Enzyme-catalyzed reactions, kinetics immobilized enzymes

Several thermodynamic and kinetic behaviors of enzyme-catalyzed reactions performed in ILs, with respect to enzymatic reactions carried out in conventional solvents, could lead to an improvement in the process performance [34—37]. ILs showed an over-stabilization effect on biocatalysts [38] on the basis of the double role played by these neoteric solvents ILs could provide an adequate microenvironment for the catalytic action of the enzyme (mass transfer phenomena and active catalytic conformation) and if they act as a solvent, ILs may be regarded as liquid immobilization supports, since multipoint enzyme-1L interactions (hydrogen. Van der Waals, ionic, etc.) may occur, resulting in a flexible supramolecular not able to maintain the active protein conformation [39]. Their polar and non-coordinating properties hold considerable potential for enantioselective reactions since profound effects on reactivities and selectivities are expected [40]. In recent years attention has been focused on the appUcation of ILs as reaction media for enantioselective processes [41—43]. 

Diffusion, partition, and enzyme,reactions influence the sensor characteristics in a complex manner. The effect of enzyme immobilization on the reaction rate is described by the following terminology. Apparent or effective kinetics are observed when internal or external diffusion affects the overall rate. Inherent kinetics prevail when only partitioning (and not mass transfer) effects are present. Intrinsic kinetics describe the enzyme-catalyzed reaction when no partitioning effects or diffusion limitation are present. 

The use of enzymes to lyse cells, hydrolyze fat emulsions, solubilize proteinaceous colloids, liquify or saccharify starch gels and granules, and degrade various components of celluloslc substrates indicates that many substrates are present in a particulate form. Kinetic forms for such enzyme catalyzed reaction rates are here noted, and will be revisited in the subsequent discussion of immobilized enzyme kinetics. 

Four methods have been developed for enzyme immobilization (1) physical adsorption onto an inert, insoluble, solid support such as a polymer (2) chemical covalent attachment to an insoluble polymeric support (3) encapsulation within a membranous microsphere such as a liposome and (4) entrapment within a gel matrix. The choice of immobilization method is dependent on several factors, including the enzyme used, the process to be carried out, and the reaction conditions. In this experiment, an enzyme, horseradish peroxidase (donor H202 oxidoreductase EC 1.11.1.7), will be imprisoned within a polyacrylamide gel matrix. This method of entrapment has been chosen because it is rapid, inexpensive, and allows kinetic characterization of the immobilized enzyme. Immobilized peroxidase catalyzes a reaction that has commercial potential and interest, the reductive cleavage of hydrogen peroxide, H202, by an electron donor, AH2 ... 

Many problems involving competitive reaction kinetics may be treated by invoking the steady-state assumption within the digital simulation this has been done in at least two instances [29-34]. The first of these involves the development of a model for enzyme catalysis in the amperometric enzyme electrode [29-31]. In this model, the enzyme E is considered to be immobilized in a diffusion medium covering an electrode that is operated at a fixed potential such that the product (P) of enzyme catalysis is electroactive under diffusion-controlled conditions. (This model has also served as the basis for the simulation of the voltammetric response of the enzyme electrode [35].) The substrate (S) diffuses through the medium that contains the immobilized enzyme and is catalyzed to form P by straightforward enzyme kinetics ... 

The numerical values for ki. .. k4 vary with RG. For instance, for RG = 10, the following values provide the analytical function Jfei = 0.40472, k2 = 1.60185, k3 = 0.58819, and k4 = -2.37294 [12]. The analytical approximations for hindered diffusion provide a way to determine d from experimental approach curves. For this purpose, one can use an irreversible reaction at the UME (often 02 reduction). In such a case, Fig. 37.2, curve 1 is obtained irrespective of the nature of the sample. Besides the mediator flux from the solution bulk, there might be a heterogeneous reaction at the sample surface during which the UME-generated species O is recycled to the mediator R. The regeneration process of the mediator might be (i) an electrochemical reaction (if the sample is an electrode itself) [9], (ii) an oxidation of the sample surface (if the sample is an insulator or semiconductor) [14], or (iii) the consumption of O as an electron acceptor in a reaction catalyzed by enzymes or other catalysts immobilized at the sample surface [15]. All these processes will increase (t above the values in curve 1 of Fig. 37.2. How much iT increases, depends on the kinetics of the reaction at the sample. If the reaction of the sample occurs with a rate that is controlled by the diffusion of O towards the sample, Fig. 37.2, curve 2 is recorded. If the sample is an electrode itself, such a curve is experimentally obtained if the sample potential... 

Kinetics of Immobilized Enzymes. Another major factor in the performance of immobilized enzymes is the effect of the matrix on mass transport of substrates and products. Hindered access to the active site of an immobilized enzyme can affect the kinetic parameters in several ways. The effective concentration of substrates and products is also affected by the chemistry of the matrix especially with regard to the respective partition coefficients between the bulk solution and the matrix. In order to understand the effects of immobilization upon the rate of an enzyme-catalyzed reaction one must first consider the relationship between the velocity of an enzyme-catalyzed reaction and the... 

Where R is the carrier radius, Defrthe effective diffusion coefficient of the substrate, E is the enzyme concentration in the carrier, and kcat and Km are the kinetic parameters of an enzyme. From a practical standpoint it is important to remember that there are no diffusional limitations as long as substrate concentration S exceeds Km. This condition normally exists at the beginning of many processes catalyzed by immobilized enzymes. At the end of the process, when a substrate is depleted and effective Km may increase because of the product inhibition, the whole reaction may be limited by diffusion. 

