Activity of immobilized enzymes

On the surface the activity assay of immobilized enzymes is quite simple and is not very dissimilar from measuring the activity of soluble enzymes. In both cases the 

General description Preparation Physical and chemical characterization Kinetics  

The complexity of the physical and catalytic properties of immobilized biocatalysts and the difficulty in comparison of effectiveness based on literature descriptions has led to the publication of guidelines for the characterization of immobilized bio-catalysts1351. The authors suggest that description of parameters listed in Table 6-4 should be the minimum required for characterization of an immobilized preparation. 

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Even though, the immobilization procedure should be engineered to maximum retained activity of immobilized enzyme, it is difficult to measure the amount of active enzyme on the carrier without an active site titration. However, this has been done in the case of immobilized trypsin, although only covalent immobilized. (Daly and Shih, 1982)... 

Barros, R.J., Wehtje, E., Garcia, F.A.P. and Adlercreutz, P (1998) Physical characterization of porous materials and correlation with the activity of immobilized enzyme in organic medium. Biocatalysis and Biotransformation, 16, 67-85. 

In the past the mineral matrix was considered as inert, only providing stabilization support for enzymes and humic substances however, due to the overwhelming amount of evidence at the molecular level, there is no doubt that minerals participate in abiotic catalysis of humification reactions in soils. Naidja et al. (2000) referred to mineral particles as the Hidden Half of enzyme-clay complexes, which not only prolong the activity of immobilized enzymes but also are readily able to participate in electron transfer reactions. Many environmental factors can negatively affect the... 

Henzler K, Haupt B, Ballauff M (2008) Enzymatic activity of immobilized enzyme determined by isothermal titration calorimetry. Anal Biochem 378 184-189... 

Another fruitful area of research has been that of the sonochemical activation of immobilized enzymes where ultrasound appears to be particularly useful in increasing the transport of substrate to the enzyme. Using a-chymotrypsin (on agarose gel) and casein as substrate, a two-fold increase in activity was observed at 20 kHz [12]. Here the origin of the enhancement was thought to be associated with increased penetration of the casein into the support gel induced by cavitational effects close to the surface. However an increase in the activity of a-amylase (on porous polystyrene) was produced on irradiation with 7 MHz ultrasound [13]. This is a very significant result since at this high-frequency cavitation cannot occur and... 

Immobilization of the enzyme may also have direct effects on its catalytic ability in that conformational changes may lead to partial inactivation which affects the Michaelis-Menten parameters. Allosteric enzymes may, moreover, loose their ability to undergo allosteric activation. Steric restrictions may also be responsible for lower activities of immobilized enzymes by preventing or hindering the access of the substrate or effectors. On the other hand the stability or activity of enzymes on a solid phase is often better than in the fluid phase, probably due to the local high concentration of enzyme. Certain solid phases may, however, directly inactivate the enzyme, such as polystyrene for horseradish POase (Berkowitz and Webert, 1981). 

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... 

Figure 4. Top, relative specific activity of immobilized enzyme versus the molar ratio (ratio of immobilized su ace coupling groups to enzyme immobilized). Key , diazotized lysozyme X. diazotized lipase A, acylated a-chymotrypsin. Bottom, relative specific activity of modified soluble enzymes versus the molar ratio (ratio of soluble coupling reagent to enzyme). Key O, diazobenzenesulfonic acid lysozyme X. diazobenzenesulfonic acid-lipase H, diazobenzenesulfonic acid-chymotrypsin A, acetic anhydride-chymotrypsin. Reproduced, with permission, from Ref. 20.

Materials Concentration Relative activity of free enzyme (%) Relative activity of immobilized enzyme (%)... 

The protection of the active sites has a positive refection on the activity of immobilized enzyme (figure 10).The reduction in the Km value and activity enhancement proved the role of misorientation but still the fact that difiusion limitation of the substrate has the main role. 

