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Ammonia pretreatment greatly improves enzymatic hydrolysis of grass biomass, but is reported to be ineffective for hardwood biomass. Here, we examined the effectiveness of ammonia pretreatment of biomass from six hardwood species with different contents of xylan and lignin. Ammonia pretreatment greatly improved enzymatic hydrolysis of polysaccharides in birch and willow, but was less effective for acacia, eucalyptus, and poplar. The effectiveness of ammonia pretreatment increased with xylan content but decreased with lignin content of the hardwood species. By adding a recombinant xylanase to the commercial enzyme digestion cocktail, the yield of enzymatic hydrolysis of ammonia-pretreated birch biomass was improved to a similar level to that obtained with grass biomass. Our results indicate that enzymatic hydrolysis of biomass from hardwood species having a relatively high xylan/lignin ratio can be achieved with a xylanase-enriched enzyme cocktail after ammonia pretreatment. [ABSTRACT FROM AUTHOR]

Fungal cellobiohydrolases act at liquid-solid interfaces. They have the ability to hydrolyze cellulose chains of a crystalline substrate because of their two-domain structure, i.e. cellulose-binding domain and catalytic domain, and unique active site architecture. However, the details of the action of the two domains on crystalline cellulose are still unclear. Here, we present real time observations of Trichoderma reesei (Tr) cellobiohydrolase I (Cel7A) molecules sliding on crystalline cellulose, obtained with a high speed atomic force microscope. The average velocity of the sliding movement on crystalline cellulose was 3.5 nm/s, and interestingly, the catalytic domain without the cellulose-binding domain moved with a velocity similar to that of the intact TrCel7A enzyme. However, no sliding of a catalytically inactive enzyme (mutant E212Q) or a variant lacking tryptophan at the entrance of the active site tunnel (mutant W4OA) could be detected. This indicates that, besides the hydrolysis of glycosidic bonds, the loading of a cellulose chain into the active site tunnel is also essential for the enzyme movement. [ABSTRACT FROM AUTHOR]

Abstract: Synthesis of cellulose in vitro is expected to afford tailor-made cellulosic materials with highly homogeneous structure compared to natural cellulosic materials. Here we report the enzymatic synthesis of cellulose II with high crystallinity from glucose and α-glucose 1-phosphate (αG1P) by cellodextrin phosphorylase (CDP). Although glucose had been believed not to act as a glucosyl acceptor of CDP, a significant amount of insoluble cellulose was precipitated without accumulation of soluble cello oligosaccharides when glucose was mixed with αG1P and CDP. This phenomenon can be explained in terms of the large difference in acceptor reactivity between glucose and cello oligosaccharides. 1H NMR spectrometric analysis revealed that this insoluble cellulose had an average degree of polymerization (DP) of nine. TEM observation, together with electron and X-ray diffraction studies, indicated that the insoluble cellulose formed platelet-shaped single lamellar crystals of cellulose II, several μm in length and several hundred nm in width; this is large compared to reported cellulose crystals. The thickness of the lamellar crystal is 4.5nm, which is equivalent to a chain length of a cello oligosaccharide with DP nine and is consistent with the 1H NMR spectroscopic results. These results suggest that cello oligosaccharides having an average DP of nine are synthesized in vitro by CDP when glucose is used as an acceptor, and the product forms highly crystalline cellulose II when it precipitates. [Copyright &y& Elsevier]

Thermoplastic elastomers (TPEs) have long been used in a wide range of industries. However, most existing TPEs are petroleum-derived polymers. To realize environmentally benign alternatives to conventional TPEs, cellulose acetate is a promising TPE hard segment because of its sufficient mechanical properties, availability from renewable sources, and biodegradability in natural environments. Because the degree of substitution (DS) of cellulose acetate governs a range of physical properties, it is a useful parameter for designing novel cellulose acetate-based TPEs. In this study, we synthesized cellulose acetate-based ABA-type triblock copolymers (AcCel x - b -PDL- b -AcCel x) containing a celloologosaccharide acetate hard A segment (AcCel x , where x is the DS; x = 3.0, 2.6, and 2.3) and a poly(δ -decanolactone) (PDL) soft B segment. Small-angle X-ray scattering showed that decreasing the DS of AcCel x - b -PDL- b -AcCel x resulted in the formation of a more ordered microphase-separated structure. Owing to the microphase separation of the hard cellulosic and soft PDL segments, all the AcCel x - b -PDL- b -AcCel x samples exhibited elastomer-like properties. Moreover, the decrease in DS improved toughness and suppressed stress relaxation. Furthermore, preliminary biodegradation tests in an aqueous environment revealed that the decrease in DS endowed AcCel x - b -PDL- b -AcCel x with greater biodegradability potential. This work demonstrates the usefulness of cellulose acetate-based TPEs as next-generation sustainable materials. [Display omitted] [ABSTRACT FROM AUTHOR]

