Fig. 9. Analysis by SDS-PAGE of the selectivity of the immobilization of poly-His-tagged P-galactosidase on different Sepabeads supports. Lane 1, molecular weight markers; lane 2, crude preparation of P-galactosidase; lane 3, proteins that are not immobilized on IDA-Co-Sepabeads; lane 4, proteins that are desorbed from glycine-blocked IDA-Co-Sepabeads derivatives; lane 5, proteins that are not immobilized on standard Sepabeads; lane 6, proteins that are desorbed from glycine-blocked standard Sepabeads derivatives. The immobilized preparations were in all cases blocked with mercaptoethanol before the desorptions.

selective than the immobilization on the conventional one. Figure 9 shows that although many protein subunits could be detected using the standard support, only a band of 67 kDa (which corresponds to the molecular weight of the monomer of Htag-BgaA) could be detected in the bifunctional support.

3. Activity standard assay was performed with o-nitrophenyl- P-D-galactopyranoside (ONPG) at 25°C. ONPG was dissolved in Novo buffer, pH 6.5, and used at a final concentration of 13.3 mM

4. One unit of P-galactosidase activity is defined as the amount of enzyme that produces 1 |imol of o-nitrophenol per minute under the conditions described.

3.7. Structural Stabilization of Poly-His-Tagged P-Galactosidase From Thermus spp. Strain T2 on Sepabeads FP-EP IDA-Epoxy Via Multipoint Covalent Attachment

1. Immobilized enzymes derivative prepared on Sepabeads FP-EP IDA-epoxy were further incubated after covalent immobilization at pH 10.0 for 24 h. This was carried out to favored multipoint covalent attachment (see Subheading 3.5.). Figure 10 shows that this derivative of Htag-BgaA was more stable than the soluble enzyme and even more stable than that prepared using the conventional epoxy supports. Moreover, this immobilized derivative retained almost 100% of the initial activity after several weeks of incubation at 50°C (temperature employed normally at industrial pasteurization processes; see Fig. 11).

Fig. 10. Inactivation of different derivatives of P-galactosidase from Thermus sp. Strain T2. (•) Soluble enzyme; (A) enzyme immobilized on standard epoxy support; (■) enzyme immobilized on IDA-CO-epoxy supports. Inactivation was carried out at pH 6.5 and 80°C.
Fig. 11. Effect of the incubation at 50°C and pH 6.5 of the enzyme immobilized on IDA-CO-epoxy supports.

4. Notes

1. The epoxy supports (Sepabeads and Eupergit 250) must be stored dried at -20°C for the epoxy stability.

2. During covalent immobilization process, some enzymes may be inactivated at alkaline pH. In these cases, some inhibitors may be added to the immobilization/ incubation buffer to protect the enzyme.

3. Stirring should be perfomed gently to prevent support breakage. This may promote some fines where diffusion problems are decreased and activity may apparently increase.

4. The poly-His-tagged GA extract was produced in E. colias described previously (19).

5. Crude extract of poly-His-tagged P -galactosidase cloned and overexpressed in E. coli (MC1116) (Htag-BgaA) was produced as previously described (20). Only fresh enzyme preparations could be used to obtain immobilized enzyme derivatives with maximum loading. That results from the tendency of Htag-BgaA from Thermus strain T2 to self-associate in solution, and therefore to form large oligo-meric species that obstruct the pores of the matrix (21).

6. The Novo buffer is designed to reproduce the composition of milk (2.7 mMsodium citrate, 7.91 citric acid, 2.99 mM potassium biphosphate, 10.84 mM potassium phosphate, 19.43 mMpotassium hydroxide, 4.08 mMmagnesium chloride, 5.1 mM calcium chloride, and 3.33 mM sodium carbonate) (22).


1. Hemdan, E. S., Zhao, Y-J., Sulkowski, E., and Porath, J. (1989) Surface topography of histidine residues: A facile probe by immobilized metal ion affinity chromatography. Proc. Nat. Acad. Sci. USA 86, 1811-1815.

