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Cross-Linked Glucose Oxidase Aggregates: Synthesis and Characterization

Year 2011, Volume: 39 Issue: 3, 241 - 251, 01.08.2011

Abstract

The aim of the submitted study is to realize carrier-free immobilization as cross-linking enzyme aggregates of Aspergillus niger Glucose Oxidase enzyme, to determinate the optimum immobilization conditions, and to investigate the properties of immobilized enzyme sets. Carrier-free enzyme immobilization was realised by forming cross-linked glucose oxidase enzyme aggregates CLEA’s . Different precipitants were used in different media conditions during forming CLEA’s. The “immobilized enzyme activity” was measured by activity determination method with the use of glucose substrate. The optimum conditions which were determined in the light of the experiments for the cross-linked enzyme aggregates can be summarized as; the initial enzyme concentration, 0.05 mg/ml; the optimum temperature, 25ºC; the precipitant type, BSA; the precipitant concentration, 5 mg/ml; the concentration of glutaraldehyde, 2% v/v . After determining the optimum conditions, glucose solutions at different concentrations 1.0-4.0 mg/dl were prepared for investigating the performance of the glucose oxidase aggregates. The kinetic parameters were calculated by Lineweaver-Burk plots such as; Km =0.0115 mM; Vm =1.206 mM.min-1 for native enzyme and Km = 0.025 mM; Vm =0.593 mM.min-1 for cross-linking enzyme aggregates.

References

  • 1. K. Mosbach, Immobilised enzymes and cells (Part B) Methods Enzymology 1987, Vol. 135.
  • 2. W. Hartmeier, Immobilised Biocatalysts, SpringerVerlag, 1988.
  • 3. S. Rocchietti, A.S.V. Urritia, M. Pregnolata, A. Tagliani, J.M. Guisan, R. Fernandez-Lafuente, M. Tereni; Influence of the enzyme derivative preparation and substrate structure on the enantioselective of penicillin G acylase, Enzyme Microb. Technol., 31 (2002) 88.
  • 4. J.M.S. Cabral, J.F. Kennedy, Immobilisation techniques for altering thermal stability of enzymes, Thermo stability of Enzymes, edited by M.N. Gupta, Berlin, Springer–Verlag, 1993, 163-179.
  • 5. T. Boller, C. Meier, S. Menzler, Eupergit oxirane acrylic beads: how to make the enzyme fit for biocatalysts. Org. Process Res. Develop, 6 (2002) 509.
  • 6. L. Cao, F. van Rantwijk, R.A. Sheldon, Cross-linked enzyme aggregates, a simple and effective method for the immobilisation of penicilin acylase. Org. Lett., 2 (2000) 1361.
  • 7. F. Rothstein, Differential precipitation of proteins: science and technology. In Protein Purification Process Engineering, Edited by R.G. Harrison, New York, Marcel Dekker, Inc., 1994, pp. 115-208.
  • 8. J. Chen, J. Zhang, B. Han, J. Li, X. Feng, Synthesis of cross-linked aggregates in CO2-expanded micellar solutions, Colloid Surf. B: Biointerface., 48 (2006) 72.
  • 9. M.K. Ram, M. Adami, S. Paddeu, C. Nicolini, Nanoassembly of glucose oxidase on the in situ selfassembled films of polypyrrole and its optical, surface and electrochemical characterizations, Nanotechnology, 11 (2000)112.
  • 10. Sigma Technical Bulletin, No.510, Sigma Chemical CO., St. Louis, 1983.
  • 11. S. Shah, A. Sharma, M.N. Gupta, Preparation of crosslinked enzyme aggregates by using bovine serum albumin as a proteic feeder, Anal. Biochem., 351 (2006) 207.
  • 12. R. Tyagi, R. Batra, M.N. Gupta, Amorphous enzyme aggregates: Stability towards heat and aqueousorganic co solvents mixtures, Enzym. Microb. Technol., 24 (1999) 348.
  • 13. C. Roberge, D. Amos, D. Pollard, P. Devine, Preparation and application of cross-linked aggregates of chloroperoxidase with enhanced hydrogen peroxide tolerance, J. Molecul. Catal. B. Enzym., 56 (2009) 41.
  • 14. P. Lopez-Serrano, L. Cao, F. van Rantwijk, R.A. Sheldon, Cross-linked enzyme aggregates with enhanced activity: application to lipases, Biotechnol. Lett., 24 (2002) 1379.
  • 15. L. Zhao, L. Zheng, G. Gao, F. Jia, S. Cao, Resolution of N-(2-ethyl-6-methylphenyl) alanine via cross-linked aggregates of Pseudomonas sp. Lipase, J. Molecul. Catal. B. Enzym., 54 (2008) 7.
  • 16. V. Bulmuş, H. Ayhan, E. Pişkin, Modified PMMA monosize microbeads for glucose oxidase immobilization, Chem. Eng. J., 65 (1996) 71.
  • 17. T. Li, Z. Yao, L. Ding, Development of an amperometric biosensor based on glucose oxidase immobilized through silica sol-gel film onto Prussian Blue modified electrode, Sens. Act. B. Chem., 101 (2004) 155.
  • 18. H. Han, Y. Li, Z. Zhou, D. Xiao, M.M.F. Choi, Clinical determation of glucose in human serum by a tomato skin biosensor, Clin. Chim. Acta, 395 (2008) 155.
  • 19. L.Y. Chu, Y. Liang, W. Chen, X. Ju, H. Wang, Preparation of glucose-sensitive microcapsules with a porous membrane and functional Gates, Colloid. Surf. B., 37 (2004) 9.
  • 20. G. Ozyilmaz, S.S. Tukel, O.Z. Alptekin, Activity and storage stability of immobilised glucose oxidase onto magnesium silicate, J. Molecul. Catal. B: Enzym., 35 (2005) 154.
  • 21. T. Li, Z. Yao, L. Ding, Development of an amperometric biosensor based on glucose oxidase immobilized through silica sol–gel film onto Prussian Blue modified electrode, Sens. Act. B. Chem., 101 (2004) 155.
  • 22. L.Y. Chu, Y.J. Liang, W.M. Chen, X.J. Ju, H.D. Wang, Preparation of glucose-sensitive microcapsules with a porous membrane and functional Gates, Colloid. Surf. B., 37 (2004) 9.

