Epoxy-Amino Groups: A New Tool for Improved Immobilization of

Biomacromolecules 2003, 4, 772-777

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Epoxy-Amino Groups: A New Tool for Improved Immobilization of Proteins by the Epoxy Method Cesar Mateo, Rodrigo Torres, Gloria Ferna´ ndez-Lorente, Claudia Ortiz, Manuel Fuentes, Aurelio Hidalgo, Fernando Lo´ pez-Gallego, Olga Abian, Jose M. Palomo, Lorena Betancor, Benevides C. C. Pessela, Jose´ M. Guisan,* and Roberto Ferna´ ndez-Lafuente* Departamento de Biocata´ lisis, Instituto de Catalisis, CSIC, Campus Universidad Autonoma, Cantoblanco, 28049 Madrid, Spain Received December 17, 2002; Revised Manuscript Received March 3, 2003

The properties of a new commercially available amino-epoxy support (amino-epoxy-Sepabeads) for immobilizing enzymes have been compared to those of conventional epoxy supports. The new support has a layer of epoxy groups over a layer of ethylenediamine that is covalently bound to the support. Thus, this support has a great anionic exchanger power and a high number of epoxy groups. We have found a number of advantages to this new heterofunctional support. Immobilization proceeds at low ionic strength using amino epoxy Sepabeads while requiring high ionic strength using conventional monofunctional epoxy supports. Immobilization is much more rapid using amino-epoxy supports than employing conventional epoxy supports. The possibility of achieving immobilized preparations in which the enzyme orientation may be different to that obtained using the traditional hydrophobic supports (with likely effects in terms of activity or stability). 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. Here we show some examples of these advantages using different enzymes (beta-galactosidases, lipase, glutaryl acylase, invertase, and glucoamylase). Introduction Epoxy-activated supports seem to be almost-ideal systems to develop very easy protocols for enzyme immobilization.1-5 Epoxy groups are very stable at neutral pH values even in wet conditions, and hence, commercial supports can be stored for long periods of time, and they can be prepared quite far from the place where the enzyme has to be immobilized. Furthermore, epoxy supports are able to react with different nucleophilic groups on the protein surface (e.g., amino, hydroxy, or thiol moieties) to form extremely strong linkages (secondary amino bonds, ether bonds, and thioether bonds) with minimal chemical modification of the protein (e.g., pK values of the new secondary amino groups are very similar to those of the preexisting primary amino groups). However, epoxy groups actually are hardly reactive for enzyme immobilization under mild experimental conditions (neutral pH, low ionic strength).6-8 Thus, the immobilization on this kind of supports take place through a two step mechanism: in the first step, the adsorption of the protein on the support surface is promoted4,9 (Scheme 1). For this reason, most commercial supports designed for protein immobilization have a fairly hydrophobic nature, and the recommended immobilization conditions on these supports include the use of high ionic strength (to force the hydro* To whom correspondence should be addressed. Dr. Roberto Ferna´ndezLafuente/Jose´ M. Guisan, Instituto de Catalisis, CSIC, Campus Universidad Auto´noma, 28049 Madrid, Spain. Fax: 34 91 585 47 60. Phone: 34 91 585 48 09. E-mail: [email protected]/[email protected].

Scheme 1. Mechanism of Immobilization of Proteins on Epoxy Supportsa

a The covalent reaction between soluble enzyme and epoxy support is extremely slow. However, the covalent reaction between previously adsorbed proteins and the support proceeds at very high apparent concentrations of reactive groups on the support and on the protein surface (an “intramolecular reaction”) giving a high covalent immobilization rate.

phobic adsorption of the proteins). In a second step, the protein which has been previously adsorbed is covalently attached to the epoxy groups present in the support surface. Occasionally, this may promote some stability problems on the immobilized enzyme, that may be, at least partially solved by a suitable chemical blocking protocol.10 Even with

10.1021/bm0257661 CCC: $25.00 © 2003 American Chemical Society Published on Web 04/10/2003

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Scheme 2. Covalent Immobilization of Enzymes on Epoxy-Amine Support Obtained by Modification of a Small Fraction of the Epoxy Groups Contained in the Supporta

Scheme 3. Covalent Immobilization of Enzymes on Epoxy-Amine Support Obtained by Modification of a Layer of Ethylenediamine with Epoxy Moieties Covalently Bound to the Support Surfacea

a The covalent immobilization is promoted via physical adsorption of amine groups contained in the modified epoxy support. In this case, suboptimal adsorption and covalent immobilization rates are obtained.

