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Recombinant pyranose dehydrogenase—A versatile enzyme possessing both mediated and direct electron transfer
Electrochemistry Communications 24 (2012) 120–122
Contents lists available at SciVerse ScienceDirect
Electrochemistry Communications journal homepage: www.elsevier.com/locate/elecom
Recombinant pyranose dehydrogenase—A versatile enzyme possessing both mediated and direct electron transfer Maria E. Yakovleva a, Anikó Killyéni a, b, Roberto Ortiz a, Christopher Schulz a, Domhnall MacAodha c, Peter Ó. Conghaile c, Dónal Leech c, Ionel Catalin Popescu b, Christoph Gonaus d, Clemens K. Peterbauer d, Lo Gorton a,⁎ a
Department of Chemistry, Lund University, P.O. Box 124, SE-22100 Lund, Sweden Department of Physical Chemistry, Babes-Bolyai University, Cluj-Napoca, Romania c School of Chemistry, National University of Ireland Galway, University Road, Galway, Ireland d Department of Food Sciences and Technology, BOKU-University, Muthgasse 18, A-1190 Wien, Austria b
a r t i c l e
i n f o
Article history: Received 25 June 2012 Received in revised form 28 August 2012 Accepted 29 August 2012 Available online 8 September 2012 Keywords: Pyranose dehydrogenase Deglycosylation Osmium polymer Direct electron transfer Biosensor
a b s t r a c t The catalytical properties of glycosylated pyranose dehydrogenase (gPDH) and deglycosylated PDH (dgPDH) from Agaricus meleagris recombinantly expressed in Pichia pastoris were studied. Both gPDH and dgPDH were “wired” to an osmium redox polymer on graphite electrodes mounted in a flow-injection system. The current from oxidation of glucose by immobilised gPDH and dgPDH was compared using flow injection amperometry and cyclic voltammetry. An increase in the current density was observed for dgPDH (190 μA cm−2) compared with that for gPDH (90 μA cm−2) due to the improved electron transfer between the active site and the electrode. Additionally, the ability of dgPDH for direct electron transfer (DET) was discovered, which is rather unique among FAD-containing enzymes. The ability to oxidise a variety of sugars at a rather low potential makes dgPDH attractive for construction of biofuel cells with high power output. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Pyranose dehydrogenase (PDH) is an extracellular monomeric fungal glycoprotein (7% glycosylation) produced by litter-degrading fungi containing one covalently bound flavin adenine dinucleotide (FAD) as prosthetic group [1], belonging to the widespread flavoprotein glucosemethanol-choline oxidoreductase superfamily [2]. PDH has attracted attention due to applications in carbohydrate, organic and analytical bioelectrochemistry. Compared with pyranose oxidase PDH has broader substrate specificity and can oxidise various mono-, di-, and oligosaccharides in their pyranose form [3]. Depending on the source of PDH and substrate structure, the enzyme can catalyse the oxidation of sugars at the C‐1, C-2, and C-3 or dioxidation at C-1,3, C-2,3 and C-3,4 positions to their corresponding aldonolactones, or (di)dehydrosugars (aldos(di) uloses) [4–6]. The diversity of substrates makes PDH attractive for production of intermediate sugar derivatives further utilised in industrial synthesis of rare sugars or drug development [4]. Also, PDH is a true dehydrogenase as it cannot utilise O2 as an electron acceptor (c.f. sugar oxidases) and this is beneficial for fabrication of membraneless enzyme biofuel cells (EBFCs), where utilisation of O2 dependent enzymes at the anode part will result in production of H2O2. Even trace amounts of H2O2 can cause oxidative damage to commonly used enzymes (laccase ⁎ Corresponding author. Tel.: +46 46 222 7582; fax: +46 46 222 4544. E-mail address: [email protected] (L. Gorton). 1388-2481/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.elecom.2012.08.029
and bilirubin oxidase) at the cathode part of the EBFC and reduce the power output. The ability of PDH to operate at physiological conditions is also advantageous, when implantable devices are to be constructed [7]. In order to use the beneficial properties of PDH in the development of EBFCs, the PDH isolated from Agaricus meleagris was electrically “wired” to various Os-polymers with formal potentials (E°′) ranging from −270 to +160 mV vs. Ag|AgCl0.1 M KCl [8,9]. This was further extended by co-immobilisation of PDH with another enzyme with the Os-polymer. By the combination of several enzymes with different site-specificity for glucose, it was possible to extract more than 2 e− per substrate molecule thus increasing the current density and Coulombic efficiency of the bioanode for use in EBFC [10]. Since the expression levels of PDH in the natural source are low and in order to meet the high demands for PDH production, the AmPDH gene was expressed in heterologous expression hosts such as Aspergillus sp. [11], Escherichia coli and Pichia pastoris [12]. Expression of AmPDH in Aspergillus sp. was genetically elaborate and time-consuming. Therefore, other microbial systems were tested. Expression in E. coli resulted in production of insoluble protein with no activity. This can be attributed to the inability of prokaryotic organisms to form the glycosylation shell essential for correct folding, long term stability, and solubility [13,14]. When using eukaryotic P. pastoris as host large-scale production combined with a simple purification procedure was established. The MW of recAmPDH was determined to be about 93 kDa corresponding to about 30% glycosylation [12]. The much higher degree of glycosylation
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121
(30% vs. 7% for the wild type AmPDH, MW 66.5 kDa [1]) resulted in 20% reduced specific activity of the recombinant enzyme compared to the wild-type. Therefore, here the recombinant enzyme was deglycosylated by endoglycosidase H (Endo H) and both gPDH and dgPDH, when immobilised onto the electrode, were characterised using amperometry and cyclic voltammetry. Both gPDH and dgPDH were “wired” with an Os-polymer and the current response for glucose was registered. Additionally, the ability of dgAmPDH to exhibit direct electron transfer (DET) was tested, as it was shown previously that depletion of the glycan moiety of an enzyme results in shortening the distance between the active centre of the enzyme and the electrode surface facilitating DET [15–18].
