Liquid phase reactions enzymes

For practical purposes it is often beneficial to use a heterogeneous system with the enzyme as a solid preparation which easily can be separated from the product in the liquid phase. Solid enzyme preparatiorrs can conveniently be used in packed bed and stirred tank reactors. As in other cases with heterogeneous catalysis, mass trarrsfer limitations can reduce the overall reaction rate, but usually this is no major problem. 

Enzymes are proteins that catalyze reactions. Thousands of enzymes have been classified and there is no clear limit as to the number that exists in nature or that can be created artificially. Enzymes have one or more catalytic sites that are similar in principle to the active sites on a solid catalyst that are discussed in Chapter 10, but there are major differences in the nature of the sites and in the nature of the reactions they catalyze. Mass transport to the active site of an enzyme is usually done in the liquid phase. Reaction rates in moles per volume per time are several orders of magnitude lower than rates typical of solid-catalyzed gas reactions. Optimal temperatures for enzymatic reactions span the range typical of living organisms, from about 4°C for cold-water fish, to about 40°C for birds and mammals, to over 100°C for thermophilic bacteria. Enzymatic reactions require very specific molecular orientations before they can proceed. As compensation for the lower reaction rates, enzymatic reactions are highly selective. They often require specific stereoisomers as the reactant (termed the substrate in the jargon of biochemistry) and can generate stereospecific products. Enzymes are subject to inhibition and deactivation like other forms of catalysis. 

To put it simple, the enzyme reactor is an agitated tank reactor in which the substrate is placed and stirred together with the enzyme for a certain period of time at a certain temperature in the liquid phase. The enzyme is added preferably in a concentration of 0.1 to 1% and is lost after completion of the reaction. 

In this chapter we provide a review of variational transition state theory with a focus on how quantum mechanical effects are incorporated. We use illustrative examples of H-transfer reactions to assist in the presentation of the concepts and to highlight special considerations or procedures required in different cases. The examples span the range from simple gas-phase hydrogen atom transfer reactions (triatomic to polyatomic systems), to solid-state and liquid-phase reactions, including complex reactions in biomolecular enzyme systems. 

In practice, we approximate the exact transmission coefficient by a mean-field-type of approximation that is we replace the ratio of averages by the ratio for an average or effective potential. For gas-phase reactions with small reaction-path curvature, this effective potential would just be the vibrationally adiabatic ground-state potential. In the liquid phase and enzymes we generalize this with the canonical mean-shape approximation. In any event, though, the transmission coefficient should not be thought of as a perturbation. The method used here may be thought of as an approximate full-dimensional quantum treatment of the reaction rate. 

Microreactors can be used for either gas-phase or liquid-phase reactions, whether catalyzed or uncatalyzed. Heterogeneous catalysts (or immobilized enzymes) can be coated onto the channel wall, although on occasion the metal wall itself can act as the catalyst. Gas-liquid contacting can be effected in the microchannels by either bubbly or slug flow of gas, an annular flow of liquid, or falling liquid films along the vertical channel walls. Contact between two immiscible liquids is also possible. The use of microreactor systems in the area of biotechnology shows much promise, not only for analytical purposes but also for small-scale production systems. 

Several homogeneous gas- and liquid-phase reactions are now also known to exhibit self oscillations and it is clear that many living organisms depend on coupled oscillatory reactions catalysed by enzymes to control biological functions.However, only heterogeneous oxidation reactions catalysed by noble metals are reviewed here. Experimental studies are first described, followed by a discussion of kinetic analyses which have been put forward to account for them. Particular attention is given to the most extensively studied system to date, the oxidation of CO over Pt catalysts. 

Most of the gas-liquid applications of monoliths have used a heterogeneous catalyst (be it supported noble metals or immobihzed enzymes) on the channel walls. Here, we also consider the use of monohths without a catalyst on the walls in gas-liquid applications, i.e. homogeneously catalyzed liquid-phase reactions. The fluid mechanics of the system do not change appreciably by lethng the reaction take place in the liquid bulk instead of in a washcoat layer, and it is interesting to consider such reactions in a discussion of mass transfer and power-input requirement. Of course, the mass-transfer behavior does change by changing the locahon where the reaction takes place, and we will discuss gas-hquid reactors and gas-liquid-solid reactors separately. 

In previous sections, different simplifications and approximations have been introduced using first-order reactions involving linear models as examples. In many real situations, kinetic models corresponding to different physicochemical systems are nonlinear. As mentioned earlier, two typical scenarios where the QSS approximation can be used are gas- or liquid-phase reactions with free radicals as intermediates and catalytic or biocatalytic reactions involving catalytic surface intermediates or substrate-enzyme complexes. Within the traditional mathematical procedure for dealing with these intermediates, three steps can be distinguished ... 

Homogeneous Many gas-phase reactions. Some liquid phase reactions, such as neutralisation of acid and alkalis Many liquid-phase reactions, such as those catalysed by acid or alkalis. Some enzyme reactions... 

The study of enzymes is important because every syndietic and degradation reaction in all living cells is controlled and catalyzed by specific enzymes. Many of these reactions are the soluble enzyme-soluble substrate type and are homogeneous in the liquid phase. 