Perhaps the obvious advantage of using polymer multilayers in biosensor design is that they offer a solution for enzyme immobilization when it is otherwise not feasible in a single-polymer layer, as is the case with polyaniline biosensors whose electropolymerization conditions in acid media tend to significantly compromise the activity of immobilized enzymes [155]. 

Enzymes, such as glucoamylase and 3-glucosidase, which differ in their secondary and tertiary stmctures, were adsorbed on SPEB. ° The activity of the enzymes was analyzed in terms of the Michaelis-Menten parameters Km and fecat. This study demonstrated that the activity of the enzymes was preserved despite the dense packing of proteins within the bmsh (Figure 31). Recently, it was shown that ITC is a useful tool to study the activity of immobilized enzymes. This technique allows studies of the enzymatic activity in turbid media, such as in concentrated suspensions of the SPEB. Moreover, ITC does not demand chromophores or fluorophores as photometric assays do. Thus, this method is very versatile because unlabeled substrates can be used. The results from ITC were in agreement with a typical photometric study, and showed that the activity of (1-glucosidase adsorbed on the SPEB is retained. 

Yoshida M, Kumakura M, Kaetsu I (1979) Immobilization of enzymes by radiation-induced polymerization of glass-forming monomers 2. Effects of cooling rate and solvent on porosity and activity of immobilized enzymes. Polymer 20 9-12... 

The use of a carrier or polymeric matrix to immobilize enzymes introduces a large noncatalytic component into the system which has the potential to interfere with the catalytic properties of the enzyme and reduce its activity compared to the same mass of free enzyme. Although this disadvantage is balanced by the reusability of immobilized enzymes, it would be even more beneficial if the activities of immobilized enzymes could match those of free enzymes. Carrier-free systems, in which enzyme molecules are linked to each other to form large complexes, may provide a solution to this problem. In a carrier-dependent system, up to 99.9% of the mass is taken up by the noncatalytic matrix. In noncarrier systems, 100% of the complex has the potential to retain catalytic activity. 

To solve the problem of diffusion limitation, the enzyme was used to be covalently immobilized on the surface of the alginate beads after activation with p-benzoquinone. The impact of different factors controlling the activation process of the alginate hydroxyl groups using /7-benzoquinone (PB(J) in addition to the immo-biUzation conditions on the activity of immobilized enzyme have been studied. The immobilized enzyme has been characterized from the bio-chemical point of view as compared with the free enzyme. 

Synowiecki J, Sikorski ZE, Naczk M (1981) The activity of immobilized enzymes on different krill chitin preparations. Biotechnol Bioeng 23 2211... 

C. Activity of Immobilized Enzymes (Reaction at Solid-Liquid Interfaces)... 

It is important to realize that biocompatibility issues are not only relevant in respect of the well being of the host, but also in respect of the requirements of the sensor itself. More specifically, the required chemical interactions between the sensor and the body must not be interfere with by interactions—either chemical or physical— between the membrane material and contacting/adhering cells. For example, an encapsulating membrane of an electrochemically based sensor must maintain appropriate mass transport conditions for the analyte and electrolyte species, and must exclude species that could interfere with the electrochemistry, or denature or inhibit the activity of immobilized enzymes. Stability of mass transport conditions is especially critic, since any change in the permeability of the membrane or the surrounding tissues can affect the sensor calibration. Satisfactory stability can not be achieved without a biocompatible encapsulation material. 

The application test of protein-engineered, thermostable glucose isomerase. The activity of immobilized enzyme is plotted as a function of time. The stability at 70 °C indicates how the enzymes will behave under industrial conditions. The variant Lys253Arg oi Actinoplanes missouriensis glucose isomerase has been shown to have (also under industrial conditions) a doubled half-life... 

As is well known, the activity of immobilized enzymes, especially covalently bound systems, is more resistant against heat and denaturing... 

An important case of solute partition is partition of hydrogen ions, since this can influence pH of the microenvironment, making it considerably different from the bulk solution pH. As a result, activity of immobilized enzymes display a shifted behavior with respect to the solution pH (Fig. 6.71). 

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