Antioxidant Biosensor Using Microbe

Authors: Dyah Iswantini, Trivadila, Novik Nurhidayat, Waras Nurcholis

Abstract:

The antioxidant compounds are needed for the food, beverages, and pharmaceuticals industry. For this purpose, an appropriate method is required to measure the antioxidant properties in various types of samples. Spectrophotometric method usually used has some weaknesses, including the high price, long sample preparation time, and less sensitivity. Among the alternative methods developed to overcome these weaknesses is antioxidant biosensor based on superoxide dismutase (SOD) enzyme. Therefore, this study was carried out to measure the SOD activity originating from Deinococcus radiodurans and to determine its kinetics properties. Carbon paste electrode modified with ferrocene and immobilized SOD exhibited anode and cathode current peak at potential of +400 and +300mv respectively, in both pure SOD and SOD of D. radiodurans. This indicated that the current generated was from superoxide catalytic dismutation reaction by SOD. Optimum conditions for SOD activity was at pH 9 and temperature of 27.50C for D. radiodurans SOD, and pH 11 and temperature of 200C for pure SOD. Dismutation reaction kinetics of superoxide catalyzed by SOD followed the Lineweaver-Burk kinetics with D. radiodurans SOD KMapp value was smaller than pure SOD. The result showed that D. radiodurans SOD had higher enzyme-substrate affinity and specificity than pure SOD. It concluded that D. radiodurans SOD had a great potential as biological recognition component for antioxidant biosensor.

Keywords: Antioxidant biosensor, Deinococcus radiodurans, enzyme kinetic, superoxide dismutase (SOD).

Digital Object Identifier (DOI): doi.org/10.5281/zenodo.1076848

References:

