binding domain (CBD)-fi-galactosidase fusion protein on cellulosic supports resulted in a fairly efficient reusable biocatalyst (28).

Recently, combinatorial peptide synthesis has made it possible to obtain peptides as affinity ligands with a predetermined activity (5). Such peptide affinity ligands, in principle, may also be used for affinity immobilization of enzymes since it will be possible to screen such ligands for binding constants in the range desirable for affinity immobilization. In this context, Hahn et al. (29) have outlined an innovative approach wherein such peptide ligands are linked only to accessible pore sites and do not occupy internal space of the matrix pores. This results in an affinity matrix with better binding capacity and ligand utilization for the target protein.

It is obvious that several innovative variants of bioaffinity immobilization are available. Hence, today, bioaffinity immobilization is a viable and good choice as far as immobilization strategies are concerned.

2. Materials

2.1. Assay of fi-Galactosidase Activity

1. E. coli fi-galactosidase (Sigma Chemical Co., St. Louis, MO)

2. Buffer 1: 100 mMsodium phosphate buffer, pH 6.5.

3. o-Nitrophenyl-fi-D-galactopyranoside (ONGP; CSIR Centre for Biochemicals, New Delhi, India).

4. Buffer 2: 300 mM sodium phosphate buffer, pH 7.5, containing 3 mM MgCl2

5. Buffer 3: 10 mM Tris acetate buffer, pH 7.5, containing 10 mM MgCl2.

6. Double distilled water.

7. 1Mfi-Mercaptoethanol (Fluka, St. Louis, MO). Solution from the bottle is diluted with double distilled water.

9. Bausch and Lomb colorimeter.

2.2. Preparation of Con A-fi-Galactosidase Conjugate and Immobilization on Sephadex G-50

1. Concanavalin A (Con A; CSIR Centre for Biochemicals, New Delhi, India).

2. 25% Glutaraldehyde solution (Reidel-Haan, St. Louis, MO). Dilute with double distilled water, as required (see Note 1).

3. Vortex mixer.

4. Sephadex G-50 gel (Pharmacia, Uppsala, Sweden). The gel is swollen and deaer-ated per the manufacturer's instructions (Handbook of Amersham Biosciences: Gel Filtration: Principles and Methods).

5. Frac 100 fraction collector (Pharmacia, Uppsala, Sweden)

3. Methods

3.1. Assay of fi-Galactosidase Activity (30)

1. Incubate the enzyme sample (200 ||L in Buffer 1) in a reaction mixture containing 1.50 mL Buffer 2, 1.65 mL double distilled water, 0.014 MONGP solution in 0.45 mL of Buffer 3), and 1 M fi-mercaptoethanol in 0.75 mL double distilled water at 25°C.

2. Stop the reaction after 5 min by adding 4 mL 1 MNa2CO3.

3. Read the absorbance of the liberated o-nitrophenol at 405 nm. One enzyme unit liberates 1 |imol of o-nitrophenol per min at 25°C, pH 6.5.

4. For estimating the enzyme activity of the Sephadex-bound enzyme, the enzyme sample is pipeted with a precision air displacement pipet. The end of the tip is cut and the conjugate is taken in 50% liquid phase (Buffer 1) to facilitate pipeting (i.e., 100 |L of enzyme-bound beads and 100 |L Buffer 1 is incubated with the substrate, shaken constantly, and assayed as above).

5. Before reading the absorbance of the solution, centrifuge at 5000^ for 5 min so that the matrix settles down.

3.2. Preparation of Con A-P-Galactosidase Conjugate and Immobilization on Sephadex Matrix

1. Prepare solutions of 4 mg/mL Con A and 1 mg/mL P-galactosidase separately in Buffer 1 containing 500 |L 1 MNaCl. Mix Con A and 100 |L P-galactosidase by gentle shaking in a vortex mixer and cool to 4°C (see Note 2).

2. Slowly add 20 |L of a cold 25% glutaraldehyde solution with constant mixing (see Note 3). Allow the mixture to stand at 4°C for 30 min (see Note 4).

