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Immobilization of enzymes

It is also possible to trap an enzyme to the sensitive component of an electrode using a dialysis membrane that prevents protein diffusion. Guibault and Shu [23] constructed a urea-sensitive enzyme electrode by spreading a suspension of urease over the surface of an electrode equipped with a nylon net, and covering the whole assembly with a dialysis membrane the enzyme electrode is then rinsed and is ready for use. [Pg.24]

Physical entrapment is now rarely used because the presence of the enzyme in solution does not give good long-term enzymatic activity. Chemical immobilization, using covalent bonds, ensures a greater long-term enzymatic stability [24], and a number of examples of this will now be described. [Pg.24]

Cross-linking is a process that uses a bi- or multifunctional agent to form a bridge between different biocatalytic species or proteins. The process results in a considerable increase in molecular weight, and the compounds or aggregates formed are insoluble. [Pg.24]

It is possible to cross-link molecules of the same enzyme, or to coreticulate two or more different proteins (enzyme with enzyme, enzyme with protein, or more than one enzyme with a load protein such as BSA, bovine serum albumin). Even organelles or entire cells can be coreticulated. Coreticulation is a very important process because it makes multienzymatic immobilization accessible. The use of a load protein like albumin improves enzymatic activity because of the better mass distribution of the various proteins, but it does not alter the [Pg.24]

It is also possible to use other bifunctional agents such as hexamethylene diisocyanate  [Pg.25]

The obstacles for the use of enzymes in electrocatalysis are their instability and the impossibility of multiple application of an enzyme which is in a homogeneous phase. Overcoming these difficulties is possible through the utilization of so-called immobilized enzymes, i.e., enzymes bound with the carrier. The most promising methods of immobilization— immobilization by way of adsorption on carriers inclusion into the space lattice of gels chemical methods of immobilization — for use in bioelectrocatalysis will be presented in the following subsections. [Pg.246]

The adsorption method is the simplest one and is often used in bioelectrocatalysis research. Essentially it involves the incubation of protein in the carrier suspension with the subsequent washing of the nonadsorbed protein. Adsorption of proteins on different types of surfaces is effected due to electrostatic, hydrophobic, and dispersion interactions. The most popular carriers are carbon, soot, clays, aluminum oxide, silica gel, and glass. The optimal inert carrier is glass. It has recently been shown that porous glass with calibrated pore size can be used for immobilization of enzymes by adsorption. An interesting method of immobilization by adsorption has been proposed in which lipid is first adsorbed on carbon or silica gel and then the enzyme is adsorbed on the so-called soft surface of the lipid. [Pg.246]

By definition, enzyme immobilization is the conversion of an enzyme to a form with artificially restricted mobility and retention of catalytic function W. This restricted mobility allows for containment and recovery of the enzyme and is often achieved by either conversion to an insoluble form (for example by linking to insoluble particles) or by containment within a semi-permeable barrier (for example entrapment within an ultrafiltration membrane). In the course of this immobilization, enzymes can acquire four advantageous properties  [Pg.163]

There are, however, a number of practical limitations on the utility of immobilized enzymes. First, the yield of protein binding is rarely quantitative. Second, in many cases, the cost of the carrier can be quite significant and may even exceed the cost of the enzyme itself. Third, the activity of the resulting immobilized enzyme is usually reduced because of chemical modification of the protein, steric hindrance and mass transfer limitations. Finally, the proportion of active enzyme to the carrier material in immobilized enzyme preparations rarely exceeds 5-10% w/w, and thus dramatically reduces catalytic activity per weight of solid. [Pg.164]

Despite the limitations, the great success of enzyme immobilization in diagnostics, pharmaceutical, food and chemical industries is undeniable12, 3. The decision whether one should use a soluble enzyme preparation or an immobilized enzyme does not have a universal solution and can be decided only on a case by case basis. Ordinarily, if the cost of an enzyme represents a significant portion of the overall cost or if isolation of the final product is complicated by the presence of the soluble protein, the cost of immobilization can be offset by the gains in productivity and improved product quality. The intent of this section is to describe, in general terms with illustrative examples, the features and considerations of these broad classes of enzyme immobilization as they impact their application to biocatalysis. Detailed experimental protocols are available in the original literature and exemplary protocols for these methods are offered in many excellent reviews and texts 14L [Pg.164]

