Immobilized enzyme, kinetics

The variety of enzyme-catalyzed kinetic resolutions of enantiomers reported ia recent years is enormous. Similar to asymmetric synthesis, enantioselective resolutions are carried out ia either hydrolytic or esterification—transesterification modes. Both modes have advantages and disadvantages. Hydrolytic resolutions that are carried out ia a predominantiy aqueous medium are usually faster and, as a consequence, require smaller quantities of enzymes. On the other hand, esterifications ia organic solvents are experimentally simpler procedures, aHowiag easy product isolation and reuse of the enzyme without immobilization. 

The Michaehs-Menten equation and other similar nonhnear expressions characterize immobihzed enzyme kinetics. Therefore, for a spherical porous carrier particle with enzyme molecules immobilized on its external as well as internal surfaces, material balance of the substrate will result in the following ... 

Enzyme linked electrochemical techniques can be carried out in two basic manners. In the first approach the enzyme is immobilized at the electrode. A second approach is to use a hydrodynamic technique, such as flow injection analysis (FIAEC) or liquid chromatography (LCEC), with the enzyme reaction being either off-line or on-line in a reactor prior to the amperometric detector. Hydrodynamic techniques provide a convenient and efficient method for transporting and mixing the substrate and enzyme, subsequent transport of product to the electrode, and rapid sample turnaround. The kinetics of the enzyme system can also be readily studied using hydrodynamic techniques. Immobilizing the enzyme at the electrode provides a simple system which is amenable to in vivo analysis. 

BRADLEY A. SAVILLE is an Associate Professor of Chemical Engineering at i the University of Toronto. He received his B.Sc. and Ph.D. in chemical engi-neering at the University of Alberta. He is the author or co-author of over 25 research articles on enzyme kinetics, pharmacokinetics, heterogeneous reactions in biological systems, and reactors for immobilized enzymes. He is a member, of the Chemical Institute of Canada, the Canadian Society of Chemical Engineering, and Professional Engineers Ontario. 

Urea in kidney dialysate can be determined by immobilizing urease (via silylation or with glutaraldehyde as binder) on commercially available acid-base cellulose pads the process has to be modified slightly in order not to alter the dye contained in the pads [57]. The stopped-flow technique assures the required sensitivity for the enzymatic reaction, which takes 30-60 s. Synchronization of the peristaltic pumps PI and P2 in the valveless impulse-response flow injection manifold depicted in Fig. 5.19.B by means of a timer enables kinetic measurements [62]. Following a comprehensive study of the effect of hydrodynamic and (bio)chemical variables, the sensor was optimized for monitoring urea in real biological samples. A similar system was used for the determination of penicillin by penicillinase-catalysed hydrolysis. The enzyme was immobilized on acid-base cellulose strips via bovine serum albumin similarly as in enzyme electrodes [63], even though the above-described procedure would have been equally effective. 

This chapter describes the different types of batch and continuous bioreactors. The basic reactor concepts are described as well as the respective basic bioreactors design equations. The comparison of enzyme reactors is performed taking into account the enzyme kinetics. The modelhng and design of real reactors is discussed based on the several factors which influence their performance the immobilized biocatalyst kinetics, the external and internal mass transfer effects, the axial dispersion effects, and the operational stabihty of the immobilized biocatalyst. 

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 ... 

An enzyme is immobilized on solid surface. Assume that the external mass-transfer resistance for substrate is not negligible and that the Michaelis-Menten equation describes the intrinsic kinetics of enzyme reaction. 

To realize glucose-, urea- and penicillin sensors in a flow injection mode (FIA) appropriate enzymes can be immobilized [13]. The measuring range is very limited in all reported cases due to enzyme kinetics and a more sophisticated set up is required. 

By using a resonant mirror biosensor, the binding between YTX and PDEs from bovine brain was studied. The enzymes were immobilized over an aminosilae surface and the association curves after the addition of several YTX concentrations were checked. These curves follow a typical association profile that fit a pseudo-first-order kinetic equation. From these results the kinetic equilibrium dissociation constant (K ) for the PDE-YTX association was calculated. This value is 3.74 p,M YTX (Pazos et al. 2004). is dependent on YTX structure since it increases when 44 or 45 carbons (at C9 chain) group. A higher value, 7 p,M OH-YTX or 23 p,M carboxy-YTX, indicates a lower affinity of YTXs analogues by PDEs. 

