Immobilization and Stabilization of Proteins by Multipoint Covalent Attachment on Novel Amino EpoxySepabeads

Cesar Mateo, Benevides C. C. Pessela, Valeria Grazu, Fernando López-Gallego, Rodrigo Torres, Manuel Fuentes, Aurelio Hidalgo, Jose M. Palomo, Lorena Betancor, Gloria Fernández-Lorente, Claudia Ortiz, Olga Abian, Jose M. Guisan, and Roberto Fernandez-Lafuente

Summary

The prospects of a new commercially available support (amino-epoxy-Sepabeads®) for enzyme immobilization are discussed in this chapter. These supports have a layer of epoxy groups over a layer of ethylenediamine that is covalently bound to the support. Thus, the support has a high anionic exchanger power and a high number of epoxy groups. Some relevant properties of this support are (1) immobilization proceeds at low ionic strength using amino-epoxy-Sepabeads, (2) immobilization is much more rapid using amino-epoxy supports than employing conventional epoxy supports, and (3) stability of the immobilized enzyme has been found to be much higher using the new support than in preparations using the conventional ones in many cases. Therefore, this support may be a good complement to the traditional hydrophobic epoxy supports.

Key Words: Heterofunctional supports; ionic adsorption plus multipoint covalent attachment; third generation of epoxy supports.

1. Introduction

Epoxy-activated supports are almost ideal matrices to perform very easy immobilization of proteins and enzymes at both laboratory and industrial scale (1-6). These activated supports are very stable during storage and during suspension in neutral aqueous media. Hence, they are easily handled before and during immobilization processes as well as over the long term. Furthermore, epoxy-activated supports are able to directly form very stable covalent linkages with different protein groups (e.g., amino, thiol, phenolic) under very mild experimental conditions

Epoxy Group Immobilization
Fig. 1. Mechanism of immobilization of proteins on epoxy supports.

such as pH 7.0. The immobilization follows a two-step mechanism: first, a rapid mild physical adsorption between the protein and the support is produced and then a covalent reaction between the adsorbed protein and the epoxy groups occurs (511) (see Fig. 1).

However, the immobilization onto conventional epoxy supports has several problems:

1. Use of high ionic strength, which can affect the stability of certain enzymes.

2. The enzymes require a hydrophobic area to be immobilized on the support.

3. The enzymes will be immobilized by their hydrophobic area, which may be not the most convenient to stabilize the enzyme.

4. Hydrophobic supports are required for this immobilization, with a likely negative effect on the enzyme stability.

1.1. Multifunctional Epoxy Supports for Protein Immobilization

Bearing in mind this two-step mechanism for covalent immobilization of proteins on epoxy supports, multifunctional epoxy supports have been proposed as a second generation of activated supports that are able to covalently immobilized enzymes, antibodies, or other molecules under very mild experimental conditions. These multifunctional supports should contain two types of functional groups: (1) groups that are able to promote the physical adsorption of proteins (such as ionic

Epoxy Protein Immobilization
Fig. 2. Some different multifunctional epoxy supports for protein immobilization.

exchangers or by metal-chelate adsorption), and (2) groups that are able to co-valently immobilize the enzymes such as epoxyde groups (12-17). The partial modification of the epoxy supports with ethylenediamine, iminodiacetic acid, metal chelates, or m-amino-phenyl-boronic acid (see Fig. 2) may be good alternatives for reaching this goal.

1.2. Third Generation of Epoxy Supports

Use of the first heterofunctional supports required substitution of some epoxy moieties by groups that could promote the physical adsorption of the proteins to the support. This resulting decrement in the number of epoxy groups available for the covalent multipoint immobilization compromised the desired solutions (see Fig. 3) (12). To avoid this, Residion srl (Milano, Italy) has developed a third generation of epoxy supports, in which the epoxy moieties have been introduced by attaching them to ethylenediamine (ED) covalently bound to the support surface (see Fig. 4) (16,17). Therefore, using these supports, there is a 1:1 ratio between the number of amino groups and the reactive epoxy groups, which determine the physical adsorption and covalent immobilization rate, respectively.

