Fig. 1. Multimeric enzymes in industrial reactors.

Inactivation of multimeric enzymes may be strongly influenced by the dissociation of their subunits (see Fig. 1). Moreover, multipoint noncovalent interactions among monomers could stabilize the structure of each individual monomer correctly assembled in the multimer. On the contrary, conformational changes promoted by any denaturing agent (e.g., heat, pH, organic solvents) on the small fraction of dissociated monomers (not stabilized by these interactions) could be much more rapid (3). Dissociation of subunits may become even more relevant when working at the industrial scale. In general, we will try to use large volumes of reaction medium for many reaction cycles (see Fig. 1). The dissociation of a small fraction of subunits per reaction cycle could promote a very rapid deactivation of the biocatalyst. At first glance, it may be assumed that stabilization of the quaternary structure of multimeric enzymes may have very profitable effects on the industrial performance of these kind of enzymes.

1.1. Stabilization ofMultimeric Enzymes Via Multisubunit Immobilization

A very intense chemical cross-linking between all the protein subunits should be one of the best approaches to stabilization of the quaternary structure as well as the stabilization of the correct enzyme assembly. In this way, an activated support may be considered as a very good cross-linking reagent. Thus, we propose the multisubunit attachment of multimeric enzymes to very rigid supports as a way to obtain the stabilization of the assembling shape and of the quaternary structure (see Fig. 2). Of course, the design of such multisubunit immobilization is not a trivial problem and it may require a careful selection of the immobilization systems. To intensify the support-enzyme interaction, it is necessary to control some parameters that affect the intensity of this interaction, and in general they will be similar to those controlling the multipoint covalent attachment (2). Among the main parameters, the activation degree of the support, the internal morphology of the support (e.g., plane surface vs thin fibers), and the immobilization conditions (e.g., pH, temperature, time, in general all the parameters that can improve the reactivity of the reactive groups of proteins and supports) have been described as parameters critical to stabilization of the multimeric enzymes.

Multimeric Enzyme
| Long im mob ¡(Nation on supports having I Large internal surface covered by \ A very dense layer of active groups

Fig. 2. Stabilization of multimeric enzymes via multisubunit immobilization.

1.2. Stabilization of the Quaternary Structure of Immobilized Multimeric Enzymes Via Multisubunit Cross-Linking With Dextran-Aldehyde

Immobilization of proteins under optimal conditions is not always enough to fully stabilize the quaternary structure of all multimeric enzymes. In some cases, the subunits are not in the same plane (e.g., tetrahedric enzymes), making it impossible to stabilize the enzymes solely via immobilization even in plane surfaces (see Fig. 3). In these cases the enzyme still can release subunits to the reaction medium and become easily deactivated.

To prevent this, cross-linking of subunits of the previously immobilized enzyme using multifunctional polymers such as polyaldehyde dextran has been described as a successful technology (see Fig. 3). Dextrans are polymers of (1-6) glucose, with some ramifications (1,2) commercially available in a broad range of molecular weights from different companies (Fluka, Sigma). Each monomer of glucose contains two points that can be easily oxidized by periodate to yield two aldehyde groups per molecule of glucose (see Fig. 4) (3). That enables the production of a polyaldehyde that can react with any primary amino group of the support or the enzyme surface. The reduction with sodium borohydride yields irreversible secondary amine bonds between the protein or the support, and the polymer. Moreover, this reduces the aldehyde groups that are presented in the dextran to a highly hydrophilic and inert polyhydroxyl.

This reagent may be very useful in intersubunit cross-linking because

• They have a high number of reactive groups able to react with primary amino group and quite frequent in the protein surface.

• Its large size may permit involvement in the cross-linking groups places in different protein subunits.

Enzym Immobilization Cross Linking
Fig. 3. An integrated approach to the stabilization of multimeric enzymes.
Dextran React With Amino Group

Fig. 4. Preparation dextran-aldehyde by sodium periodate oxidation.

Dextran Aldehyde-dextran

Fig. 4. Preparation dextran-aldehyde by sodium periodate oxidation.

• Its large size decrease the competition between one-point chemical modification and protein cross-linking.

2. Materials

1. Epoxy Sepabeads® (EP-2 or 3) from Resindion SRL (Milan, Italy; see Note 1.)

2. Eupergit® 250 from Degussa (see Note 1).

3. Commercial dextran (from Leucosnostoc mesenteroides) from Sigma or Fluka.

4. Glycidol (2,3-epoxy-propanol) sodium metaperiodate and sodium borohydride (Sigma, St. Louis, MO).

5. Dialysis membrane (MW cut: 12,000 Da) (Medicell Ltd., London, England).

6. Immobilization buffer: 1Msodium phosphate at pH 8.0 adjusted with 5MNaOH (see Note 2).

7. Incubation buffer: 100 mM sodium phosphate 9.0 adjusted at pH 9.0 with 5 M NaOH. Some additives could be added.

8. Blocking solution: 3 Mglycine, pH 8.5.

9. Dextran to be oxidized was dissolved in distilled water.

10. Cross-linking buffer: 500 mM sodium phosphate, pH 7.0; adjusted with 5MNaOH.

11. Reduction buffer: 100 mM sodium carbonate adjusted at pH 10.0 with 1 MNaOH at 4°C.

12. NOVO buffer: 2.7 mM sodium citrate, 7.91 mM citric acid, 1.03 mM potassium sulfate, 2.99 mM sodium dihydrogen phosphate, 10.84 mM disodium hydrogen phosphate, 19.43 mM potassium hydroxide, 4.08 mM magnesium chloride, 5.1 mM calcium chloride, 3.33 mM sodium hydrogen carbonate, and sodium hydroxide to achieve a pH value of 6.5.

