Cesar Mateo, Benevides C. C. Pessela, Manuel Fuentes, Rodrigo Torres,
Claudia Ortiz, Fernando López-Gallego, Lorena Betancor,
Noelia Alonso-Morales, Jose M. Guisan, and Roberto Fernandez-Lafuente
In this chapter, the properties of tailor-made anionic exchanger resins based on films of large polyethylenimine polymers (e.g., molecular weight 25,000) as supports for strong but reversible immobilization of proteins is shown. The polymer is completely coated, via covalent immobilization, the surface of different porous supports. Proteins can interact with this polymeric bed, involving a large percentage of the protein surface in the adsorption. Different enzymes have been very strongly adsorbed on these supports, retaining enzyme activities. On the other hand, adsorption is very strong and the derivatives may be used under a wide range of pH and ionic strengths. These supports may be useful even to stabilize multimeric enzymes, by involving several enzyme subunits in the immobilization.
Key Words: Polymeric beds; volume effect; nondistorting but strong adsorption; reversible immobilization.
Many protocols for enzyme immobilization involve the irreversible cova-lent binding between the enzyme and a pre-existing support. Usually, when the immobilized enzyme becomes inactivated during its industrial application both the enzyme and the support should be eliminated as wastes (1-8). In this way, these conventional protocols for enzyme immobilization have two important drawbacks: (1) a relatively high cost resulting from the use of large amounts of expensive supports and the performance of more or less complex immobilization protocols; and (2) the production of large amounts of waste when the immobilized derivatives become inactivated (such as the inactivated enzymes covalently immobilized on polymeric resins). Hence, these protocols for enzyme immobili-
From: Methods in Biotechnology: Immobilization of Enzymes and Cells, Second Edition Edited by: J. M. Guisan © Humana Press Inc., Totowa, NJ
zation are only economically sustainable when the immobilized derivative is very stable and can be reused for many reaction cycles for the catalysis of high value-added processes.
From this point of view, the reversible immobilization of enzymes on pre-existing supports could be a very convenient protocol for the immobilization of many industrial enzymes. Thus, reversibility means the possibility of promoting the complete desorption of the enzyme away from the support when the immobilized derivative becomes inactivated and it cannot be further used in the industrial reactor. The supports can then be recovered fully intact and protein free, and they become ready to be used again for a new immobilization of a fresh solution of soluble enzyme. The support, even a fairly expensive one, can be used indefinitely, and the only waste produced is a solution of inactivated enzyme. We can summarize a number of advantages of such methods for reversible immobilization of industrial enzymes (e.g., via physical adsorption of enzyme on activated supports (1-12):
1. Activated supports are chemically inert and hence they are very stable during transport, storage.
2. Protocols for immobilization are usually very simple and carried out under very mild conditions.
3. After enzyme inactivation, the enzyme can be desorbed away from the support and this support can be reused for many times with the subsequent reduction of costs and wastes.
4. Complex enzyme reactors such as monolith reactors for treatment of large effluent volumes can be "in sitU' regenerated after enzyme inactivation.
A number of protocols for this reversible immobilization of industrial enzymes have been reported in recent years (9-15). However, the most popular, simplest, and oldest protocol for reversible immobilization of enzymes is the adsorption of enzymes on ionic exchange resins. In fact, more than 25 yr ago, the first industrial process catalyzed by immobilization of enzymes (resolution of D,L-amino acids by amino acylase) was developed using this type of immobilized preparation (6,7). This reversible immobilization of enzymes is frequently fairly mild, and most of the proteins are therefore fully desorbed from such matrices at moderate and even low ionic strength (0.2-0.3 MNaCl). In this way, high concentrations of ionizable substrates, changes of pH during the reaction, and so forth can promote undesirable leakage of immobilized enzyme away from the support, promoting an apparent inactivation of the enzyme derivative and a certain contamination of the product (1-4).
The criteria for the selection and development of new anion exchange supports for immobilization of industrial enzymes are very different from the most adequate criteria to design supports for purification of enzymes and proteins by ion exchange chromatography. Approaches, immobilization, or purification may have very different requirements:
1. The key objective of enzyme and protein chromatography is the recovery of fully intact enzymes and proteins after adsorption on the support. The supports should be designed to permit a mild desorption of native proteins.
2. The key objective of the reversible immobilization of industrial enzymes is the prevention of enzyme leakage during catalytic operation and the further recovery of fully intact supports after enzyme inactivation. Therefore, the promotion of a very strong adsorption (and simultaneously, a difficult desorption) of the proteins on the supports is now very convenient. Desorption of the enzymes has only to be performed after enzyme inactivation and hence the mild recovery of intact proteins is not necessary. Now, the necessity of using drastic conditions such as very high ionic strength or extreme pHs for the desorption of inactivated proteins preserving supports is not a problem but it becomes an important advantage.