2. Hernaiz, M. J. and Crout, D. H. G. (2000) Immobilization-stabilization on Eupergit C of the b-galatosidase from B. circulans and an a-galactosidase from A. oryzae. Enzyme Microb. Technol. 27, 26-32

3. Hernandez-Justiz, O., Fenandez-Lafuente, R., Guisan, J. M., et al. (1997) One pot chemoenzymatic synthesis of 3 functionalized cephalosporins (cephazolin) by three consecutive biotransformations in fully aqueous medium J. Org. Chem. 62, 9099-9106.

4. Hubert, H. and Porath, J. (1980) Metal chelate affinity chromatography. I. Influence of various parameters on the retention of nucleotides and related compounds. J. Chromatogr. 198, 247-255.

5. Johnson, R. D. and Arnold, F. H. (1995) Multipoint binding and heterogeneity in immobilized metal affinity chromatography. Biotechnol. Bioeng. 48, 437-443.

6. Katchalski-Katzir, E. and Kraemer, D. (2000) Eupergit C, a carrier for immobilization of enzymes of industrial potential. J. Mol. Catal. B: Enzymatic 10, 157-176.

7. Kennedy, J. F., Melo, E. H. M., and Jumel, K. (1990) Immobilized enzymes and cells. Chem. Eng. Progr. 45, 81-89.

8. Klibanov, A.M. (1983) Immobilized enzymes and cells as practical catalysts. Science 219, 722-727.

9. Mateo, C., Abian, O., Fernandez-Lafuente, R., and Guisan, J. M. (2000) Increase in conformational stability of enzymes immobilized on epoxy activated supports by favoring additional multipoint covalent attachment. Enzyme Microb. Technol. 26,509-515.

10. Mateo, C., Abian, O., Fernandez-Lorente, G., Pedroche, J., Fernandez-Lafuente, R., and Guisan, J. M. (2002) Sepabeads: A novel epoxy-support for stabilization of industrial enzyme via very intense multipoint covalent attachment. Biotechnol. Prog. 18, 629-634.

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

12. Smalla, K., Turkova, J., Coupek, J., and Herman, P. (1988) Influence of salts on the covalent immobilization of proteins to modified copolymers of 2-hydroxyethyl methacrylate with ethylene dimetacrylate. Biotechnol. Appl. Biochem, 10, 21-31.

13. Wheatley, J.B. and Schmidt, D.E. (1993) Salt induced immobilization of proteins on a high-performance liquid chromatographic epoxide affinity support. J. Chromat. 644, 11-16.

14. Wheatley, J.B. and Schmidt, D.E. (1999) Salt induced immobilization of affinity ligands onto epoxide-activated supports. J. Chromat. A, 849, 1-12.

15. Mateo, C., Fernández-Lorente, G. Abian, O. Fernández-Lafuente, R., and Guisán, J. M. (2000) 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 1, 739-745.

16. Mateo, C. Fernández-Lorente, G. Cortes, E. Garcia, J .L., Fernández-Lafuente, R., and Guisán, J. M. (2001) One Step purification, covalent immobilization and additional stabilization of poly-His-tagged proteins using novel heterofunctional chelate-epoxy supports. Biotechnol. Bioeng. 76, 269-277.

17. Bradford, M. M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 76, 248.

18. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277, 680-685.

19. Armisén, P., Mateo C., Cortés, E., et al. (1999) Selective adsorption of poly-His tagged glutaryl acylase on tailor-made metal chelate supports. J. Chromatogr A. 848,61-70.

20. Pessela, B. C., Vian, A., Mateo, C., Fernández-Lafuente, R., García, J. L., Guisán, J. M., and Carrascosa, A. V. (2003) Overproduction of Thermus sp. Strain T2 b-galactosidase in Escherichia coli and preparation by using tailor-made chelate supports. Appl. Environ. Microbiol. 69 (4), 1967-1972.

21. 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 from Thermus sp. Strain T2 by using novel heterofunctional chelate-epoxy sepabeads. Biomacromolecules 4, 107-113.

22. Novo, commercial information, 1979.

Was this article helpful?

0 0
Detoxify the Body

Detoxify the Body

Need to Detoxify? Discover The Secrets to Detox Your Body The Quick & Easy Way at Home! Too much partying got you feeling bad about yourself? Or perhaps you want to lose weight and have tried everything under the sun?

Get My Free Ebook

Post a comment