Çapraz Bağlı Glukoz Oksidaz Agregatları: Sentez ve Karakterizasyonu

Year 2011, Volume: 39 Issue: 3, 241 - 251, 01.08.2011

Abstract

S unulan çalışmanın amacı Aspergillus niger Glukoz Oksidaz enziminin taşıyıcısız immobilizasyonunu çapraz bağlı enzim agregatları olarak gerçekleştirmek, en uygun immobilizasyon koşullarını bulmak ve immobilize enzim kümelerinin özelliklerini belirlemektir. Taşıyıcısız enzim immobilizasyonu çapraz bağlı glukoz oksidaz enzim agregatları ÇBEA oluşturarak uygulanmıştır. ÇBEA’nın oluşumu sırasında farklı çöktürücüler yine farklı ortamlarda kullanılmıştır. “İmmobilize enzim aktivitesi” aktivite tayin yöntemine göre glukoz substratı kullanılarak ölçülmüştür. Deneylerin sonunda çapraz bağlı enzim agregatları için bulunan ve en yüksek enzim aktivitesini veren en uygun koşullar: başlangıç enzim derişimi, 0.5 mg/ml; en uygun sıcaklık, 25oC; çöktürücü türü, Bovin Serum Albümin BSA ; çöktürücü derişimi, 5 mg/ml; ve glutaraldehit derişimi, % 2 v/v . En uygun koşulların belirlenmesinden sonra glukoz oksidaz agregatlarının performanslarını belirlemek üzere farklı derişimlere sahip glukoz çözeltileri hazırlandı 1.0 - 4.0 mg/dl . Verilerin Lineweaver-Burk doğrusu olarak çizimi ile kinetik parametreler Km= 0.0115 mM ve Vm=1.206 mM.min-1 serbest enzim için ve ÇBEA için Km= 0.025 mM; V=0.593 mM.min-1 olarak hesaplanmıştır. m

