Info

aGA modified with 1 MED in the presence of 10-2 MEDAC at pH 6.0.

aGA modified with 1 MED in the presence of 10-2 MEDAC at pH 6.0.

Enzyme Immobilization

Fig. 5. Thermal inactivation of different GA glyoxyl derivatives prepared at pH 10.0. (A) Glutaryl acylase immobilized on glyoxyl agarose; (■) glutaryl acylase immobilized on glyoxyl acylase and then modified with 1 MED and 10-2 MEDAC; (•) glutaryl acylase modified with 1 MED and 10-2 MEDAD and then immobilized onto glyoxyl agarose; (♦) soluble glutaryl acylase. Experiments were carried out at 45°C, pH 7.0).

Fig. 5. Thermal inactivation of different GA glyoxyl derivatives prepared at pH 10.0. (A) Glutaryl acylase immobilized on glyoxyl agarose; (■) glutaryl acylase immobilized on glyoxyl acylase and then modified with 1 MED and 10-2 MEDAC; (•) glutaryl acylase modified with 1 MED and 10-2 MEDAD and then immobilized onto glyoxyl agarose; (♦) soluble glutaryl acylase. Experiments were carried out at 45°C, pH 7.0).

2. The stability of the derivatives immobilized at pH 9.0 was lower than the ones in which the immobilization was carried out at pH 10.0. However, the stability was improved if, after immobilization at pH 9.0, the pH value was increased at pH 10.0, yielding a similar value to the enzyme directly immobilized at pH 10.0 (see Fig. 6).

4. Notes

1. It has been reported that the use of 0.1 MED at pH 4.7 and 10 mMEDAC allows the full modification of the carboxylic groups of the protein surface, while using 1 mMEDAC in 1 MED at pH 4.7 the modification degree is only between 40 and 50% (28).

2. Store between 0 and 5°C. Caution: toxic!

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

4. The choice of the amount of EDAC to be used is done by evaluating its impact on the activity and stability of the previously immobilized enzyme. Thus, modification conditions which decrease the stability of the modified enzymes are avoided (28).

Immobilized Enzyme Stability

Fig. 6. Thermal inactivation courses of GA derivatives immobilized at different pH values. (♦) Aminated GA immobilized at pH 9.0 onto glyoxyl-agarose; (•) aminated GA immobilized at pH 10.0 onto glyoxyl-agarose; (■) aminated GA immobilized at pH 9.0 onto glyoxyl-agarose and incubated at pH 10.0 for 3 h.

Fig. 6. Thermal inactivation courses of GA derivatives immobilized at different pH values. (♦) Aminated GA immobilized at pH 9.0 onto glyoxyl-agarose; (•) aminated GA immobilized at pH 10.0 onto glyoxyl-agarose; (■) aminated GA immobilized at pH 9.0 onto glyoxyl-agarose and incubated at pH 10.0 for 3 h.

5. Avoid magnetic stirring of agarose, especially during long reaction times.

6. Glycidol addition must be very slow to prevent the temperature rising over 25°C.

7. Oxidation of glycols with sodium periodate is a stoichiometric reaction. Therefore, the activation degree of the support can be easily controlled through the periodate concentration used. The protocol described enables an activation degree of 75 and 200 |imol/mL. To achieve this level, 112.5 and 300 mL, respectively, of oxidation solution have to be added to completely oxidize agarose 6 and 10BCL.

8. If the enzyme activity decreases during the course of immobilization resulting from enzyme inactivation, this effect must be distinguished from loss of the supernatant resulting from immobilization.

9. Supernatant was achieved by using pipet filter or by centrifugation of the suspension.

10. Rigidification of the protein structure via the formation of multipoint covalent linkages between its nonionic amine groups and the reactive groups of the support could be obtained by keeping the suspension at pH 10.0 for a fairly long interaction time at 25°C. The optimum multi-interaction time is the shortest one that provides the maximal stability of the enzyme derivative.