Epoxy Protein Immobilization
Fig. 3. Covalent immobilization of enzymes on epoxy-amine support obtained by modification of a small fraction of the epoxy groups contained in the support.
Eupergit Protein Immobilization
Fig. 4. Covalent immobilization of enzymes on epoxy-amine supports obtained by modification of a layer of ethylenediamine with epoxy moieties covalently bound to the support surface.

2. Materials

1. Commercial EC-HFA (new amino-epoxy supports) was purchased from Resindion SRL (Milan, Italy; see Note 1).

2. Immobilization buffer: 5 mM sodium phosphate buffer, pH 7.0.

3. Incubation buffer: 5 mM sodium bicarbonate buffer, pH 9.0 to 10.0.

4. Blocking solution: 3 M glycine, pH 8.5.

5. Desorption buffer: 1 M sodium phosphate, pH 7.0.

3. Methods

3.1. Preparation of Amino-Epoxy Supports and Enzyme Solution

1. Wash the support 10 times with five volumes of 5 mM sodium phosphate buffer, pH 7.0, at 4°C using a Büchner flask with glass-sintered funnel connected to a vacuum line.

2. Dissolve the proteins in 5 mM sodium phosphate, pH 7.0, take a sample as reference blank and test the enzyme activity (see Note 2).

3.2. Immobilization of Proteins on Amino-Epoxy Supports

1. Add the support to the enzyme solution prepared as described in Subheading 2., step 1.

2. Gently stir the suspension (enzyme and gel) at 25°C (see Note 3).

3. Periodically, samples of supernatant and suspension were taken for assay of enzyme activity. Supernatant was achieved by using a tip filter or by centrifugation of the suspension (see Note 4).

4. Assay the enzyme activity of reference solution at the same time intervals as in step 3. The immobilization process is finished when the activity of the supernatant is zero.

3.3. Desorption of Proteins Noncovalently Immobilized on the Support

1. 2.5 mL of enzymatic suspension were taken and dried by filtration under vacuum.

2. The dried support was resuspended in 2.5 mL of 1 M sodium phosphate buffer, pH 7.0. The suspension was gently stirred for 30 min at 20°C.

3. The enzyme activity or the protein concentration of the supernatant was checked.

4. If there are no proteins released, covalent attachment was considered.

3.4. Multi-Interaction Step on Enzyme Immobilization

1. Then, the immobilized preparation was washed five times with three volumes of 5 mM sodium phosphate solution and then resuspended in three volumes of 100 mM sodium bicarbonate buffer, pH 9.0 to 10.0. Stirring is not necessary in this step.

2. The immobilized protein was left to interact with the support for different times (ranging from 1 d to 1 wk) before incubation with 3 M glycine, pH 8.5. Activity of the immobilized preparations could be followed all along the incubation (i.e., every day; see Note 5).

Enzyme Immobilisatiobn

Fig. 5. P-galactosidase from A. oryzae immobilization on Sepabeads EC-EP1 (A) and Sepabeads EC-HFA (B) supports: (•) activity of the suspension; (■) activity of the supernatant. The enzyme was immobilized in presence of 1 M sodium phosphate when using Sepabeads EC-EP1 and 5 mM sodium phosphate for Sepabeads EC-HFA. All the enzyme immobilizations were carried out at 20°C, pH 7.0) as described in Subheading 3.

Fig. 5. P-galactosidase from A. oryzae immobilization on Sepabeads EC-EP1 (A) and Sepabeads EC-HFA (B) supports: (•) activity of the suspension; (■) activity of the supernatant. The enzyme was immobilized in presence of 1 M sodium phosphate when using Sepabeads EC-EP1 and 5 mM sodium phosphate for Sepabeads EC-HFA. All the enzyme immobilizations were carried out at 20°C, pH 7.0) as described in Subheading 3.

