Immobilized, enzymes

Enzymes in solution are usually used only once. The repeated use of enzymes fixed to a carrier is more economical. The use of enzymes in 

The catalytic properties of an immobilized enzyme can be characterized using the Michaelis-Menten model. The exact form of the model will depend on the type of enzyme reactor used. In general, whenever non-steady-state conditions prevail, the integrated form of the Michaelis-Menten model is used  

The three main types of immobilized enzyme reactors used are batch (Fig. 9.1), plug-flow (Fig. 9.2), and continuous-stirred (Fig. 9.3). In both batch and plug-flow reactors, non-steady-state reaction conditions prevail, while in continuous-stirred reactors, steady-state reaction conditions are prevalent. 

Of all the FIA methods using packed reactors, systems with immobilized enzymes are far the most common (Table 7.1). They offer the selectivity of enzymatic reactions and the economy gained by immobilizing the often costly catalysts. To illustrate the versatility of this technique selected examples of published applications comprising use of immobilized enzyme reactors are given below. 

Petersson et al. [1160] recently described a miniaturized flow-injection system for the determination of four different substrates, which each could be degraded enzymatically by means of appropriate oxidases according 

More elaborate systems incorporating a series of reactors have been described by a Swedish group in Lund, which has also conducted a number of theoretical studies on optimization of the design of packed reactors in FIA systems [344, 345]. Thus, Olsson et al. [1067] have devised a procedure for determination of sucrose based on the following sequence of reactions  

Using the manifold shown in Fig. 4.49, the injected sample of sucrose is first directed to a packed reactor (MGC) containing coimmobilized mu-tarotase, glucose oxidase, and catalase, which decomposes any glucose already present in the sample, and then on to a second reactor (IMG) containing coimmobilized invertase, mutarotase, and glucose oxidase in which the sucrose via the reactions described above is degraded to hy- 

The same Swedish group has designed a FIA system containing even more packed reactors and, indeed, a dialysis unit [758]. Aimed at the determination for galactose, the manifold for this system is shown in Fig. 4.50. Again, the final measurement is based on determination of hydrogen peroxide generated by an enzymatic reaction of an oxidase  

In comparing the chemical and enzymatic routes, the former route comprises three reaction steps with dirty and expensive chemicals/organic solvents under relatively extreme conditions. The enzymatic route requires only one reaction step under mild conditions the enzyme is immobilized (operating lifetime 1000 h productivity up to 2000 kg 6-APA per kg immobilized enzyme). In this case, the biocatalytic route has largely replaced the chemical route [169,170], 

In Japan, continuous production processes using immobilized living cells are currently introduced in such classical areas as beer (Kirin Brewery Co.) and sake brewing (Ohzeki, Co. Ltd), vinegar production (Kewpie Jyozo, Co. Ltd), and in the production of soy sauce (Kik-koman Co.). 

The other subunit has also been converted into the square form, which can now more readily accept a second substrate molecule. In other words, the equilibrium constant for 

Theories of interacting subunits are often referred to as allosteric theories, but the use of this word is unfortunate and should be avoided. The word allostery (Greek alios, other stereos, solid) refers to the possibility that substances (known as modifiers or effectors) can be attached at sites other than the site for the attachment of substrate. This is a completely different type of phenomenon and is of great importance in connection with the regulation of metabolic processes, but should be sharply distinguished from subunit interactions. However, in some enzymes the two effects are found together. 

In biological systems certain enzymes occur in free solution. Examples are the proteolytic enzymes, which bring about hydrolysis in the digestive system. Other enzymes, however, are attached to structural material in the living system and do not have the mobility they would have if they were in free solution. Such enzymes are said to be immobilized, or supported. 

The kinetic aspects of immobilized enzymes are rather complicated. A typical situation is when the enzyme is immobilized within some polymeric material, which may be cut into slices and immersed in a suitably buffered solution of the substrate. This is the type of situation that occurs in a biological system, an example being a muscle (in which the enzyme myosin is immobilized) surrounded by a solution of the substrate ATP. For reaction to occur, the substrate has to diffuse through the polymeric material in order to reach the enzyme. Reaction then occurs and the products must diffuse out into the free solution. Since diffusion in polymeric materia occurs more slowly than in water, there is now a greater possibility of diffusion control (see p. 403) the overall rate of reaction may depend to some extent on the rates with which these diffusion processes occur. 