Thousands of publications and patents detail the immobilization of specific enzymes using an impressive array of strategies. The majority of these immobilization techniques can be divided into four broadly defined groups  [Pg.164]

The first three classes involve the use of a solid matrix to support or entrap the enzyme and to confer the desirable mechanical properties of the solid carrier (Fig. 6-1 A-D). The last method entails covalent linking of the enzyme to itself with no additional support (Fig. 6-1 E). Each of the covalent methods requires one or more covalent bonds between reactive groups on the enzyme surface with complementary groups on the carrier, either directly or through the action of a multivalent cross-linking reagent. Covalent attachment methods result in direct chemical modification [Pg.164]


The assumption of the association of Hb in the pores of carboxylic cation exchangers has been advanced in Ref. [47] on the basis of electron microscopy at the maximum filling, almost all the pore surface is filled with Hb associates which are ordered star-shaped structures. Interprotein interaction in the adsorption immobilization of enzymes have been reported in Refs. [74, 75]. [Pg.26]

The immobilization of enzymes with the formation of insoluble forms is usually intended for the development of specific catalysts for technical purposes. Here, we consider another medico-biological problem of the preparation of insoluble enzymatic systems based on crosslinked polyelectrolytes, used in the replacement therapy for oral administration. [Pg.34]

The next two examples illustrate more complex surface reaction chemistry that brings about the covalent immobilization of bioactive species such as enzymes and catecholamines. Poly [bis (phenoxy)-phosphazene] (compound 1 ) can be used to coat particles of porous alumina with a high-surface-area film of the polymer (23). A scanning electron micrograph of the surface of a coated particle is shown in Fig. 3. The polymer surface is then nitrated and the arylnitro groups reduced to arylamino units. These then provided reactive sites for the immobilization of enzymes, as shown in Scheme III. [Pg.170]

For most applications, enzymes are purified after isolation from various types of organisms and microorganisms. Unfortunately, for process application, they are then usually quite unstable and highly sensitive to reaction conditions, which results in their short operational hfetimes. Moreover, while used in chemical transformations performed in water, most enzymes operate under homogeneous catalysis conditions and, as a rule, cannot be recovered in the active form from reaction mixtures for reuse. A common approach to overcome these limitations is based on immobilization of enzymes on solid supports. As a result of such an operation, heterogeneous biocatalysts, both for the aqueous and nonaqueous procedures, are obtained. [Pg.100]

The importance of proper immobilization of enzymes can be shown in the kinetic resolution of racemic a-acetoxyamides. This group of compounds is an important class of chemicals since they can be readily transformed into a-amino acids [17], N-methylated amino acids, and tripeptide mimetics [18], amino alcohols [19], 1,2-diols [20], 1,2-diamines [21], and enantiopure l,4-dihydro-4-phenyl isoquinolinones [22]. [Pg.100]

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. [Pg.31]

Polyamines and their ammonium salts have been of interest because they are known to have potential applications as chelating agents (1-3), ion exchange resins (4-6), flocculants (7,8), and other industrial uses (9). Recent biomedical applications have constituted another important use of polymeric amines they have been investigated for use as biocompatable materials, polymeric drugs, immobilization of enzymes, cell-culture substratum and cancer chemotherapeutic agents (10-12). [Pg.127]

Table 6-1. Immobilization of enzymes and affinity ligands on polysaccharide matrices with CD1. Table 6-1. Immobilization of enzymes and affinity ligands on polysaccharide matrices with CD1.
The three different enzymes used in combination in this system were FateDH, FaldDH, andADH. FateDH catalyzes the initial reduction of C02 to formate, FaldDH the reduction of formate to formaldehyde, and ADH the reduction of formaldehyde to methanol. Interestingly, the enzymes when immobilized were more active than a combination of the free enzymes, which is claimed to be due to a reduction of spatial interference among the different enzymes. Moreover, due to the immobilization of enzymes within the microreactor, the intermediate species have significantly reduced distances to travel between active sites [21, 22]. [Pg.141]

Shchipunov, Yu.A., Karpenko, T.Yu., Bakunina, I.Yu., Burtseva, Yu.V. and Zvyagintseva, T.N. (2004) A new precursor for the immobilization of enzymes inside sol-gel derived hybrid silica nanocomposites containing polysaccharides. Journal of Biochemical and Biophysical Methods, 58, 25-38. [Pg.106]

