Skip to main content
SpringerLink
Log in

Characterization of secondary structure and fad conformational state in free and sol–gel immobilized glucose oxidase

  • Original Paper
  • Published:
Journal of Sol-Gel Science and Technology Aims and scope Submit manuscript

Abstract

Immobilization procedures are a fundamental step in the technological use of enzymes. Among the different immobilization procedures sol–gel technique is widely recognised as a valuable approach to obtain very high quality catalytic supports. In this paper different optical techniques have been used and compared to investigate structural and dynamic properties of glucose oxidase (GOD) prior and after sol–gel immobilization process. In particular, using Fourier Transform infrared micro-spectroscopy and time-resolved fluorescence the secondary structure of GOD and the flavin adenine dinucleotide (FAD) conformational changes have been respectively investigated. Infrared spectroscopy measurements have confirmed that enzymatic activity is preserved and a predominant β-sheet subcomponent is retained by immobilized GOD. By time-resolved FAD fluorescence a three-exponential decaying behaviour has been observed for both free and immobilized enzymes with three different lifetimes, each being characteristic of a peculiar conformational state of the FAD structure. The comparison between lifetime values for free and immobilized GOD has not shown significant differences, while the fractional steady-state intensities of the single exponential components have been changed by immobilization procedure. All results reported and discussed in this paper have confirmed once again the efficacy of the adopted sol–gel immobilization procedure for enzymes and proteins. In addition, the joint use of different optical spectroscopic techniques has shown to be a very valuable tool for getting a better insight into structural and dynamic properties of immobilized enzymes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Brinker J, Scherer GW (1990) The physics and chemistry of sol gel processing. Academic Press, New York

    Google Scholar 

  2. Dave BC, Dunn B, Valentine JS, Zink JI (1998) Sol–gel matrices for protein entrapment. In: Cass T, Ligier FS (eds) Immobilized biomolecules in analysis, a practical approach. Oxford University, Oxford

    Google Scholar 

  3. Pierre AC (2004) The sol–gel encapsulation of enzymes. Biocatal Biotransform 22:145–170

    Article  Google Scholar 

  4. Mac Craith BD, McDonagh C, McEvoy AK, Butler T, O’Keeffe G, Murphy V (1997) Optical chemical sensors based on sol–gel materials: recent advances and critical issues. J Solgel Sci Technol 8:1053–1061

    Article  Google Scholar 

  5. Jin W, Brennan JD (2002) Properties and applications of proteins encapsulated within sol–gel derived materials. Anal Chim Acta 461:1–36

    Article  Google Scholar 

  6. Sakka S (2005) Handbook of Sol-gel science and technology: processing, characterization and applications. Kluwer Academic Publisher, New York (USA)

    Google Scholar 

  7. Prodromis MI, Karayannis MI (2002) Enzyme based amperometric biosensors for food analysis. Electroanalysis 14:241–261

    Article  Google Scholar 

  8. Chaudhury NK, Bhardwaj R, Murari BM (2003) Fluorescence spectroscopic study to characterize and monitor TEOS based sol–gel process for development of optical biosensors. Curr Appl Phys 3:177–184

    Article  Google Scholar 

  9. Tatsu Y, Yamashita K, Yamaguchi M, Yamamura S, Yamamoto H, Yoshikawa S (1992) Entrapment of glucose oxidase in silica gel by the sol–gel method and its application to glucose sensor. Chem Lett 21:1615–1618

    Article  Google Scholar 

  10. Narang U, Prasad PN, Bright FV, Ramanathan K, Kumar DK, Malhotra BD, Kamalahasan MN, Chandra S (1994) Glucose biosensor based on a sol–gel-derived platform. Anal Chem 66:3139–3144

    Article  Google Scholar 

  11. Portaccio M, Lepore M, Della Ventura B, Stoilova O, Manolova N, Rashkov I, Mita DG (2009) Fiber-optic glucose biosensor based on glucose oxidase immobilised in a silica gel matrix. J Solgel Sci Technol 50:437–448

    Article  Google Scholar 

  12. Esposito R, Della Ventura B, De Nicola S, Altucci C, Velotta R, Mita DG, Lepore M (2011) Glucose sensing based on the intrinsic time-resolved visible fluorescence of sol–gel immobilized glucose oxidase. Sensors 11:3483–3497

    Article  Google Scholar 

  13. Lim J, Malati P, Bonet F, Dunn B (2007) Nanostructured sol–gel electrodes for biofuel cells. J Electrochem Soc 154:A140–A145

    Article  Google Scholar 

  14. Devadas B, Mani V, Chen SM (2012) A glucose/O2 biofuel cell based on graphene and multiwalled carbon nanotube composite modified electrode. Int J Electrochem Sci 7:8064–8075

