controlled local reactions in limited volume and to organize catalytic ensembles of controlled sizes (nanoparticles). 

Enzyme immobilization techniques have been revealed as a powerful tool to enhance most enzyme properties such as sta-bihty, activity, specificity and selectivity, and reduction of inhibition. Several methods had been used including cross-linked enzyme aggregates (CLEAs) (Wang et al. 2011), entrapment, and support-based immobilization (Brady and Jordaan 2009). 

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. 

The conversion of benzaldehyde by the encapsulated HNLs afforded mandelo-nitrile in 96-98% yield and 97-99% for all three enzymes. Free and entrapped HfoHNL catalyzed the conversion of hexanal with 94% , whereas ee-values of only 85 and 87% could be achieved with MeHNL and PaHNL preparations, respectively, limited by the intrinsic enantioselectivity of the enzymes (Table 9.4). Furthermore, the CLEAs from HfoHNL and PaHNL suffered from activity loss under the reaction conditions in contrast to MeHNL CLEA, indicating that the cross-linked aggregates from MeHNL are particularly robust and necessitate only traces of water to keep the catalytic activity [87]. 

I. V. Berezin, and K. Martinek, Catalysis by enzymes entrapped into hydrated surfactant aggregates having lamellar or cylindrical (hexagonal) or ball-shaped (cubic) structure in organic solvents,... 

In some biochemical systems the limiting mass transfer step shifts from a gas-liquid or solid-liquid interface (as discussed earlier) to the interior of solid particles. The most important cases are solid substrates and cell aggregates (such as microbial floes, cellular tissues, etc.) and immobilized enzymes (gel-entrapped or supported in solid matrices). In the former, diffusion of oxygen (or other nutrients) through the particle limits metabolic rates, while in the latter, substrate reactant or product diffusion into or out of the enzyme carrier often limits the overall global bioreaction rates. 

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]. 

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