Surface enzymic reaction

A Box-Behnken design was employed to investigate statistically the main and interactive effects of four process variables (reaction time, enzyme to substrate ratio, surfactant addition, and substrate pretreatment) on enzymatic conversion of waste office paper to sugars. A response surface model relating sugar yield to the four variables was developed on the basis of the experimental results. The model could be successfully used to identify the most efficient combination of the four variables for maximizing the extent of sugar production. 

The final method of coupling enzyme reactions to electrochemistry is to immobilize an enzyme directly at the electrode surface. Enzyme electrodes provide the advantages already discussed for immobilization of enzymes. In addition, the transport of enzyme product from the enzyme active site to the electrode surface is greatly enhanced when the enzyme is very near to the electrode. The concept of combining an enzyme reaction with an amperometric probe should offer all of the advantages discussed earlier for ion-selective (potentiometric) electrodes with a much higher sensitivity. In addition, since the response of amperometric electrodes is linear, background can be selected. 

Both platinum and an enzyme can be considered heterogeneous catalysts in the sense that the catalytic activity occurs on their surfaces or near them. Even so, some would classify an enzyme as a homogeneous catalyst. The most important difference, however, is one of specificity. Platinum is a rather nonspecific catalyst, catalyzing many different reactions. An enzyme, however, is quite specific, usually catalyzing only one reaction rather than all reactions of a given class. 

Conducting reactions in nanospace where the dimensions of the reaction vessel are comparable to those of the reactants provides a new tool that can be used to control the selectivity of chemical transformations.1 This dimensional aspect of nano-vessels has been referred to as shape selectivity.2 The effect of spatial confinement can potentially be exerted at all points on the reaction surface but its influence on three stationary points along the reaction coordinate (reactants, transition states, and products) deserve special attention.3,4 (1) Molecular sieving of the reactants, excluding substrates of the incorrect dimension from the reaction site can occur (reactant selectivity). (2) Enzyme-like size selection or shape stabilization of transition states can dramatically influence reaction pathways (transition state selectivity). (3) Finally, products can be selectively retained that are too large to be removed via the nano-vessel openings/pores (product selectivity). 

Fig. 20c. 1. ELISA assay, (a) Antibodies to the drug of interest are secured to a solid substratum such as a test tube or micro-well plate. The sample containing the analyte antigen is added to the reaction surface, (b) After the analyte has bound to the antibody, the vessel is rinsed to remove unbound antibody. A second antibody to the analyte is added. This antibody has a bound enzyme which has been chosen because its reaction produces a colored product which can be detected spectrophotometrically. (c) After this second antibody has bound to the first antibody-antigen complex, the surface is again rinsed to remove unbound-antibody enzyme. The enzyme substrate is added in sufficient excess such that the rate of product formed is proportional to the amount of enzyme present. The enzyme-linked assays are very sensitive, since each enzyme can rapidly catalyze thousands of substrate to product reactions.

Certain constituents when added to the reaction mixture, slow down the rate of reaction. This phenomena is called inhibition and constituent called inhibitor. Such an effect is similar to the negative catalysis. But the constituent usually undergoes chemical change, inhibition is the preferred term. Inhibition may occur in chain reactions, enzyme catalysed reactions, surface reactions or many reversible or irreversible reactions. A trace amount of an inhibitor may cause a marked decrease in the rate of reaction. The inhibitor sometimes combines with a catalyst and prevents it from catalyzing the reaction. 

Locating minima is not always straightforward since a reaction surface is usually complex, and a geometry optimization calculation will only locate minima close to the starting point. It is usually not feasible to systematically explore all possible conformers, so chemical intuition and corroborative evidence from experiment play important roles. A nice example is the identification of the coordination geometry of oxo-iron(IV) intermediate in TauD (22). As mentioned above, during optimization of enzyme active sites, key atoms are sometimes fixed to mimic the constraints that the protein environment exerts on the active site (20). 

Enzymes are structurally complex, highly specific catalysts each enzyme usually catalyzes only one type of reaction. The enzyme surface binds the interacting molecules, or substrates, so that they are favorably disposed to react with one another (fig. 1.15). The specificity of enzyme catalysis also has a selective effect, so that only one of several potential reactions takes place. For example, a simple amino acid can be used in the synthesis of any of the four major classes of macromolecules or can simply be secreted as waste product (fig. 1.16). The fate of the amino acid is determined as much by the presence of specific enzymes as by its reactive functional groups. 

For an ELISA enzymatic reaction occurring for 1 min, calculate the product concentration in the following two cases (given that the surface enzyme concentration is 1 fmol/cm2 and a turnover rate is 1000 substrate molecules per second) ... 

Figure 9.1. Response surface plot showing the effect of substrate molar ratio, reaction temperature, and their mutual interaction on biodiesel synthesis. Other synthesis parameters (reaction time, enzyme concentration, and added water amount) are constant at 0 levels.

