Carrier Free Cross-linked Peroxidase Aggregates: Synthesis and Characterization

Yıl 2008, Cilt: 36 Sayı: 3, 255 - 261, 01.08.2008

Ceren Topçular Hakan Ayhan

Öz

In this study, cross-linked Horseradish Peroxidase HRP aggregates were prepared. Bovin Serum Albumin BSA ,a proteic feeder, and glutaraldehyde, a bifunctional reactive, a crosslinker agent has also been used. The initialenzyme concentration 0.05 mg/ml , BSA 5 mg and glutaraldehyde GA 4 µl/ml amounts were optimized andmaximum immobilized activities were obtained. The kinetic parameters of free and immobilised enzyme have beencalculated by using Lineweaver-Burk plots. The saturation constant Km and the maximum reaction rate Vm offree enzyme were found as 2.33 mM and 0.670mmol/l.sec, respectively. The apparent kinetic parameters Kmapp andVmapp of cross linked enzyme aggregates have been calculated as 33.4 mM and 0.127 mmol/l.sec, respectively

Anahtar Kelimeler

cross-linked enzyme aggregates, horseradish peroxidase, bovine serum albumin, proteic feeder

Kaynakça

• 1. K. Mosbach, Immobilized enzymes and cells (Part B) Methods Enzymology, Vol. 135, 1987. 2. W. Hartmeier, Immobilized Biocatalysts, Springer-Verlag, 1988.
• 3. D.S. Clarc, Can immobilization be exploited to modify enzyme activity? Trends Biotechnology, 12: 439-443, 1994.
• 4. J.M.S. Cabral, J.F. Kennedy, Immobilization techniques for altering thermal stability of enzymes, Thermo stability of Enzymes, edited by Gupta M. N. Berlin, Springer-Verlag , 163- 179, 1993.
• 5. S. Rocchietti, A.S.V. Urritia, M. Pregnolata, A. Tagliani, J.M. Guisan, R. Fernandez-Lafuente, M. Terreni, Influence of the enzyme derivative preparation and substrate structure on the enantioselective of penicillin G acylase, Enzyme Microb. Technol, 31:88-93, 2002.
• 6. L. Cao, L.V. Langen, R.A. Sheldon, Immobilised enzymes: carrier-bound or carrier –free, Current opinion in Biotechnology, 14: 387-394, 2003.
• 7. N.L. St Clair, M.A. Navia, Cross-linked enzyme crystals as robust biocatalysts, J. Am. Chem. Soc., 114: 7314-7316, 1992.
• 8. L. Cao, Immobilised enzymes: science or art?, Current opinion in Chemical Biology, 9: 217- 226, 2005.
• 9. J. Chen, J. Zhang, B. Han, Z. Li, J. Li, X. Feng, Synthesis of cross-linked aggregates (CLEAs) in CO2-expanded micellar solutions, Colloids and Surfaces B : Biointerfaces, 48: 72-76, 2006.
• 10. S.S. Caramori, K.F. Fernandes, Covalent immobilization of horseradish peroxidase onto poly(ethylene terephalate) – poly(aniline) composite, Process Biochemistry, 39: 883-888, 2004.
• 11. S. Shah, A. Sharma, M.N. Gupta, Preparation of cross-linked enzyme aggregates by using bovine serum albumin as a proteic feeder, Analytical Biochemistry, 351: 207-213, 2006.
• 12. R. Tyagi, R. Batra, M.N. Gupta, Amorphous enzyme aggregates: Stability towards heat and aqueous-organic co solvents mixtures, Enzyme and Microbial Technology, 24 : 348- 354, 1999.
• 13. B. Halphin, R. Pressey, J. Jen, N.J. Mondy, Purification and characterization of peroxidase isoenzyme from green peas ( Pisum sativum ), J.Food Sci., 54 : 44-49, 1989.
• 14. F. Rothstein, Differential precipitation of proteins: science and technology, In Protein Purification Process Engineering, Edited by Harrison R. G. New York: Marcel Decker, Inc, 115-208, 1994.
• 15. G.H. Broun, Chemically aggregated enzymes, in; Mosbach K. (Ed.), Methods in enzymology, Academic Press, New York, 263-269, 1997.
• 16. C. Topcular, H. Ayhan, Immobilisation of horseradish peroxidase onto monodisperse poly(glycidyl methacrylate) microspheres, J. Biomater. Sci. Polymer Edn. 18; 5; 595-607, 2007