Dispersion Immobilized enzymes

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 same group reported in 1986 a sensitive and selective HPLC method employing CL detection utilizing immobilized enzymes for simultaneous determination of acetylcholine and choline [187], Both compounds were separated on a reversed-phase column, passed through an immobilized enzyme column (acetylcholine esterase and choline oxidase), and converted to hydrogen peroxide, which was subsequently detected by the PO-CL reaction. In this period, other advances in this area were carried out such as the combination of solid-state PO CL detection and postcolumn chemical reaction systems in LC [188] or the development of a new low-dispersion system for narrow-bore LC [189],... 

The modeling of real immobilized-enzyme column reactors, mainly the fluidized-bed type, has been described (Emeiy and Cardoso, 1978 Allen, Charles and Coughlin, 1979 Kobayashi and Moo-Young, 1971) by mathematical models based on the dispersion concept (Levenspiel, 1972), by incorporation of an additional term to account for back-mixing in the ideal plug-flow reactor. This term describes the non-ideal effects in terms of a dispersion coefficient. 

Adsorption on solid matrices represents a quite simple and inexpensive method for enzyme immobilization. Enzyme dispersion is improved, reducing the diffusion limitations and favoring the accessibility of substrate to the enzyme [12]. On the other hand, because of the weak binding, the system can suffer from catalyst leaching, and there is little stabilization of the enzyme. The most common carriers... 

In fermentation reactors, cell growth is promoted or maintained to produce metabolite, biomass, transformed substrate, or purified solvent. Systems based on macro-organism cultures are usually referred as tissue cultures. Those based on dispersed non-tissue forming cultures of micro-organisms are loosely referred as microbial reactors. In enzyme reactors, substrate transformation is promoted without the life-support system of whole cells. Frequently, these reactors employ immobilized enzymes, where an enzyme is supported on inert solids so that it can be reused in the process. Virtually all bioreactors of technological importance deal with a heterogeneous system involving more than two phases. 

A liquid-phase resistance inside a dispersed solid phase such as cell floes, immobilized enzymes, etc. 

A great savings in enzyme consumption can be achieved by immobilizing the enzyme in the reactor. In addition to the smaller amount of enzyme required, immobilization often increases the stability of the enzyme. Several designs of immobilized-enzyme reactors (lERs) have been reported, with open tubular and packed bed being the most popular. Open-tubular reactors offer low dispersion, but have a relatively small surface area for enzyme attachment. Packed-bed reactors provide extremely high surface areas and improved mass transport at the cost of more dispersion. 

By chemical modification of the silica surface it has become possible to design new highly-selective adsorbents and catalysts, active polymer fillers, efficient thickeners of dispersive media. Interest in the modified silicas, in particular, in the activated matrices based on functional organosilicas has quickened in the past few years as a result of the favorable prospects for their application for various kinds of chromatographic separation, preparation of grafted metal complex catalysts, immobilized enzymes and other biologically active compounds [1]. 

Immobilization, dehned as the physical confinement or localization of an enzyme into a specihc micro-environment, has been a very common approach to prepare enzymes for aqueous as well as nonaqueous applications. For nonaqueous enzymol-ogy, immobilization improves storage and thermal stability, facilitates enzyme recovery, and enhances enzyme dispersion. In addition, immobilized enzymes are readily incorporated in packed bed bioreactors, allowing for continuous operation of reactions. Moreover, lyophilized enzyme powders often aggregate and attach to reactor walls, particularly when the water activity is moderately high. The major disadvantage of immobilization is low activity, induced by pore diffusion mass transfer limitations and by alteration of protein stmcture. For enzymes in nonaqueous media, the following broad categories of immobilization exist ... 

Another immobilization technique proposed is nanoentrapment into NPs. In this method, a water-in-oil microemulsion system is used for the fabrication of NPs and for the dispersion of enzyme. This procedure leads to the creation of discrete NPs through polymerization in the water phase or on the interface, in which the enzyme is dispersed [195, 196], One of the challenges of this approach is the difficulty in controlling the size of reverse micelles, as well as the number of enzyme molecules within each reverse micelle, which will directly affect the final properties of enzyme-entrapped nanoparticles [6],... 

