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

UV-visible absorption spectra of the apo- and holo-forms of DH_PDH.

<p>Dotted line, apo-form of DH_PDH; black solid line, holo-form of DH_PDH; blue solid line, reduced form by addition of 1 mM l-fucose. All spectra were recorded in 50 mM HEPES buffer, pH 7.0 at room temperature.</p

MOESM1 of Development of simple random mutagenesis protocol for the protein expression system in Pichia pastoris

Additional file 1. Additional Tables and Figures

Effect of Deglycosylation of Cellobiose Dehydrogenases on the Enhancement of Direct Electron Transfer with Electrodes

Cellobiose dehydrogenase (CDH) is a monomeric extracellular flavocytochrome composed of a catalytic dehydrogenase domain (DH_CDH) containing flavin adenine dinucleotide (FAD), a cytochrome domain (CYT_CDH) containing heme <i>b</i>, and a linker region connecting the two domains. In this work, the effect of deglycosylation on the electrochemical properties of CDH from Phanerochaete chrysosporium (<i>Pc</i>CDH) and Ceriporiopsis subvermispora (<i>Cs</i>CDH) is presented. All the glycosylated and deglycosylated enzymes show direct electron transfer (DET) between the CYT_CDH and the electrode. Graphite electrodes modified with deglycosylated <i>Pc</i>CDH (d<i>Pc</i>CDH) and <i>Cs</i>CDH (d<i>Cs</i>CDH) have a 40–65% higher <i>I</i>_max value in the presence of substrate than electrodes modified with their glycosylated counterparts. <i>Cs</i>CDH trapped under a permselective membrane showed similar changes on gold electrodes protected by a thiol-based self-assembled monolayer (SAM), in contrast to <i>Pc</i>CDH for which deglycosylation did not exhibit any different electrocatalytical response on SAM-modified gold electrodes. Glycosylated <i>Pc</i>CDH was found to have a 30% bigger hydrodynamic radius than d<i>Pc</i>CDH using dynamic light scattering. The basic bioelectrochemistry as well as the bioelectrocatalytic properties are presented

Structure of d-glucosone (A) and l-fucose (B) in a ¹C₄ conformation.

<p>Structure of d-glucosone (A) and l-fucose (B) in a ¹C₄ conformation.</p

Trade-off between Processivity and Hydrolytic Velocity of Cellobiohydrolases at the Surface of Crystalline Cellulose

Analysis of heterogeneous catalysis at an interface is difficult because of the variety of reaction sites and the difficulty of observing the reaction. Enzymatic hydrolysis of cellulose by cellulases is a typical heterogeneous reaction at a solid/liquid interface, and a key parameter of such reactions on polymeric substrates is the processivity, i.e., the number of catalytic cycles that can occur without detachment of the enzyme from the substrate. In this study, we evaluated the reactions of three closely related glycoside hydrolase family 7 cellobiohydrolases from filamentous fungi at the molecular level by means of high-speed atomic force microscopy to investigate the structure–function relationship of the cellobiohydrolases on crystalline cellulose. We found that high moving velocity of enzyme molecules on the surface is associated with a high dissociation rate constant from the substrate, which means weak interaction between enzyme and substrate. Moreover, higher values of processivity were associated with more loop regions covering the subsite cleft, which may imply higher binding affinity. Loop regions covering the subsites result in stronger interaction, which decreases the velocity but increases the processivity. These results indicate that there is a trade-off between processivity and hydrolytic velocity among processive cellulases

Multiple alignments of the amino acid sequences of CBM1 of <i>Cc</i>PDH and other known CBM1s.

<p>Residues in bold are highly conserved and those in boxes with a black background are perfect matches. Aromatic residues that are candidates for carbohydrate binding are indicated by a filled arrow, and two pairs of cysteines forming disulfide bonds are indicated by filled and open circles, respectively. <i>Tr</i>CBHI, cellobiohydrolase I (Cel7A) from <i>Trichoderma reesei</i> (accession no. P62694); <i>Pc</i>CBHII, cellobiohydrolase II (Cel6A) from <i>Phanerochaete chrysosporium</i> (Q02321); <i>Pc</i>BGL3A, glucan β-1,3-glucosidase (Bgl) from <i>P</i>. <i>chrysosporium</i> (Q8TGC6); <i>Pc</i>CBHI, cellobiohydrolase I-2 (Cel7D) from <i>P</i>. <i>chrysosporium</i> (Q09431); <i>Tr</i>CBHII, cellobiohydrolase II (Cel6A) from <i>T</i>. <i>reesei</i> (P07987); <i>Pc</i>CBCytb562, carbohydrate-binding cytochrome <i>b</i>₅₆₂ from <i>P</i>. <i>chrysosporium</i> (Q66NB8); <i>Mt</i>CDH, cellobiose dehydrogenase from <i>Myceliophthora thermophila</i> (O74240).</p

UV-visible absorption spectra of <i>Cc</i>PDH.

<p>Black solid line, oxidized form; gray solid line, reduced form by addition of L-fucose; dotted line, reduced form prepared by addition of ascorbic acid. All spectra were recorded in 50 mM HEPES buffer, pH 7.0, at room temperature.</p

Vibrational frequencies (cm^-1) of <i>Cc</i>PDH and cytochrome domain of CDH.

<p>Vibrational frequencies (cm^-1) of <i>Cc</i>PDH and cytochrome domain of CDH.</p

Specificity constant values of <i>Cc</i>PDH for various monosaccharides.

<p>Specificity constant values of <i>Cc</i>PDH for various monosaccharides.</p