Effects of Immobilization

In order to decide which immobilization technique to use it is first important to understand the changes in physical and chemical properties that an enzyme would be expected to undergo upon immobilization. Changes have been observed in the stability of enzymes and in their kinetic properties because of the microenvironment imposed upon them by a supporting matrix and also by the products of their own action. There is usually a decrease in specific activity of an enzyme upon immobilization that leads to the enzyme insolubilization; this can be attributed to denaturation of the enzyme protein caused by the coupling process.

With immobilized enzymes, the measured reaction rate depends not only on the substrate concentration and the kinetic constants Km (Michaelis constant) and Vmax (maximum velocity) but also on so-called immobilization effects. These effects are results of the following alterations of the enzyme by the immobilization process.

1.4.1. Changes in Enzyme Conformation

Conformation changes of the enzyme caused by immobilization usually decrease its affinity for the substrate (increase of Km). Furthermore, a partial inactivation of all—or complete inactivation of part—of an enzyme molecule may occur (decrease of Vmax). These two cases of a conformation-induced drop of Vmax may be distinguished by measuring the activity of the resolubilized enzyme by titration of the active center with an irreversible inhibitor.

1.4.2. Changes in Microenvironment

Ionic, hydrophobic, or other interactions between the enzyme and the matrix (microenvironmental effects) may also result in changed Km and Vmax values. These essentially reversible effects are normally caused by variations in the dissociation equilibrium of charged groups of the active center. Once an enzyme has been insolubilized, however, it finds itself in a microenvironment that may be drastically different from that existing in a free solution. The new microenvironment may be a result of the physical and chemical character of the support matrix alone or it may result from interactions of the matrix with substrates or products involved in the enzymatic reaction.

1.5. Nonuniform Distribution

A nonuniform distribution of substrate and/or product between the enzyme matrix and the surrounding solution affects the measured (apparent) kinetic constants.

1.5.1. Diffusion Limitations

In biosensors, the biocatalyst and the signal transducer are spatially combined (i.e., the enzyme reaction proceeds in a layer separated from the measuring solution). The substrate molecules reach the membrane system of the biosensor by convective diffusion from the solution.

Diffusion limitations are observed in various degrees in all immobilized enzyme systems. This occurs because the substrate must diffuse from the bulk solution up to the surface of the immobilized enzyme prior to reaction. The rate of diffusion relative to enzyme reaction determines whether there are limitations on intrinsic enzyme kinetics. The rate at which the substrate passes over the insoluble particle affects the thickness of the diffusion film, which in turn determines the concentration of the substrate in the vicinity of the enzyme and hence the rate of the reaction.

Molecular weight of the substrate may also play a large role. Diffusion of large molecules will obviously be limited by steric interactions with the matrix. This is reflected by the fact that the relative activity of bound enzymes toward high-molecular-weight substrates has generally been found to be lower than that towards low-molecular-weight substances. This may, however, be an advantage in some cases, because the immobilized enzymes may be protected from attack by large inhibitors molecules.

1.6. Enzyme Immobilization for Acetylcholinesterase-Biosensor Construction

1.6.1. General Considerations

Acetylcholinesterases (AChEs) are enzymes that are present in both invertebrates and insects and participate in nervous impulse transmission processes. These enzymes hydrolyze the natural neurotransmitter acetylcholine (ACh) and are inhibited by determined substances, among them organophosphorus and carbamate pesticides.

Amperometric biosensors based in immobilized AChEs have been used for the detection of these pesticides in several matrices (e.g., standard solutions, water, food) and their functionality is based on enzyme inhibition that is proportional to the pesticide amount present in the sample or solution.

Recently, the screen-printed electrodes have become the most widely used devices for construction of sensitive biosensors and, depending on the working electrode mediator used, the working potential may be of variable values. Different enzyme immobilization procedures have been used for the fixation of AChE enzyme on the analytical electrode, including physical entrapment with glutaral-dehyde and occlusion by polymerization in PVA with styrylpyridinium groups (PVA-SbQ).

In the method described here, 7,7,8,8-tetracyanoquinodimethano (TCNQ)-modified AChE-based biosensors will be constructed and used for detection of organophosphorus (OPs) insecticides. Immobilization procedure is based on polymerization with PVA-SbQ.

2. Materials

2.1. Preparation of the Screen-Printed Electrodes

1. DEK 248 printing Machine (DEK Printing Machines Ltd, www.dek.com).

2. PVC sheets.

3. Printing pastes: Electrodag® PF 410, Electrodag 423SS, and Electrodag 603SS (Acheson, Plymouth, UK).

4. Graphite T15 (Lonza, Switzerland).

5. Hydroxyl ethyl cellulose (HEC) (Fluka, Germany).

6. TCNQ: 7,7,8,8 Tetracyanoquinodimethane (Aldrich-Sigma, France).

7. Acetone: sodium dodecyl sulfate (SDS).

8. PVA-SbQ: polyvinylalcohol bearing styryl pyridinium groups (Toyo Gosei, Japan www.toyogosei.net).

9. AChE: acetylcholinesterases from electric eel (Sigma-Aldrich, www.sigma aldrich.com).

2.2. Tests of the Prepared Biosensor

1. 641 Metrohm detector (Metrohm, Zurich, Switzerland).

2. Flat bed recorder BD40 (Kipp and Zonen, the Netherlands).

3. Phosphate buffer solution (PBS) containing 0.05 MKCl, pH 7.5.

4. ATChCl : acetylthiocholine chloride (Sigma-Aldrich).

5. Paraoxon ethyl and dichlorvos (Dr. Ehrenstorfer, www.ehrenstorfer.de).

2.3. Solutions

1. Stock and working pesticide solutions. The pesticides paraoxon ethyl and dichlorvos will be used in this experiment. Prepare a 10-2 M stock solution by dissolving in acetone; use distilled water in order to prepare a more diluted working solution (see Note 1).

2. PBS containing KCl 0.05 M, pH 7.5: prepare the PBS solution by dissolving 18.2 g Na2HPO4, 3.6 g KH2PO4, and 3.0 g KCl in 1 L of distilled water. After solution homogenization, measure and adjust the pH to 7.5 with diluted NaOH (see Note 2).

3. Stock and working enzyme solutions: a commercially available AChE from electric eel [(ee)AChE] will be used. Prepare the AChE stock solution by dissolving the powder enzyme in an adequate 0.9% NaCl solution (see Notes 3 and 4). Working solutions can be prepared by dilution of the stock solution with PBS

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  • Cheryl
    What is the effect of enzyme immobilisation on the rate of reaction?
    8 years ago

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