3. Methods

3.1. Immobilization of Proteins on Epoxy-Supports

1. The proteins were dissolved in the immobilization buffer (see Subheading 2.) and a sample was taken as reference (see Note 2).

2. Then, the support was added to the enzyme solution and stirred gently (see Note 3).

3. Samples of supernatant and suspension were periodically taken. Supernatant was achieved by using pipet filter or by centrifugation of the suspension (see Note 4).

4. After 24 h, the immobilized preparation was washed five times with three volumes of incubation buffer, and then resuspended in three volumes of that buffer. Stirring is not necessary in this step.

5. The immobilized protein was left to interact with the support for different times (anywhere from 1 d to 1 wk). Activity of the immobilized preparations could be followed all along the incubation.

6. The immobilized preparation was vacuum dried and resuspended in three volumes of blocking solution and stirred gently for 24 h.

7. Finally, the enzyme preparation was washed with distilled water and stored at 4°C.

3.2. Preparation of Aldehyde-Dextran

1. Dissolve 1.25 g of dextran in 37.5 mL of distilled water (this gives an equivalent glucose concentration of 184.8 mM).

2. Add 3 g of 370 mM solid sodium periodate while stirring to dissolve the salt.

3. After 3 h, put the solution in a dialysis bag, and dialyze this against 5 L, changing the water every 3 h during the first 6 h and then changing every 8 h for a total of 22 h.

4. Use this solution after preparation to prevent any undesired reaction (see Note 5).

3.3. Modification of the Immobilized Enzyme With Aldehyde-Dextran

1. Wash 4 g of the immobilized enzyme five times with three volumes of cross-linking buffer.

2. Resuspend the immobilized enzyme in 40 mL of aldehyde-dextran solution.

3. Check enzyme activity along the process.

4. Periodically, take 4 mL samples of the suspension and add to 20 mL of reduction buffer.

5. Add 24 mg of sodium borohydride. Wash with distilled water after 30 min.

3.4. Physical Determination of the Stabilization of the Quaternary Structure of Multimeric Enzymes

The enzyme-support bonds are very stable secondary amino bonds, ethers, or thioether; therefore, no protein-subunit bond to the support should be released to the medium. Thus, to asses the stabilization of the quaternary structure, the following protocol was followed:

1. Incubate 1 g of immobilized proteins in 2 mL 4% (w/v) sodium dodecyl sulfate (SDS), in the presence of 10% mercaptoethanol.

2. Boil this suspension for 5 min to promote the release of any noncovalently immobilized protein.

3. Take samples of the supernatant and analyze using SDS-polyacrylamide gel electrophoresis (PAGE) techniques following standard protocols.

3.5. Functional Determination of the Stabilization of the Quaternary Structure of Multimeric Enzymes

From an applied point of view, the main effect of the stabilization of multimeric enzymes should be that the enzyme concentration has no effect on the enzyme stability. To asses that:

1. Add 10 mL of the desired inactivation buffer to 1 g of immobilized enzyme.

2. Stir that suspension and, using a pipet with a cut tip, remove 1 mL and add it to 10 mL of the same inactivation buffer.

3. Perform the inactivation experiment.

3.6. Stabilization of a Multimeric fi-Galactosidase From Thermus sp. Strain T2 by Immobilization on Novel Heterofunctional Epoxy Supports Plus Aldehyde-Dextran Cross-Linking

The immobilized fi-galactosidase derivatives (on Sepabeads-epoxy supports partially modified with boronate, iminodiacetic, etc) showed improved stabilities after favoring multipoint covalent attachment by long-term alkaline incubation. The enzyme immobilized on Sepabeads-epoxy-boronic was the most stable. This derivative had some subunits of the enzyme not covalently attached to the support (detected by SDS-PAGE). To determine the amount of protein subunits not covalently attached to the support in the fi-galactosidase-boronic-epoxy-Sepabeads derivative (prepared using a crude preparation), the immobilized preparation was boiled in 2% SDS, and the proteins released to the supernatant were analyzed by SDS-PAGE. This analysis revealed that several subunits of multimeric proteins were not covalently attached to the support (see Fig. 5).

The quaternary structure of fi-galactosidase from Thermus spp. was completely stabilized after incubation of the fi-galactosidase-boronic-epoxy-Sepabeads derivative with aldehyde-dextrans under standard conditions (5,6) (see Fig. 5, lane 3).

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