When the mass of carrier material is large relative to that of the enzyme, the physical and chemical properties of the carrier (Table 6-5) will, in large part, determine properties of the resultant immobilized enzyme. Often, the carrier will impart mechanical strength to the enzyme, allowing repetitive recovery by simple filtration of the solid particles and reuse of the enzyme. The degree of porosity and pore volume will determine the resistance to diffusion and molecular size selectivity of the biocatalyst. When used in non-aqueous media, dispersion of the enzyme over a large surface area can greatly increase its activity. Table 6-3 summarizes many of the key properties and considerations for enzyme carrier materials. 

To overcome the disparity in the optimal pH s for the isomerization and fermentation, our group [29, 35, 36] proposed a novel scheme of isomerization that incoiporates urease co-immobilized with xylose isomerase. This technique uses XI immobilized in a porous pellet for isomerization and the immobilized urease enzyme for pH control (Fig. 1). These co-immobilized enzyme pellets are dispersed in a fermentation broth, which contains urea in addition to the other necessary ingredients for fermentation. Theoretically, it is possible to sustain a significant pH gradient between the bulk liquid and the core region of the pellet... 

Because enzymes are not soluble in SCFs it should be possible to disperse free enzyme in the SCF and recover the enzyme without the need to immobilize it on a support. 

Briefly, the flow-through reactor has the advantages of essentially no external or internal mass transfer limitations, no substrate holdup, and no channeling all of which contribute to a more efficient utilization of immobilized enzyme. The substrate must contact all reactive sites while passing through the pores therefore eliminating dispersion or diffusion related problems. One can perform sequential reactions, since there is no holdup of reactants and products from one reaction step to the next. 

In traditional synthetic transformations, enzymes are normally used in aqueous or organic solvents at relatively low temperatures to preserve their activity. Therefore, it is not surprising that some of these reactions require long reaction times. In view of the newer developments that immobilize enzymes on solid supports [148], these reactions are now amenable to operation at higher temperature with adequate pH control. The application of MW irradiation has been investigated with Pseudomonas lipase dispersed in Hyflo Super Cell, which essentially consists of diato-maceous silica around pH 8.5-9.0 and commercially available SP 435 Novozym (Candida antarctica lipase grafted on an acrylic resin) [149]. 

The biologically active enzymes can be used as catalysts either in a soluble, dispersed form or in a carrier-bound form. Because of the need for the isolation step and losses of enzyme activity, enzyme processes are sparingly used at the present time. Immobilized enzymes show promise for minimizing these activity losses and for facilitating enzyme recovery (Pitcher, 1978). 

In other applications, proteins are adsorbed by purpose, for example, as immobilized enzymes in biosensors and bioreactors, immunoglobulins in immunoassays, drugs in drug targeting and controlled release systems, and as stabilizers of dispersions, emulsions, and foams in foodstuffs, pharmaceuticals, and cosmetics. 

To improve an analytical immobilized enzyme packed reactor one of the most advantageous approaches is optimization of the support size. A decrease in carrier diameter would result in three advantages decreased dispersion, a decrease in internal diffusion with an increase in efficiency, and an increased surface area to volume ratio which would result in increased external mass transfer rates. To date, the smallest particles commonly used in analytical applications are 400 mesh (37 /im I.D.) The smaller particles may require a large driving force with increased cost and mechanical complexity. The pressure drop and column dimensions are related so that the final system parameters will be determined by the specific application requirements. 

Consider the situation of catalytic particles dispersed in a reasonably thin polymeric film, where the substrate/product reaction occurs via Michaelis-Menten kinetics. This problem is directly relevant to the associated problems of immobilized enzyme catalysis and diffusion/chemical reaction processes in chemical engineering. Aspects of the theory presented in here have recently been described by Albery and coworkers for enzyme electrodes. 

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