    Google Scholar 

  15. Hartnett AM, Ingersoll CM, Baker GA, Bright FV (1999) Kinetics and thermodynamics of free flavins and the flavins-based redox active site within glucose oxidase dissolved in solution or sequestered within a sol–gel derived glass. Anal Chem 71:1215–1224

    Article  Google Scholar 

  16. Przybyt M (2003) Behaviour of glucose oxidase during formation and ageing of silica gel studied by fluorescence spectroscopy. Mater Sci 21:397–416

    Google Scholar 

  17. Han K, Wu Z, Lee J, Ahn I-S, Park JW, Min BR, Lee K (2005) Activity of glucose oxidase entrapped in mesoporous gels. Biochem Eng J 22:161–166

    Article  Google Scholar 

  18. Przybyt M, Miller E, Szreder T (2011) Thermostability of glucose oxidase in silica gel obtained by sol–gel method and in solution studied by fluorimetric method. J Photochem Photobiol B Biol 103:22–28

    Article  Google Scholar 

  19. Portaccio M, Della Ventura B, Mita DG, Manolova N, Stoilova O, Rashkov I, Lepore M (2011) FT-IR microscopy characterization of sol–gel layers prior and after glucose oxidase immobilization for biosensing applications. J Solgel Sci Technol 57:204–211

    Article  Google Scholar 

  20. Delfino I, Portaccio M, Della Ventura B, Mita DG, Lepore M (2013) Enzyme distribution and secondary structure of sol–gel immobilized glucose oxidase by means of micro-attenuated total reflection FT-IR spectroscopy. Mater Sci Eng C 33:304–310. doi:10.1016/j.msec.2012.08.044

    Article  Google Scholar 

  21. Zuo S, Teng Y, Yuan H, Lan M (2008) Direct electrochemistry of glucose oxidase on screen-printed electrodes through one-step enzyme immobilization process with silica sol–gel/polyvinyl alcohol hybrid film. Sens Actuators B 133:555–560

    Article  Google Scholar 

  22. Kong T, Chen Y, Ye Y, Zhang K, Wang Z, Wang X (2009) An amperometric glucose biosensor based on the immobilization of glucose oxidase on the ZnO nanotubes. Sens Actuators B 138:344–350

    Article  Google Scholar 

  23. Dirix C, Duvetter T, van Loey A, Hendrickx M, Heremans K (2005) The in situ observation of the temperature and pressure stability of recombinant Aspergillus aculeatus pectin methylesterase with Fourier transform IR spectroscopy reveals an unusual pressure stability of b-helices. Biochem J 392:565–571. doi:10.1042/BJ20050721

    Article  Google Scholar 

  24. Zhao H-Z, Sun J–J, Song J, Yang Q-Z (2010) Direct electron transfer and conformational change of glucose oxidase on carbon nanotube-based electrodes. Carbon 48:1508–1514

    Article  Google Scholar 

  25. Smith AW (2012) Lipid–protein interactions in biological membranes: a dynamic perspective. Biochim Biophys Acta 118:172–177

    Article  Google Scholar 

  26. Engelborghs Y (2001) The analysis of time resolved protein fluorescence in multi-tryptophan proteins. Spectrochim Acta A Mol Biomol Spectrosc 57:2255–2270

    Article  Google Scholar 

  27. Esposito R, Delfino I, Lepore M (2013) Time-resolved flavin adenine dinucleotide fluorescence study of the interaction between immobilized glucose oxidase and glucose. J Fluoresc 23:947–955. doi:10.1007/s10895-013-1220-z

    Article  Google Scholar 

  28. Shao Q, Wu P, Xu X, Zhang H, Cai C (2012) Insight into the effects of graphene oxide sheets on the conformation and activity of glucose oxidase: towards developing a nanomaterial-based protein conformation assay. Phys Chem Chem Phys 14:9076–9085

    Article  Google Scholar 

  29. Perkin Elmer Inc. (2004) Applications and Design of a Micro-ATR objective: Technical Note http://www.perkinelmer.com/CMSResources/Images/44-4861TCH_MicroATRObjective.pdf

  30. Levenberg K (1944) A method for the estimation of certain non-linear problems in least squares. Q Appl Math 2:164–168

    Google Scholar 

  31. Marquardt DW (1963) An algorithm for least squares estimation of nonlinear parameters. ESIAM J Appl Math 11:431–441

    Article  Google Scholar 

  32. Zhong D, Zewail AH (2001) Femtosecond dynamics of flavoproteins: charge separation and recombination in riboflavine (Vitamin B2)-binding protein and in glucose oxidase enzyme. Proc Natl Acad Sci USA 98:11867–11872