Free enzyme versus immobilized enzyme can influence the yield of lOS, additionally an immobilized system would be favorable economically as the biocatalyst can be reused, enables continuous production and the end product is free of contamination. Kim et al. [275] intended to make a comparison between the reaction kinetics of free and immobilized endo-inulinases in a batch reactor however significant differences were observed in the reaction behavior and product composition due to the form of enzyme used and the initial concentration of substrate. Yun et al. [276] investigated the effect of inulin concentration on the production of lOS by free and immobilized endo-inulinase from Pseudomonas sp. Their findings corroborate those of Kim et al. [275] whereby different products are formed depending on the form of enzyme a soluble enzyme yielded inulobiose and DP3 products, whereby the immobilized form predominantly produced inulobiose. As the concentration of inulin increased the yield of lOS did not increase in the soluble system and in the immobilized the yield remained the same. Although the enzyme was derived from Pseudomonas the immobilized form required a differ-... 

The effects of this concentration gradient are most significant at low bulk concentrations of the substrate, since substrate is converted to product as soon as it reaches the surface of the particle, so that the surface concentration of substrate is zero. At very high bulk substrate concentrations, the enzymatic reaction rate is limited by enzyme kinetics rather than mass transport, so that surface concentrations do not differ significantly from those in the bulk. Because of the concentration gradient, however, enzyme saturation with substrate occurs at much higher bulk substrate concentrations than required to saturate the soluble enzyme. Apparent Km values (K m) for immobilized enzymes are larger than Km values obtained for the native soluble enzymes. 

In addition to the analytical applications, there was sporadic work on the employment of flow calorimetry for the investigation of enzyme kinetics [23,24]. In 1985 Owusu et al. [25] published the first report on the use of flow microcalorimetry for the study of immobilized enzyme kinetics approaching... 

EIA often makes use of solid phases. The relative merits and disadvantages of such techniques, as well as ways to optimize them will be discussed. An important aspect, which has not yet been investigated in detail for EIA, is the influence immobilization of enzymes has on enzyme kinetics. The solid-phase may cause strong local differences in the microenvironment of the enzyme, the implications of which can only be inferred from studies on immobilized enzymes in solid-phase biochemistry (Chapter 9). Major or minor flaws in EIA design, which may discredit an otherwise perfectly valid EIA, will be discussed. 

By comparison with feedback methods, generation collection offers greater sensitivity to low activities of immobilized enzyme. An estimate of the minimum catalytic rate, kCM, of the immobilized enzyme which can still be detected and quantified can be made on the basis of the analytical sensitivity of the tip collector. If c is the detection limit of the tip, then enzyme kinetic data can be obtained if... 

A feature of SECM is the quantitative theory available based on reaction-diffusion models (Chapter 5). The SECM may be used for kinetic studies in either the feedback or generation-collection modes. These two possibilities are described below from the point of view of studying immobilized enzyme kinetics. 

Feedback. When an oxidoreductase enzyme is immobilized at the specimen surface, a redox mediator present in solution may be recycled by the diffusion-limited electrochemical process at the tip and electron exchange with the enzyme active site as described in Sec. I.C. The mass transport rate is defined by the tip radius and height of the tip above the specimen. The tip current depends on the mass transport rate and the enzyme kinetics. Kinetic information may therefore be obtained from the dependence of tip current on height, i.e., an approach curve. When the mediator is fed... 

The choice of an appropriate reactor for applications of immobilized enzymes as well as for soluble enzymes depends on the kinetics of the reaction. Kinetics of immobilized enzymes are not only a function of enzyme activity but also of substrate transport to the enzyme, which is affected by the matrix used for immobilization. For a description of immobilized enzyme kinetics the reader is referred to the comprehensive literature in this field[35 40, 138 14H. Additionally, the use of immobilized enzymes is treated in Chap. 6 of this book. 

The fixed bed reactor, behaving as a plug flow reactor, is most often used for immobilized enzyme reactions. Typically, the reactor is used with an upward direction of the flow to avoid compression of the bed and to release gas bubbles generated during the reaction. Reactor design may be done readily without knowing the detailed enzyme kinetics. Kinetic measurements are performed with a recirculation reactor and the data are plotted in the form 1/v = f(%) (see above). From this plot, the residence time necessary to reach a desired conversion x can be calculated as described. The different enzyme concentrations in the recirculation reactor and in the plug flow reactor have to be considered. 

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