Mixed-linkage glucans are major components of grassy cell-walls and cereal endosperm. Recently identified plant endo -β-glucanase from the EG16 family cleaves MLGs with strong specificity towards regions with at least four sequential β(1,4)-linked glucose residues. This activity yields a low molecular-weight MLG with a repeating structure of β(1,3)-linked cellotriose that gels rapidly at concentrations as low as 1.0 % w / v. To understand the gelation mechanism, we investigated the structure and behavior using rheology, microscopy, X-ray scattering, and molecular dynamics simulations. Upon digestion, the material's rheological behavior changes from typical polymeric material to a fibrillar network behavior seen for e.g. cellulose nanofibrils. Scanning electron microscopy and confocal microscopy verifies these changes in micro- and nanostructure. Small-angle X-ray scattering shows in-solution self-assembly of MLG through ~10 nm elemental structures. Wide-angle X-ray scattering data indicate that the polymer association is similar to cellulose II, with dominant scattering at d -spacing of 0.43 nm. Simulations of two interacting glucan chains show that β(1,3)-linkages prevent the formation of tight helices that form between β(1,4)-linked d -glucan chains, leading to weaker interactions and less ordered inter-chain assembly. Overall, these data indicate that digestion drives gelation not by enhancement of interactions driving self-assembly, but by elimination of unproductive interactions hindering self-assembly. VvEG16 digested MLG (Dig. MLG) gels rapidly whereas the intact MLG is a solutions. The digested MLG gel forms a nanofibrillar network that show a cellulose II type crystal assembly. [Display omitted] [ABSTRACT FROM AUTHOR]

Cellulose-binding domains (CBDs) play a vital role in cellulose degradation by enzymes. Despite the strong ability of brown-rot fungi to degrade cellulose in wood, they have been considered to lack or have a low number of enzymes with CBD. Here, we report the C-terminal domain of a lytic polysaccharide monooxygenase from the brown-rot fungus Gloeophyllum trabeum (Gt LPMO9A-2) functions as a CBD, classified as a new family of carbohydrate-binding module, CBM104. The amino acid sequence of Gt CBM104 shows no similarity to any known CBDs. A BLAST search identified 84 homologous sequences at the C-terminus of some CAZymes, mainly LPMO9, in basidiomycetous genomes. Binding experiments revealed Gt CBM104 binds selectively to native crystalline cellulose (cellulose I), but not to artificially modified crystalline or amorphous cellulose, while the typical fungal CBD (CBM1) bound to all cellulosic materials tested. The adsorption efficiency of Gt CBM104 to cellulose I was >20-times higher than that of CBM1. Adsorption tests and microscopic observations strongly suggested that Gt CBM104 binds to the hydrophilic regions of cellulose microfibrils, while CBM1 recognizes the hydrophobic surface. The discovery of Gt CBM104 strongly suggests that the contribution of CBD to the cellulose enzymatic degradation mechanism of brown-rot fungi is much larger than previously thought. [Display omitted] [ABSTRACT FROM AUTHOR]

Xanthine dehydrogenase (XDH) from the bacterium Rhodobacter capsulatus catalyzes the hydroxylation of xanthine to uric acid with NAD+ as the electron acceptot. R. capsulatus XDH forms an (αβ)2 heterotetramer and is highly homologous to homodimeric eukaryotic xanthine oxidoreductases. Here we first describe reductive titration and steady state kinetics on recombinant wild-type R. capsulatus XDH purified from Escherichia coli, and we then proceed to evaluate the catalytic importance of the active site residues Glu-232 and Glu-730. The steady state and rapid reaction kinetics of an E232A variant exhibited a significant decrease in both kcat and kred as well as increased Km and Kd values as compared with the wild-type protein. No activity was determined for the E730A, E730Q, E730R, and E730D variants in either the steady state or rapid reaction experiments, indicating at least a 107 decrease in catalytic effectiveness for this variant. This result is fully consistent with the proposed role of this residue as an active site base that initiates catalysis. [ABSTRACT FROM AUTHOR]