3. Wash 10 mL (settled volume) of Sephadex G-50 gel in a sintered glass funnel connected to a Buchner funnel, under mild suction.

4. Wash the gel thoroughly with double distilled water (5 portions of 30 mL each).

5. Transfer this moist gel cake to a beaker and add sufficient volume of Buffer 1 so that the gel forms a thick slurry.

6. Transfer this slurry to a glass column (1 x 15 cm) using a glass rod aligned diagonally across the column (see Fig. 2) so that the gel is transferred to the column in one smooth motion (see Note 5).

7. Wash the packed bed of gel with 5 bed volumes of Buffer 1.

8. Load the enzyme-conjugate mixture directly on the column. Elute the unbound components with Buffer 1 containing 0.1 M NaCl at 22 mL/h (see Note 6).

4. Notes

1. Glutaraldehyde polymerizes in solution. Its cross-linking efficiency is, in fact, dependent upon the existence of oligomers. Hence, old preparations give better cross-linking results. Equally important, use the same glutaraldehyde preparation for consistent results.

2. Conjugation at pH 7.0 leads to precipitation of P-galactosidase. This is the reason why pH 6.5 has been chosen for intermolecular cross-linking.

3. A large number of cross-linking reagents are available. Some of these may turn out to be more efficient in terms of conjugation efficiency and are likely to give more consistent results. However, glutaraldehyde was used (and continues to be used) as a cross-linking reagent by many researchers because it is readily available and turns out to be quite economical.

4. If the amount of P-galactosidase is increased to 200 |g and the cross-linking is continued for 30 min, the protein precipitates. In this case, better results are obtained by limiting the cross-linking time to 15 min.

Glass Sephadex Column

Fig. 2. Packing of a column with Sephadex G-50 gel for bioaffinity immobilization of Con A-P-galactosidase conjugate.

5. This is required so that no air bubbles get trapped in the gel matrix.

6. In some applications, it is possible that the cost of the matrix is comparable to the enzyme. In many such cases, it may be desirable to strip the enzyme/conjugate off the matrix and reload the fresh lot of enzyme/conjugate on the matrix.

7. It is necessary to check that the affinity ligand is also stable under operational conditions. Here, 50°C has been used as the process temperature, where Con A is known to be stable (25).

8. Immobilization, often (but not always), enhances thermal stability. Thus, it may be possible to work at higher temperatures provided the affinity ligand is also stable (see Note 7). Similarly, it is necessary to check the optimum pH of the immobilized enzyme before deciding the process parameters.

9. In some designs, it is possible that the product causes dissociation of the enzyme/ enzyme conjugate from the matrix. However, suitable reactor design such as packed bed/fluidized bed reactors may avoid local high concentration of the product to that level.


The authors thank Dr. S. K. Khare (IIT Delhi) for the work described in this chapter. Financial support from the Department of Science and Technology, the

Department of Biotechnology, and the Council for Scientific and Industrial Research, all Government of India organizations, is gratefully acknowledged.


1. Gupta, M. N. and Roy, I. (2002) Affinity-based separation: An overview. In: Methods for Affinity-Based Separation of Proteins/Enzymes, (Gupta, M. N., ed.), Birkhauser Verlag, Basel, pp. 1-15.

2. Mattiasson, B. (1988) Affinity immobilization. Methods Enzymol. 137, 647-656.

3. Saleemuddin, M. (1999) Bioaffinity based immobilization of enzymes. Adv. Biochem. Eng. Bioeng. 64, 203-226.

4. Saleemuddin, M. and Husain, Q. (1991) Concanavalin A: a useful ligand for glycoenzyme immobilization—a review. Enzyme Microb. Technol. 13, 290-295.

5. Zhao, H. and Arnold, F. H. (1997) Combinatorial protein design: Strategies for screening protein libraries. Curr. Opin. Struct. Biol. 7, 480-485.

6. Sulkowski, E. (1985) Purification of proteins by IMAC. Trends Biotechnol. 3, 1-8.

7. Clonis, Y. D. (1988) The applications of reactive dyes in enzyme and protein downstream processing. Crit. Rev. Biotechnol. 7, 263-269.