[1] S. Chevion, M. A. Roberts, M. Chevion, “The use of cyclic voltammetry for the evaluation antioxidant capacity,” Free Rad. Biol. Med. vol. 6, no. 28, pp. 860-870, 2000.
[2] R. Dinkov, G. Hristov, D. Stratiev, V. B. Aldayri, “Effect of commercially available antioxidants over biodiesel/diesel blends stability,” Fuel. vol. no. 88, pp.732-737, 2009.
[3] L. D. Mello, L.T. Kubota, “Biosensor as a tool for antioxidant status evaluation,” Talanta, vol. no. 72, pp. 335-348, 2007.
[4] B. Prieto-Simon, M. Cortina, M. Campas, C. Calas-Blanchard, “Electrochemical biosensor as a tool for antioxidant capacity assessment,” Sens. Actuators B, vol. no. 129, pp. 459-466, 2008.
[5] L. Campanella, G Favero, L. Persi, M. Tomasetti, “Evaluation of radical scavenging properties of several plants, fresh or from a herbalist’s using superoxide dismutase biosensor,” J. Pharm. Biomed. Anal, vol. 24, pp. 1055-1064, 2001.
[6] L. Campanella, A. Bonanni, G. Favero, M. Tomassetti, “Determination of antioxidant properties of aromatic herbs, olives and fresh fruit using an enzymatic sensor,” Anal. Bioanal. Chem, vol. 375, pp. 1011-1016, 2003.
[7] L. Campanella, A. Bonanni, M. Tomassetti, “Determination of antioxidant capacity of samples of different types of tea, or of beverages based on tea or other herbal product, using a superoxide dismutase biosensor,” J. Pharm. Biomed. Anal, vol. 32, pp. 725-736, 2003.
[8] L. Campanella, A. Bonanni, E. Finotti, M. Tomassetti, “Biosensors for determination of total and natural antioxidant capacity of red and white wines: comparison with other spectrophotometric and fluorimetric methods,” Biosens. Bielectron, vol. 19, pp. 641-651, 2003.
[9] L. Campanella, E. Martini, M. Tomassetti, “Antioxidant capacity of the algae using a biosensor method,” Talanta, vol. 66, pp. 902-911, 2005.
[10] L. Campanella, G. Favero, L. Persi, M. Tomasetti, “New biosensor for superoxide radical used to evidence molecules of biomedical and pharmaceutical interest having radical scavenging properties,” J. Pharm. Biomed. Anal, vol. 23, pp. 69-76, 2000.
[11] L. Campanella, A. Bonanni, D. Bellantoni, G. Favero, M. Tomassetti, “Comparison of fluorimetric, voltammetric and biosensor methods for the determination of total antioxidant capacity of drug products containing acetylsalicylic acid,” J. Pharm. Biomed. Anal, vol. 36, pp. 91-99, 2004.
[12] L. Campanella, A. Bonanni, D. Bellantoni, M. Tomassetti, “Biosensor for determination of total antioxidant capacity of phytotherapeutic integrators: comparison with other spectrophotometric, fluorimetric andvoltammetric methods,” J. Pharm. Biomed. Anal, vol. 35, pp. 303- 320, 2004.
[13] L. Campanella, S. De Luca, G. Favero, L. Persi, M. Tomassetti, “Superoxide dismutase biosensor working in non-aqueous solvent,” Fresenius J. Anal. Chem, vol. 369, pp. 594-600, 2001.
[14] A. Bonanni, L. Campanella, T. Gatta, E. Gregori, M. Tomassetti, “Evaluation of the antioxidant and prooxidant properties of several commercial dry spices by different analytical methods,” Food Chem,vol. 102, pp. 751-758, 2007.
[15] L. T. Benov, W. F. Beyer Jr, R. D. Stevens, I. Fridovich, “Purification and characterization of the Cu,Zn SOD from Escherichia coli,” Free Rad Bio Med, vol. 21, no. 1, pp. 117-121, 1996.
[16] M. B. Angelova, S. B. Pashova, L. S. Slokoska, “Comparison of antioxidant enzyme biosynthesis by free and immobilized Aspergillus niger cells,” Enzyme Microb. Tech, vol. 26, pp. 544-549, 2000.
[17] E. Zyracka et al., “Ascorbate abolishes auxotrophy caused by the lack of superoxide dismutase in Saccharomyces cerevisiae Yeast can be a biosensor for antioxidant,” J Biotech, vol. 115, pp. 271-278, 2005.
[18] R. J. Dennis et al., “Structure of the manganese superoxide dismutase from Deinococcus radiodurans in two crystal forms,” Acta cryst, vol. F62, pp. 325-329, 2006.
[19] S. Y. Young, N. L. Young, “Production of superoxide dismutase by Deinococcus radiophylus,” J. Biochem. Mol. Biol, vol. 36, no. 3, pp. 282-287, 2003.
[20] J. Jia-Ying, S. N. Keeney, E. M. Gregory, “Reconstitution of the Deinococcus radiodurans aposuperoxide dismutase,” Arch Biochem Biophysics, vol.286, no. 1, pp. 257-263, 1991.
[21] G. Fang-Xian, E. Shi-Jin, L. Shou-An, C. Jing, L. Duo-Chuan, “Purification and characterization of a thermostable MnSOD from the thermophilic fungus Chaetomium thermophilum,” Mycologia, vol. 100, no. 3, pp. 375-380, 2008.
[22] J. M. McCord, I. Fridovich, “Superoxide dismutase An enzymic for erythrocuprein (hemocuprein),” J. Biol. Chem, vol. 244, no. 22, pp. 6049-6055, 1969.
[23] L. Campanella, G. Favero, M. Tomasetti, “A modified amperometric electrode for the determination of free radical,” Sens. Actuators B, vol. 44, pp. 559-565, 1997.
[24] T. Ikeda et al., ”Electrochemical monitoring of in vivo reconstitution of glucose dehydrogenase in Escherichia coli cells with externally added pyrroloquinoline quinine,” J. Electroanal. Chem, vol. 449, pp. 219-224, 1998.
[25] A. M. Earl, M. M. Mohundro, I. S. Mian, J. R. Battista, “The irrE protein of Deinococcus radiodurans R1 is a novel regulator of iA expression,” J. Bacteriol, vol. 184, no. 22, pp. 6216-6224, 2002.
[26] Y. Zhang et al., “irrE, an exogenous gene from Deinococcus radiodurans, improves the growth of and ethanol production by a Zymomonas mobilis strain under ethanol and acid stress,” J. Microbiol. Biotechnol, vol. 20, no. 7, pp. 1156-1162, 2010.
[27] F. Yamakura, “Purification, crystallization, and properties of ironcontaining superoxide dismutase from Pseudomonas ovalis,” Biochim. Biophys. Act, vol. 422, pp. 280-294, 1976.
[28] J.R. Battista, “Against all odds: the survival strategies of Deinococcus radiodurans,” Annu, Rev. Microbiol, vol. 51, pp. 203-224, 1997.
[29] D. Iswantini, K. Kato, K. Kano, T. Ikeda, “Electrochemical measurements of glucose dehydrogenase activity exhibited by Escherichia coli cells; effects of the addition of pyrroloquinoline quinone, magnesium or calcium ions and ethylenediaminetetraacetic acid, ”Bioelectrochemistry and Bioenergetics, vol. 46, pp.249-254, 1998.
[30] K. Endo et al., “Development of superoxide sensor by immobilization of superoxide dismutase,” Sens. Actuators B, vol. 83, pp. 30-34, 2002.
[31] J. Di, S. Peng, C. Shen, Y. Gao, Y. Tu, “One-step method embedding superoxide dismutase and gold nanoparticles in silica sol-gel network in the presence of cysteine for construction of third-generation biosensor,” Biosens. Bioelectron, vol. 23, pp. 88-94, 2007.
[32] R. Antiochia, K. Movassaghi,P Lipone, L. Campanella, “Determination of the antioxidant capacity of different types of bread and flour using a superoxide dismutase biosensor, J. Chem. Chem. Eng, vol. 6, pp. 199- 208, 2012.
[33] I. A. Abreu, D. E. Cabelli, “Superoxide dismutases-a review of the metal-associated mechanistic variations,” BBAPAP, vol. 1804, pp. 263- 274, 2010.
[34] I.A. Abreu et al., “The kinetic mechanism of manganese-containing superoxide dismutase from Deinococcus radiodurans : A specialized enzyme for the elimination of high superoxide concentrations,” Biochem, vol. 47, pp. 2350-2356, 2008.
[35] A. Rigo, P. Viglino, G Rotilio, “Kinetic study of O2 - dismutation by bovine superoxide dismutase. Evidence for saturation of the catalytic sites by O2 -,” Biochem Biophysic Res Comm, vol. 63, no. 4, pp. 1013- 1018, 1975.