Highlights • The first eukaryotic PQQ-dependent dehydrogenase was discovered in a plant saprophytic fungus. • This multi-domain enzyme, Cc PDH, defines a new family of PQQ-dependent eukaryotic enzymes. • Cc PDH oxidizes 2-keto- d -glucose, l -fucose, and rare pyranoses, such as d -arabinose and l -galactose. • The dehydrogenase domain of Cc PDH is the founding member of family AA12 in CAZy. • Cc PDH can activate on LPMOs. In 2014, the first fungal pyrroloquinoline-quinone (PQQ)-dependent enzyme was discovered as a pyranose dehydrogenase from the basidiomycete Coprinopsis cinerea (Cc PDH). This discovery laid the foundation for a new Auxiliary Activities (AA) family, AA12, in the Carbohydrate-Active enZymes (CAZy) database and revealed a novel enzymatic activity potentially involved in biomass conversion. This review summarizes recent progress made in research on this fungal oxidoreductase and related enzymes. Cc PDH consists of the catalytic PQQ-binding AA12 domain, an N-terminal cytochrome b AA8 domain, and a C-terminal family 1 carbohydrate-binding module (CBM1). Cc PDH oxidizes 2-keto- d -glucose (d -glucosone), l -fucose, and rare sugars such as d -arabinose and l -galactose, and can activate lytic polysaccharide monooxygenases (LPMOs). Bioinformatic studies suggest a widespread occurrence of quinoproteins in eukaryotes as well as prokaryotes. [ABSTRACT FROM AUTHOR]

Neutron diffraction provides different information from X-ray diffraction, because neutrons are scattered by atomic nuclei, whereas X-rays are scattered by electrons. One of the key advantages of neutron crystallography is the ability to visualize hydrogen and deuterium atoms, making it possible to observe the protonation state of amino acid residues, hydrogen bonds, networks of water molecules and proton relay pathways in enzymes. But, because of technical difficulties, less than 100 enzyme structures have been evaluated by neutron crystallography to date. In this review, we discuss the advantages and disadvantages of neutron crystallography as a tool to investigate the functional structure of glycoside hydrolases, with some examples. [ABSTRACT FROM AUTHOR]

The glycoside hydrolase family 7 cellobiohydrolase Cel7A from Trichoderma reesei is one of the best studied cellulases with the ability to degrade highly crystalline cellulose. The catalytic domain and the cellulose-binding domain (CBD) are both necessary for full activity on crystalline substrates. Our previous high-speed atomic force microscopy studies showed that mutation of Trp-40 at the entrance of the catalytic tunnel drastically decreases the ability to degrade crystalline cellulose. Here, we examined the activities of the WT enzyme and mutant W40A (with and without the CBD) for various substrates. Evaluation and comparison of the specific activities of the enzymes (WT, W40A, and the corresponding catalytic subunits (WTcat and W40Acat)) adsorbed on crystalline cellulose indicated that Trp-40 is involved in recruiting individual substrate chains into the active site tunnel to initiate processive hydrolysis. This was supported by molecular dynamics simulation study, i.e. the reducing end glucose unit was effectively loaded into the active site of WTcat, but not into that of W40Acat, when the simulation was started from subsite - 7. However, when similar simulations were carried out starting from subsite - 5, both enzymes held the substrate for 50 ns, indicating that the major difference between WTcat and W40Acat is the length of the free chain end of the substrate required to allow initiation of processive movements; this also reflects the difference between crystalline and amorphous celluloses. The CBD is important for enhancing the enzyme population on crystalline substrate, but it also decreases the specific activity of the adsorbed enzyme, possibly by attaching the enzyme to non-optimal places on the cellulose surface and/or hindering processive hydrolysis. [ABSTRACT FROM AUTHOR]