8. Labrou, N. E. (2003) Design and selection of ligands for affinity chromatography. J. Chromatogr. B 790, 67-78.

9. Hermanson, G. T., Mallia, A. K., and Smith, P. K. (1992) Immobilized Affinity Ligand Technique, Academic Press, San Diego, CA.

10. Roy, I. and Gupta, M. N. (2003) Selectivity in affinity chromatography. In: Isolation and Purification ofProteins, (Mattiasson, B. and Kaul-Hatti, R., eds.), Marcel Dekker, New York, NY, pp. 57-94.

11. Bilkova, Z., Churacek, J., Kucerova, Z., and Turkova, J. (1997) Purification of anti-chymotrypsin antibodies for the preparation of a bioaffinity matrix with oriented chymotrypsin as immobilized ligand. J. Chromatogr. B 689, 273-279.

12. Turkova, J. (1999) Oriented immobilization of biologically active proteins as a tool for revealing protein interactions and function. J. Chromatogr. B722, 11-31.

13. Wilchek, M., and Bayer, E. A. (eds.). (1990) Methods in Enzymology, Vol. 184, Academic Press, New York, NY.

14. Tyagi, R. and Gupta, M. N. (1998) Chemical modification and chemical crosslinking for protein/enzyme stabilization. Biochemistry(Mosc) 63, 334-344.

15. Ong, E., Greenwood, J. M., Gilkes, N. R., Kilburn D. G., Miller Jr., R. C., and Warren, R. A. J. (1989) The cellulose-binding domains of cellulases: tools for biotechnology. Trends Biotechnol. 7, 239-243.

16. Cha, H. J., Dalal, N. G., Pham, M. Q., and Bentley, W. E. (1999) Purification of human interleukin-2 fusion protein produced in insect larvae is facilitated by fusion with green fluorescent protein and metal affinity ligand. Biotechnol. Prog. 15, 283-286.

17. Farooqi, M., Saleemuddin, M., Ulber, R., Sosnitza, P., and Scheper, T. (1997) Bioaffinity layering: A novel strategy for the immobilization of large quantities of glycoenzymes. J. Biotechnol. 55, 171-179.

18. Farooqi, M., Sosnitza, P. Saleemuddin, M., Ulber, R., and Scheper, T. (1999) Immunoaffinity layering of enzymes: stabilization and use in flow injection analysis of glucose and hydrogen peroxide. Appl. Microbiol. Biotechnol. 52, 373-379.

19. Gemeiner, P., Docolomansky, P., Nahalka, J., Stefuca, V. and Danielsson, B. (1996) New approaches for verification of kinetic parameters of immobilized con-canavalin A: invertase preparations investigated by flow microcalorimetry. Biotechnol. Bioeng. 49, 26-35.

20. Jan, U., Husain, Q., and Saleemuddin, M. (2001) Preparation of stable, highly active and immobilized glucose oxidase using the anti-enzyme antibodies and F(ab)'2. Biotechnol. Appl. Biochem. 34, 13-17.

21. Hale, J. E. (1995) Irreversible, oriented immobilization of antibodies to cobalt-iminodiacetate resin for use as immunoaffinity media. Anal. Biochem. 231, 46-49.

22. Lehtio, J., Wernerus, H., Samuelson, P., Teeri, T. T., and Stahl, S. (2001) Directed immobilization of recombinant staphylococci on cotton fibers by functional display of a fungal cellulose-binding domain. FEMSMicrobiol. Lett. 195, 197-204.

23. Wernerus, H., Lehtio, J., Teeri, T., Nygren, P. A., and Stahl, S. (2001) Generation of metal-binding staphylococci through surface display of combinatorially engineered cellulose-binding domains. Appl. Environ. Microbiol. 67, 4678-4684.

24. Johnson, C. P., Jensen, I. E., Prakasam, A., Vijayendran, R., and Leckband, D. (2003) Engineered Protein A for the oriented control of immobilized poteins. Bioconjug. Chem. 14, 974-978.

