Enzymatic reactions and immobilization techniques

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. 

The production of both specialty and cammodlty chemicals by enzyme reaction has become a reality due to recent advances In Immobilization. These Immobilization techniques have provided an econcmlcal system for reuse of enzyme and thus provide a route to optical Iscmiers In high enantiomeric yields. This provides specific stereoisomers for agricultural synthesis at reasonable cost. The advantages of stereoisomers Include high activity levels as well as reduced toxicity due to the absence of the Incorrect stereoisomer. Methods of Immobilization will be reviewed with emphasis on Immobilization by polyazetldlne. Enzymatic reaction via immobilization enzymes and Immobilized whole cells will be reviewed with emphasis on the production of agrlcultaral chemicals. 

This book starts with a review of the tools and techniques used in kinetic analysis, followed by a short chapter entitled How Do Enzymes Work , embodying the philosophy of the book. Characterization of enzyme activity reversible and irreversible inhibition pH effects on enzyme activity multisubstrate, immobilized, interfadal, and allosteric enzyme kinetics transient phases of enzymatic reactions and enzyme... 

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 Section 11.1.2. 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, that is, an approach curve. When the mediator is fed back from the specimen at a diffusion-controlled rate, the approach curve will be identical to that above a metallic conductor. In the opposite situation, when the flux of mediator fed back from the specimen is much less than the flux of mediator to the tip from bulk solution, the approach curve will correspond to that above an insulating surface, that is, pure negative feedback. In between these two limits, the approach curve will contain information on the steady-state rate of the enzymatic reaction and the shape of the approach curve as a function of substrate and cosubstrate concentrations may be used to investigate the reaction order (Figure 11.3). A detailed study of GOx with several redox mediators and immobilization techniques has been reported [15]. The enzyme reaction kinetics was... 

Immobilization techniques have been applied in the preparation of immobilized CL reagents, with specific advantages such as reusability, improved stability, and increased efficiency. These strategies have been applied in the development of CL sensors, which today constitute the most important tools in analytical chemistry because of the high sensitivity offered. Optical fibers have been used to transfer light in order to improve the quality of detection, and new types of flow-through cells have been introduced in the construction of CL sensors. Also, selectivity has been considerably improved by the utilization of enzymatic or antigen-antibody reactions. 

Affinity capture-release electrospray ionization mass spectrometry (ACESIMS) is another recently introduced technique for quantification of proteins, and to date has most often been applied to clinical enzymology.60 The product conjugates of the enzymatic reaction between the synthetic substrate and targeted enzyme are captured by immobilized affinity reagents, purified, released into solution, and analyzed by ESI-MS. 

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. 

Enzymatic reactions are commonly observed or practiced in various kinds of food and biotechnology products. With the goals of reducing operating costs and improving product quality, a number of enzyme immobilization techniques have been developed in recent decades [Woodward, 1985]. The availability of robust membranes, particularly porous inorganic membranes, has improved the enzyme immobilization technology. One type of membrane bioieactors immobilizes enzyme in the membrane pores by dead-end filtration of the enzyme solution. 

Due to high biocompability and large surface are of cobalt oxide nanoparticles it can be used for immobilization of other biomolecules. Flavin adenine FAD is a flavoprotein coenzyme that plays an important biological role in many oxidoreductase processes and biochemical reactions. The immobilized FAD onto different electrode surfaces provides a basis for fabrication of sensors, biosensors, enzymatic reactors and biomedical devices. The electrocatalytic oxidation of NADH on the surface of graphite electrode modified with immobilization of FAD was investigated [276], Recently we used cyclic voltammetry as simple technique for cobalt-oxide nanoparticles formation and immobilization flavin adenine dinucleotide (FAD) [277], Repeated cyclic voltammograms of GC/ CoOx nanoparticles modified electrode in buffer solution containing FAD is shown in Fig.37A. 

During the last ten years enzyme technology has moved mainly towards the development of new immobilization techniques and the improvement of those already existing. In turn, the attention of applied research has been focused on the engineering of systems based on immobilized biocatalysts. Enzymes involved in this development were enzymes catalyzing simple reactions that normally require no cofactors. A number of drawbacks affected the use of immobilized enzymatic preparations. An often dramatic reduction of initial enzyme activity due to the binding process, and the existence of diffusional resistances limits this approach with low activity enzymes, with macromolecular substrates and in general with enzymes whose cata-... 

Layer-by-layer (LBL) technique was used for assembling AChE on a CNT transducer (Figure 2). The principle of the biosensor is based on the inhibition of enzyme activity by OPs. The inhibition can be electroehemieally measured. Positively charged poly(diallyldimethylammonium chloride) (PDDA) and negatively charged AChE enzyme were assembed on the CNT surface by LBL technique. Here, CNTs play dual significant roles in the transduction and enzyme-immobilization events. As carriers, CNTs provide a suitable microenviromnent to retain the AChE activity. As a transducer, CNTs amplify the electrochemical signal of the product of the enzymatic reaction. 

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