Temperature dependence, immobilized enzymes

The methods of gel synthesis, immobilization of monomer conjugated enzyme, assay of enzyme activity, and determination of gel water content have been published elsewhere (4,5). A schematic of the synthesis is shown in Fig. 1. The gel compositions are identified as NA-100" (100% NIPAAm), "NA-95" (95% NIPAAm, 5% AAm), NA-90 (90% NIPAAm, 10% AAm) and "NA-85" (85% NIPAAm, 15% AAm) all are based on mole percents of monomers. Total monomer concentration was always 1.75 M. The experiment to determine the temperature dependence of enzyme activity was carried out after the enzyme reversibility experiment. 

Enzyme activity has a certain dependence on temperature, and the general enzyme has an optimal temperature, and immobilized enzyme is no exception. Compared to the solution enzyme, the optimum temperature of immobilized enzyme shows ups and downs. The study found that the aminoacylase was bound to DEAE-cellulose and DEAE-dextran using ion-binding, or embedded in crosslinked polyacrylamide gel. Thus, the immobihzed enzyme was prepared, and its optimum temperature was somewhat higher than that before the immobilization. When aminoacylase was immobilized by iodine acetyl cellulose with covalent binding, its optimum temperature was somewhat lower than that before the immobilization. When the glucose isomerase was boimd to porous resin with covalent binding. 

Depending on the immobilization procedure the enzyme microenvironment can also be modified significantly and the biocatalyst properties such as selectivity, pH and temperature dependence may be altered for the better or the worse. Mass-transfer limitations should also be accounted for particularly when the increase in the local concentration of the reaction product can be harmful to the enzyme activity. For instance H2O2, the reaction product of the enzyme glucose oxidase, is able to deactivate it. Operationally, this problem can be overcome sometimes by co-immobilizing a second enzyme able to decompose such product (e.g. catalase to destroy H202). 

Figure 5. Temperature dependence of immobilized enzyme activity in LCST hydrogels. (Reproduced with permission from Ref. 5. Copyright 1986 Elsevier Science.)...

Figure 7. Dependence of immobilized enzyme activity on composition of LCST hydrogels at 30 and 40 first temperature cycle.

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. 

Fig. 6. Temperature dependency of activity yield in the immobilization of enzymes by radiation polymerization. Enzyme O ot-amylase in 50% HEMA A glucoamyla.se in 50% HEMA cellulase in 50% HEMA glucose oxidase in 50% HEMA A glucoamylase in 30% HEMA a-glucosidase in 50% HEMA. Monomer HEMA (2-hydroxyethyl methacrylate). Irradiation. 1 x 106 rad, in vacuo. % monomen in buffer solution...

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... 

All results reviewed herein demonstrate that the microgel particles may serve as nanoreactors for the immobilization of catalytically active nanostructures, namely for metal nanoparticles and enzymes. In both cases, the resulting composites particles are stable against coagulation and can be easily handled. Moreover, the catalytic activity of metal nanoparticles can be modulated through the volume transition that takes place within the thermosensitive microgel carrier system. Similar behavior has been also observed for the temperature dependence of enzymatic activity. Thus, the microgel particles present an active carrier system for applications in catalysis. 

The activity of the immobilized enzyme bioreactor plays an important role in the quality of the spectra. Immobilized enzyme activity is dependent on pH, temperature, solvents and buffers used for the hydrolysis. Extremes in any one condition can irreversibly destroy the bioreactor activity. Hydrolysis at conditions far from optimal can lead to no-hydrolysis or partial hydrolysis of a peptide, providing limited information on the peptide. In order to successfully employ an immobilized enzyme column on-line with HPLC/thermospray MS, organic modifiers must be kept minimal (less than 30-50%), pH must be between 6.5 and 8.5 and buffer (ammonium acetate) concentration around 0.05-0.1M [12]. 

In a diffusion-free enzyme reaction the reaction rate increases up to a certain critical value exponentially and is described by the Arrhenius equation [82]. In diffusion-controlled reactions the reaction rate is a matter of the efficiency factor ri [see Eqs. (3 - 5)]. In more detail, the maximum reaction rate is expressed within the root of Eq. (4). Conclusively, the temperature dependence is a function of the square root of the enzyme activity. In practice, immobilized enzymes are much less temperature dependent when their reaction rate is diffusion controlled. 

Along with enhanced stability, the activity profile of the enzyme is broadened over a larger temperature range as a result of the immobilization. The activity maximum is shifted to a slightly higher temperature, from T = 30°C for the free enzyme to T = 37°C for the Sepharose-immobilized enzyme (Figure 4). This result may reflect the temperature dependence on... 

In this paper a mathematical model will be presented describing the conversion process in a fixed bed reactor. The model allows the calculation of the temperature dependence of the initial acitivity of the immobilized enzyme. It also predicts the stability of that activity as a function of the operating tenperature. 

In table I the kinetic constants and their temperatures dependences for the present immobilized enzyme and some other relevant data have been summarized. 

Enzymes are exploited as catalysts in many industrial, biomedical, and analytical processes. There has been considerable interest in the development of carrier systems for enzyme immobilization because immobilized enzymes have enhanced stability compared to soluble enzymes, and can easily be separated from the reaction. This leads to significant savings in terms of reduced enzyme consumption, and the ability to use such enzymes in continuous processes. The activity and stability of enzymes depends largely on the particular operating and storage conditions, and is strongly influenced by factors such as the chemical environment, temperature, pH, and solvent properties. Most enzymes are water soluble and a certain amount of water is always required for their solubilization. Additionally, many enzymes require the presence of a cofactor, which may be attached firmly to the enzyme or may need to be added separately as a coenzyme. When the immobilization of an enzyme... 

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