Immobilized enzyme properties

Influence of Contact Time and Enzyme Concentration on the Immobilized Enzyme Properties... 

Numerous different immobilization methods have been reported that take advantage of various enzyme properties such as size, chemically reactive functionality, ionic groups or hydrophobic domains.Based on these properties, enzyme immobilization can be split into three main classes (which are also applicable to the immobilization of cell cultures) ... 

Effect of immobilization on the dynamic properties of enzymes. Examples of three different experimentally imposed conditions include (a) enzyme and substrate are present in an aqueous solution (b) enzyme and substrate are immobilized on or within a solid support and (c) immobilized enzyme acts on a substrate present within an aqueous solution. The rate laws and curves that are presented below each diagram indicate the expected rate behavior. (From K. J. Laidler P. S. Bunting (1982) Meth. Enzymol. 64, 229.)... 

Although the kinetics of immobilized enzymes is especially relevant to industrial process chemistry, there is growing recognition that enzymes in crowded solutions may likewise experience diffusional control of the catalytic properties. 

The first insoluble derivatives of bioluminescent enzymes were prepared by Erlanger et al. by reacting luciferases they investigated the properties of these immobilized enzyme preparations and their potential for studying the mechanism of bioluminescence [54]. 

In Figure 10.1 the time course of thermodynamically and kinetically controlled processes catalysed by biocatalysts are compared. The product yield at the maximum or end point is influenced by pH, temperature, ionic strength, and the solubility of the product. In the kinetically controlled process (but not in the thermodynamically controlled process) the maximum yield also depends on the properties of the enzyme (see next sections). In both processes the enzyme properties determine the time required to reach the desired end point. The conditions under which maximum product yields are obtained do not generally coincide with the conditions where the enzyme has its optimal kinetic properties or stability. The primary objective is to obtain maximum yields. For this aim it is not sufficient to know the kinetic properties of the enzyme as functions of various parameters. It is also necessary to know how the thermodynamically or the kinetically controlled maximum is influenced by pH, temperature and ionic strength, and how this may be influenced by the immobilization of the biocatalysts on different supports. 

Existing uses of proteases in foods have been discussed in the foregoing section. Expanding such applications in the future depends upon our ability to control both the processes themselves and their costs. The development of continuous reactors utilizing free or immobilized enzymes will address each of these constraints. Furthermore, our understanding of the chemical basis for the various functional properties of proteins must be expanded... 

Before an immobilized enzyme can be used for an industrial process, it is essential to characterize it in terms of its catalytic and kinetic properties. A quantitative assay must be developed to measure the activity, kinetic parameters, and stability of the enzyme. In a coupling reaction, H202 rapidly reacts with phenol and 4-aminoantipyrine (electron donor) in the presence of peroxidase to produce a quinoneimine chromogen (Equation E12.2, Figure El 1.2), which is intensely colored with a maximum absorbance at 510 nm. (This is the same as the product formed in the analysis of cholesterol in Experiment 11.)... 

A main adimntage of immobilized enzyme is that it can be reused since it can be easily separated from the reaction solution and can be easily retained in a continuous-flow reactor. Furthermore, immobilized enzyme may show selectively altered chemical or physical properties and it may simulate the realistic natural environment where the enzyme came from, the cell. 

Immobilized enzyme systems can be differentiated according to mode of immobilization, carrier properties, and rate-determining step. 

From the previous paragraph it can be discerned that the half-life of a biocatalyst is determined not only by the deactivation properties of the free enzyme but also by the mass-transfer properties of an immobilisate. Figure 19.7 reveals the factor by which the dependence on temperature increases operating stability in the case of immobilized enzyme. 

Because of interesting and specific properties of the gel, curdlan has many potential uses not only in food industry but also as a film, a fiber, a support for immobilizing enzymes and a binding agent in tobacco product etc.(2). Besides these, an antitumor activity(3) has been reported. 

The biocatalyst a-chymotrypsin s ability to hydrolyze 20 is inhibited in the presence of copolymer 19a loaded with 0.2 mol% of the triphenyl carbinol units. 47b Photoirradiation of 19a results in heterolytic bond cleavage and the formation of the cationic copolymer 19b. In this polymer structure, the biocatalyzed hydrolysis of 20 is activated (V = 1.0 pM min-1). The polymer-induced photostimulated activation and deactivation of a-chymotrypsin in the different membrane environments correlates with the permeability and transport properties of the substrate 20 through the different structures of the polymer membranes.1471 Flow dialysis experiments showed that the polymer states 17a, 18a, and 19a are nonpermeable to 20, and hence the biocata-lytic functions of the immobilized enzyme are blocked. The polymer structures 17b,... 

Having briefly outlined the historical development of cross-linked enzyme crystals, a discussion of their properties as compared to soluble and immobilized enzymes is in order. 

Enzyme immobilization has been reported to improve the thermal stability of enzymes (1,2) and may also affect binding of substrates and inhibitors to the enzyme, thereby affecting the Michaelis constant and enzyme inhibition. Several previous studies have considered the advantages of immobilized enzymes with soluble substrates, and a few studies have also investigated the properties of immobilized enzymes with insoluble substrates. The main objective of the present work was to establish the effect of immobilization on the thermal stability of these enzymes, so that they may be used at elevated temperatures without significant activity loss. The immobilization conditions were varied, and their effect on the performance of the immobilized enzymes was analyzed with reference to their physiochemical and structural properties. 

Batch studies for evaluating immobilized enzyme activity and properties of the "bioplastic" (urease entrapped in PDMS) material were conducted in 250-mL shake flasks in an environmentally controlled shaker/ incubator. 

Enzyme immobilization on a hydrophobin layer does not appear to significantly decrease enzyme properties, and these enzymes display similar substrate affinity to free enzymes. The specific activity of these enzymes may be lower than enzymes that are free in solution, but the immobilized enzymes also have the advantage of increased stability from 1 to 3 months (HRP and GOX, respectively). 

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