With all of this in mind, it has been proposed that the preparation and selection of a new generation of matrices for a very strong ionic adsorption of enzymes is very suitable for the design of reversible immobilization of industrial enzymes (9-12). Composites based on the full coating of rigid porous supports with flexible polymers containing a very high concentration of ion exchange moieties are proposed as new matrices that are very promising to get a mild and very strong adsorption of industrial enzymes (although very inadequate for protein purification; see Fig. 1). In this way, the adsorption of industrial enzymes on a very high concentration of ionic exchange moieties placed on a flexible bed that is coating a rigid surface may be much stronger than adsorptions promoted on conventional ionic exchange resins (rigid surfaces containing a much lower concentration of ionic exchange moieties).
Supports activated with different groups (e.g., glyoxyl, amino, epoxy) can be covalently coated with different ionic polymers such as polyethyleneimine, polyalylamine, and aldehyde-aspartic dextran. Supports with ionic groups (e.g., carboxymethyl, sulfonic, amino) may be ionically coated with polymers of the opposite charge (e.g., amino support with sulfate-dextran). The coating conditions (e.g., time, polymer size and concentration, pH, and ionic strength) have been proven to be critical to this goal, because a polymeric bed formed by the full coating of the surface with very flexible immobilized ionic polymers is desired (see Figs. 2 and 3) (9-12).
1. Sepabeads® EC-EP (epoxy-activated supports) and Sepabeads-MANAE (primary amino-activated supports) supplied by Mitsubishi Chemical Co. (Milano, Italy).
2. Sulfate-dextran (different molecular weights) supplied from Sigma Chemical Co. (St. Louis, MO).
3. Polyethylenimine (PEI; different molecular weights) from Sigma.
4. Carboxymethyl cellulose (CMC) from Sigma.
5. Sulfate-dextran coating buffer: 25 mM sodium phosphate buffer, pH 7.0.
6. Sodium periodate supplied by Merck (Darmstadt, Germany)
7. Sodium borohydride supplied by Sigma.
8. Immobilization buffer: 5 mM sodium phosphate buffer 5 mM, pH 7.0, or 5 mM sodium acetate buffer, pH 5.0.
9. Desorption buffers: 5 mM sodium phosphate buffer at different pH values (from 5.0 to 9.0) with different NaCl concentrations.
10. Washing solution to prepare PEI support: 100 mMsodium acetate buffer, pH 4.0, and 100 mM sodium borate buffer, pH 9.0, and water.
11. Washing solution to prepare sulfate-dextran support: 100 mM sodium phosphate buffer, pH 7.0, and water.
1. Epoxy supports were incubated in 10 volumes of 100 mM sulfuric acid for 24 h to open all epoxy groups. Then, the support was equilibrated with 0.1 M sodium phosphate, pH 7.0, in batch during 2 h, and then filtered and washed with an excess of distilled water. Until the washing keep the initial pH value.
2. The support was added to 10 volumes of 50 mM sodium periodate for 2 h. Then, the aldehyde-support was washed with an excess of distilled water.
3. Next, aldehyde-supports were incubated at pH 11.0 with 10 volumes of 10% PEI (w/v) for 12 h under mild stirring at 25°C.
4. The support was reduced by adding 20 mg of solid sodium borohydride/mL of suspension during 1 h.
5. After, the PEI-supports were washed with 100 mM sodium acetate buffer, pH 4.0, and 100 mM sodium borate buffer, pH 9.0. Finally, the PEI-Sepabeads complex was washed exhaustively with distilled water.
1. 10 g MANAE-Sepabeads were incubated in 20 mL of dextran-sulfate solution (8 g of dextran-sulfate [500 kDa] dissolved in 20 mL of 25 mM sodium phosphate buffer, pH 7.0) for 16 h at 25°C, under mild stirring.
2. Then, the composites were washed with 1 L of 100 mM sodium phosphate buffer, pH 7.0, under mild stirring for 1 h to decrease sulfate-dextran viscosity.
3. After that, the composites were washed with abundant water to eliminate the excess of sulfate-dextran.
3.3. Immobilization/Adsorption of Proteins on Ionic Exchanger Supports
1. For each of the different supports 5 g were suspended in 25 mL of an enzymatic solution (1 mg/mL or 1 U/mL was the maximum concentration in order to avoid diffusion problems) in sodium phosphate 5 mM at pH 5.0 or 7.0 at 20°C. The suspension was stirred gently.
2. Periodically, samples of supernatant and suspension were taken and the enzymatic activity and/or the protein concentration was measured by using the Bradford method.
3. After the immobilization process the preparations were washed with the same buffer use in the immobilization process.
3.4. Desorption of Proteins on Ionic Exchanger Supports
1. 5 g of composites containing the immobilized proteins were suspended in 5 mM sodium phosphate buffer, pH 5.0 to 9.0. Increasing concentrations of NaCl were then added.
2. Samples were taken from the supernatant 30 min after the NaCl addition (longer incubations times up to 3 h did not result in significant increments in the des-orbed protein) and the protein concentrations were determined by Bradford method. When studying enzymes, desorption was checked via enzyme activity determinations. A reference solution with soluble enzyme was submitted to the same treatment to detect any possible effect the NaCl upon activity of the enzyme. The percentage of desorbed enzyme is referred to the amount of adsorbed one.
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