The kinetic investigations so far carried out on immobilized enzymes have indeed provided evidence for various degrees of diffusion control. Enzymes that obey the Michaelis-Menten equation (equation (10.25)) when they are in free solution generally obey it to a good approximation when they are immobilized, but the Michaelis constant is usually significantly different. The rate equation for the immobilized system is 

Immobilization by adsorption onto surfaces such as activated carbon or an ion exchange resin gives a reversible and relatively weak bond, but this can be sufficient to increase the retention time in a flow system to acceptable levels. Recall Section 

where it was shown that the residence time of an adsorbed species can be much larger than that of the mobile phase, in essence giving more time for catalysis. 

Immobilization by chemical bonding gives strong, irreversible attachments to a solid support. The bonds are normally covalent but can be electrostatic. Typical supports are functionalized glass and ceramic beads and fibers. Enzymes are sometimes crosslinked to form a gel. Occasionally, enzymes can be flocculated while retaining catalytic activity. 

All these immobilization techniques run the risk of altering activity compared to the native enzyme. Improved activity is occasionally reported, but this is the exception. The immobilization techniques fisted above are in approximate order of loss in activity. Physical entrapment normally causes no change. Adsorption will distort the shape of the molecule compared to the native state. The effect of covalent bonding depends on the location of the bond relative to an active site. If remote from the site, it may have no effect. The chemical nature of the support can affect activity. Crosslinking requires two covalent attachments per enzyme molecule and is thus likely to distort the shape of the enzyme to the point that catalytic activity is lost. Such distortions are even more likely, but not inevitable, for coagulated or flocculated enzymes. On the positive side, immobilization tends to stabilize enzymes against deactivation. 

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  

All these immobilization techniques run the risk of altering activity compared with the native enzyme. Improved activity is occasionally reported, but this is the exception. The immobilization techniques listed above are in approximate order of loss in activity. Physical entrapment normally causes no change. Adsorption will distort the shape of the molecule compared with the native 

This definition recognizes that immobilization—e.g., at cellular membranes— is the native state for some enzymes. Although interesting mathematics are 

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An electrode that responds to the concentration of a substrate by reacting the substrate with an immobilized enzyme, producing an ion that can be monitored with an ion-selective electrode. 

Mifflin, T. E. Andriano, K. M. Robbins, W. B. Determination of Penicillin Using an Immobilized Enzyme Electrode, /. Chem. Educ. 1984, 61, 638-639. 

Enzyme catalysis Enzyme electrode Enzyme immobilization Enzyme immunoassay Enzyme inhibitors... 

Immiscible blends Immiscible liquids Immiticide Immobileliquids Immobdines Immobilized enzymes... 

In most cases, hoUow fibers are used as cylindrical membranes that permit selective exchange of materials across their waUs. However, they can also be used as containers to effect the controUed release of a specific material (2), or as reactors to chemically modify a permeate as it diffuses through a chemically activated hoUow-fiber waU, eg, loaded with immobilized enzyme (see Enzyme applications). 

Immobilized Enzymes. The immobilized enzyme electrode is the most common immobilized biopolymer sensor, consisting of a thin layer of enzyme immobilized on the surface of an electrochemical sensor as shown in Figure 6. The enzyme catalyzes a reaction that converts the target substrate into a product that is detected electrochemicaHy. The advantages of immobilized enzyme electrodes include minimal pretreatment of the sample matrix, small sample volume, and the recovery of the enzyme for repeated use (49). Several reviews and books have been pubHshed on immobilized enzyme electrodes (50—52). 

Fig. 6. Diagram of an immobilized enzyme electrode, where S is the substrate and P is the enzyme-bound substrate product.

The response of the immobilized enzyme electrode can be made independent of the enzyme concentration by using a large excess of enzyme at the electrode surface. The electrode response is limited by the mass transport of the substrate. Using an excess of enzyme often results in longer electrode lifetimes, increased linear range, reduced susceptibiUty to pH, temperature, and interfering species (58,59). At low enzyme concentrations the electrode response is governed by the kinetics of the enzyme reaction. 

Multienzyme Electrodes. Coupling the reactions of two or more immobilized enzymes increases the number of analytes that can be measured. An electro-inactive component can be converted by an enzyme to a substrate that is subsequentiy converted by a second enzyme to form a detectable end product (57). For example, a maltose [69-79-4] sensor uses the enzymes glucoamylase and glucose oxidase, which convert... 

The resonant frequency of the crystal is inversely proportional to the mass of the Pmssian blue coating. When the immobilized enzyme acts on its substrate, glucose-6-phosphate [54010-71-8] (4), electrons are transferred to the Pmssian blue. In order to maintain electrical neutraUty, cations... 