a The covalent immobilization is promoted via physical adsorption of amine groups contained in the “bottom” layer of epoxy amine support. In this case, a high correlation between adsorption and covalent immobilization rates are reached.

these limitations, standard epoxy-supports have been successfully used to stabilize enzymes via multipoint covalent attachment.10,11 Finally, to finish the protein-support reaction, epoxy groups can be easily blocked by reaction with very different thiol or amine compounds under mild conditions,12 preventing further uncontrolled reaction between the support and the enzyme that could decrease its stability. Having in mind this two-step mechanism for the covalent immobilization of proteins on epoxy supports, multifunctional supports have been recently proposed as a second generation of activated supports that are able to covalently immobilize enzymes, antibodies, and other proteins under very mild experimental conditions.9,13,14 In general, these multifunctional supports should contain two types of functional groups: i. groups that are able to promote the physical adsorption of proteins (e.g., by ionic exchange, by adsorption on immobilized metal chelates); and ii. groups that are able to covalently immobilize the enzyme (e.g., epoxy groups). In this way, enzymes are physically adsorbed on the support first via different phenomena and then some covalent linking could take place between nucleophilic groups of the protein (amino, thiol, and hydroxyl) and the epoxy groups on the support surface. In this first approach, such heterofunctional supports were prepared by the modification of some epoxy groups of the support with the compounds able to promote the adsorption9 and some interesting results have been achieved, immobilizing and purifying some enzymes.14,15 However, these first heterofunctional supports require a very small modification of the epoxy groups on the support surface (e.g., to modify 20% of the epoxy groups with amino groups) in order to preserve the maximum amount of epoxy groups to achieve the covalent attachment between the protein and the support. Thus, a compromise solution in the modification of the epoxy groups in the support between physical adsorption rate and covalent immobilization rate has to be reached. (Scheme 2).11

In this paper, we present a first evaluation of a support activated with these new epoxy-amino groups (Sepabeads EC-HFA) as an alternative to conventional epoxy supports in the immobilization of industrial enzymes.16 This support is commercially available from Resindion srl. In this case, the epoxy moieties have been introduced by modification of a layer of ethylenediamine covalently bound to the support surface (Scheme 3).17 Therefore, using these supports, there is a 1:1 ratio between the number of amino groups and the reactive epoxy groups (that determine the physical adsorption and covalent immobilization rate, respectively). To compare both, conventional and novel amino-epoxy supports, we have evaluated their performance in the immobilization of very different enzymes, following parameters of industrial relevance, such as immobilization rate, immobilization yield, intrinsic activity of the immobilized derivative, and stability of the final enzyme preparation. Materials and Methods Materials. Epoxy-Sepabeads (EC-EP1, EC-EP2, and ECEP3), ethylenediamine (EDA)-Sepabeads (FP-EA), and Epoxy-EDA-Sepabeads supports (EC-HFA) were kindly donated by Resindion S. R.L, (Mitsubishi Chemical Corporation; Milan, Italy). β-Galactosidase from Aspergillus oryzae (Grade XI) and lipase from Candida rugosa (Type VII) were purchased from Sigma Chemical Co. β-Galactosidase from Thermus sp. was produced as published elsewhere (Pessela et al., 2002). Glutaryl acylase was kindly donated from Bioferma Murcia S.A (Murcia, Spain). Invertase from baker’s yeast and glucoamylase from Aspergillus niger were kindly supplied by Novo Nordisk. Maltose, sucrose, p-nitrophenyl propionate (p-NPP), and o-nitrophenyl-β-D-galactopyronoside (o-NPG) were purchased from Sigma Chemical Co. Glutaryl 7-amino-cephalosporanic acid (7-ACA) was kindly donated by Bioferma Murcia S. A. All other reagents were of analytical grade.