temperature. For MET 2 μl of an aqueous Os-polymer solution (10 mg ml − 1) and 1 μl of a freshly prepared aqueous 68% (v/v) PEGDGE solution were placed on the top of the electrodes. After 10 min 5 μl of enzyme solution (50.8 U cm − 2) was added. The electrodes were left overnight at 4 °C for complete cross-linking [9]. For DET 5 μl of enzyme solution with different enzyme loadings (1.8–38.8 U cm − 2) was evenly distributed on the top of the electrodes and allowed to adsorb overnight at 4 °C. For both DET and MET the excess of the enzyme was washed away with the buffer.
2. Material and methods
Based on previous studies [8] the Os-polymer with an E°′ of +32 mV vs Ag|AgCl0.1 M KCl was chosen for electrical “wiring” of recombinant AmPDH to the electrode. The E°′-value of the Os-polymer is slightly higher than that of AmPDH (−140 mV vs. Ag|AgCl0.1 M KCl, pH 7.4 [21]), which from a thermodynamic point of view represents the ideal case for efficient electron transfer from the enzyme to the electrode [22]. To study the effect of deglycosylation, both gPDH and dgPDH were separately cross-linked to the Os-polymer on the electrodes. Equal amounts of gPDH and dgPDH (in terms of activity) were used for the construction of the electrodes. As shown in Fig. 1 at least a 2-fold increase in the maximal catalytic current density (Jmax) for the electrodes modified with dgPDH (Vmax; 13.15 ± 0.40 μA, Jmax; 190 μA cm − 2) compared with that for gPDH (Vmax; 6.37 ± 0.081 μA, Jmax; 90 μA cm − 2) was observed. The linear range for both modified electrodes was the same, between 0.1 and 1 mM, however, the lower Κapp Μ value for dgPDG: 2.57 ± 0.30 mM (7.93 ± 0.32 mM for gPDH) indicates a higher affinity of the Os-polymer for dgPDH. The increase in current response and affinity for the dgPDG modified electrode might be attributed to a better electron transfer between the Os-polymer and the bound FAD becoming more accessible after deglycosylation or altered substrate access through the osmium–polymer enzyme network. However, it may also be partially attributed to a higher concentration of Ospolymer around the smaller dgPDH [15,23]. Deglycosylation of AmPDH resulted in DET between dgPDH and graphite. Glycosylation acts as an insulator on the protein surface and increases the distance between the active centre and the electrode making electron transfer (ET) less efficient or not at all [24,25]. Thus studies were performed in order to chemically or enzymatically remove the glycan shell and improve ET. So far except for deglycosylated peroxidases [16–18] only a few published papers on FAD-containing glucose oxidase
2.1. Materials Pyranose dehydrogenase from A. meleagris (AmPDH) was recombinantly expressed in P. pastoris and later deglycosylated with Endo Hf (New England Biolabs, Bionordiska AB, Stockholm, Sweden) as described previously [12]. Poly(ethylene glycol) (400) diglycidyl ether (PEGDGE), ferricenium hexafluorophosphate and D(+)-glucose were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). The Os redox polymer [Os(4,4′-dimethyl2,2′-bipyridine)2poly(N-vinylimidazole)10Cl] +2/+, E°′; +32 mV vs. Ag|AgCl0.1 M KCl, was synthesised as described in [19]. Water was purified with a Milli-Q purification system (Millipore, Bedford, MA, USA). 2.2. Equipment Flow injection measurements were performed with a flow-through wall-jet amperometric cell, containing a Pt wire as the counter and an Ag|AgCl0.1 M KCl, +288 mV vs. NHE as a reference electrode. The applied potential was controlled by a three-electrode potentiostat (Zäta Electronics, Höör, Sweden). The response of graphite rod electrodes (Ringsdorff Werke GmbH, Bonn, Germany, type RW001, 3.05 mm diameter, 13% porosity) was registered with a recorder (BD 112, Kipp & Zonen, Utrecht, The Netherlands). For the introduction of the samples an injector (Rheodyne, type 7125 LabPR, Cotati, CA, USA) with a 50 μl loop was used. The kinetic parameters were calculated by fitting the data using the Michaelis–Menten equation. A 50 mM phosphate buffer (pH 7.4) containing 137 mM NaCl and a 0.1 M phosphate buffer (pH 7.4) was used as a carrier at 0.45 ml min−1 for MET and 0.5 ml min−1 for DET, respectively. Before use the carrier was degassed for 20 min. CVs were performed with a computer controlled AutoLab PGSTAT30 electrochemical system (Metrohm Nordic AB, Bromma, Sweden) using the modified graphite electrode as a working electrode, an Ag|AgClsat. KCl, +199 mV vs. NHE as reference and a Pt foil as counter electrode. Before measurement Ar was purged through the solution for 10 min.