The dye is excited by light suppHed through the optical fiber (see Fiber optics), and its fluorescence monitored, also via the optical fiber. Because molecular oxygen, O2, quenches the fluorescence of the dyes employed, the iatensity of the fluorescence is related to the concentration of O2 at the surface of the optical fiber. Any glucose present ia the test solution reduces the local O2 concentration because of the immobilized enzyme resulting ia an iacrease ia fluorescence iatensity. This biosensor has a detection limit for glucose of approximately 100 ]lM , response times are on the order of a miaute. 

In the early years of the chemical industry, use of biological agents centered on fermentation (qv) techniques for the production of food products, eg, vinegar (qv), cheeses (see Milk and milk products), beer (qv), and of simple organic compounds such as acetone (qv), ethanol (qv), and the butyl alcohols (qv). By the middle of the twentieth century, most simple organic chemicals were produced synthetically. Fermentation was used for food products and for more complex substances such as pharmaceuticals (qv) (see also Antibiotics). Moreover, supports were developed to immobilize enzymes for use in industrial processes such as the hydrolysis of starch (qv) (see Enzyme applications). 

The formulation of an enzyme is normally considered a way to store and transport the enzyme until its appHcation. One common exception is immobilized enzymes where formulation is an active part of their appHcation. 

Immobilization. Enzymes, as individual water-soluble molecules, are generally efficient catalysts. In biological systems they are predorninandy intracellular or associated with cell membranes, ie, in a type of immobilized state. This enables them to perform their activity in a specific environment, be stored and protected in stable form, take part in multi-enzyme reactions, acquire cofactors, etc. Unfortunately, this optimization of enzyme use and performance in nature may not be directiy transferable to the laboratory. 

A significant advantage of immobilized enzymes is the total absence of catalytic activity in the product. Moreover, the degree of substrate-to-product conversion can be controlled during processing, eg, by adjusting the flow rate through a packed-bed column reactor of immobilized enzyme. 

Because enzymes can be intraceUularly associated with cell membranes, whole microbial cells, viable or nonviable, can be used to exploit the activity of one or more types of enzyme and cofactor regeneration, eg, alcohol production from sugar with yeast cells. Viable cells may be further stabilized by entrapment in aqueous gel beads or attached to the surface of spherical particles. Otherwise cells are usually homogenized and cross-linked with glutaraldehyde [111-30-8] to form an insoluble yet penetrable matrix. This is the method upon which the principal industrial appHcations of immobilized enzymes is based. 

Membrane reactors, where the enzyme is adsorbed or kept in solution on one side of an ultrafHtration membrane, provides a form of immobilized enzyme and the possibiHty of product separation. 

Immobilized enzyme Glucose isomerase Penicillin V acylase... 

The second most important group of immobilized enzymes is stiU the penicillin G and V acylases. These are used in the pharmaceutical industry to make the intermediate 6-aminopenici11anic acid [551-16-6] (6-APA), which in turn is used to manufacture semisynthetic penicillins, in particular ampicilHn [69-53-4] and amoxicillin [26787-78-0]. This is a remarkable example of how a complex chemical synthesis can be replaced with a simple enzymatic one ... 

When selecting a suitable feed symp, the main criteria are optimization of enzyme productivity and minimization of the formation of by-products. Typical feed symp specifications are shown in Table 5. Higher symp concentration and higher viscosity results in a reduced isomerization rate due to diffusion resistance in the pores of the immobilized enzyme. A deaeration step is desirable to remove dissolved oxygen that would otherwise iacrease the formation of by-products. The pH is adjusted to the optimum level for the productivity of the enzyme. 

During operation, the immobilized enzyme loses activity. Most commercial enzymes show decay as a function of time (Eig. 12). The glucose isomerase ia a reactor is usually replaced after three half-Hves, ie, when the activity has dropped to around 12.5% of the initial value. The most stable commercial glucose isomerases have half-Hves of around 200 days ia practical use. To maintain the same fmctose content ia the finished symp, the feed-flow rate is adjusted according to the actual activity of the enzyme. With only one isomerization reactor ia operation, the result would be excessive variations ia the rate of symp production. To avoid this, several reactors at different stages ia the cycle of enzyme decay are operated ia combiaation. 

An immobilized enzyme-carrier complex is a special case that can employ the methodology developed for evaluation of a heterogeneous cat ytic system. The enzyme complex also has external diffusional effects, pore diffusional effects, and an effectiveness factor. When carried out in aqueous solutions, heat transfer is usually good, and it is safe to assume that isothermal conditions prevail for an immobihzed enzyme complex. 

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