Carbohydrate structures are modified and degraded in the biosphere by a myriad of mostly hydrolytic enzymes. Recently, lytic polysaccharide mono-oxygenases (LPMOs) were discovered as a new class of enzymes for cleavage of recalcitrant polysaccharides that instead employ an oxidative mechanism. LPMOs employ copper as the catalytic metal and are dependent on oxygen and reducing agents for activity. LPMOs are found in many fungi and bacteria, but to date no basidiomycete LPMO has been structurally characterized. Here we present the three-dimensional crystal structure of the basidiomycete Phanerochaete chrysosporium GH61D LPMO, and, for the first time, measure the product distribution of LPMO action on a lignocellulosic substrate. The structure reveals a copper-bound active site common to LPMOs, a collection of aromatic and polar residues near the binding surface that may be responsible for regio-selectivity, and substantial differences in loop structures near the binding face compared with other LPMO structures. The activity assays indicate that this LPMO primarily produces aldonic acids. Last, molecular simulations reveal conformational changes, including the binding of several regions to the cellulose surface, leading to alignment of three tyrosine residues on the binding face of the enzyme with individual cellulose chains, similar to what has been observed for family 1 carbohydrate-binding modules. A calculated potential energy surface for surface translation indicates that P. chrysosporium GH61D exhibits energy wells whose spacing seems adapted to the spacing of cellobiose units along a cellulose chain. [ABSTRACT FROM AUTHOR]

Arabinogalactan proteins are proteoglycans found on the cell surface and in the cell walls of higher plants. The carbohydrate moieties of most arabinogalactan proteins are composed of β-1,3-galactan main chains and β-1,6-galactan side chains, to which other auxiliary sugars are attached. For the present study, an endo-β-1,3-galactanase, designated FvEn3GAL, was first purified and cloned from winter mushroom Flammulina velutipes. The enzyme specifically hydrolyzed β-1,3-galactan, but did not act on β-1,3-glucan, β-1,3:1,4-glucan, xyloglucan, and agarose. It released various β-1,3-galactooligosaccharides together with Gal from β-1,3-galactohexaose in the early phase of the reaction, demonstrating that it acts on β-1,3-galactan in an endo-fashion. Phylogenetic analysis revealed that FvEn3GAL is member of a novel subgroup distinct from known glycoside hydrolases such as endo-β-1,3-glucanase and endo-β-1,3:1,4-glucanase in glycoside hydrolase family 16. Point mutations replacing the putative catalytic Glu residues conserved for enzymes in this family with Asp abolished activity. These results indicate that FvEn3GAL is a highly specific glycoside hydrolase 16 endo-β-1,3-galactanase. [ABSTRACT FROM AUTHOR]

Abstract: A polar and hydrophilic ionic liquid, choline dihydrogen phosphate ([ch][dhp]), has been evaluated here as a medium for a bioelectrocatalytic process for generating cellulosic materials. The [ch][dhp] was mixed with water in proportions that best the competing requirements of solubilization and stabilization of enzymes, solution properties, and process conditions. Fructose dehydrogenase (FDH) and cellobiose dehydrogenase (CDH) were immobilized on gold or carbon nanoparticle-modified electrodes, and their biocatalytic oxidation was detected electrochemically in the hydrated [ch][dhp] (Hy[ch][dhp]). An improvement of the shelf life was found (>3 weeks) of CDH immobilized on the electrode in Hy[ch][dhp]. [Copyright &y& Elsevier]

Abstract: Arabinogalactan-proteins (AGPs) are a family of plant proteoglycans having large carbohydrate moieties attached to core-proteins. The carbohydrate moieties of AGPs commonly have β-(1→3)(1→6)-galactan as the backbone, to which other auxiliary sugars such as l-Ara and GlcA are attached. For the present study, an α-l-arabinofuranosidase belonging to glycoside hydrolase family (GHF) 54, NcAraf1, and an endo-β-(1→6)-galactanase of GHF 5, Nc6GAL, were identified in Neurospora crassa. Recombinant NcAraf1 (rNcAraf1) expressed in Pichia pastoris hydrolyzed radish AGPs as well as arabinan and arabinoxylan, showing relatively broad substrate specificity toward polysaccharides containing α-l-arabinofuranosyl residues. Recombinant Nc6GAL (rNc6GAL) expressed in P. pastoris specifically acted on β-(1→6)-galactosyl residues. Whereas AGP from radish roots was hardly hydrolyzed by rNc6GAL alone, β-(1→6)-galactan side chains were reduced to one or two galactan residues by a combination of rNcAraf1 and rNc6GAL. These results suggest that the carbohydrate moieties of AGPs are degraded by the concerted action of NcAraf1 and Nc6GAL secreted from N. crassa. [Copyright &y& Elsevier]