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Methods Determination of the Enzyme Activity. 1. β-Galactosidases. A. Determination of the ActiVity of β-Galactosidase from A. oryzae. Activity was assayed by controlling the increase in the absorbance at 405 nm caused by the hydrolysis of 10 mM o-nitrophenyl β-D-galactopyranoside (o-NPG) in 0.1 M sodium acetate pH 4.5 at 25 °C in a stirred and temperature controlled cuvette. B. Determination of the ActiVity of β-Galactosidase from Thermus sp. Enzyme activity was assayed at pH 6.8 and 25 °C using 10 mM o-NPG in 50 mM potassium phosphate, containing 0.2 M KCl and 2 mM MgCl2. The rate of formation of free ortho-nitrophenol was determined spectrophotometrically at 405 nm using a stirred and temperature controlled cuvette. 2. Determination of InVertase ActiVity from Baker’s Yeast. Invertase activity was determined monitoring the hydrolysis of 5 mL of 100 mM sucrose in 0.1 M acetate buffer at pH 4.5 and 25 °C in a stirred and thermostatized vessel. At different times, 100 µL samples were withdrawn from the reaction solution. When using soluble enzymes, 20 µL of 0.1 M NaOH was added to stop the enzymatic reaction, followed by addition of 20 µL of 0.1 M HCl. Glucose produced by sucrose hydrolysis was measured spectrophotometrically at 505 nm using an enzymatic method (Glucose Trinder, Sigma Chemical Co). 3. Determination of Glucoamylase ActiVity from A. niger. Glucoamylase was determined monitoring the hydrolysis of 5 mL of 100 mM maltose in 0.1 M acetate buffer at pH 4.5 and 25 °C in a stirred and thermostatized vessel. At different times, 100 µL samples from the reaction solution were withdrawn. When using soluble enzymes, to stop the enzymatic reaction, 20 µL of 0.1 M NaOH was added to inactivate the glucoamylase, followed by addition of 20 µL of 0.1 M HCl. Glucose produced by maltose hydrolysis was measured spectrophotometrically at 505 nm using an enzymatic method (Glucose Trinder, Sigma Chemical Co). 4. Determination of Glutaryl Acylase ActiVity. Glutaryl acylase was measured using a pH-stat by titration of the glutaric acid released in the hydrolysis of 20 mL of 10 mM glutaryl 7-ACA in 100 mM sodium phosphate at pH 7.5 and 25 °C, using a stirred and thermostatized vessel. 25 mM NaOH was used as titrating reagent. 5. Determination of Lipase ActiVity from C. rugosa. Lipase activity was determined spectrophotometrically by controlling the increase in the absorbance at 405 nm caused by the hydrolysis of 0.4 mM p-NPP in 25 mM sodium phosphate pH 7 at 25 °C in a stirred and temperature controlled cuvette. Protein Determination. Soluble protein was determined by Bradford’s method19 using bovine serum albumin as protein standard. Desorption of the Proteins Physically Adsorbed to the Support but Not Covalently Bound. To test the covalent attachment of the proteins, the conditions of desorption of the proteins that were only physically adsorbed on the different supports (where the epoxy-groups have been completely destroyed by incubation on 100 mM sulfuric acid for 24 h) were studied.

Mateo et al.

Enzyme Immobilization on Epoxy-Supports. A total of 5 g of support were suspended in 45 mL of protein or enzyme solutions (maximum protein concentration was 0.1 mg/mL) in sodium phosphate pH 7, using different buffer concentrations (from 5 mM to 1 M) at 20 °C. This low enzyme load was chosen to prevent diffusion problems. Periodically, samples of the supernatants were withdrawn and analyzed for enzyme activity determination. In some cases, the immobilized enzyme was incubated under conditions in which the physically adsorbed protein molecules were released, to check that the immobilization was actually covalent. After immobilization, enzyme preparations were washed under those conditions to eliminate any protein molecule that had not been covalently attached to the support. Finally, to completely block the epoxy groups, 5 g of the support or enzyme-support preparations were incubated in 25 mL of 3 M glycine for 16 h at 20 °C (Mateo et al., 2002). Then, the enzyme preparations were washed with an excess of distilled water. Enzyme Stability Assays. Enzyme preparations (soluble and immobilized) were incubated under the specified conditions (depending upon enzyme preparation) and samples were periodically withdrawn and their remaining activities were assayed as described previously. Results and Discussion Enzyme Immobilization using Standard Sepabeads and Novel Amino Epoxy Sepabeds. Immobilization of proteins on conventional epoxy supports proceeds more rapidly at high ionic strength,10 whereas in the new amino-epoxy support, immobilization proceeds more rapidly at low ionic strength (results not shown). Thus, immobilization on the new amino-epoxy Sepabeads did not occur at 1 M sodium phosphate while immobilization on conventional Sepabeads did not occur using less than 250 mM of sodium phosphate.11 This could evidence a different mechanism of enzyme immobilization on the different supports, as described in ref 9. Figure 1 shows the immobilization courses of several enzymes (beta-galactosidases from A. oryzae and Thermus sp., and Invertase from baker’s yeast) using epoxy-Sepabeads and epoxy-amino Sepabeads under their respective optimal conditions. In all cases, immobilization on epoxy-amino Sepabeads was more rapid than immobilization on conventional epoxy Sepabeads. Using epoxy-amino Sepabeads, the presence of amino groups allows a very rapid ionic adsorption of the proteins onto the support at low ionic strength. On the other hand, using epoxy Sepabeads, enzyme adsorption requires high ionic strength and proceeds via the hydrophobic interaction between the external hydrophobic pockets of the enzyme and the hydrophobic surface of the support, which yielded moderate immobilization rates. In fact, in some examples, a fraction of the offered enzyme was immobilized using the standard support after 24 h, whereas all of the enzyme was immobilized on the new amino-epoxy supports (see Table 1). Significant differences in the recovery of activity after immobilization on the different supports may be also

