Enzymes confinement

Dunker, A.K. and Fernandez, A., Engineering productive enzyme confinement. Trends Biotechnol., 2007, 25, 189-190. 

For DMDBT oxidation, CPO physically immobilized on SBA-16 of 90 A had a higher thermostability than the free enzyme, retaining 50% of its activity at 45°C after 187 h while the free enzyme was half-inactivated after 68 h. This could be due to the restricted movement of the immobilized enzyme confined in the pores of this material. In contrast, the immobilization in material with a larger pore of 117 A did not improve the thermostability of the enzyme, probably due to the fact that larger pores did not prevent the increased conformational flexibility of the enzyme at this temperature. 

Consider that initially uncharged species M and O move into the cell, where M is reacted and transformed into N. Some of the N flows out of the cell. The transformation is mediated by the action of an enzyme confined to the interior of the cell. The component O does not take part in the transformation. However, the flow of O is coupled with the flow of M. The linear phenomenological equations are... 

Figure 21.23. Glycogen-Engorged Lysosome. This electron micrograph shows skeletal muscle from an infant with type II glycogen-storage disease (Pompe disease). The lysosomes are filled with glycogen because of a deficiency in a-1,4-glucosidase, a hydrolytic enzyme confined to lysosomes. The amount of glycogen in the cytosol is normal. [From H.-G. Hers and F. Van Hoof, Eds. Lysosomes and Storage Diseases (Academic Press, 1973), p. 205.]...

Most of the above problems could be overcome using "immobilized enzymes", or in other words "enzymes confined or compartmentalized in a well defined region of space, which retain their catalytic properties and can be repeatedly and continuously used".3 Several advantages can derive from the use of immobilized enzymes or microbial cells, such as ... 

It has been demonstrated by several groups that the catalytic activity of enzymes confined in microemulsion droplets varies with overall water content. For a variety of enzymes it has been found that there is a bell-shaped dependence of activity on IVq [31-35]. Figure 4 shows a representative example. 

Immobilized enzyme systems, (a) Enzyme non-covalently adsorbed to an insoluble particle (b) enzyme covalently attached to an insoluble particle (c) enzyme entrapped within an insoluble particle by a cross-linked polymer [Pg.10]

In the experiments summarized in Fig. 1, the thinnest reaction layer, which corresponds to the largest plateau current, is of the order of 20 gm, that is, ca 2000 times the size of the enzyme, indicating that such experiments sense enzymes dispersed in the solution rather than a layer of enzyme confined onto the electrode surface. The peak current increases with the scan rate up to a situation where the plateau current becomes negligible (under these conditions, the current response is governed solely by diffusion, the enzymatic reaction being too slow to compete). 

Johnson knew that the total and specific activities of maltase, sucrase, lactase, trehalase, palatinase, and peroxidase decrease in the intestines of parenterally fed rats. To determine the effect of pentagastrin upon the enzymes, Johnson measured the specific activities of the two brush border enzymes, maltase and sucrase, and of peroxidase, an enzyme confined to the lamina propria. He found that pentagastrin addition to intravenous feeding prevented the decline in the brush border enzymes but not in the lamina propria enzyme. This, as well as the effect of pentagastrin on oxyntic mucosal weight, Johnson concluded, is the result of the trophic action of gastrin, not of its ability to stimulate secretion, for histamine in all instances failed to have the effect of pentagastrin. " ... 

FIGURE 14.11 The pH activity profiles of four different enzymes. Trypsin, an intestinal protease, has a slightly alkaline pH optimnm, whereas pepsin, a gastric protease, acts in the acidic confines of the stomach and has a pH optimmn near 2. Papain, a protease found in papaya, is relatively insensitive to pHs between 4 and 8. Cholinesterase activity is pH-sensitive below pH 7 but not between pH 7 and 10. The cholinesterase pH activity profile suggests that an ionizable group with a pK near 6 is essential to its activity. Might it be a histidine residue within the active site ... 

Uncovering of the three dimentional structure of catalytic groups at the active site of an enzyme allows to theorize the catalytic mechanism, and the theory accelerates the designing of model systems. Examples of such enzymes are zinc ion containing carboxypeptidase A 1-5) and carbonic anhydrase6-11. There are many other zinc enzymes with a variety of catalytic functions. For example, alcohol dehydrogenase is also a zinc enzyme and the subject of intensive model studies. However, the topics of this review will be confined to the model studies of the former hydrolytic metallo-enzymes. 