3.5. Blocking of Epoxy Groups of the Supports

1. The immobilized preparations were vacuum dried and resuspended in three volumes of 3 M glycine, pH 8.5, and stirred gently at 20°C for 8 h.

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

3.6. Immobilization of fi-Galactosidase From A. oryzae on Sepabeads® EC-HFA1

1. Dissolve 500 mg of solid fi-galactosidase (Sigma, grade XII) in 90 mL of 5 mM sodium phosphate solution, pH 7.0.

2. Assay the catalytic activity of this solution. Add 10 g of Sepabeads EC-HFA1 and assay the enzyme activity of both suspension and supernatant after 15 min. If any activity remains in the supernatant, stir the suspension for an additional 15 min, then repeat again the enzyme assays (see Fig. 5).

3. Stir this suspension very gently for 24 h at 25°C.

4. After this, evaluate covalent immobilization as described earlier (see Subheading 3.3.).

Enzyme Immobilization

Fig. 6. Thermal stability of different P-galactosidase preparations covalently immobilized on Sepabeads EC-HFA. (•) Soluble enzyme. The enzyme was immobilized in 5 mM sodium phosphate, pH 7.0 at 20°C for 24 h and then blocked with glycine (■) or the pH was adjusted at 10.0 and the reaction enzyme-support reaction was left to proceed for 8 h before blocking (♦). Inactivation of enzyme derivatives was carried out at 55°C, pH 4.5.

Fig. 6. Thermal stability of different P-galactosidase preparations covalently immobilized on Sepabeads EC-HFA. (•) Soluble enzyme. The enzyme was immobilized in 5 mM sodium phosphate, pH 7.0 at 20°C for 24 h and then blocked with glycine (■) or the pH was adjusted at 10.0 and the reaction enzyme-support reaction was left to proceed for 8 h before blocking (♦). Inactivation of enzyme derivatives was carried out at 55°C, pH 4.5.

5. Increase and adjust the enzyme suspension at pH 10.0 and gently stir the enzymesupport reaction for 8 h (see Note 5 and Fig. 6).

6. Filter the suspension and then block the epoxy groups as described in Subheading 3.5., step 1.

7. Then wash and filter the suspension with 25 mM phosphate buffer, pH 7.0, and the distilled water as described in Subheading 3.5., step 2. Finally, filter it to dryness

3.7. Immobilization of Glutaryl Acylase on Sepabeads EC-HFA2

1. Mix 5 mL of a glutaryl acylase (GA) from Roche with 20 mL of 5 mM sodium phosphate solution, pH 7.0.

2. Assay the catalytic activity of this solution. Add 10 g of Sepabeads EC-HFA2 to 20 mL of the previous GA and assay the enzyme activity of both suspension and supernatant after 15 min. If any activity remains in the supernatant, stir the suspension for an additional 15 min, then repeat again the enzyme assays (see Fig. 7).

3. Stir this suspension very gently for 24 h at 25°C.

4. After this, evaluate covalent immobilization as described above (see Subheading 3.3.)

5. Increase and adjust the enzyme suspension at pH 10.0, gently stir the enzymesupport reaction for 7 d at 4°C (see Note 5). (See effect on enzyme stability in Fig. 8.)

6. Filter the suspension and then block the epoxy groups as described in Subheading 3.5., step 1.

7. Wash and filter the suspension with 25 mM phosphate buffer, pH 7.0, and the distilled water as described in Subheading 3.5., step 2. Finally, filter it to eliminate all inter-particle water.

Enzyme Immobilization

Fig. 7. Immobilization course of GA onto EC-HFA2 at different ionic strenghs. (A) Immobilization was carried out in 25 mMpotassium phosphate buffer at 25°C, pH 7.0. (♦) Supernatant. (■) Suspension. (A) Control (soluble enzyme in immobilization conditions). (B) Immobilization was carried out in 1Mpotassium phosphate buffer at pH 7.0. (♦) Supernatant. (■) Suspension. (A) Control (soluble enzyme in immobilization conditions).