11. The already immobilized enzymes were incubated under the conditions reported to yield the maximum stability for the nonmodified enzyme (4).

12. Commercial preparation of GA (purchased from Roche) was diluted fivefold (v/ v) in 25 mM potassium phosphate buffer, pH 7.0, and then dialyzed threefold against 100 volumes of 25 mMpotassium phosphate buffer, pH 7.0. The dialyzed enzyme was then centrifuged (24,149^ for 30 min at 4°C) and the supernatant (containing 16 U/mL and 11 mg of protein/mL) were used as the enzymatic preparation for further experiments. More than 90% of initial activity was recovered after this process.

References

1. Haki, G. D. and Rakshit, S. K. (2003) Developments in industrially important thermostable enzymes: a review. Bioresource Technol. 89, 17-34

2. Wong, S. S and Wong, L. J. (1992) Chemical crosslinking and the stabilization of proteins and enzymes. Enzyme Microb Technol. 14, 866-874

3. Klibanov, A. M. (1983) Immobilized enzymes against termal inactivation. Adv. Appl. Microbiol. 29, 1-28.

4. Alvaro, G., Fernández-Lafuente, R., Blanco, R. M., and Guisán, J. M. (1990) Immobilization-stabilization of penicillin acylase from E. coli. Appl. Biochem. Biotech. 26, 181-195.

5. Guisán, J. M., Alvaro, G., Fernández-Lafuente, R., Rosell, C. M., Garcia-Lopez, J. L., and Tagliatti, A. (1993) Stabilization of a heterodymeric enzyme by multipoint covalent immobilization: Penicillin G acylase from Kluyvera citrophila. Biotechnol. Bioeng. 42, 455-464.

6. Blanco, R. M. and Guisán J. M. (1988) Protecting effect of competitive inhibitors during very intense insolubilized enzyme-activated support multipoint attachments: trypsin (amine)-agarose (aldehyde) system. Enzyme Microb. Technol. 10, 227-232.

7. Guisán, J. M., Bastida, A., Cuesta, A. C., Fernández-Lafuente, R., and Rosell, C. M. (1991) Immobilization-stabilization of chymotrypsin by covalent attachment to aldehyde agarose gels. Biotechnol. Bioeng. 39, 75-84.

8. Tardioli, P. W., Pedroche, J., Giordano, R. L., Fernández-Lafuente, R., and Guisán, J. M. (2003) Hydrolysis of Proteins by Immobilized-Stabilized Alcalase®-Glyoxyl Agarose. Biotechnol. Progr. 19, 352-360.

9. Pedroche, J., Yust, M. M., Girón-Calle, J., et al. (2002) Stabilization-immobilization of carboxypeptidase to aldehyde-agarose gels. A practical example in the hydrolysis of casein. Enzyme Microb. Technol. 31, 711-718.

10. Tardioli, P. W., Fernández-Lafuente, R., Guisán, J. M., and Giordano, R. L. C. (2003) Design of New Immobilized-Stabilized Carboxypeptidase A Derivative for Production of Aromatic Free Hydrolysates of Proteins. Biotechnol. Prog. 19, 565-574.

11. Bes, T., Gomez-Moreno, C., Guisán, J. M., and Fernández-Lafuente, R. (1995) Selective enzymetic oxidations: stabilization by multipoint covalent attachment of ferredoxin NAD-reductasa: an interesting cofactor recycling enzyme" J. Mol. Catal. 98, 161-169.

12. Fernández-Lafuente, R., Cowan, D.A., and Wood, A.N.P. (1995) Hyperstabilization of a thermophilic esterase by multipoint covalent attachment. Enzyme Microb. Technol. 17, 366-372.

13. Guisán, J. M, Polo, E., Agudo, J., Romero, M.D., Alvaro, G., and Guerra, M.J. (1997) Immobilization-stabilization of thermolysin onto activated agarose gels. Biocatal. Biotrans. 15, 159-173.