25. Khare, S. K., and Gupta, M. N. (1988) Preparation of Concanavalin A-ß-galac-tosidase conjugate and its application in lactose hydrolysis. J. Biosci. 13, 47-54.

26. Anspach, F. B. and Altmann-Hasse, G. (1994) Immobilized metal-chelate regen-erable carriers: (I) Adsorption and stability of penicillin G aminohydrolase from Escherichia coli. Biotechnol. Appl. Biochem. 20, 313-322.

27. Baneyx, F., Schmidt, C., and Georgiou, G. (1990) Affinity immobilization of a genetically engineered bifunctional hybrid protein. Enzyme Microb. Technol. 12, 337-342.

28. Ong, E., Gilkes, N. R., Warren, R. A. J., Miller Jr., R. C., and Kilburn, D. G. (1989) Enzyme immobilization using the cellulose-binding domain of a Cellulomonas fimi exoglucanase. Biotechnology 7, 604-607.

29. Hahn, R., Berger, E., Pflegel, K., and Jungbauer, A. (2003) Directed immobilization of peptide ligands to accessible pore sites by conjugation with a placeholder molecule. Anal. Chem. 75, 543-548.

30. Craven, G. R., Steers Jr., E., and Anfinsen, C. B. (1965) Purification, composition and molecular weight determination of the b-galactosidase of Escherichia coli K12. J. Biol. Chem. 240, 2468-2471.

31. Woodward, J. (1985) Immobilized enzymes: Adsorption and covalent coupling. In: Immobilized Cells and Enzymes: A Practical Approach, (Woodward, J., ed.), IRL Press, Oxford, UK, pp. 3-17.

32. Phelps, M. R., Hobbs, J. B., Kilburn, D. G., and Turner, R. F. B. (1995) An auto-clavable biosensor for microbial fermentation monitoring and control. Biotechnol. Bioeng. 46, 514-524.

33. Koneke, R., Menzel, C., Ulber, R., Schugerl, K., and Saleemuddin, M. (1996) Reversible coupling of glucoenzymes on fluoride-sensitive FET biosensors based on lectin-glucoprotein binding. Biosens. Bioelectron. 11, 1229-1236.

34. Mauro, J. M., Cao, L. K., Kondracki, L. M., Walz, S. E., and Campbell, J. R. (1996) Fiber-optic fluorometric sensing of polymerase chain reaction-amplified DNA using an immobilized DNA capture protein. Anal. Biochem. 235, 61-72.

35. Habibi-Rezaei, M. and Nemat-Gorgani, M. (1997) Adsorptive immobilization of submitochondrial particles on concanavalin A Sepharose-4B. Appl. Biochem. Biotechnol. 67, 165-181.

36. Sloan, D. D., Barrett, R. W., Tate, E. H., and England, B. P. (1997) Expression of an epitope-tagged human C5a receptor and antibody-mediated immobilization of detergent-solubilized receptor for drug discovery screening. Protein Expr. Purif. 11, 119-124.

37. Catimel, B., Scott, A. M., Lee, F. T., et al. (1998) Direct immobilization of gan-gliosides onto gold-carboxymethyldextran sensor surfaces by hydrophobic interaction: applications to antibody characterization. Glycobiology 8, 927-938.

38. Sosnitza, P., Farooqi, M., Saleemuddin, M., Ulber, R., and Scheper, T. (1998) Application of reversible immobilization techniques for biosensors. Anal. Chim. Acta 368, 197-203.

39. Quinn, J., Patel, P., Fitzpatrick, B., et al. (1999) The use of regenerable, affinity-ligand-based surfaces for immunosensor applications. Biosens. Bioelectron. 14, 587-595.

40. Patel, N., Bhandari, R., Shakesheff, K. M., et al. (2000) Printing patterns of biospecifically-adsorbed protein. J. Biomater. Sci. Polym. Ed. 11, 319-331.

41. Willner, I. I. and Katz, E. (2000) Integration of layered redox proteins and conductive supports for bioelectronic pplications. Angew. Chem. Int. Ed. Engl. 39, 1180-1218.

42. Cosnier, S. (2003) Biosensors based on electropolymerized films: new trends.

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