Enzymes mass transfer

Several thermodynamic and kinetic behaviors of enzyme-catalyzed reactions performed in ILs, with respect to enzymatic reactions carried out in conventional solvents, could lead to an improvement in the process performance [34—37]. ILs showed an over-stabilization effect on biocatalysts [38] on the basis of the double role played by these neoteric solvents ILs could provide an adequate microenvironment for the catalytic action of the enzyme (mass transfer phenomena and active catalytic conformation) and if they act as a solvent, ILs may be regarded as liquid immobilization supports, since multipoint enzyme-1L interactions (hydrogen. Van der Waals, ionic, etc.) may occur, resulting in a flexible supramolecular not able to maintain the active protein conformation [39]. Their polar and non-coordinating properties hold considerable potential for enantioselective reactions since profound effects on reactivities and selectivities are expected [40]. In recent years attention has been focused on the appUcation of ILs as reaction media for enantioselective processes [41—43]. 

Enzymes can be immobihzed in sheets. One design had discs of enzymes fastened to a rotating shaft to improve mass transfer, and an alternate design had the feed stream flowing back and forth through sandwiches of sheets with enzyme. However, volumetric efficiency of such reactors is low because sheets with finite spacing offer less area than that of packed particles. 

Enzymatic reactions frequently undergo a phenomenon referred to as substrate inhibition. Here, the reaction rate reaches a maximum and subsequently falls as shown in Eigure 11-lb. Enzymatic reactions can also exhibit substrate activation as depicted by the sigmoidal type rate dependence in Eigure 11-lc. Biochemical reactions are limited by mass transfer where a substrate has to cross cell walls. Enzymatic reactions that depend on temperature are modeled with the Arrhenius equation. Most enzymes deactivate rapidly at temperatures of 50°C-100°C, and deactivation is an irreversible process. 

Immobilization can give rise to mass transfer limitations that do not occur for freely suspended enzymes in their native state. As a formality, these limitations can be incorporated into an effectiveness factor ... 

Like enzymes, whole cells are sometime immobilized by attachment to a surface or by entrapment within a carrier material. One motivation for this is similar to the motivation for using biomass recycle in a continuous process. The cells are grown under optimal conditions for cell growth but are used at conditions optimized for transformation of substrate. A great variety of reactor types have been proposed including packed beds, fluidized and spouted beds, and air-lift reactors. A semicommercial process for beer used an air-lift reactor to achieve reaction times of 1 day compared with 5-7 days for the normal batch process. Unfortunately, the beer suffered from a mismatched flavour profile that was attributed to mass transfer limitations. 

In a biphasic medium, two situations are distinguished for the reaction. Biocatalysis occurs at the liquid-liquid interface [42,43] or in the bulk of the aqueous phase [25,27]. Models have been developed for both types, and interaction between mass transfer and enzyme-catalyzed reactions has been also studied. 

This system displays a two-enzyme kinetic model in which bioconversion is controlled by the interaction between the two reactions and the mass transfer. This situation offers a more realistic model for the conditions occurring in vivo, in which some pathways of intermediary metabolism consist of linear sequences of reactions. These pathways take place in highly organized compartments. 

Quantitative analytical treatments of the effects of mass transfer and reaction within a porous structure were apparently first carried out by Thiele (20) in the United States, Dam-kohler (21) in Germany, and Zeldovitch (22) in Russia, all working independently and reporting their results between 1937 and 1939. Since these early publications, a number of different research groups have extended and further developed the analysis. Of particular note are the efforts of Wheeler (23-24), Weisz (25-28), Wicke (29-32), and Aris (33-36). In recent years, several individuals have also extended the treatment to include enzymes immobilized in porous media or within permselective membranes. The important consequence of these analyses is the development of a technique that can be used to analyze quantitatively the factors that determine the effectiveness with which the surface area of a porous catalyst is used. For this purpose we define an effectiveness factor rj for a catalyst particle as... 

At low temperatures, the nonenzymatic reaction is reduced to a larger extent than the enzymatic reaction. The mass transfer rate is reduced to a smaller extent. Mass transfer limitation is required for high enantiomeric excess and determines the conversion rate. Therefore, the volumetric productivity decreases at lower temperatures. The equilibrium constant is considerably higher at low temperatures, resulting in a higher extent of conversion or a lower HCN requirement. Both the volumetric productivity and the required enzyme concentration increase by increasing the reaction temperature and aqueous-phase volume while meeting the required conversion and enantiomeric excess [44]. The influence of the reaction medium (solvent and water activity) is much more difficult to rationalize and predict [45],... 

Depending on the immobilization procedure the enzyme microenvironment can also be modified significantly and the biocatalyst properties such as selectivity, pH and temperature dependence may be altered for the better or the worse. Mass-transfer limitations should also be accounted for particularly when the increase in the local concentration of the reaction product can be harmful to the enzyme activity. For instance H2O2, the reaction product of the enzyme glucose oxidase, is able to deactivate it. Operationally, this problem can be overcome sometimes by co-immobilizing a second enzyme able to decompose such product (e.g. catalase to destroy H202). 

Nomenclature, 17 384-413 basic scheme of, 17 384-385 biochemical, 17 401-402 computerized approaches to, 17 400-401 elastomer, 21 761t enzyme, 10 258-260 for ionic liquids, 26 840-841 glossaries related to, 17 404 inorganic, 17 387-394 macromolecular (polymers), 17 403 404 organic, 17 394-401 polymer, 20 390-395 pump, 21 88 quinone, 21 236-237 reactor technology, 21 358 related to mass transfer, 15 731-737 reverse osmosis, 21 674-676 Society of Rheology, 21 704 spray-related, 23 199t systematic, 17 394... 

The expression for the effectiveness factor q in the case of zero-order kinetics, described by the Michaelis-Menten equation (Eq. 8) at high substrate concentration, can also be analytically solved. Two solutions were combined by Kobayashi et al. to give an approximate empirical expression for the effectiveness factor q [9]. A more detailed discussion on the effects of internal and external mass transfer resistance on the enzyme kinetics of a Michaelis-Menten type can be found elsewhere [10,11]. 

As can be concluded from this short description of the factors influencing the overall reaction rate in liquid-solid or gas-solid reactions, the structure of the stationary phase is of significant importance. In order to minimize the transport limitations, different types of supports were developed, which will be discussed in the next section. In addition, the amount of enzyme (operative ligand on the surface of solid phase) as well as its activity determine the reaction rate of an enzyme-catalyzed process. Thus, in the following sections we shall briefly describe different types of chromatographic supports, suited to provide both the high surface area required for high enzyme capacity and the lowest possible internal and external mass transfer resistances. 

Because enzymes are insoluble in organic solvent, mass-transfer limitations apply as with any heterogeneous catalyst. Water-soluble enzymes (which represent the majority of enzymes currently used in biocatalysis) have hydrophilic surfaces and so tend to form aggregates or stick to reaction vessel walls rather than form the fine dispersions that are required for optimum efficiency. This can be overcome by enzyme immobilization, as discussed in Section 1.5. 

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