Stability improvement by crosslinking of previously immobilized glucose oxidase on carbon nanotube-based bioanode

Bioanode stability with glucose oxidase was enhanced significantly by covalent crosslinking without substantialenzymatic activity and affinity loss. Initially, glucose oxidase was immobilized by aldehyde groups on the electrode that was developed using ferrocenecarboxaldehyde, polyethyleneimine, multiwall carbon nanotubes, and carbon cloth for biofuel cell applications. The glucose oxidase half-life was extended by more than 4 times, from 27.2 to 124.7 h, after the electrode was crosslinked. Enzymatic kinetic parameters were determined for the crosslinked enzyme and they were compared to the noncrosslinked immobilized enzyme parameters on the electrode. The apparent substrate affinity of the crosslinked enzyme electrode was decreased (i.e. kM was increased) by 16%, while the maximum reaction rate was decreased by only 3%, by the crosslinking process. Moreover, effects of the electrolyte type (i.e. buffer type) and concentration on the performance of the crosslinked enzyme electrode were evaluated and appropriate conditions were determined.

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1. Kim J, Jia H, Wang P. Challenges in biocatalysis for enzyme-based biofuel cells. Biotechnology Advances 2006; 24 (3): 296-308.
2. Kim BC, Zhao X, Ahn HK, Kim JH, Lee HJ et al. Highly stable enzyme precipitate coatings and their electrochemical applications. Biosensors and Bioelectronics 2011; 26 (5): 1980-1986.
3. Minteer SD, Liaw BY, Cooney MJ. Enzyme-based biofuel cells. Current Opinion in Biotechnology 2007; 18 (3): 228-234.
4. Moore CM, Akers NL, Hill AD, Johnson ZC, Minteer SD. Improving the environment for immobilized dehydrogenase enzymes by modifying Nafion with tetraalkylammonium bromides. Biomacromolecules 2004; 5 (4): 1241-1247.
5. Gallaway JW. Mediated enzyme electrodes. In: Luckarift HR, Atanassov P, Johnson GR (editors). Enzymatic Fuel Cells: From Fundamentals to Applications. Hoboken, NJ, USA: Wiley, 2014, pp. 146-180.
6. Kavanagh P, Leech D. Mediated electron transfer in glucose oxidizing enzyme electrodes for application to biofuel cells: recent progress and perspectives. Physical Chemistry Chemical Physics 2013; 15 (14): 4859-4869.
7. Ivnitski D, Branch B, Atanassov P, Apblett C. Glucose oxidase anode for biofuel cell, based on direct electron transfer. Electrochemistry Communications 2006; 8 (8): 1204-1210.
8. Kuwahara T, Ohta H, Kondo M, Shimomura M. Immobilization of glucose oxidase on carbon paper electrodes modified with conducting polymer and its application to a glucose fuel cell. Bioelectrochemistry 2008; 74 (1): 66-72.
9. Gouda MD, Singh SA, Rao AGA, Thakur MS, Karanth NG, Thermal Inactivation of Glucose Oxidase. Journal of Biological Chemistry 2003; 278 (27): 24324-24333.
10. Betancor L, Lopez-Gallego F, Hidalgo A, Alonso-Morales N, Dellamora-Ortiz G et al. Preparation of a very stable immobilized biocatalyst of glucose oxidase from Aspergillus niger. Journal of Biotechnology 2006; 121 (2): 284-289.
11. Betancor L, Lopez-Gallego F, Hidalgo A, Alonso-Morales N, Dellamora-Ortiz G et al. Different mechanisms of protein immobilization on glutaraldehyde activated supports: effect of support activation and immobilization conditions. Enzyme and Microbial Technology 2006; 39 (4): 877-882.
12. Mateo C, Palomo JM, Fuentes M, Betancor L, Grazu V et al. Glyoxyl agarose: a fully inert and hydrophilic support for immobilization and high stabilization of proteins. Enzyme and Microbial Technology 2006; 39 (2): 274-280.
13. Schoevaart R, Wolbers MW, Golubovic M, Ottens M, Kieboom APG et al. Preparation, optimization, and structures of crosslinked enzyme aggregates (CLEAs). Biotechnology and Bioengineering 2004; 87 (6): 754-762.
14. Cao L, van Langen L, Sheldon RA. Immobilised enzymes: carrier-bound or carrier-free. Current Opinion in Biotechnology 2003; 14 (4): 387-394.
15. Bahar T. Preparation of a ferrocene mediated bioanode for biofuel cells by MWCNTs, polyethylenimine and glutaraldehyde: glucose oxidase immobilization and characterization. Asia-Pacific Journal of Chemical Engineering 2016; 11 (6): 981-988.
16. Bahar T, Yazici MS. Immobilized glucose oxidase biofuel cell anode by MWCNTs, ferrocene and polyethylenimine: electrochemical performance. Asia-Pacific Journal of Chemical Engineering 2018; 13 (1): e2149. doi: 10.1002/apj.2149
17. Chuang CL, Wang YJ, Lan HL. Amperometric glucose sensors based on ferrocene containing B-polyethylenimine and immobilized glucose oxidase. Analytica Chimica Acta 1997; 353 (1): 37-44.
18. Bahar T. Clinoptilolite particles as a carrier for biocatalysts immobilization: invertase immobilization and characterization. Asia-Pacific Journal of Chemical Engineering 2014; 9 (1): 31-38.
19. Ohnishi ST, Barr JK. A simplified method of quantitating protein using the biuret and phenol reagents. Analytical Biochemistry 1978; 86 (1): 193-200.
20. Cardoso JP, Emery AH. A new model to describe enzyme inactivation. Biotechnology and Bioengineering 1978; 20 (9): 1471-1477.
21. Laider KJ, Bunting PS. The kinetics of immobilized enzyme systems. In: Purich DL (editor). Methods in Enzymology 64. San Diego, CA, USA: Academic Press, 1980, pp. 227-248.
22. Bahar T, Yazici MS. Performance assessment of a perfluorosulfonic acid-type membrane (i.e. Nafion 115) for an enzymatic fuel cell, Electroanalysis 2019; 31: 1656-1663. doi: 10.1002/elan.201900171
23. Yeager HL. Cation exchange selectivity of a perfluorosulfonate membrane. In: Eisenberg A, Yeager HL (editors). Perfluorinated Ionomer Membranes. 2nd ed. New York, NY, USA: ACS Symposium Series, 1982, pp. 41-64.
24. Merchant SA, Meredith MT, Tran TO, Brunski DB, Johnson MB et al. Effect of mediator spacing on electrochemical and enzymatic response of ferrocene redox polymers. Journal of Physical Chemistry C 2010; 114 (26): 11627-11634.
25. Sakai H, Nakagawa T, Tokita Y, Hatazawa T, Ikeda T et al. High-power glucose/oxygen biofuel cell operating under quiescent conditions. Energy & Environmental Science 2009; 2: 133-139.
26. Voet JG, Coe J, Epstein J, Matossian, V, Shipley T. Electrostatic control of enzyme reactions: effect of ionic strength on the pKa of an essential acidic group on glucose oxidase. Biochemistry 1981; 20 (25): 7182-7185.