Glycoside hydrolase family 55 consists of β-1,3-glucanases mainly from filamentous fungi. A β-1,3-glucanase (Lam55A) from the Basidiomycete Phanerochaete chrysosporium hydrolyzes β-1,3-glucans in the exo-mode with inversion of anomeric configuration and produces gentiobiose in addition to glucose from β-1,3/1,6-glucans. Here we report the crystal structure of Lam55A, establishing the three-dimensional structure of a member of glycoside hydrolase 55 for the first time. Lam55A has twoβ3-helical domains in a single polypeptide chain. These two domains are separated by a long linker region but are positioned side by side, and the overall structure resembles a rib cage. In the complex, a gluconolactone molecule is bound at the bottom of a pocket between the two β-helical domains. Based on the position of the gluconolactone molecule, Glu-633 appears to be the catalytic acid, whereas the catalytic base residue could not be identified. The substrate binding pocket appears to be able to accept a gentiobiose unit near the cleavage site, and a long cleft runs from the pocket, in accordance with the activity of this enzyme toward various β-1,3-glucan oligosaccharides. In conclusion, we provide important features of the substrate-binding site at the interface of the two β-helical domains, demonstrating an unexpected variety of carbohydrate binding modes. [ABSTRACT FROM AUTHOR]

Abstract: The carbohydrate moieties of arabinogalactan-proteins (AGPs), which are mainly composed of Gal, l-Ara, GlcA, and 4-Me-GlcA residues, are essential for the physiological functions of these proteoglycans in higher plants. For this study, we have identified two genes encoding family 79 β-glucuronidases, designated AnGlcAase and NcGlcAase, in Aspergillus niger and Neurospora crassa, respectively, based on the amino acid sequence of a native β-glucuronidase purified from a commercial pectolytic enzyme preparation from A. niger. Although the deduced protein sequences of AnGlcAase and NcGlcAase were highly similar, the recombinant enzymes expressed in Pichia pastoris exhibited distinct substrate specificity toward 4-Me-GlcA residues of AGPs: recombinant AnGlcAase (rAnGlcAase) substantially liberated both GlcA and 4-Me-GlcA residues from radish AGPs, whereas recombinant NcGlcAase (rNcGlcAase) activity on the 4-Me-GlcA residues of AGPs was very low. Maximum activity of rAnGlcAase hydrolyzing PNP β-GlcA occurred at pH 3.0–4.0, whereas the maximum rNcGlcAase activity was at pH 6.0. The apparent K m values of rAnGlcAase were 30.4μM for PNP β-GlcA and 422μM for β-GlcA-(1→6)-Gal, and those of rNcGlcAase were 38.3μM and 378μM, respectively. Similar to the native enzyme, rAnGlcAase was able to catalyze the transglycosylation of GlcA residues from PNP β-GlcA to various monosaccharide acceptors such as Glc, Gal, and Xyl. We propose that both AnGlcAase and NcGlcAase are instances of a novel type of β-glucuronidase with the capacity to hydrolyze β-GlcA and 4-Me-β-GlcA residues of AGPs, although they differ significantly in their preferences. [Copyright &y& Elsevier]

An exo-β-1,3-galactanase gene from Phanerochaete chrysosporium has been cloned, sequenced, and expressed in Pichia pastoris. The complete amino acid sequence of the exo-β-1,3-galactanase indicated that the enzyme consists of an N-terminal catalytic module with similarity to glycoside hydrolase family 43 and an additional unknown functional domain similar to carbohydrate-binding module family 6 (CBM6) in the C-terminal region. The molecular mass of the recombinant enzyme was estimated as 55 kDa based on SDS-PAGE. The enzyme showed reactivity only toward β-1,3-linked galactosyl oligosaccharides and polysaccharide as substrates but did not hydrolyze β-1,4-linked galacto-oligosaccharides, β-1,6-linked galacto-oligosaccharides, pectic galactan, larch arabinogalactan, arabinan, gum arabic, debranched arabinan, laminarin, soluble birchwood xylan, or soluble oat spelled xylan. The enzyme also did not hydrolyze β-1,3-galactosyl galactosaminide, β-1,3-galactosyl glucosaminide, or β-1,3-galactosyl arabinofuranoside, suggesting that it specifically cleaves the internal β-1,3-linkage of two galactosyl residues. High performance liquid chromatographic analysis of the hydrolysis products showed that the enzyme produced galactose from β-1,3-galactan in an exo-acting manner. However, no activity toward p-nitrophenyl β-galactopyranoside was detected. When incubated with arabinogalactan proteins, the enzyme produced oligosaccharides together with galactose, suggesting that it is able to bypass β-1,6-linked galactosyl side chains. The C-terminal CBM6 did not show any affinity for known substrates of CBM6 such as xylan, cellulose, and β-1,3-glucan, although it bound β-1,3-galactan when analyzed by affinity electrophoresis. Frontal affinity chromatography for the CBM6 moiety using several kinds of terminal galactose-containing oligosaccharides as the analytes clearly indicated that the CBM6 specifically interacted with oligosaccharides containing a β-1,3-galactobiose moiety. When the degree of polymerization of galactose oligomers was increased, the binding affinity of the CBM6 showed no marked change. [ABSTRACT FROM AUTHOR]