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Table 2. Activity Recovery of the Different Enzymes Immobilized on Epoxy-Sepabeads and Epoxy-Amino-Sepabeads

enzyme (source)

epoxy Sepabeads recovered activity (%)a

epoxy-aminoSepabeads recovered activity (%)a

β-galactosidase (A. oryzae) β-galactosidase (Thermus sp.) invertase (baker’s yeast) glucoamylase (A. niger) lipase (C. rugosa) glutaryl acylase (n.a.)

15 50 90 87 5 100

100 100 70 75 65 100

a Recovered activity: Percentage of enzyme activity exhibited by the immobilized enzyme when compared to the soluble form. Immobilizations were performed in 5 mM and 1 M sodium phosphate pH 7 at 20 °C for 24 h with epoxy-amino and epoxy supports, respectively.

Figure 1. Immobilization courses of different enzymes on (A) Sepabeads EC-EP3 and (B) Sepabeads EC-HFA: (circles) β-galactosidase from A. oryzae (squares) β-galactosidase from Thermus sp. (triangles) Invertase from baker’s yeast. The enzymes were immobilized at pH 7.0 and 20 °C, using 1 M of sodium phosphate for standard Sepabeads and 5 mM for Sepabeads EC-HFA. Other specifications as described in Methods. Table 1. Immobilization Yields of the Different Enzymes Immobilized on Epoxy-Sepabeads and Epoxy-Amino-Sepabeads

enzyme (source)

epoxy Sepabeads immobilization yield (%)a

epoxy-aminoSepabeads immobilization yield (%)a

β-galactosidase (A. oryzae) β-galactosidase (Thermus sp.) invertase (baker’s yeast) glucoamylase (A. niger) lipase (C. rugosa) glutaryl acylase (n.a.)

36 58 60 85 85 75

100 100 100 100 88 100

a

Immobilization yield: Activity of the supernatant of the immobilization suspension compared to a reference suspension. Immobilizations were performed in 5 mM and 1 M sodium phosphate pH 7 at 20 °C for 24 h with epoxy-amino and epoxy supports, respectively.

observed (Table 2). For example, the lipase from C. rugosa (Figure 2), or β-galactosidase from A. oryzae were almost fully inactivated when using the conventional epoxy supports, whereas a high recovering of activity was detected when using the epoxy-amino support (see some examples in Table 2). This different effect on the enzyme activity of the immobilization when using the different supports (even although having both of them epoxy groups as reactive group) may be related to different enzyme orientations of the enzyme on the support. This implies that the enzyme likely reacts with the support via different areas (the areas of the protein surface that have more hydrophobic residues or the areas of the protein that have more negative charge) with concomitant different effects on enzyme activity. Moreover, the different nature of the support (hydrophobic

Figure 2. Effect of the type of support on the recovery of enzyme activity during immobilization of lipase from C. rugosa: (A) Sepabeads EC-EP3 (B) Sepabeads EC-HFA. Immobilizations were performed at pH 7.0 and 4° C as described in methods (squares) Suspension activity (circles) Supernatant Activity.

