Properties of Immobilized Enzymes

The activity of an enzyme immobilized by one of the chemical methods is determined by numerous factors the properties of the enzyme, the properties of the carrier, the method of formation of the covalent bond with the carrier, etc. Therefore, it is difficult to predict the properties of the enzyme in every given case. The methods of immobilization, as well as specific examples of enzyme fixation, are discussed elsewhere.  

Immobilization is, as a rule, accompanied by a declining activity of the enzymes. However, at present there are numerous examples of successful immobilization with the preservation of enzyme specificity and activity within 10 to 90% of its activity in the native state. This permits us to think that the problem of obtaining enzymic preparations which are close in physicochemical properties to heterogeneous catalysts and electrocatalysts, particularly as regards the preservation of enzymatic activity and specificity, can be successfully solved in each specific case. 

The behavior of immobilized enzymes differs from that of dissolved enzymes because of the effects of the support material, or matrix, as well as conformational changes in the enzyme that result from interactions with the support and covalent modification of amino acid residues. Properties observed to change significantly upon immobilization include specific activity, pH optimum, Km, selectivity, and stability.23 Physical immobilization methods, especially entrapment and encapsulation, yield less dramatic changes in an enzyme s catalytic behavior than chemical immobilization methods or adsorption. The reason is that entrapment and encapsulation result in the enzyme remaining essentially in its native conformation, in a hydrophilic environment, with no covalent modification. 

Chemical immobilization methods may alter the local and net charges of enzymes, through covalent modification of charged residues such as lysine (NH4), aspartate, and glutamate (COO-). Conformational changes in secondary and tertiary protein structure may occur as a result of this covalent modification, or as a result of electrostatic, hydrogen-bonding or hydrophobic interactions with the support material. Finally, activity losses may occur as a result of the chemical transformation of catalytically essential amino acid residues. 

The specific activity of an enzyme almost always decreases on immobilization. The active sites are less accessible to substrate, and the diffusion of substrates and products across the stagnant layer of solution at the particle surface, and within polymer networks, lowers apparent values of Vmax and raises apparent Km values. The activity of an immobilized enzyme should be expressed as specific activity 

The primary amine groups that remain underivatized exist in the protonated form, and these positively charged —NHj groups attract OH- from the bulk solution. This increases the local [OH-] relative to that in the bulk, so that 

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. 

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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. 

Rialdi, G. and Battistel, E. (1993) Thermodynamic properties of immobilized enzymes, Calorim. Anal. Therm. 24, 401-404. 

Our group has demonstrated that the catalytic properties of immobilized enzymes can be manipulated by the temperature-dependent swelling behavior of the microgel. The hydrolytic activity of adsorbed and native P-D-glucosidase was determined as a function of temperature. Desorption of immobilized enzyme upon... 

Thus, the results shown here demonstrate that thermosensitive microgel particles can serve as superior carriers for the adsorption of enzymes in which the activity of adsorbed enzymes are preserved. The catalytic activity of adsorbed P-D-glucosidase from almonds is increased by a factor of more than three. Moreover, the catalytic properties of immobilized enzymes can be manipulated by the volume transition of the microgel. Hence, such microgels present a novel class of active nanoreactors for biocatalysis. 

Investigation of Catalytic Properties of Immobilized Enzymes and Cells by Flow Microcalorimetry... 

The properties of immobilized enzyme preparations are governed by the properties of both the enzyme and the carrier material. The specific interaction between the latter provides an immobilized enzyme with distinct chemical, biochemical, mechanical and kinetic properties (Fig. 1). 

Immobilization by chemical cross-linking without the addition of an inert carrier or matrix can provide the means to stabilize and reuse a biocatalyst without dilution of volumetric activity. A major deficiency in all of the aforementioned immobilization methods is that a substantial amount of a catalytically inert carrier or matrix is used to bind or contain the biocatalyst. In many cases, the amount of carrier is two orders of magnitude higher than the protein catalyst. Unfortunately, direct cross-linking of the enzyme, followed by precipitation of an amorphous solid often results in low activity and poor mechanically properties and so this method is not often used. Recently, however, cross-linked enzyme crystals have been reported to give many of the desirable properties of immobilized enzymes without the need for a support material (Sect. 6.4.1). 

Stefuca, VC, Gemeiner, P Investigation of Catalytic Properties of Immobilized Enzymes and Cells by Flow Microcalorimetry. Vol. 64, p. 69... 

The first volume of a new series entitled Applied Biochemistry and Bioengineering deals with the principles of immobilized enzymes, which, among other topics, are also discussed in one of the latest volumes of Methods in Enzymology . Recent reviews have covered the uses of immobilized enzymes and immobilized microbial cells in industry, the design of immobilized-enzyme flow reactors, the properties of immobilized enzymes, the reactions used to immobilize enzymes, and the uses of immobilized enzymes in clinical diagnosis, detoxification, and therapy, etc. and of immobilized microbial cells. 

A review on the subject of immobilized enzymes in biochemical analysis covers preparation and properties of immobilized enzymes assay methods using immobilized enzymes (spectrophotomatic assays, automated methods in biochemical analysis, enzyme electrodes, and colorimetric analyses using immobilized enzymes) and applications of immobilized enzymes in biochemical analysis. ... 

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