Fan,., Lei,., Wang, L Yu, C Tu, B. and Zhao, D. (2003) Rapid and high-capacity immobilization of enzymes based on mesoporous silicas with controlled morphologies. Chemical Communications, 2140-2141. [Pg.266]

In order to overcome some limitations of the adsorption process due to surface accessibility or diffusional hindering, immobilization of enzymes by direct in situ encapsulation has been investigated. When inorganic supports can be prepared in mild conditions compatible with the enzyme stability, then such processes allow... [Pg.449]

Clay minerals or phyllosilicates are lamellar natural and synthetic materials with high surface area, cation exchange and swelling properties, exfoliation ability, variable surface charge density and hydrophobic/hydrophilic character [85], They are good host structures for intercalation or adsorption of organic molecules and macromolecules, particularly proteins. On the basis of the natural adsorption of proteins by clay minerals and various clay complexes that occurs in soils, many authors have investigated the use of clay and clay-derived materials as matrices for the immobilization of enzymes, either for environmental chemistry purpose or in the chemical and material industries. [Pg.454]

Immobilization of Enzymes in 3-D Inorganic Hosts 15.4.2.1 Immobilization in Si02... [Pg.464]

The immobilization of enzymes for sensing purposes frequently provides several important advantages an increase of its stability, operational reusability and greater efficiency in consecutive multistep reactions. Sometimes immobilization is accompanied by a certain degree of denaturalization however, the enzyme-matrix interactions may assist in stabilization preventing conformational transitions that favor such process. In some cases excessive bond formation affects the conformation of the active site and the steric hindrances caused by the polymer matrix may render an inactive sensor. [Pg.338]

Hearn, M.T.W. (1987) l,l -Carbonyldiimidazole-mediated immobilization of enzymes and affinity ligands. In Methods in Enzymology, (K. Mosbach, ed.), Vol. 135, pp. 102-117. Academic Press, Orlando, FL. [Pg.1072]

S. Andreescu, V. Magearu, A. Lougarre, D. Fournier, and J.L. Marty, Immobilization of enzymes on screen-printed sensors via a histidine tail. Application to the detection of pesticides using modified cholinesterase. Anal. Lett. 34, 529-540 (2001). [Pg.73]

Vertically aligned CNT-modified electrodes are based on a more elaborated technique than other methods, and microscopic images are used to characterize the integrity of this type of electrode. The technique has been applied for the immobilization of enzymes and DNA, and the sensors based on this technique have shown a lower detection limit than those based on other methods. More research activities using this technique, particularly with low density CNT arrays, are expected in the near future because of its sensitivity and versatility. [Pg.516]

Y. Kurokawa, T. Sano, H. Ohta, and Y. Nakagawa, Immobilization of enzyme onto cellulose-titanium oxide composite fiber. Biotech. Bioenerg. 42, 394—397 (1993). [Pg.551]

G. Fortier, R. Beliveau, E. Leblond, and D. Belanger, Development of biosensors based on immobilization of enzymes in Eastman AQ polymer coated with a layer of Nafion. Anal. Lett. 23, 1607-1619 (1990). [Pg.593]

C.X. Lei, H. Wang, G.L. Shen, and R.Q. Yu, Immobilization of enzymes on the nano-Au film modified glassy carbon electrode for the determination of hydrogen peroxide and glucose. Electroanalysis 16, 736-740 (2004). [Pg.601]


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Activity of immobilized enzymes

Analytical Applications of Immobilized Enzymes

Edman degradation immobilization of enzymes

Effects of Diffusion within Immobilized Enzyme Particles

Enzyme immobilization

Immobilization of Enzymes Cross-linked Enzyme Aggregates (CLEAs)

Immobilization of Enzymes in 2-D Inorganic Hosts

Immobilization of the enzymes using non-conventional media

Immobilization, of redox enzymes

Immobilized enzymes

Industrial applications of immobilized enzymes

Kinetic of immobilized enzymes

Kinetics of Immobilized Enzymes

Kinetics of Soluble and Immobilized Enzymes

Modification and Immobilization of Proteins (Enzymes)

Preparation of immobilized enzyme

Probing the Distribution of Immobilized Enzyme Within Hierarchical Structures

Properties and application of immobilized enzyme

Properties of Immobilized Enzymes

Stability of immobilized enzymes

Structure and Catalytic Behavior of Immobilized Enzymes

The catalytic properties of immobilized enzyme

Use of immobilized enzymes

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