The predominant activity in the study of enzymes has been in relation to biological reactions. This is because specific enzymes have bodi controlled and catalyzed syndietic and degradation reactions in all living cells. Many of diese reactions are homogeneous in the liquid phase (i.e., type 3 reactions). 

It is not unusual for the full chemical potential of a reaction to be diminished by slower transport processes (i.e., to be transport limited). In fast liquid phase enzyme reactions, mechanical stirring rates can have a strong influence on the observed kinetics that may be limited by the rate of contacting of the reactants and enzymes. Most heterogeneous catalytic reactions take... 

Enhanced thermal stability enlarges the areas of application of protein films. In particular it might be possible to improve the yield of reactors in biotechnological processes based on enzymatic catalysis, by increasing the temperature of the reaction and using enzymes deposited by the LB technique. Nevertheless, a major technical difficulty is that enzyme films must be deposited on suitable supports, such as small spheres, in order to increase the number of enzyme molecules involved in the process, thus providing a better performance of the reactor. An increased surface-to-volume ratio in the case of spheres will increase the number of enzyme molecules in a fixed reactor volume. Moreover, since the major part of known enzymatic reactions is carried out in liquid phase, protein molecules must be attached chemically to the sphere surface in order to prevent their detachment during operation. 

TS-1-catalyzed processes are advantageous from the environmental point of view as the oxidant is aqueous hydrogen peroxide, which turns into water, and the reactions are operated in liquid phase under mild conditions, showing very high selectivity and yields, thus reducing problems and the costs of by-product treatments. Confinement of the metal species in the well-defined MFl pore system endows TS-1 with shape selectivity properties analogous to enzymes. For these features the application of the terms mineral enzyme or zeozyme to TS-1 is appropriate [42]. 

The volumetric ratio of the two liquid phases (j6 = Forg/ Faq) can affect the efficiency of substrate conversion in biphasic media. The biocatalyst stability and the reaction equilibrium shift are dependent on the volume ratio of the two phases [29]. In our previous work [37], we studied the importance of the nonpolar phase in a biphasic system (octane-buffer pH 9) by varying the volume of solvent. The ratio /I = 2/10 has been the most appropriate for an improvement of the yield of the two-enzyme (lipase-lipoxygenase) system. We found that a larger volume of organic phase decreases the total yield of conversion. Nevertheless, Antonini et al. [61] affirmed that changes in the ratios of phases in water-organic two-phase system have little effect upon biotransformation rate. 

Thermal reaction techniques enable a quantification of the influence of solvation on reactivities.1,2,19 One particular reaction which is a good example of how solvation can affect the nature of a core ion reaction site comes from a study118 of the interaction of OH with C02. The gas-phase reaction between the individual species is quite exothermic and can only take place by a three-body association mechanism. The reaction proceeds very slowly in the liquid phase and has been calculated119 to have a barrier of about 13 kcal mol-1. In biological systems, the reaction rate is enhanced by about 4 orders of magnitude through the enzyme carbonic anhydrase. Recent studies carried out in our laboratory provide detailed... 

In the past, general chapters and reviews have been published, related to the characteristics of CL as analytical technique [7-9], mainly in the liquid phase [10-14], and its use as detection mode in flowing streams and immunoassay [15-17]. Two extensive reviews reported on the specific application of CL reactions according to the nature of the analyte (inorganic species, enzymes and nucleotides, acids and amines, carbohydrates, steroids, polycyclic aromatic compounds, and drugs) and covering the literature from 1983 to 1991 [18] and from 1991 to mid-1995 [19]. 

Chromatographic fixed-bed reactors consists of a single chromatographic column containing a solid phase on which adsorption and reaction take place. Normally a pulse of reactant is injected into the reactor and, while traveling through the reactor, simultaneous conversion and separation take place (Fig. 3). Since an extensive overview of the models and applications of this type of reactor was presented by Sardin et al. [ 132], only a few recent results will be discussed here. Most of the practical applications have been based on gas-liquid systems, which are not applicable for the enzyme reactions, but a few reactions were also reported in the liquid phase. One of these studies, performed by Mazzotti and co-workers [ 141 ], analyzed the esterification of acetic acid into ethyl acetate according to the reaction ... 

In this chapter chromatographic bioreactors are considered as chromatographic reactors where the reaction is catalyzed by an enzyme or enzyme system, which can be present in pure form or as a cell component. The enzyme can be immobilized on the matrix or it can be dissolved in a liquid phase. Therefore, the reaction can take place in either phase. Several different bioreactions were performed in chromatographic reactors of different types. In the following part some pertinent examples are presented according to their type of reaction. 

Fig. 12. Outlet concentration profiles from a batch chromatographic bioreactor for enzyme catalyzed esterification. Water, which when in the liquid phase irreversibly inhibits the reaction, is adsorbed. The profiles of water (open circle), propionic acid (filled square), isoamyl alcohol (filled triangle) and isoamyl propionate (open square) at the reactor outlet are presented. (Reprinted with permission from [178])...

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