14. Betancor, L., Hidalgo,A., Fernández-Lorente, G., et al. (2003) The use of physi-cochemical tools to solve enzyme stability problems alters the choice of the optimal enzyme: stabilization of D-aminoacid oxidase. Biotechnol. Prog. 19, 784-788.

15. Betancor, L., Hidalgo, A., Fernández-Lorente, G., et al. (2003) Preparation of a stable biocatalyst of bovine liver catalase. Biotechnol. Prog. 19, 763-767.

16. Hidalgo, A., Betancor, L., Lopez-Gallego, F., et al. (2003) Preparation of a versatile biocatalyst of immobilized and stabilized catalase from Thermus thermophilus. Enzyme Microb. Technol. 33, 278-285.

17. Otero, C., Ballesteros, A., and Guisán, J. M. (1991) Immobilization/stabilization of lipase from Candida rugosa. Appl. Biochm. Biote chnol. 19, 163-175.

18. Palomo, J. M., Muñoz, G., Fernández-Lorente, G., Mateo, C., Fernández-Lafuente, R., and Guisán, J. M. (2002) Interfacial adsorption of lipases on very hydrophobic support (octadecyl-Sepabeads): Immobilization, hyperactivation and stabilization of the open form of lipases. J. Mol. Cat B Enzymatic. 19,20, 279286.

19. Suh, C-W, Choi, G-S, and Lee, E-K. (2003) Enzymatic cleavage of fusion protein using immobilized urokinase covalently conjugated to glyoxyl-agarose. Biotecnol. Appl. Biochem. 37, 149-155.

20. Toogood, H. S., Taylor, I. N., Brown, R. C., Taylor, S. J. C., McCague, R., and Littlechild, J.A. (2002) Immobilisation of the Thermostable L-aminoacylase from Thermus litotalis to generate a Reusable Industrial Biocatalyst. Biocatal. Biotrans. 20, 241-249.

21. Ichikawa, S., Takano, K., Kuroiwa, T., Hiruta, O., Sato S., and Mukataka, S. (2002) Immobilization and stabilization of chitosanase by multipoint attachment to agar gel support J. Bioscien. Bioeng. 93, 201-206.

22. Kuroiwa, T., Ichikawa, S., Sato, S., and Mukataka, S. (2003) Improvement of the yield of physiologically active oligosaccharides in continuous hydrolysis of chitosan using immobilized chitosanases. Biotechnol. Bioeng. 84, 121-127.

23. Kuroiwa, T., Ichikawa, S., Sosaku, H., Sato, S., and Mukataka, S. (2002) Factors affecting the composition of oligosaccharides produced in chitosan hydrolysis using immobilized chitosanases. Biotechnol. Prog. 18, 969-974.

24. Guisán, J. M., Bastida, A., Blanco, R. M., Fernández-Lafuente, R., and García-Junceda, E. (1997) Immobilization of enzymes on glyoxyl supports:. strategies for nzyme stabilization by multipoint covalent attachment. In: Immobilization of Enzymes and Cells, Methods in Biotechnology (Bickerstaff, G. S., ed.) vol 1, Humana Press, Totowa, NJ, pp. 277-288.

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

26. Mateo, C., Abian, O., Bernedo, M., et al. (2005) Special mechanism of immobilization of proteins in glyoxyl supports. Enzyme Microb. Technol. 37, 456-46Z

27. Abian, O., Grazú, V., Hermoso, J., et al. (2004) Stabilization of penicillin G acylase from Escherichia coli: site-directed mutagenesis of the protein surface to increase multipoint covalent attachment. Appl Environ Microb, 70, 1249-1251.

28. López-Gallego, F., Montes, T., Fuentes, M., et al. (2005) Improved stabilization of chemically aminated enzymes via multipoint covalent attachment on glyoxyl supports. J. Biotech. 116, 1-10.

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