The enzymatic hydrolysis of barley beta-glucan, konjac glucomannan and carboxymethyl cellulose by a β-1,4-D-endoglucanase MeCel45A from blue mussel, Mytilus edulis , which belongs to subfamily B of glycoside hydrolase family 45 (GH45), was compared with GH45 members of subfamilies A (Humicola insolens HiCel45A), B (Trichoderma reesei TrCel45A) and C (Phanerochaete chrysosporium PcCel45A). Furthermore, the crystal structure of MeCel45A is reported. Initial rates and hydrolysis yields were determined by reducing sugar assays and product formation was characterized using NMR spectroscopy. The subfamily B and C enzymes exhibited mannanase activity, whereas the subfamily A member was uniquely able to produce monomeric glucose. All enzymes were confirmed to be inverting glycoside hydrolases. MeCel45A appears to be cold adapted by evolution, as it maintained 70% activity on cellohexaose at 4 °C relative to 30 °C, compared to 35% for TrCel45A. Both enzymes produced cellobiose and cellotetraose from cellohexaose, but TrCel45A additionally produced cellotriose. [Display omitted] • GH45 subfamily A, B and C members are inverting enzymes. • GH45 subfamily B and C members exhibit mannanase activity on glucomannan. • Cel45A possibly adapted to cold in northern mollusc. • Differences in linkage preference on glucomannan among GH45 subfamilies A, B and C [ABSTRACT FROM AUTHOR]

• The PQQ domain can undergo direct bioelectrocatalysis without its cytochrome domain. • The AuE modified with SAM composed of 2-mercaptoethanol is suited for DET reaction. • Catalytic currents depended on the distance between the enzyme and electrode surface. • High current densities of 1.6 mA/cm2 was achieved on AuNPs modified electrode. • Nanostructured electrodes with AuNPs increased the long-term stability of the PQQ domain. Direct electron transfer (DET) between oxidoreductases and electrodes is crucial for understanding the fundamental features of redox proteins as well as developing mediator-free bioelectronics devices. Bioelectrocatalysis with pyrroloquinoline quinone (PQQ)-dependent dehydrogenases is promising for both biosensors and biofuel cells. However, a limited number of studies have reported DET between PQQ in the enzyme and the electrode. Here, the PQQ domain (containing only the PQQ cofactor) of the fungal PQQ-dependent pyranose dehydrogenase from Coprinopsis cinerea , was studied in DET with various electrodes. A 2-mercaptoethanol self-assembled monolayer (SAM)-coated polycrystalline gold electrode was found to give excellent DET for the PQQ domain, resulting in 10-times the catalytic current density compared to bare glassy carbon. The amount of immobilized PQQ domain was determined to be 8.2 ± 0.4 pmol/cm2 by using a 27 MHz quartz-crystal microbalance (QCM), suggesting an approximate protein monolayer formation on the SAM modified gold surface. To improve current density, gold nanoparticles (AuNPs) were modified on top of polycrystalline gold electrodes. Importantly, high catalytic current densities of 1.6 mA/cm2 for the oxidation of l -fucose were achieved under optimized conditions. Together, these results demonstrate highly efficient DET to PQQ in the active site of the fungal PQQ-dependent dehydrogenase. Thus, our PQQ-domain/SAM/AuNPs coated polycrystalline gold electrode could serve as an exquisitely sensitive biosensor and a highly active sugar oxidizing anode for biofuel cells. Image, graphical abstract [ABSTRACT FROM AUTHOR]