Article pubs.acs.org/ac
Effect of Deglycosylation of Cellobiose Dehydrogenases on the Enhancement of Direct Electron Transfer with Electrodes Roberto Ortiz,†,◆ Hirotoshi Matsumura,†,‡,◆ Federico Tasca,† Kawah Zahma,§ Masahiro Samejima,‡ Kiyohiko Igarashi,‡ Roland Ludwig,§ and Lo Gorton*,† †
Department of Analytical Chemistry/Biochemistry and Structural Biology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden Department of Biomaterials Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan § Food Biotechnology Laboratory, Department of Food Sciences and Technology, BOKUUniversity of Natural Resources and Life Sciences, Vienna, Muthgasse 18, A-1190 Vienna, Austria ‡
S Supporting Information *
ABSTRACT: Cellobiose dehydrogenase (CDH) is a monomeric extracellular flavocytochrome composed of a catalytic dehydrogenase domain (DHCDH) containing flavin adenine dinucleotide (FAD), a cytochrome domain (CYTCDH) containing heme b, and a linker region connecting the two domains. In this work, the effect of deglycosylation on the electrochemical properties of CDH from Phanerochaete chrysosporium (PcCDH) and Ceriporiopsis subvermispora (CsCDH) is presented. All the glycosylated and deglycosylated enzymes show direct electron transfer (DET) between the CYTCDH and the electrode. Graphite electrodes modified with deglycosylated PcCDH (dPcCDH) and CsCDH (dCsCDH) have a 40−65% higher Imax value in the presence of substrate than electrodes modified with their glycosylated counterparts. CsCDH trapped under a permselective membrane showed similar changes on gold electrodes protected by a thiol-based self-assembled monolayer (SAM), in contrast to PcCDH for which deglycosylation did not exhibit any different electrocatalytical response on SAM-modified gold electrodes. Glycosylated PcCDH was found to have a 30% bigger hydrodynamic radius than dPcCDH using dynamic light scattering. The basic bioelectrochemistry as well as the bioelectrocatalytic properties are presented. Despite increased research activities in the field, the original problem of insufficient communication between a redox enzyme and the supportive electrode material remains,3 and thus, it is difficult to obtain a high current density based on DET, which is needed both for high sensitivity of a biosensor and high power output of a biofuel cell (BFC).4 In order to improve the characteristics of such biodevices four different strategies are commonly used: (i) coimmobilization of redoxactive compounds, using a mediated electron-transfer (MET) approach where the mediator acts as a potential electrontransferring bridge between the electrode and the enzyme,5 (ii) increase in the electrode surface area using nano- or
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ne of the major challenges in electrochemical research focusing on the communication between redox enzymes and electrodes is to obtain efficient electron transfer. In many instances this can only be reached by the use of redox mediators facilitating the electron transfer between the redoxactive sites in the biological material and the electrode. Not so frequently, however, is efficient direct electron transfer (DET) between the biocomponent and the electrode reported, even if almost 40 years have passed since the first examples were reported.1 DET is believed to occur at considerable rates only over distances shorter than 20 Å between prosthetic groups and electrode surfaces and is reported for only about 50 redox enzymes out of 3500.2 For the application of redox enzymes in biosensors and biofuel cells, an efficient DET is certainly a great feature to achieve leading to a simple electrode setup and avoiding expensive or harmful mediators. © 2012 American Chemical Society
Received: August 16, 2012 Accepted: October 29, 2012 Published: October 29, 2012 10315
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Article
subvermispora CDH (gCsCDH) and their deglycosylated counterparts (dPcCDH and dCsCDH) were compared. PcCDH represents the most well-studied variant of CDH,18b,20 with the crystal structures of its individual domains previously published,21 and it is approximately 9% glycosylated for the enzyme produced by the fungi. CsCDH is for the first time electrochemically characterized and with 16% of the total mass glycosylated when expressed from the fungus, one of the most heavily glycosylated CDHs.22