So far the economic feasibility of co-enzyme dependent biocatalyses is confined to relatively small market niches comprising products with high added value. 

Free Enzymes in Flow Reactors. Substitute t = zju into the DDEs of Example 12.5. They then apply to a steady-state PFR that is fed with freely suspended, pristine enzyme. There is an initial distance down the reactor before the quasisteady equilibrium is achieved between S in solution and S that is adsorbed on the enzyme. Under normal operating conditions, this distance will be short. Except for the loss of catalyst at the end of the reactor, the PFR will behave identically to the confined-enzyme case of Example 12.4. Unusual behavior will occur if kfis small or if the substrate is very dilute so Sj Ej . Then, the full equations in Example 12.5 should be (numerically) integrated. 

The case for a CSTR is similar. Under normal operating conditions, the solution in Example 12.3 will apply to free enzymes as well as to confined enzymes. Like the PFR case, unusual behavior will occur if kfis small or if the substrate is very dilute so Sj Ej . 

Waals interactions with the PI Cys, and also located basic Arg and Lys residues in position to interact with the P6 acidic residue. More detail of the binding of the N-terminal (i.e., P residues) part of the substrate was subsequently inferred from NMR studies showing a p-sheet interaction between this N-terminal segment of the substrate and enzyme largely confined to within the C-terminal p-barrel (Cicero et al. 1999). 

The effects of the intramicellar confinement of polar and amphiphilic species in nanoscopic domains dispersed in an apolar solvent on their physicochemical properties (electronic structure, density, dielectric constant, phase diagram, reactivity, etc.) have received considerable attention [51,52]. hi particular, the properties of water confined in reversed micelles have been widely investigated, since it simulates water hydrating enzymes or encapsulated in biological environments [13,23,53-59]. 

Figure 48-12. Schematic illustration of some aspects of the role of the osteoclast in bone resorption. Lysosomal enzymes and hydrogen ions are released into the confined microenvironment created by the attachment between bone matrix and the peripheral clear zone of the osteoclast. The acidification of this confined space facilitates the dissolution of calcium phosphate from bone and is the optimal pH for the activity of lysosomal hydrolases. Bone matrix is thus removed, and the products of bone resorption are taken up into the cytoplasm of the osteoclast, probably digested further, and transferred into capillaries. The chemical equation shown in the figure refers to the action of carbonic anhydrase II, described in the text. (Reproduced, with permission, from Jun-queira LC, Carneiro J BasicHistology. Text Atlas, 10th ed. McGraw-Hill, 2003.)...

Many enzyme activities have been proposed for diagnosis, in serum, urine, cerebrospinal fluid and other body fluids, and the proposed methods have been reviewed elsewhere (17). Here we will confine ourselves to the tried and most commonly used enzyme activities which yield useful information in ambulatory or hospital patients. 

This gene is broadly distributed in skeletal muscle, heart, uterus, and in a variety of non-muscle cells. The mRNA levels are particularly high in intestine, lung and spleen, whereas they are very low in liver, testes, kidney and pancreas. In the muscle tissue SERCA3 may be confined primarily to non-muscle cells (vascular smooth muscle, endothelial cells, etc.). The C-terminus of SERCA3 is Asp-Gly-Lys Lys-Asp-Leu-Lys (Table I) it may serve as a sorting signal for retention of the enzyme in the endoplasmic reticulum [57]. 

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 entrapment method is based on confining the enzyme within the lattice of a polymeric matrix. Polyacrylamide gels have successfully yielded stable enzyme films with a high retention of activity... 

Enzymes are efficient catalysts for cathodic and anodic reactions relevant to fuel cell electrocatalysis in terms of overpotential, active site activity, and substrate/reaction specificity. This means that design constraints (e.g., fuel containment and anode-cathode separation) are relaxed, and very simple devices that may take up ambient fuel or oxidant from their environment are possible. While operation is generally confined to conditions close to ambient temperature, pressure, and pH, and power densities over about 10 mW cm are rarely achieved, enzyme fuel cells may be particularly useM in niche environments, for example scavenging trace H2 released into air, or sugar and O2 from blood. Thus, trace or unusual fuels become viable for energy production. 

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