or ionic) may have also some relevance to explain the different results.10 Stability of the Immobilized Enzyme Preparations. Immobilization on both supports permitted the stabilization of the enzyme, (Figures 3-5). Interestingly, amino-epoxy immobilized enzyme preparations are more stable than the epoxy immobilized enzymes in many instances. For example, the immobilization on the epoxy-amino support allowed us to increase by around 5-fold the half-life of the glutaryl acylase compared to the immobilized preparation on epoxy support enzyme (Figure 5). In a similar way, half-lives of invertase (Figure 3) and β-galactosidase from Thermus sp. (Figure 4) were 4 and 3 times longer when immobilized on amino-epoxy supports than when immobilized on the epoxy conventional Sepabeads. We have not found any case in which the stability was higher using the epoxy support (glucoamylase stability was similar after immobilization on both supports and similar to the soluble enzyme and lipase and beta-galactosidase from A. oryzae were almost inactive).

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In any case, the new supports seems to yield enzyme preparation with better properties than conventional supports. Conclusion

Figure 3. Thermal stability of different preparations of Invertase from baker’s yeast: (triangles) soluble enzyme (squares) Sepabeads ECEP3 (circles) Sepabeads EC-HFA. Inactivation courses of soluble and immobilized enzyme preparations were performed in 50 mM sodium acetate at pH 4.5 and 55 °C.

Figure 4. Thermal stability of different beta-galactosidase from Thermus sp. preparations: (triangles) soluble enzyme (squares) Sepabeads EC-EP3 (circles) Sepabeads EC-HFA. Inactivation courses of soluble and immobilized enzyme preparations were performed in NOVO Buffer at pH 6.5 and 70 °C.

Figure 5. Thermal stability of different preparations of glutaryl acylase: (triangles) soluble enzyme (squares) Sepabeads EC-EP1 (circles) Sepabeads EC-HFA. Inactivation courses of soluble and immobilized enzyme preparations were performed in 25 mM Sodium Phosphate Buffer at pH 7.5 and 45 °C.

This higher stability should be explained by the differences between both kind of supports: i. the orientation of enzyme molecules onto the support surface (in conventional epoxy supports immobilization occurs via the most hydrophobic areas, whereas in this new supports occurs via the most negatively charged areas); ii. different possibilities of multipoint covalent attachment (the spacer arm using aminoepoxy is a little longer than using standard epoxy supports); iii. different hydrophobicity of the support surface even after blocking (Mateo et al., 2002).

Epoxy-amino Sepabeads have shown to be an interesting support for enzyme immobilization. The different immobilization mechanism (ionic adsorption) and the possibility to carry out the enzyme adsorptions at low ionic strength are advantages that have allowed us to obtain both better immobilization yields and enzyme activity recoveries. Moreover, the incorporation of a layer of amino groups in their epoxy-support structures can dramatically improve immobilization rates. Moreover, in many instances, the stability of the immobilized enzyme may be better using these supports than using traditional epoxy supports. Acknowledgment. The authors gratefully recognize the financial support and the supply of matrixes from Resindion SRL. This work has been partially sponsored by the Spanish CICYT (Projects BIO2001-2259 and PPQ 2002-01231). We thank a PhD fellowship from Universidad Industrial de Santander, Colombia for R. Torres; a Postdoctoral fellowship for G. Ferna´ndez-Lorente from CAM; a postdoctoral fellowship for A. Hidalgo from Basque Government; and a I3PBPG2001 fellowship for M. Fuentes from CSIC. References and Notes (1) Hartmeier, W. Immobilized biocatalysts-from simple to complex systems. Trends Biotechnol. 1985, 3, 149-153. (2) Katchalski-Katzir, E. Immobilized enzymes: Learning from past successes and failures. Trends Biotechnol. 1993, 11, 471478. (3) Bickerstaff, G. F. Immobilization of enzymes and cells. Methods in Biotechnology 1; Humana Press: Totowa, 1997. (4) Wheatley, J. B.; Schmidt, D. E. Salt induced immobilization of affinity ligands onto epoxide-activated supports. J. Chromatogr., A 1999, 849, 1-12. (5) Katchalski-Katsir, E.; Kraemer, D. Eupergit C, a carrier for immobilization of enzymes of industrial enzymes. J. Mol. catalysis B: Enzymatic 2000, 10, 157-176. (6) Hannibal-Friederich, O.; Chun, M.; Sernertz, M. Immobilization of galactosidase, albumin and globulin on epoxy-activated acrylic beads. Biotechnol. Bioeng. 1980, 22, 157-175. (7) Smalla, K.; Turkova, J.; Coupek, J.; Herman, P. Influence of salts on the covalent immobilization of proteins to modified copolymers of 2-hydroxyethyl methacrylate with ethylene dimethacrylate. Biotechnol. Appl. Biochem. 1988, 10, 21-31. (8) Melander, W.; Corradini, D.; Hoorvath. Salt-mediated retention of proteins in hydrophobic-interaction chromatography. Application of solvophobic theory. J. Chromatogr. 1984, 317, 67-85. (9) Mateo, C.; Ferna´ndez-Lorente, G.; Abian, O.; Ferna´ndez-Lafuente, R.; Guisan, J. M. Multifunctional Epoxy Supports: A new tool to improve the covalent immobilization of proteins: the promotion of physical adsorptions of proteins on the supports before their covalent linkage. Biomacromolecules 2000, 1, 739-745. (10) Mateo, C.; Abian, O.; Ferna´ndez-Lorente, G.; Pedroche, J.; Ferna´ndez-Lafuente, R.; Guisan, J. M.; Tam, A.; Daminati, A. Epoxy Sepabeads: A novel Epoxy Support for stabilization of industrial enzymes via very intense multipoint covalent attachment. Biotechnol. Prog. 2002, 18, 629-634. (11) Mateo, C.; Abian, O.; Ferna´ndez-Lafuente, R.; Guisan, J. M. Increase in conformational stability of enzymes immobilized on epoxyactivated supports by favoring additional multipoint covalent attachment. Enzyme Microbial Technol. 2000A, 26, 509-515.