References

  • 1. K. Mosbach, Immobilised enzymes and cells (Part B) Methods Enzymology 1987, Vol. 135.
  • 2. W. Hartmeier, Immobilised Biocatalysts, SpringerVerlag, 1988.
  • 3. S. Rocchietti, A.S.V. Urritia, M. Pregnolata, A. Tagliani, J.M. Guisan, R. Fernandez-Lafuente, M. Tereni; Influence of the enzyme derivative preparation and substrate structure on the enantioselective of penicillin G acylase, Enzyme Microb. Technol., 31 (2002) 88.
  • 4. J.M.S. Cabral, J.F. Kennedy, Immobilisation techniques for altering thermal stability of enzymes, Thermo stability of Enzymes, edited by M.N. Gupta, Berlin, Springer–Verlag, 1993, 163-179.
  • 5. T. Boller, C. Meier, S. Menzler, Eupergit oxirane acrylic beads: how to make the enzyme fit for biocatalysts. Org. Process Res. Develop, 6 (2002) 509.
  • 6. L. Cao, F. van Rantwijk, R.A. Sheldon, Cross-linked enzyme aggregates, a simple and effective method for the immobilisation of penicilin acylase. Org. Lett., 2 (2000) 1361.
  • 7. F. Rothstein, Differential precipitation of proteins: science and technology. In Protein Purification Process Engineering, Edited by R.G. Harrison, New York, Marcel Dekker, Inc., 1994, pp. 115-208.
  • 8. J. Chen, J. Zhang, B. Han, J. Li, X. Feng, Synthesis of cross-linked aggregates in CO2-expanded micellar solutions, Colloid Surf. B: Biointerface., 48 (2006) 72.
  • 9. M.K. Ram, M. Adami, S. Paddeu, C. Nicolini, Nanoassembly of glucose oxidase on the in situ selfassembled films of polypyrrole and its optical, surface and electrochemical characterizations, Nanotechnology, 11 (2000)112.
  • 10. Sigma Technical Bulletin, No.510, Sigma Chemical CO., St. Louis, 1983.
  • 11. S. Shah, A. Sharma, M.N. Gupta, Preparation of crosslinked enzyme aggregates by using bovine serum albumin as a proteic feeder, Anal. Biochem., 351 (2006) 207.
  • 12. R. Tyagi, R. Batra, M.N. Gupta, Amorphous enzyme aggregates: Stability towards heat and aqueousorganic co solvents mixtures, Enzym. Microb. Technol., 24 (1999) 348.
  • 13. C. Roberge, D. Amos, D. Pollard, P. Devine, Preparation and application of cross-linked aggregates of chloroperoxidase with enhanced hydrogen peroxide tolerance, J. Molecul. Catal. B. Enzym., 56 (2009) 41.
  • 14. P. Lopez-Serrano, L. Cao, F. van Rantwijk, R.A. Sheldon, Cross-linked enzyme aggregates with enhanced activity: application to lipases, Biotechnol. Lett., 24 (2002) 1379.
  • 15. L. Zhao, L. Zheng, G. Gao, F. Jia, S. Cao, Resolution of N-(2-ethyl-6-methylphenyl) alanine via cross-linked aggregates of Pseudomonas sp. Lipase, J. Molecul. Catal. B. Enzym., 54 (2008) 7.
  • 16. V. Bulmuş, H. Ayhan, E. Pişkin, Modified PMMA monosize microbeads for glucose oxidase immobilization, Chem. Eng. J., 65 (1996) 71.
  • 17. T. Li, Z. Yao, L. Ding, Development of an amperometric biosensor based on glucose oxidase immobilized through silica sol-gel film onto Prussian Blue modified electrode, Sens. Act. B. Chem., 101 (2004) 155.
  • 18. H. Han, Y. Li, Z. Zhou, D. Xiao, M.M.F. Choi, Clinical determation of glucose in human serum by a tomato skin biosensor, Clin. Chim. Acta, 395 (2008) 155.
  • 19. L.Y. Chu, Y. Liang, W. Chen, X. Ju, H. Wang, Preparation of glucose-sensitive microcapsules with a porous membrane and functional Gates, Colloid. Surf. B., 37 (2004) 9.
  • 20. G. Ozyilmaz, S.S. Tukel, O.Z. Alptekin, Activity and storage stability of immobilised glucose oxidase onto magnesium silicate, J. Molecul. Catal. B: Enzym., 35 (2005) 154.
  • 21. T. Li, Z. Yao, L. Ding, Development of an amperometric biosensor based on glucose oxidase immobilized through silica sol–gel film onto Prussian Blue modified electrode, Sens. Act. B. Chem., 101 (2004) 155.
  • 22. L.Y. Chu, Y.J. Liang, W.M. Chen, X.J. Ju, H.D. Wang, Preparation of glucose-sensitive microcapsules with a porous membrane and functional Gates, Colloid. Surf. B., 37 (2004) 9.
There are 22 citations in total.