mesoporous structures to increase the amount of biomaterial in contact with the electrode possibly also increasing the percentage of enzyme molecules oriented for DET,6 (iii) tailoring the electrode surface through modifications to achieve a better orientation or attachment of the enzyme facilitating DET,7 or (iv) protein engineering of the enzyme to improve its catalytic properties, its orientation on the electrode, or to minimize the distance to the redox-active cofactor(s) of the enzyme and the electrode surface.8 In previous research it was shown that deglycosylation of horseradish peroxidase (HRP) and tobacco peroxidase shortens the distance between the prosthetic group (heme b) and the electrode and enhances the DET rate.8d,9 More recently, different laboratories have used this strategy on glucose oxidase (GOx). GOx from Aspergillus niger is a glycosylated dimeric flavoenzyme containing two FAD (flavin adenine dinucleotide) cofactor units buried ∼15 Å below the protein surface.10 The removal of the sugar residues from the enzyme surface allows a closer contact of the bound FAD in the active site and the electrode, which resulted in clear DET and glucose electrooxidation. When using a redox polymer the electrode modified with deglycosylated GOx also exhibited a higher electrooxidation current compared to electrodes modified with the fully glycosylated GOx, due to the downsizing of the enzyme’s dimensions on the electrode surface.11 Some other authors also used this approach on GOx but did not show clear direct electrocatalysis of glucose.8b,12 Cellobiose dehydrogenase [CDH, (cellobiose:acceptor) 1oxidoreductase, EC 1.1.99.18] is a monomeric enzyme secreted by a variety of fungi from the phyla of Basidiomycota and Ascomycota.13 CDH belongs, like GOx, to the glucose− methanol−choline (GMC) oxidoreductase superfamily14 and is the only extracellular flavocytochrome known today. It is composed of a larger catalytic flavodehydrogenase domain (DHCDH) containing one noncovalently bound FAD molecule as cofactor and a smaller cytochrome domain (CYTCDH) containing heme b as cofactor, which are connected through a flexible linker.15 CDHs from a variety of origins have shown efficient DET; however, the fact that CDH is glycosylated (about 9−16%, similar to GOx) implies that an improvement of its bioelectrocatalytic properties can be expected after deglycosylation. Because of their different catalytic properties, various CDHs have been used for the development of amperometric biosensors and biofuel cells based on both DET16 and mediated electron transfer (MET).5c,16a,17 Reviews on this work can be found in literature.14,18 The reaction catalyzed by CDHs starts at the DHCDH domain, which oxidizes cellobiose, cello-oligosaccharides, and some other carbohydrates (e.g., lactose) to their corresponding δ-lactones. The two electrons stored in the reduced FAD can be transferred onto small one- or two-electron acceptors, e.g., ferricyanide or quinones. Alternatively, the electrons can be transferred sequentially from the reduced FAD in the DHCDH to the heme b in the CYTCDH by an intramolecular electron-transfer (IET) step and further on to a terminal electron acceptor like cytochrome c. If CDH is immobilized in a favorable position, an electrode can also function as terminal electron acceptor. DET between CYTCDH and the electrode has been shown to occur at a slower rate than the rate of the internal electron transfer (IET), identified to be the rate-limiting step of CDH in solution.8e,19 In this contribution, the DET of the glycosylated forms of Phanerochaete chrysosporium CDH (gPcCDH) and Ceriporiopsis
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EXPERIMENTAL SECTION Chemicals. 11-Mercaptoundecanol (MUOH) was obtained from Sigma-Aldrich GmbH (Sigma-Aldrich Chemicals, Steinheim, Germany). 11-Amino-1-undecanothiol (MUNH2) was obtained from Dojindo Laboratories (Kumamoto, Japan). Absolute ethanol (>99% vol) used to prepare the thiol solutions was obtained from Solveco Chemicals AB (Stockholm, Sweden). Glutaraldehyde (GA) solution in water (50% weight), β-lactose (min 99%), cytochrome c (cyt c) from bovine heart, and hydrogen peroxide (30% w/w) were obtained from Sigma-Aldrich GmbH (Sigma-Aldrich Chemicals, Steinheim, Germany). Sulfuric acid (analytical grade) was obtained from VWR International (Poole, England). Buffer salts were of analytical grade and obtained from Fluka (Buchs, Germany). Water purified with a Milli-Q apparatus (Millipore, Bedford, MA, U.S.A.) to a specific resistivity (18.2 MΩ cm) was used to prepare all aqueous solutions. Enzyme Preparation. Recombinant wild-type gPcCDH was heterologously