3. Results and discussion
2.3. Enzyme assay The activities of gPDH and dgPDH were determined by monitoring the reduction of ferricenium (Fc+) to ferrocene at 300 nm, ε= 4.3 mM−1 cm−1, for 3 min at 20 °C in a standard photometric assay [20]. One millilitre of standard solution contained 100 μmol of phosphate buffer, pH 7.4, 50 μmol of D-glucose, 0.4 μmol of Fc+PF6− (prepared daily by dissolving 3.3 mg of salt in 5 ml of 5 mM HCl) and an appropriately diluted enzyme. One unit of enzyme activity was defined as the amount of enzyme necessary for the reduction of 2 μmol of Fc+ per 1 min at 20 °C. 2.4. Preparation of electrodes Graphite electrodes (geometric area of 0.071 cm 2) were polished on wet emery paper, rinsed with Milli-Q water, and dried at room
Fig 1. Calibration curves for glucose for gPDH/Os-polymer and dgPDH/Os polymer-modified electrodes. E appl ; + 200 mV vs. Ag|AgCl 0.1 M KCl , 50 mM phosphate buffer, containing 137 mM NaCl, pH 7.4, 50 μl injected sample, flow rate 0.45 ml min − 1. Insert: calibration curve for glucose of a dgPDH-modified electrode. E appl ; + 180 mV vs. Ag|AgCl0.1 M KCl , 0.1 M phosphate buffer, pH 7.4, 50 μl injected sample, flow rate 0.5 ml min − 1 .
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further applications of PDH as a biocatalyst in the development of EBFCs.
20 15 10
Acknowledgements
I/A
5 0 -5 1.8 U/cm2 (5µL) in 0.1 M PB, pH 7.4
-10
1.8 U/cm2 (5µL) + 25 mM glucose
-15
3.7 U/cm2 (5µL) + 25 mM glucose 9.2 U/cm2 (5µL) + 25 mM glucose
-20
36.8 U/cm2 (5µL + 5 µL) + 25 mM glucose
-25 -0.6
-0.4
-0.2
0.0
0.2
Financial support was obtained from the following agencies: (AK) the Sectoral Operational Programme for Human Resources Development 2007–2013, co-financed by the European Social Fund (POSDRU/107/1.5/ S/76841 “Modern Doctoral Studies: Internationalization and Interdisciplinarity”), (LG) the Swedish Research Council (project 2010–5031), (DL, DMA and LG) the European Commission (“3D-Nanobiodevice”, NMP4-SL-2009-229255) and “Chebana” FP7-PEOPLE-2010-ITN-264772), (POC) an ERA-Chemistry award, (CG and CP) the Austrian Science Foundation, Translational Research Program project number TRP 218-BL and (AK and ICP) the project PN II-IDPCCE 140/2008.
0.4
Eappl. / V vs. Ag/AgCl (sat. KCl) Fig 2. CV of dgPDH-modified electrodes with different enzyme loadings in the presence and absence of 25 mM glucose (scan rate; 10 mV s−1, 0.1 M phosphate buffer, pH 7.4).