Kellogg, Feringa and co-workers have achieved successful dynamic kinetic resolution reactions using cyclic hemiacetals as substrates[13, 14l The enzyme-catalyzed acetylation of 6-hydroxypyranone shown in Fig. 9-6 has been achieved with reasonable enantioselectivity with essentially complete conversion. The racemisation of the hemiacetal is presumed to proceed via reversible ring-opening of the pyranone1 1. The rate of reaction was found to greatly increase when the enzyme, lipase PS (Pseudomonas sp.) was immobilized on Hyflo Super Cell (HSC). 

In many cases it is not possible or desirable to register the reaction catalyzed by an enzyme. It may be that the product is difficult to detect or that the sensitivity in the analysis one needs to apply is not high enough. Then the use of one or more additional enzymes is quite common. The strategy has been worked out for soluble enzymes, and, in the flow systems, the enzymes are either immobilized separately, or coimmobilized. The latter approach has certain advantages in the sense that a better kinetic performance can be observed in a coimmobilized enzyme sequence as compared to when the enzymes are immobilized separately [52]. However, since most assays are based on the use of an excess of immobilized enzymes, no dramatic differences are observed. 

We are presuming that the intrinsic reaction kinetics of the immobilized enzyme catalyzed reaction is of the Michaelis-Menten type, therefore eq. (9.231) takes the form... 

Chemferm is one of among several companies which apply penicillin acylase for the kinetically controlled industrial synthesis of semisynthetic antibiotics in aqueous environments (Scheme 37) [109-111]. Ampicillin (119) and amoxicillin (120) can be obtained by the enzyme-catalyzed condensation of 6-aminopenicillic acid (6-APA, 117) with the amide or ester of D-(-)-4-hydrox-yphenylglycine and D-(-)-phenylglycine, respectively. In a similar way, cephalexin (121) can be obtained by reaction of D-(-)-phenylglycine with 7-aminodesacetoxycephalosporanic acid (7-ADCA, 118). Penicillin acylase from diverse microbial strains such as E. colU Klyveromyces citrophiluy and Bacillus megabacterium was successfully applied for this transformation and was used in its immobilized form based on a gelatin carrier. The immobilization allows an easy separation from the reaction medium and the reuse of the enzyme for at least 50 cycles. Impressive characteristics of this transformation are yields >90%, a selectivity of >95%, and an optical purity of >99% ee. The industrial manufacture takes place in repetitive batch reactors at many locations worldwide with an annual production volume of 2,000 t. 

The products of the reaction they catalyze may inhibit many enzymes through Michaelis-Menten kinetic retroaction. Protons, which are involved as products or reactants in a number of cases, may also influence the enzymatic kinetics. The course of the reaction may therefore be altered by the attending production or depletion of protons. It is thus interesting to examine whether these phenomena may be revealed by the effect they might have on the electrochemical responses of immobilized enzyme films under appropriate conditions [92]. A first clue of the existence of such inhibition effects is the observation of hysteresis behaviors of the type shown in Fig. 18(a) where data obtained with 10 glucose oxidase monolayers with ferrocene methanol as cosubstrate have been taken as example. In the absence of inhibition, the forward and reverse traces should be exactly superimposed. Hysteresis increases to the point of making a peak appear on the forward trace as the scan rate decreases and as the concentration of the buffer decreases, as illustrated in Fig. 18c, c , c , c by comparison with Fig. 18(a). 

Kinetics of the Reactions Catalyzed by Immobilized Enz)mies. The kinetics of the reactions catalyzed by the immobilized enz3mies is influenced by a series of factors inexistent for the free enzymes, such as conformational and steric transformations of the proteic chain induced by the carrier, the diffusion of the substrate and of the reaction products to and from the active centers, the composition of the microsystem created by the carrier. The diffusion effects are reflected by the values of the Michaelis constants (K ). The of an immobilized enzyme is generally higher identical and only in few cases lower than that of the free enz3nne. Higher % values are generally due to diffusion effects. 

Enzyme and microbial kinetics involve the study of reaction rates and the variables that affect these rates. It is a topic, that is critical for the analysis of enzyme and microbial reacting systems. The rate of a biochemical reaction can be described in many different ways. The most commonly used definition is similar to that employed for traditional reactors. It involves the time change in the amount of one of the components participating in the reaction or of one of the products of the reaction this rate is also based on some arbitrary factor related to the system size or geometry, such as volume, mass or interfacial area. In the case of immobilized enzyme catalyzed reactions, it is common to express the rate per unit mass or per unit volume of the catalyst. 

The problem of pore diffusion is only limited to immobilized enzyme catalysts, and not enzyme catalyzed reactions in which the enzyme is used in the native or soluble form. Immobilized enzymes are supported catalysts in which the enzyme is supported or immobilized on a suitable inert support such as alumina, kiesulguhr, silica, or microencapsulated in a suitable polymer matrix. The shape of the immobilized enzyme pellet may be spherical, cylindrical, or rectangular (as in a slab). If the reaction follows Michaelis-Menten kinetics discussed previously, then a shell balance around a spherical enzyme pellet results in the following second order differential equation ... 

Kinetic models to describe lipase-catalyzed reaction mechanisms have been proposed, and most have been extensions of the model developed by Michaelis and Menten (1913). However, normal Michaelis-Menten kinetics do not apply to lipase-induced changes, because the substrates (lipids) are not water-soluble and the enzyme operates at an interface (Brockman, 1984). However, rate expressions for the hydrolysis of emulsified lipids catalyzed by immobilized lipases resemble the rate expressions modeled with Michaelis-Menten mechanisms (Benzonana and Desnuelle, 1965). The kinetics and mechanisms of reactions catalyzed by immobilized lipases have been reviewed by Malcata et al. (1990 1992). 

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