    Article  Google Scholar 

  33. Lepore M, Portaccio M, De Tommasi E, De Luca P, Bencivenga U, Maiuri P, Mita DG (2004) Glucose concentration determination by means of fluorescence emission spectra of soluble and insoluble glucose oxidase: some useful indications for optical fibre-based sensors. J Mol Catal B Enzym 31:151–158

    Article  Google Scholar 

  34. Trettnak W, Wolfbeis OS (1989) Fully reversible fiber-optic glucose biosensor based on the intrinsic fluorescence of glucose oxidase. Anal Chim Acta 211:195–203

    Article  Google Scholar 

  35. Started G (1997) MATLAB The Language of Technical Computing. Components, L(7):186

  36. Qingwen L, Guoan L, Yiming W, Xingrong Z (2000) Immobilization of glucose oxidase in sol–gel matrix and its application to fabricate chemiluminescent glucose sensor. Mater Sci Eng C 11:67–70. doi:10.1016/S0928-4931(00)00130-2

    Article  Google Scholar 

  37. Barth A (2007) Infrared spectroscopy of proteins. Biochim Biophys Acta Bioenerg 1767:1073–1101

    Article  Google Scholar 

  38. Krimm S, Bandekar J (1986) Vibrational spectroscopy and conformation of peptides, polypeptides, and proteins. Adv Protein Chem 38:181–364

    Article  Google Scholar 

  39. Susi H, Byler DM (1986) Resolution-enhanced Fourier transform infrared spectroscopy of enzymes. Methods Enzymol 130:290–311

    Article  Google Scholar 

  40. Mei Y, Miller L, Gao W, Gross RA (2003) Imaging the distribution and secondary structure of immobilized enzymes using infrared microspectroscopy. Biomacromolecules 4:70–74. doi:10.1021/bm025611t

    Article  Google Scholar 

  41. Jurgen-Lohmann DL, Nacke C, Legge RL, Simon LC (2009) Preparation and methodolodgy for chemical mapping of sol–gel thin films containing lysozyme. J Solgel Sci Technol 50:77–86

    Article  Google Scholar 

  42. Kong T, Chen Y, Ye Y, Zhang K, Wang Z, Wang X (2009) An amperometric glucose biosensor based on the immobilization of glucose oxidase on the ZnO nanotubes. Sens Actuators B 138:344–350

    Article  Google Scholar 

  43. Liang R-P, Jiang J-L, Qiu J-D (2008) Preparation of GOD/Sol–Gel silica film on prussian blue modified electrode for glucose biosensor application. Electroanalysis 20:2642–2648. doi:10.1002/elan.200804370

    Article  Google Scholar 

  44. Li J, Wang YB, Qiu J-D, Sun DC, Xia XH (2005) Biocomposites of covalently linked glucose oxidase on carbon nanotubes for glucose biosensor. Anal Bioanal Chem 383:918–922

    Article  Google Scholar 

  45. Jackson M, Mantsch HH (1995) The use and misuse of FTIR spectroscopy in the determination of protein structure. Crit Rev Biochem Mol Biol 30:95–120. doi:10.3109/10409239509085140

    Article  Google Scholar 

  46. Hecht HJ, Kalisz HM, Hendle J, Schmid RD, Schomburg D (1993) Crystal structure of glucose oxidase from Aspergillus niger refined at 2.3 A resolution. J Mol Biol 229:153–172

    Article  Google Scholar 

  47. van de Weert M, Haris PI, Hennink WE, Crommelin DJ (2001) Fourier transform infrared spectrometric analysis of protein conformation: effect of sampling method and stress factors. Anal Biochem 297:160–169

    Article  Google Scholar 

  48. Gouda MD, Singh SA, Rao AGA, Thakurt MS, Karanth NG (2003) Thermal inactivation of glucose oxidase: mechanism and stabilization using additives. J Biol Chem 278:24324–24333. doi:10.1074/jbc.M208711200

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Dipartimento di Medicina Sperimentale, Seconda Università di Napoli, Naples, Italy

    Marianna Portaccio & Maria Lepore

  2. Dipartimento di Scienze Fisiche, Università “Federico II”, Naples, Italy

    Rosario Esposito

  3. Dipartimento di Scienze Ecologiche e Biologiche, Università della Tuscia, I-01100, Viterbo, Italy

    Ines Delfino

Authors
  1. Marianna Portaccio
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rosario Esposito
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ines Delfino
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Maria Lepore
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ines Delfino.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Portaccio, M., Esposito, R., Delfino, I. et al. Characterization of secondary structure and fad conformational state in free and sol–gel immobilized glucose oxidase. J Sol-Gel Sci Technol 71, 580–588 (2014). https://doi.org/10.1007/s10971-014-3408-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10971-014-3408-3

Keywords

Search

Navigation