expressed in the methylotropic yeast Pichia pastoris and purified as described previously.23 The purified enzyme was stored in 50 mM sodium citrate buffer (pH 5.5) at −20 °C. The specific cyt c activity for gPcCDH and dPcCDH was 17.1 and 12.5 U mg−1, respectively. The gCsCDH was produced and purified as described previously.22 The purified enzyme was stored in 50 mM sodium citrate buffer (pH 5.0) at −20 °C. gCsCDH and dCsCDH exhibited a specific cyt c activity of 19.5 and 10.6 U mg−1, respectively. The purified glycosylated CDHs were treated with α-1,2-mannosidase and endoglycosidase H (New England Biolabs, Ipswich, MA, U.S.A.) according to the manufacturer’s instructions to obtain the deglycosylated CDHs a procedure previously described by Hallberg et al.21a Kinetic Measurements. The cyt c assay was performed to determine the homogeneous activity of the enzymes used by following the reduction of 20 μM cyt c (ε550 = 19.6 mM−1 cm−1) in 80 mM sodium acetate buffer, pH 4.50, containing 30 mM lactose at 30 °C. One unit of enzymatic activity is defined as the amount of enzyme that oxidizes 1 μmol of lactose per min under the assay conditions. The protein concentration was determined by the method of Bradford24 using a prefabricated assay from Bio-Rad Laboratories (Hercules, CA, U.S.A.) and bovine serum albumin as the standard. An Applied Photophysics (Leatherhead, U.K.) SX-20 stopped-flow apparatus in the single-wavelength mode was used to measure the presteady-state reduction rates at 30 °C of FAD and heme b at 447 and 563 nm, respectively. Both substrate and enzyme solution were buffered in 80 mM sodium acetate buffer, pH 4.5. The final concentrations in the reaction cell were 50 mM lactose and 6 μM gPcCDH or 4 μM dPcCDH. The observed rates (kobs) for both reactions are averages of three shots. Preparation of Graphite Electrodes. Rods of spectrographic graphite (SPGE, Ringsdorff Werke GmbH, Bonn, Germany, type RW001, Ø 3.05 mm) were used as working 10316
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Netherlands). A three-electrode cell was used with the gCDHor dCDH-modified electrode as the working electrode, a Ag| AgCl, KCl(S) (+197 mV vs NHE) as the reference electrode, and a Pt foil as the auxiliary electrode. All CV measurements were performed in 100 mM acetate buffer (pH 4.50). Anaerobicity was achieved by purging with pure argon gas for at least 20 min before making measurements. For detailed information about flow injection analysis (FIA) and dynamic light scattering (DLS) see the Supporting Information. The prediction of the potential N-glycosylation sites was done by the NetNGlyc 1.0 server (www.cbs.dtu.dk/services/ NetNGlyc)27 for the two CDHs tested.
electrodes. The surface of the SPGE was prepared by polishing with fine emery paper (Tufback Durite, P1500), then thoroughly rinsed with Milli-Q water, and finally allowed to dry. The gCDH- and dCDH-modified electrodes were prepared by placing an aliquot of 5 μL of enzyme solution (all enzyme solutions were diluted to an equal volumetric activity of 18.2 U mL−1) on the entire disk surface of the electrode. The electrodes were let to dry at room temperature and then stored overnight at 4 °C. Before each measurement, the electrodes were thoroughly rinsed with acetate buffer to remove weakly adsorbed enzyme molecules. Preparation of Permselective Membrane Gold Electrodes. Gold disk electrodes (BASi, West Lafayette, IN, U.S.A., Ø 1.6 mm) were exposed for 3 min into freshly prepared Piranha solution (3/1 v/v H2SO4/H2O2; CAUTION: Piranha solution is especially dangerous, corrosive, and may explode if contained in a closed vessel; it should be handled with special care) followed by polishing in aqueous alumina slurry (0.1 μm, Struers, Copenhagen, Denmark), ultrasonicated in Milli-Q water for 5 min, and finally activated electrochemically by cyclic voltammetry (CV) in 0.5 M H2SO4 (300 mV s−1, −0.1 to 1.7 V vs Ag|AgCl, KCl(s), 20 cycles). In order to confirm the cleanliness of the electrodes the last scan of the CV was compared to that previously reported as clean gold.25 The electrode was rinsed abundantly with water between every step. A self-assembled monolayer (SAM) of thiols was prepared by immersion into a 1 mM ethanolic thiol solution for 1 h at room temperature followed by rinsing with ethanol and drying with Ar. Modification of the electrode with CDH was made by trapping 11 μL of CDH solution in a Teflon holder between the electrode and a soaked permselective dialysis membrane (PME) (MWCO