Enzyme-substrate complexes have been studied by kinetic analysis, chemical modification, inhibition of enzymes by specific compounds that interact with active sites, detection of characteristic spectral absorption bands during reaction of enzymes with substrates, and X-ray crystallographic analysis of enzymes combined with compounds which are in similar structure to the natural substrates. The interaction between enzymes and substrates has been analyzed by the concepts of lock-and-key" and "induced fit". The former presumes that the substrate surface must fit the enzyme surface like a key in a lock, while the latter refined theory assumes that binding of the substrate induces ( informational changes in the enzyme to provide a better fit. 

The results of an EVB/FEP for proton transfer from Cys to His were presented by Warshel (1991), based on a calibration of the protein reaction surface with solution results, which amount to 6 kcal/mol difference (in favor of SH relative to ImH+) due to the pKa values of the two conjugate acids. The protein is found to invert the stabilization of covalent and ionic state relative to their order in solution. This is a result of the stronger solvation in the enzyme, compared to water, and due to the orientation of protein dipoles. 

Microsomal oxidative reactions constitute the most prominent phase I biotransformation pathway for a wide variety of structurally unrelated drugs (Table 1.4). Some drugs (e.g. amphetamine, diazepam, propranolol, lignocaine) simultaneously undergo more than one type of microsomal-mediated oxidative reaction. Microsomal enzymes are located primarily in liver cells, where they are associated with the smooth-surface (without ribosomes) endoplasmic reticulum (Fouts, 1961). Lipid solubility is a prerequisite for drug access to the... 

The electrons provided in the light reaction, however, may also be directly exported from the cells and used to reduce a variety of extracellular substrates. This electron export is effected by surface enzymes (called transplasmamembrane reductases) spanning the plasmamembrane from the inside surface to the outside. They transfer electrons from an internal electron donor [chiefly NADH and NADPH see Crane et al. (1985)] to an external electron acceptor. Direct reduction of extracellular compounds by transplasmamembrane electron transport proteins is prevalent in all cells thus far examined (Fig. 2.2). Although the function of this redox system is still subject to speculation, in phytoplankton it shows considerable activity, relative to other biochemical processes. A host of membrane-impermeable substrates, including ferricyanide, cytochrome c, and copper complexes, are reduced directly at the cells surface by electrons originating from within the cell. In phytoplankton, where the source of electrons is the light reactions of photosynthesis, the other half-redox reaction is the evolution of ()2 from H20. In heterotrophs, the electrons originate in the respiration of reduced substances. 

Apart from releasing photosynthetically reduced carbon and exporting elec-Irons, microbes also catalyze extracellular redox and hydrolysis reactions on I heir surfaces using external substrates. Of particular interest in our discussion is i class of surface enzymes in phytoplankton, deaminases, that catalyze the iclease of NII4 by oxidation of nitrogen containing compounds (Palcnik, 1989)... 

In some cases, mechanisms 2.1 and 2.3 are combined to yield a sequence of reactions where reductants excreted by the cell participate in redox reactions catalyzed by ectoenzymes (Fig. 2.3). Although no such reaction to our knowledge is yet characterized for aquatic microbes, in higher plants a cell surface enzyme that oxidizes malate to reduce extracellular NAD+ operates in this manner. 

A surface enzymic reaction presumes two simultaneous processes adsorption of the enzyme at the interface and the disappearance of the substrate from the interface (2). Therefore, it is essential to know at every moment the surface concentration of the substrate and of the enzyme. 

Compared to batch processes, continuous processes often show a higher space-time yield. Reaction conditions may be kept within certain limits more easily. For easier scale-up of some enzyme-catalyzed reactions, the Enzyme Membrane Reactor (EMR) has been developed. The principle is shown in Fig. 7-26 A. The difference in size between a biocatalyst and the reactants enables continuous homogeneous catalysis to be achieved while retaining the catalyst in the vessel. For this purpose, commercially available ultrafiltration membranes are used. When continuously operated, the EMR behaves as a continuous stirred tank reactor (CSTR) with complete backmixing. For large-scale membrane reactors, hollow-fiber membranes or stacked flat membranes are used 129. To prevent concentration polarization on the membrane, the reaction mixture is circulated along the membrane surface by a low-shear recirculation pump (Fig. 7-26 B). 

Enzymes are covalently immobilized primarily onto the surface of the membrane exposed to the feed solution, known as the "active side" of the asymmetric membrane. In general, it is not clear whether reaction between enzymes and polymeric membranes via coupling agents simply results in enzyme attachment to the membrane, or if it leads to an enzyme-carrier network inside the polymer matrix. For the sake of simplicity let us assume that asymmetric membranes are used, that suitable active groups are available on the polymeric surface and that the membrane molecular weight cut-off is such that the active layer is enzyme-impermeable. In this way, even though their activity is often drastically reduced, surface bound enzymes are in close proximity to the substrate solution-thus reducing the mass transfer resistance to that associated with the boundary layer. When enzymes are covalently immobilized in the... 

Despite the fact that catalysis is a kinetic phenomenon, there are quite many issues in catalysis which are not related to kinetics. Mechanisms of catalytic reactions, elementary reactions, surface reactivity, adsorption of reactants on the solid surfaces, synthesis and structure of solid materials, enzymes, or organometallic complexes, not to mention engineering aspects of catalysis are obviously outside the scope of chemical kinetics. 

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