showed that deglycosylation improved ET [13,26]. Here the glycan moiety was removed by endoglycosidase H (Endo H). As is seen from the CV (Fig. 2) a clear increase in the oxidation wave occurs in the presence of glucose at − 50 mV vs. Ag|AgClsat. KCl close to the E°′ of AmPDH. As FAD in PDH is covalently bound, it cannot disappear into the contacting solution and/or adsorb onto the electrode and therefore it is clear that DET in our case is accomplished by the bound FAD. A calibration curve for glucose for dgPDH adsorbed on graphite is shown in Fig. 1. The applied potential was set to +180 mV vs. Ag| AgCl0.1 M KCl, the lowest potential at which maximum current response can be obtained for a 5 mM glucose solution (from a polarisation curve, data not shown). Approximately the same Κapp Μ for adsorbed dgPDH ( Κapp = 7.41) was obtained as in the case of gPDH in combination Μ with MET (Fig. 1). Maximum current response for adsorbed dgPDH (Vmax; 423.6 nA, Jmax; 6 μA cm − 2) was much lower compared with that for dgPDH “wired” to the Os-polymer, however, DET still seems promising in the development of EBFCs, as simplification of the system towards reducing the number of component is always desirable. Another important aspect is related to the general decrease in the BFC voltage, and as a consequence power output, when using redox mediators, compared to a DET-based approach [22]. 4. Conclusions In the present study graphite electrodes were modified with recombinant gPDH and dgPDH, which were “wired” with an Ospolymer. The response of the electrodes was compared using glucose as a substrate. A two-fold increase in current for MET and a significant decrease in the Κapp Μ value were observed for dgPDH compared with gPDH. It was discovered that dgPDH is able to directly transfer electrons from its active centre to graphite when most of the glycosylation was removed by Endo H. The current obtained for DET was much lower than that for MET. Nevertheless, both approaches seem promising for
References [1] C. Sygmund, R. Kittl, J. Volc, P. Halada, E. Kubatova, D. Haltrich, C.K. Peterbauer, Journal of Biotechnology 133 (2008) 334–342. [2] J. Volc, E. Kubatova, G. Daniel, P. Sedmera, D. Haltrich, Archives of Microbiology 176 (2001) 178–186. [3] P. Sedmera, P. Halada, E. Kubatova, D. Haltrich, V. Prikrylova, J. Volc, Journal of Molecular Catalysis B: Enzymatic 41 (2006) 32–42. [4] C.K. Peterbauer, J. Volc, Applied Microbiology and Biotechnology 85 (2010) 837–848. [5] J. Volc, P. Sedmera, P. Halada, V. Prikrylova, G. Daniel, Carbohydrate Research 310 (1998) 151–156. [6] J. Volc, P. Sedmera, P. Halada, G. Daniel, V. Prikrylova, D. Haltrich, Journal of Molecular Catalysis B: Enzymatic 17 (2002) 91–100. [7] S.C. Barton, J. Gallaway, P. Atanassov, Chemical Reviews 104 (2004) 4867–4886. [8] M.N. Zafar, F. Tasca, S. Boland, M. Kujawa, I. Patel, C.K. Peterbauer, D. Leech, L. Gorton, Bioelectrochemistry 80 (2010) 38–42. [9] F. Tasca, S. Timur, R. Ludwig, D. Haltrich, J. Volc, R. Antiochia, L. Gorton, Electroanalysis 19 (2007) 294–302. [10] M. Shao, M.N. Zafar, D.A. Guschin, R. Ludwig, P. Clemensbauer, W. Schuhmann, L. Gorton, Biosensors and Bioelectronics (in press), http://dx.doi.org/10.1016/j.bios. 2012.07.069. [11] I. Pisanelli, M. Kujawa, D. Gschnitzer, O. Spadiut, B. Seiboth, C. Peterbauer, Applied Microbiology and Biotechnology 86 (2010) 599–606. [12] C. Sygmund, A. Gutmann, I. Krondorfer, M. Kujawa, A. Glieder, B. Pscheidt, D. Haltrich, C. Peterbauer, R. Kittl, Applied Microbiology and Biotechnology 94 (2012) 695–704. [13] H.M. Kalisz, H.J. Hecht, D. Schomburg, R.D. Schmid, Biochimica et Biophysica Acta 1080 (1991) 138–142. [14] G. Kern, N. Schulke, F.X. Schmid, R. Jaenicke, Protein Science 1 (1992) 120–131. [15] A. Prevoteau, O. Courjean, N. Mano, Electrochemistry Communications 12 (2010) 213–215. [16] A. Lindgren, M. Tanaka, T. Ruzgas, L. Gorton, I. Gazaryan, K. Ishimori, I. Morishima, Electrochemistry Communications 1 (1999) 171–175. [17] G. Presnova, V. Grigorenko, A. Egorov, T. Ruzgas, A. Lindgren, L. Gorton, T. Börchers, Faraday Discussions 116 (2000) 281–289. [18] E.E. Ferapontova, V.G. Grigorenko, A.M. Egorov, T. Börchers, T. Ruzgas, L. Gorton, Journal of Electroanalytical Chemistry 509 (2001) 19–26. [19] S. Rengaraj, P. Kavanagh, D. Leech, Biosensors and Bioelectronics 30 (2011) 294–299. [20] M. Kujawa, J. Volc, P. Halada, P. Sedmera, C. Divne, C. Sygmund, C. Leitner, C. Peterbauer, D. Haltrich, FEBS Journal 274 (2007) 879–894. [21] F. Tasca, L. Gorton, M. Kujawa, I. Patel, W. Harreither, C.K. Peterbauer, R. Ludwig, G. Nöll, Biosensors and Bioelectronics 25 (2010) 1710–1716. [22] A. Heller, Physical Chemistry Chemical Physics 6 (2004) 209–216. [23] O. Courjean, V. Flexer, A. Prevoteau, E. Suraniti, N. Mano, ChemPhysChem 11 (2010) 2795–2797. [24] S. Demin, E.A.H. Hall, Bioelectrochemistry 76 (2009) 19–27. [25] R.A. Marcus, N. Sutin, Biochimica et Biophysica Acta 811 (1985) 265–322. [26] O. Courjean, F. Gao, N. Mano, Angewandte Chemie International Edition 48 (2009) 5897–5899.