Amino Epoxy-Supports for Enzyme Immobilization (12) Kramer, D. M.; Lehman, K.; Pennewiss, H.; Plainer, H. Oxirane acrylic beads for protein immobilization: a novel matrix for biocatalysis and biospecific adsorption. 26 International IUPAC Symposium on macromolecules, 1979. (13) Abad, J. M.; Ve´lez, M.; Santamarı´a, C.; Guisan, J. M.; Matheus, P. R.; Va´zquez, L.; Gazaryan, I.; Gorton, L.; Gibson, T.; Fernandez, V. M. Immobilization of peroxidase glycoprotein on gold electrodes modified with mixed epoxy-boronic acid monolayers. J. Am. Chem. Soc. 2002, 124 (43), 12845-12853. (14) Mateo, C.; Ferna´ndez-Lorente, G.; Cortes, E.; Garcı´a, J. L.; Ferna´ndez-Lafuente, R.; Guisan, J. M. One step purification, covalent immobilization and additional stabilization of poly-hys-tagged proteins using a novel heterofunctional chelate-epoxy supports. Biotechnol. Bioeng. 2001, 76, 269-277. (15) Pessela, B. C.; Mateo, C.; Carrascosa, A. V.; Vian, A.; Garcı´a, J. L.; Guisan, J. M.; Ferna´ndez-Lafuente, R. One step purification, covalent immobilization and additional stabilization of a thermophilic polyhis-tagged beta-galactosidase of Thermus sp. strain t2, novel heterofunctional chelate-epoxy supports. Biomacromolecules 2003, 4, 107-113.

Biomacromolecules, Vol. 4, No. 3, 2003 777 (16) Guisan, J. M.; Ferna´ndez-Lafuente, R.; Mateo, C.; Torres, R.; Ferna´ndez-Lorente, G.; Ortiz, C.; Fuentes, M.; Hidalgo, A.; Palomo, J. M.; Lo´pez-Gallego, F.; Betancor, L.; Pessela, B. C. C. Nuevo metodo de inmovilizacion de enzimas y otras bio-macromoleculas sobre soportes activados con grupos epoxido conteniendo grupos ionizados en el brazo espaciador que une cada grupo epoxido a la superficie del soporte. Spanish patent application No. 200,300,428, 2003. (17) Tam, A.; Re, D.; Cami, P.; Daminati, M. Supporti per l’immobilizzazione covalente di enzimi. Italian Patent No. MI2002A000729, 2002. (18) Pessela, B. C. Ch.; Vian, A.; Mateo, C.; Ferna´ndez-Lafuente, R.; Garcı´a, J. L.; Guisa´n, J. M.; Carrascosa, A. V. Over production of Thermus sp. T2 β-galactosidase in Escherichia coli and purification using tailor-made metal chelate supports. Appl. EnViron. Microbiol. 2003B, in press. (19) Bradford, M. M. A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem. 1976, 72, 248-254.

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