Direct Electrochemistry of Phanerochaete chrysosporium Cellobiose Dehydrogenase Covalently Attached onto Gold Nanoparticle Modified Solid Gold Electrodes

Achieving efficient electrochemical communication between redox enzymes and various electrode materials is one of the main challenges in bioelectrochemistry and is of great importance for developing electronic applications. Cellobiose dehydrogenase (CDH) is an extracellular flavocytochrome composed of a catalytic FAD containing dehydrogenase domain (DH_CDH), a heme <i>b</i> containing cytochrome domain (CYT_CDH), and a flexible linker region connecting the two domains. Efficient direct electron transfer (DET) of CDH from the basidiomycete Phanerochaete chrysosporium (<i>Pc</i>CDH) covalently attached to mixed self-assembled monolayer (SAM) modified gold nanoparticle (AuNP) electrode is presented. The thiols used were as follows: 4-aminothiophenol (4-ATP), 4-mercaptobenzoic acid (4-MBA), 4-mercaptophenol (4-MP), 11-mercapto-1-undecanamine (MUNH₂), 11-mercapto-1-undecanoic acid (MUCOOH), and 11-mercapto-1-undecanol (MUOH). A covalent linkage between <i>Pc</i>CDH and 4-ATP or MUNH₂ in the mixed SAMs was formed using glutaraldehyde as cross-linker. The covalent immobilization and the surface coverage of <i>Pc</i>CDH were confirmed with surface plasmon resonance (SPR). To improve current density, AuNPs were cast on the top of polycrystalline gold electrodes. For all the immobilized <i>Pc</i>CDH modified AuNPs electrodes, cyclic voltammetry exhibited clear electrochemical responses of the CYT_CDH with fast electron transfer (ET) rates in the absence of substrate (lactose), and the formal potential was evaluated to be +162 mV vs NHE at pH 4.50. The standard ET rate constant (<i>k</i>_s) was estimated for the first time for CDH and was found to be 52.1, 59.8, 112, and 154 s^–1 for 4-ATP/4-MBA, 4-ATP/4-MP, MUNH₂/MUCOOH, and MUNH₂/MUOH modified electrodes, respectively. At all the mixed SAM modified AuNP electrodes, <i>Pc</i>CDH showed DET only via the CYT_CDH. No DET communication between the DH_CDH domain and the electrode was found. The current density for lactose oxidation was remarkably increased by introduction of the AuNPs. The 4-ATP/4-MBA modified AuNPs exhibited a current density up to 30 μA cm^–2, which is ∼70 times higher than that obtained for a 4-ATP/4-MBA modified polycrystalline gold electrode. The results provide insight into fundamental electrochemical properties of CDH covalently immobilized on gold electrodes and promote further applications of CDHs for biosensors, biofuel cells, and bioelectrocatalysis

Real-Time Dynamic Adsorption Processes of Cytochrome <i>c</i> on an Electrode Observed through Electrochemical High-Speed Atomic Force Microscopy

<div><p>An understanding of dynamic processes of proteins on the electrode surface could enhance the efficiency of bioelectronics development and therefore it is crucial to gain information regarding both physical adsorption of proteins onto the electrode and its electrochemical property in real-time. We combined high-speed atomic force microscopy (HS-AFM) with electrochemical device for simultaneous observation of the surface topography and electron transfer of redox proteins on an electrode. Direct electron transfer of cytochrome <i>c</i> (cyt <i>c</i>) adsorbed on a self-assembled monolayers (SAMs) formed electrode is very attractive subject in bioelectrochemistry. This paper reports a real-time visualization of cyt <i>c</i> adsorption processes on an 11-mercaptoundecanoic acid-modified Au electrode together with simultaneous electrochemical measurements. Adsorbing cyt <i>c</i> molecules were observed on a subsecond time resolution simultaneously with increasing redox currents from cyt <i>c</i> using EC-HS-AFM. The root mean square roughness (<i>R</i>_RMS) from the AFM images and the number of the electrochemically active cyt <i>c</i> molecules adsorbed onto the electrode (<i>Γ</i>) simultaneously increased in positive cooperativity. Cyt <i>c</i> molecules were fully adsorbed on the electrode in the AFM images when the peak currents were steady. This use of electrochemical HS-AFM significantly facilitates understanding of dynamic behavior of biomolecules on the electrode interface and contributes to the further development of bioelectronics.</p></div

AFM images show (A) the MUA-modified gold surface and (B) cyt <i>c</i> adsorbed on the MUA SAM at 450 sec.