Contents lists available at SciVerse ScienceDirect
Electrochemistry Communications journal homepage: www.elsevier.com/locate/elecom
Recombinant pyranose dehydrogenase—A versatile enzyme possessing both mediated and direct electron transfer Maria E. Yakovleva a, Anikó Killyéni a, b, Roberto Ortiz a, Christopher Schulz a, Domhnall MacAodha c, Peter Ó. Conghaile c, Dónal Leech c, Ionel Catalin Popescu b, Christoph Gonaus d, Clemens K. Peterbauer d, Lo Gorton a,⁎ a
Department of Chemistry, Lund University, P.O. Box 124, SE-22100 Lund, Sweden Department of Physical Chemistry, Babes-Bolyai University, Cluj-Napoca, Romania c School of Chemistry, National University of Ireland Galway, University Road, Galway, Ireland d Department of Food Sciences and Technology, BOKU-University, Muthgasse 18, A-1190 Wien, Austria b
a r t i c l e
i n f o
Article history: Received 25 June 2012 Received in revised form 28 August 2012 Accepted 29 August 2012 Available online 8 September 2012 Keywords: Pyranose dehydrogenase Deglycosylation Osmium polymer Direct electron transfer Biosensor
a b s t r a c t The catalytical properties of glycosylated pyranose dehydrogenase (gPDH) and deglycosylated PDH (dgPDH) from Agaricus meleagris recombinantly expressed in Pichia pastoris were studied. Both gPDH and dgPDH were “wired” to an osmium redox polymer on graphite electrodes mounted in a flow-injection system. The current from oxidation of glucose by immobilised gPDH and dgPDH was compared using flow injection amperometry and cyclic voltammetry. An increase in the current density was observed for dgPDH (190 μA cm−2) compared with that for gPDH (90 μA cm−2) due to the improved electron transfer between the active site and the electrode. Additionally, the ability of dgPDH for direct electron transfer (DET) was discovered, which is rather unique among FAD-containing enzymes. The ability to oxidise a variety of sugars at a rather low potential makes dgPDH attractive for construction of biofuel cells with high power output. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Pyranose dehydrogenase (PDH) is an extracellular monomeric fungal glycoprotein (7% glycosylation) produced by litter-degrading fungi containing one covalently bound flavin adenine dinucleotide (FAD) as prosthetic group [1], belonging to the widespread flavoprotein glucosemethanol-choline oxidoreductase superfamily [2]. PDH has attracted attention due to applications in carbohydrate, organic and analytical bioelectrochemistry. Compared with pyranose oxidase PDH has broader substrate specificity and can oxidise various mono-, di-, and oligosaccharides in their pyranose form [3]. Depending on the source of PDH and substrate structure, the enzyme can catalyse the oxidation of sugars at the C‐1, C-2, and C-3 or dioxidation at C-1,3, C-2,3 and C-3,4 positions to their corresponding aldonolactones, or (di)dehydrosugars (aldos(di) uloses) [4–6]. The diversity of substrates makes PDH attractive for production of intermediate sugar derivatives further utilised in industrial synthesis of rare sugars or drug development [4]. Also, PDH is a true dehydrogenase as it cannot utilise O2 as an electron acceptor (c.f. sugar oxidases) and this is beneficial for fabrication of membraneless enzyme biofuel cells (EBFCs), where utilisation of O2 dependent enzymes at the anode part will result in production of H2O2. Even trace amounts of H2O2 can cause oxidative damage to commonly used enzymes (laccase ⁎ Corresponding author. Tel.: +46 46 222 7582; fax: +46 46 222 4544. E-mail address: [email protected] (L. Gorton). 1388-2481/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.elecom.2012.08.029
and bilirubin oxidase) at the cathode part of the EBFC and reduce the power output. The ability of PDH to operate at physiological conditions is also advantageous, when implantable devices are to be constructed [7]. In order to use the beneficial properties of PDH in the development of EBFCs, the PDH isolated from Agaricus meleagris was electrically “wired” to various Os-polymers with formal potentials (E°′) ranging from −270 to +160 mV vs. Ag|AgCl0.1 M KCl [8,9]. This was further extended by co-immobilisation of PDH with another enzyme with the Os-polymer. By the combination of several enzymes with different site-specificity for glucose, it was possible to extract more than 2 e− per substrate molecule thus increasing the current density and Coulombic efficiency of the bioanode for use in EBFC [10]. Since the expression levels of PDH in the natural source are low and in order to meet the high demands for PDH production, the AmPDH gene was expressed in heterologous expression hosts such as Aspergillus sp. [11], Escherichia coli and Pichia pastoris [12]. Expression of AmPDH in Aspergillus sp. was genetically elaborate and time-consuming. Therefore, other microbial systems were tested. Expression in E. coli resulted in production of insoluble protein with no activity. This can be attributed to the inability of prokaryotic organisms to form the glycosylation shell essential for correct folding, long term stability, and solubility [13,14]. When using eukaryotic P. pastoris as host large-scale production combined with a simple purification procedure was established. The MW of recAmPDH was determined to be about 93 kDa corresponding to about 30% glycosylation [12]. The much higher degree of glycosylation
M.E. Yakovleva et al. / Electrochemistry Communications 24 (2012) 120–122
121
(30% vs. 7% for the wild type AmPDH, MW 66.5 kDa [1]) resulted in 20% reduced specific activity of the recombinant enzyme compared to the wild-type. Therefore, here the recombinant enzyme was deglycosylated by endoglycosidase H (Endo H) and both gPDH and dgPDH, when immobilised onto the electrode, were characterised using amperometry and cyclic voltammetry. Both gPDH and dgPDH were “wired” with an Os-polymer and the current response for glucose was registered. Additionally, the ability of dgAmPDH to exhibit direct electron transfer (DET) was tested, as it was shown previously that depletion of the glycan moiety of an enzyme results in shortening the distance between the active centre of the enzyme and the electrode surface facilitating DET [15–18].