Details

Primary Language English
Journal Section Research Article
Authors

Fatma Ayhan This is me

Yasemin İspirli Doğaç This is me

Hakan Ayhan This is me

Publication Date August 1, 2011
Published in Issue Year 2011 Volume: 39 Issue: 3

Cite

APA Ayhan, F., Doğaç, Y. İ., & Ayhan, H. (2011). Cross-Linked Glucose Oxidase Aggregates: Synthesis and Characterization. Hacettepe Journal of Biology and Chemistry, 39(3), 241-251.
AMA Ayhan F, Doğaç Yİ, Ayhan H. Cross-Linked Glucose Oxidase Aggregates: Synthesis and Characterization. HJBC. August 2011;39(3):241-251.
Chicago Ayhan, Fatma, Yasemin İspirli Doğaç, and Hakan Ayhan. “Cross-Linked Glucose Oxidase Aggregates: Synthesis and Characterization”. Hacettepe Journal of Biology and Chemistry 39, no. 3 (August 2011): 241-51.
EndNote Ayhan F, Doğaç Yİ, Ayhan H (August 1, 2011) Cross-Linked Glucose Oxidase Aggregates: Synthesis and Characterization. Hacettepe Journal of Biology and Chemistry 39 3 241–251.
IEEE F. Ayhan, Y. İ. Doğaç, and H. Ayhan, “Cross-Linked Glucose Oxidase Aggregates: Synthesis and Characterization”, HJBC, vol. 39, no. 3, pp. 241–251, 2011.
ISNAD Ayhan, Fatma et al. “Cross-Linked Glucose Oxidase Aggregates: Synthesis and Characterization”. Hacettepe Journal of Biology and Chemistry 39/3 (August 2011), 241-251.
JAMA Ayhan F, Doğaç Yİ, Ayhan H. Cross-Linked Glucose Oxidase Aggregates: Synthesis and Characterization. HJBC. 2011;39:241–251.
MLA Ayhan, Fatma et al. “Cross-Linked Glucose Oxidase Aggregates: Synthesis and Characterization”. Hacettepe Journal of Biology and Chemistry, vol. 39, no. 3, 2011, pp. 241-5.
Vancouver Ayhan F, Doğaç Yİ, Ayhan H. Cross-Linked Glucose Oxidase Aggregates: Synthesis and Characterization. HJBC. 2011;39(3):241-5.

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