<p>Continuous AFM images of adsorbing cyt <i>c</i> molecules with real-time labels. Frame rate, 2 frames/s; image area, 150 × 150 nm². The cross sections of the images in (A) and (B), along the short white line, are shown in the lower right. </p

CVs of cyt <i>c</i> adsorbed on the electrode.

<p>(A) CVs of cyt <i>c</i> molecules adsorbed on the MUA electrode in a 10 mM phosphate buffer solution (pH 7.0) from 333 to 522 s (from −0.35 V to 0.1 V, each segment is 4.5 s). (B) Background-subtracted CVs from the voltammogram from 313 to 324 s and 333 to 522 s (from inside to outside, each segment is 4.5 s). The voltammograms were collected at a scan rate of 100 mVs⁻¹. Ag wire was used as a reference electrode. </p

Real-time cyt <i>c</i> desorption processes from the MUA-modified gold electrode.

<p>(A) Continuous AFM images of desorbing cyt <i>c</i> molecules. Frame rate, 2 frames/s; image area, 150 × 150 nm². (B) Time evolution of <i>R</i>_RMS values at the higher ionic strengths. (C) Schematic of desorbing cyt <i>c</i> molecules from the MUA-modified electrode. </p

Time-course analysis of <i>R</i>_RMS and <i>Γ</i> values.

<p>(A) Time evolution of <i>R</i>_RMS values from the HS-AFM images (open circle) and the level of electrochemically active cyt <i>c</i> (<i>Γ</i>) from the cyclic voltammograms (open square). (B) Schematic of the adsorbing cyt <i>c</i> molecules on the MUA-modified electrode at each time point.</p

A protease/peptidase from culture medium of <i>Flammulina velutipes</i> that acts on arabinogalactan-protein

<p>Arabinogalactan-proteins (AGPs) are highly diverse plant proteoglycans found on the plant cell surface. AGPs have large arabinogalactan (AG) moieties attached to a core-protein rich in hydroxyproline (Hyp). The AG undergoes hydrolysis by various glycoside hydrolases, most of which have been identified, whereas the core-proteins is presumably degraded by unknown proteases/peptidases secreted from fungi and bacteria in nature. Although several enzymes hydrolyzing other Hyp-rich proteins are known, the enzymes acting on the core-proteins of AGPs remain to be identified. The present study describes the detection of protease/peptidase activity toward AGP core-proteins in the culture medium of winter mushroom (<i>Flammulina velutipes</i>) and partial purification of the enzyme by several conventional chromatography steps. The enzyme showed higher activity toward Hyp residues than toward proline and alanine residues and acted on core-proteins prepared from gum arabic. Since the activity was inhibited in the presence of Pefabloc SC, the enzyme is probably a serine protease.</p> <p>The degradation of the core-protein of AGPs by a protease/peptidase from winter mushroom.</p

Enzyme concentration dependence of the amount of adsorbed <i>Cc</i>PDH.

Closed circle, highly crystalline cellulose from Cladophora; open circle, PASC. The adsorption of CcPDH was measured after incubation for 120 min with 1 mg/mL of cellulose at 30°C as described in the Experimental Procedures.</p

Cyclic voltammogram of <i>Cc</i>PDH immobilized on a plastic-formed carbon electrode modified with 27 nm carbon nanoparticles.

The voltammogram was obtained in 100 mM HEPES buffer, pH 7.0, at a scan rate of 20 mV/s.</p

Titration of the apo-form of DH_PDH with PQQ.

The purified apo-form of DHPDH (50 nM) was pre-incubated with various concentrations of PQQ in 50 mM MES buffer, pH 6.5, containing 1 mM CaCl2. After 1 min, the enzyme activity was determined according to the procedure described in the Experimental Procedures.</p