temperature. For MET 2 μl of an aqueous Os-polymer solution (10 mg ml − 1) and 1 μl of a freshly prepared aqueous 68% (v/v) PEGDGE solution were placed on the top of the electrodes. After 10 min 5 μl of enzyme solution (50.8 U cm − 2) was added. The electrodes were left overnight at 4 °C for complete cross-linking [9]. For DET 5 μl of enzyme solution with different enzyme loadings (1.8–38.8 U cm − 2) was evenly distributed on the top of the electrodes and allowed to adsorb overnight at 4 °C. For both DET and MET the excess of the enzyme was washed away with the buffer.
2. Material and methods
Based on previous studies [8] the Os-polymer with an E°′ of +32 mV vs Ag|AgCl0.1 M KCl was chosen for electrical “wiring” of recombinant AmPDH to the electrode. The E°′-value of the Os-polymer is slightly higher than that of AmPDH (−140 mV vs. Ag|AgCl0.1 M KCl, pH 7.4 [21]), which from a thermodynamic point of view represents the ideal case for efficient electron transfer from the enzyme to the electrode [22]. To study the effect of deglycosylation, both gPDH and dgPDH were separately cross-linked to the Os-polymer on the electrodes. Equal amounts of gPDH and dgPDH (in terms of activity) were used for the construction of the electrodes. As shown in Fig. 1 at least a 2-fold increase in the maximal catalytic current density (Jmax) for the electrodes modified with dgPDH (Vmax; 13.15 ± 0.40 μA, Jmax; 190 μA cm − 2) compared with that for gPDH (Vmax; 6.37 ± 0.081 μA, Jmax; 90 μA cm − 2) was observed. The linear range for both modified electrodes was the same, between 0.1 and 1 mM, however, the lower Κapp Μ value for dgPDG: 2.57 ± 0.30 mM (7.93 ± 0.32 mM for gPDH) indicates a higher affinity of the Os-polymer for dgPDH. The increase in current response and affinity for the dgPDG modified electrode might be attributed to a better electron transfer between the Os-polymer and the bound FAD becoming more accessible after deglycosylation or altered substrate access through the osmium–polymer enzyme network. However, it may also be partially attributed to a higher concentration of Ospolymer around the smaller dgPDH [15,23]. Deglycosylation of AmPDH resulted in DET between dgPDH and graphite. Glycosylation acts as an insulator on the protein surface and increases the distance between the active centre and the electrode making electron transfer (ET) less efficient or not at all [24,25]. Thus studies were performed in order to chemically or enzymatically remove the glycan shell and improve ET. So far except for deglycosylated peroxidases [16–18] only a few published papers on FAD-containing glucose oxidase
2.1. Materials Pyranose dehydrogenase from A. meleagris (AmPDH) was recombinantly expressed in P. pastoris and later deglycosylated with Endo Hf (New England Biolabs, Bionordiska AB, Stockholm, Sweden) as described previously [12]. Poly(ethylene glycol) (400) diglycidyl ether (PEGDGE), ferricenium hexafluorophosphate and D(+)-glucose were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). The Os redox polymer [Os(4,4′-dimethyl2,2′-bipyridine)2poly(N-vinylimidazole)10Cl] +2/+, E°′; +32 mV vs. Ag|AgCl0.1 M KCl, was synthesised as described in [19]. Water was purified with a Milli-Q purification system (Millipore, Bedford, MA, USA). 2.2. Equipment Flow injection measurements were performed with a flow-through wall-jet amperometric cell, containing a Pt wire as the counter and an Ag|AgCl0.1 M KCl, +288 mV vs. NHE as a reference electrode. The applied potential was controlled by a three-electrode potentiostat (Zäta Electronics, Höör, Sweden). The response of graphite rod electrodes (Ringsdorff Werke GmbH, Bonn, Germany, type RW001, 3.05 mm diameter, 13% porosity) was registered with a recorder (BD 112, Kipp & Zonen, Utrecht, The Netherlands). For the introduction of the samples an injector (Rheodyne, type 7125 LabPR, Cotati, CA, USA) with a 50 μl loop was used. The kinetic parameters were calculated by fitting the data using the Michaelis–Menten equation. A 50 mM phosphate buffer (pH 7.4) containing 137 mM NaCl and a 0.1 M phosphate buffer (pH 7.4) was used as a carrier at 0.45 ml min−1 for MET and 0.5 ml min−1 for DET, respectively. Before use the carrier was degassed for 20 min. CVs were performed with a computer controlled AutoLab PGSTAT30 electrochemical system (Metrohm Nordic AB, Bromma, Sweden) using the modified graphite electrode as a working electrode, an Ag|AgClsat. KCl, +199 mV vs. NHE as reference and a Pt foil as counter electrode. Before measurement Ar was purged through the solution for 10 min.