Fig. 7. Immobilization course of GA onto EC-HFA2 at different ionic strenghs. (A) Immobilization was carried out in 25 mMpotassium phosphate buffer at 25°C, pH 7.0. (♦) Supernatant. (■) Suspension. (A) Control (soluble enzyme in immobilization conditions). (B) Immobilization was carried out in 1Mpotassium phosphate buffer at pH 7.0. (♦) Supernatant. (■) Suspension. (A) Control (soluble enzyme in immobilization conditions).

4. Notes

1. Sepabeads EC-EP is a commercially available epoxy support with different pore sizes (Resindion SRL) and epoxy groups concentration (EC-EP1 [little], EC-EP2 [medium], and EC-EP3 [large]).

2. If it is necessary, verify reference enzyme activity to evaluate yield and recovery activity of enzyme immobilization process.

Enzyme Immobilization

Fig. 8. Effect of the incubation at alkaline pH value (before blocking the remaining epoxy in the support) on the stability of immobilized glutaryl acylase EC-HFA2. The derivative was incubated in 25 mM sodium bicarbonate at 4°C, pH 10.0 (A) Suspension. All derivatives were thermally inactivated at 45°C in potassium phosphate buffer, pH 7.0, and the relative stability was defined as the ratio between the half-lives of the derivatives and soluble enzyme, respectively (A). The percentage of lost activity during the incubation at pH 10.0 (■) was analyzed at different times.

Fig. 8. Effect of the incubation at alkaline pH value (before blocking the remaining epoxy in the support) on the stability of immobilized glutaryl acylase EC-HFA2. The derivative was incubated in 25 mM sodium bicarbonate at 4°C, pH 10.0 (A) Suspension. All derivatives were thermally inactivated at 45°C in potassium phosphate buffer, pH 7.0, and the relative stability was defined as the ratio between the half-lives of the derivatives and soluble enzyme, respectively (A). The percentage of lost activity during the incubation at pH 10.0 (■) was analyzed at different times.

3. Amounts, kind of EC-HFA support ratios Vgel/Vsuspension used, and reaction times must be established for each enzyme. Avoid magnetic stirring to reduce abrasion of the support.

4. If the enzyme activity is decreased because of enzyme inactivation it must be distinguished from loss in enzyme activity of the supernant resulting from immobilization process.

5. Multipunctual derivatives can be reached through long-time incubation of enzyme suspension after the immobilization has concluded. Additional bonds may be formed by keeping the suspension at pH 10.0. The optimum multi-interaction time required for each enzyme must be established in every case. For this purpose, it is necessary to prepare enzyme derivatives with different multi-interaction times and check the thermal stability of each. The time of choice is a compromise between the shortest time that provides both an optimal stability and enzyme recovery.

References

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10. Melander, W., Corradini, D., and Hoorvath, C. (1984) Salt-mediated retention of proteins in hydrophobic-interaction chromatography. Aplication of solvophobic theory. J. Chromatogr. 317, 67-85.

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13. Pessela, B. C. C., Mateo, C., Carrascosa, A. V., et al. (2003). One step purification, covalent immobilization and additional stabilization of a thermophilic poly-his-tagged P-galactosidase of Thermus sp. strain t2, novel heterofunctional chelate-epoxy supports. BiomacromoJecuJes. 4, 107-113.

14. Mateo, C., Archelas, A., Fernandez-lafuente, R., Guisan, J. M., and Furstoss, R. (2003) Enzymatic transformations. Immobilized A. niger epoxide hydrolase as a novel biocatalytic tool for repeated-batch hydrolytic kinetic resolution of ep-oxides. Org. BiomoJ. Chem. 1, 2739-2743.

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16. Torres, R., Mateo, C., Fernandez-Lorente, G., et al. (2003) A novel heterofunctional epoxy-amino Sepabeads for a new enzyme immobilization protocol: immobilization-stabilization of beta-galactosidase from AspergiJJus oryzae. BiotechnoJ. Progr. 19, 1056-1060.

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