3. Results and discussion
2.3. Enzyme assay The activities of gPDH and dgPDH were determined by monitoring the reduction of ferricenium (Fc+) to ferrocene at 300 nm, ε= 4.3 mM−1 cm−1, for 3 min at 20 °C in a standard photometric assay [20]. One millilitre of standard solution contained 100 μmol of phosphate buffer, pH 7.4, 50 μmol of D-glucose, 0.4 μmol of Fc+PF6− (prepared daily by dissolving 3.3 mg of salt in 5 ml of 5 mM HCl) and an appropriately diluted enzyme. One unit of enzyme activity was defined as the amount of enzyme necessary for the reduction of 2 μmol of Fc+ per 1 min at 20 °C. 2.4. Preparation of electrodes Graphite electrodes (geometric area of 0.071 cm 2) were polished on wet emery paper, rinsed with Milli-Q water, and dried at room
Fig 1. Calibration curves for glucose for gPDH/Os-polymer and dgPDH/Os polymer-modified electrodes. E appl ; + 200 mV vs. Ag|AgCl 0.1 M KCl , 50 mM phosphate buffer, containing 137 mM NaCl, pH 7.4, 50 μl injected sample, flow rate 0.45 ml min − 1. Insert: calibration curve for glucose of a dgPDH-modified electrode. E appl ; + 180 mV vs. Ag|AgCl0.1 M KCl , 0.1 M phosphate buffer, pH 7.4, 50 μl injected sample, flow rate 0.5 ml min − 1 .
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further applications of PDH as a biocatalyst in the development of EBFCs.
20 15 10
Acknowledgements
I/A
5 0 -5 1.8 U/cm2 (5µL) in 0.1 M PB, pH 7.4
-10
1.8 U/cm2 (5µL) + 25 mM glucose
-15
3.7 U/cm2 (5µL) + 25 mM glucose 9.2 U/cm2 (5µL) + 25 mM glucose
-20
36.8 U/cm2 (5µL + 5 µL) + 25 mM glucose
-25 -0.6
-0.4
-0.2
0.0
0.2
Financial support was obtained from the following agencies: (AK) the Sectoral Operational Programme for Human Resources Development 2007–2013, co-financed by the European Social Fund (POSDRU/107/1.5/ S/76841 “Modern Doctoral Studies: Internationalization and Interdisciplinarity”), (LG) the Swedish Research Council (project 2010–5031), (DL, DMA and LG) the European Commission (“3D-Nanobiodevice”, NMP4-SL-2009-229255) and “Chebana” FP7-PEOPLE-2010-ITN-264772), (POC) an ERA-Chemistry award, (CG and CP) the Austrian Science Foundation, Translational Research Program project number TRP 218-BL and (AK and ICP) the project PN II-IDPCCE 140/2008.
0.4
Eappl. / V vs. Ag/AgCl (sat. KCl) Fig 2. CV of dgPDH-modified electrodes with different enzyme loadings in the presence and absence of 25 mM glucose (scan rate; 10 mV s−1, 0.1 M phosphate buffer, pH 7.4).
showed that deglycosylation improved ET [13,26]. Here the glycan moiety was removed by endoglycosidase H (Endo H). As is seen from the CV (Fig. 2) a clear increase in the oxidation wave occurs in the presence of glucose at − 50 mV vs. Ag|AgClsat. KCl close to the E°′ of AmPDH. As FAD in PDH is covalently bound, it cannot disappear into the contacting solution and/or adsorb onto the electrode and therefore it is clear that DET in our case is accomplished by the bound FAD. A calibration curve for glucose for dgPDH adsorbed on graphite is shown in Fig. 1. The applied potential was set to +180 mV vs. Ag| AgCl0.1 M KCl, the lowest potential at which maximum current response can be obtained for a 5 mM glucose solution (from a polarisation curve, data not shown). Approximately the same Κapp Μ for adsorbed dgPDH ( Κapp = 7.41) was obtained as in the case of gPDH in combination Μ with MET (Fig. 1). Maximum current response for adsorbed dgPDH (Vmax; 423.6 nA, Jmax; 6 μA cm − 2) was much lower compared with that for dgPDH “wired” to the Os-polymer, however, DET still seems promising in the development of EBFCs, as simplification of the system towards reducing the number of component is always desirable. Another important aspect is related to the general decrease in the BFC voltage, and as a consequence power output, when using redox mediators, compared to a DET-based approach [22]. 4. Conclusions In the present study graphite electrodes were modified with recombinant gPDH and dgPDH, which were “wired” with an Ospolymer. The response of the electrodes was compared using glucose as a substrate. A two-fold increase in current for MET and a significant decrease in the Κapp Μ value were observed for dgPDH compared with gPDH. It was discovered that dgPDH is able to directly transfer electrons from its active centre to graphite when most of the glycosylation was removed by Endo H. The current obtained for DET was much lower than that for MET. Nevertheless, both approaches seem promising for
References [1] C. Sygmund, R. Kittl, J. Volc, P. Halada, E. Kubatova, D. Haltrich, C.K. Peterbauer, Journal of Biotechnology 133 (2008) 334–342. [2] J. Volc, E. Kubatova, G. Daniel, P. Sedmera, D. Haltrich, Archives of Microbiology 176 (2001) 178–186. [3] P. Sedmera, P. Halada, E. Kubatova, D. Haltrich, V. Prikrylova, J. Volc, Journal of Molecular Catalysis B: Enzymatic 41 (2006) 32–42. [4] C.K. Peterbauer, J. Volc, Applied Microbiology and Biotechnology 85 (2010) 837–848. [5] J. Volc, P. Sedmera, P. Halada, V. Prikrylova, G. Daniel, Carbohydrate Research 310 (1998) 151–156. [6] J. Volc, P. Sedmera, P. Halada, G. Daniel, V. Prikrylova, D. Haltrich, Journal of Molecular Catalysis B: Enzymatic 17 (2002) 91–100. [7] S.C. Barton, J. Gallaway, P. Atanassov, Chemical Reviews 104 (2004) 4867–4886. [8] M.N. Zafar, F. Tasca, S. Boland, M. Kujawa, I. Patel, C.K. Peterbauer, D. Leech, L. Gorton, Bioelectrochemistry 80 (2010) 38–42. [9] F. Tasca, S. Timur, R. Ludwig, D. Haltrich, J. Volc, R. Antiochia, L. Gorton, Electroanalysis 19 (2007) 294–302. [10] M. Shao, M.N. Zafar, D.A. Guschin, R. Ludwig, P. Clemensbauer, W. Schuhmann, L. Gorton, Biosensors and Bioelectronics (in press), http://dx.doi.org/10.1016/j.bios. 2012.07.069. [11] I. Pisanelli, M. Kujawa, D. Gschnitzer, O. Spadiut, B. Seiboth, C. Peterbauer, Applied Microbiology and Biotechnology 86 (2010) 599–606. [12] C. Sygmund, A. Gutmann, I. Krondorfer, M. Kujawa, A. Glieder, B. Pscheidt, D. Haltrich, C. Peterbauer, R. Kittl, Applied Microbiology and Biotechnology 94 (2012) 695–704. [13] H.M. Kalisz, H.J. Hecht, D. Schomburg, R.D. Schmid, Biochimica et Biophysica Acta 1080 (1991) 138–142. [14] G. Kern, N. Schulke, F.X. Schmid, R. Jaenicke, Protein Science 1 (1992) 120–131. [15] A. Prevoteau, O. Courjean, N. Mano, Electrochemistry Communications 12 (2010) 213–215. [16] A. Lindgren, M. Tanaka, T. Ruzgas, L. Gorton, I. Gazaryan, K. Ishimori, I. Morishima, Electrochemistry Communications 1 (1999) 171–175. [17] G. Presnova, V. Grigorenko, A. Egorov, T. Ruzgas, A. Lindgren, L. Gorton, T. Börchers, Faraday Discussions 116 (2000) 281–289. [18] E.E. Ferapontova, V.G. Grigorenko, A.M. Egorov, T. Börchers, T. Ruzgas, L. Gorton, Journal of Electroanalytical Chemistry 509 (2001) 19–26. [19] S. Rengaraj, P. Kavanagh, D. Leech, Biosensors and Bioelectronics 30 (2011) 294–299. [20] M. Kujawa, J. Volc, P. Halada, P. Sedmera, C. Divne, C. Sygmund, C. Leitner, C. Peterbauer, D. Haltrich, FEBS Journal 274 (2007) 879–894. [21] F. Tasca, L. Gorton, M. Kujawa, I. Patel, W. Harreither, C.K. Peterbauer, R. Ludwig, G. Nöll, Biosensors and Bioelectronics 25 (2010) 1710–1716. [22] A. Heller, Physical Chemistry Chemical Physics 6 (2004) 209–216. [23] O. Courjean, V. Flexer, A. Prevoteau, E. Suraniti, N. Mano, ChemPhysChem 11 (2010) 2795–2797. [24] S. Demin, E.A.H. Hall, Bioelectrochemistry 76 (2009) 19–27. [25] R.A. Marcus, N. Sutin, Biochimica et Biophysica Acta 811 (1985) 265–322. [26] O. Courjean, F. Gao, N. Mano, Angewandte Chemie International Edition 48 (2009) 5897–5899.