Electrochemical Monitoring of Heterogeneous Peroxygenase Reactions Unravels LPMO Kinetics.

Biological conversion of plant biomass depends on peroxygenases and peroxidases acting on insoluble polysaccharides and lignin. Among these are cellulose- and hemicellulose-degrading lytic polysaccharide monooxygenases (LPMOs), which have revolutionized our concept of biomass degradation. Major obstacles limiting mechanistic and functional understanding of these unique peroxygenases are their complex and insoluble substrates and the hard-to-measure H2O2 consumption, resulting in the lack of suitable kinetic assays. We report a versatile and robust electrochemical method for real-time monitoring and kinetic characterization of LPMOs and other H2O2-dependent interfacial enzymes based on a rotating disc electrode for the sensitive and selective quantitation of H2O2 at biologically relevant concentrations. The H2O2 sensor works in suspensions of insoluble substrates as well as in homogeneous solutions. Our characterization of multiple LPMOs provides unprecedented insights into the substrate specificity, kinetics, and stability of these enzymes. High turnover and total turnover numbers demonstrate that LPMOs are fast and durable biocatalysts.

Investigating lytic polysaccharide monooxygenase-assisted wood cell wall degradation with microsensors.

Lytic polysaccharide monooxygenase (LPMO) supports biomass hydrolysis by increasing saccharification efficiency and rate. Recent studies demonstrate that H2O2 rather than O2 is the cosubstrate of the LPMO-catalyzed depolymerization of polysaccharides. Some studies have questioned the physiological relevance of the H2O2-based mechanism for plant cell wall degradation. This study reports the localized and time-resolved determination of LPMO activity on poplar wood cell walls by measuring the H2O2 concentration in their vicinity with a piezo-controlled H2O2 microsensor. The investigated Neurospora crassa LPMO binds to the inner cell wall layer and consumes enzymatically generated H2O2. The results point towards a high catalytic efficiency of LPMO at a low H2O2 concentration that auxiliary oxidoreductases in fungal secretomes can easily generate. Measurements with a glucose microbiosensor additionally demonstrate that LPMO promotes cellobiohydrolase activity on wood cell walls and plays a synergistic role in the fungal extracellular catabolism and in industrial biomass degradation.

Amperometric Biosensors Based on Direct Electron Transfer Enzymes.

The accurate determination of analyte concentrations with selective, fast, and robust methods is the key for process control, product analysis, environmental compliance, and medical applications. Enzyme-based biosensors meet these requirements to a high degree and can be operated with simple, cost efficient, and easy to use devices. This review focuses on enzymes capable of direct electron transfer (DET) to electrodes and also the electrode materials which can enable or enhance the DET type bioelectrocatalysis. It presents amperometric biosensors for the quantification of important medical, technical, and environmental analytes and it carves out the requirements for enzymes and electrode materials in DET-based third generation biosensors. This review critically surveys enzymes and biosensors for which DET has been reported. Single- or multi-cofactor enzymes featuring copper centers, hemes, FAD, FMN, or PQQ as prosthetic groups as well as fusion enzymes are presented. Nanomaterials, nanostructured electrodes, chemical surface modifications, and protein immobilization strategies are reviewed for their ability to support direct electrochemistry of enzymes. The combination of both biosensor elements-enzymes and electrodes-is evaluated by comparison of substrate specificity, current density, sensitivity, and the range of detection.

Real-Time Measurement of Cellobiose and Glucose Formation during Enzymatic Biomass Hydrolysis.

Enzymatic hydrolysis of lignocellulosic biomass for biofuel production relies on complex multi-enzyme ensembles. Continuous and accurate measurement of the released key products is crucial in optimizing the industrial degradation process and also investigating the activity and interaction between the involved enzymes and the insoluble substrate. Amperometric biosensors have been applied to perform continuous cellobiose measurements during the enzymatic hydrolysis of pure cellulose powders. The oxygen-sensitive mediators used in these biosensors restricted their function under physiological or industrial conditions. Also, the combined measurements of the hydrolysis products cellobiose and glucose require a high selectivity of the biorecognition elements. We employed an [Os(2,2'-bipyridine)2Cl]Cl-modified polymer and cellobiose dehydrogenase to fabricate a cellobiose biosensor, which can accurately and specifically detect cellobiose even in the presence of oxygen and the other main product glucose. Additionally, a glucose biosensor was fabricated to simultaneously measure glucose produced from cellobiose by ß-glucosidases. The cellobiose and glucose biosensors work at applied potentials of +0.25 and +0.45 V versus Ag|AgCl (3 M KCl), respectively, and can selectively detect their substrate. Both biosensors were used in combination to monitor the hydrolysis of pure cellulose of low crystallinity or industrial corncob samples. The obtained results correlate with the high-performance liquid chromatography pulsed amperometric detection analysis and demonstrate that neither oxygen nor the presence of redox-active compounds from the lignin fraction of the corncob interferes with the measurements.

Heterologous expression of Phanerochaete chrysosporium cellobiose dehydrogenase in Trichoderma reesei.

BACKGROUND: Cellobiose dehydrogenase from Phanerochaete chrysosporium (PcCDH) is a key enzyme in lignocellulose depolymerization, biosensors and biofuel cells. For these applications, it should retain important molecular and catalytic properties when recombinantly expressed. While homologous expression is time-consuming and the prokaryote Escherichia coli is not suitable for expression of the two-domain flavocytochrome, the yeast Pichia pastoris is hyperglycosylating the enzyme. Fungal expression hosts like Aspergillus niger and Trichoderma reesei were successfully used to express CDH from the ascomycete Corynascus thermophilus. This study describes the expression of basidiomycetes PcCDH in T. reesei (PcCDHTr) and the detailed comparison of its molecular, catalytic and electrochemical properties in comparison with PcCDH expressed by P. chrysosporium and P. pastoris (PcCDHPp). RESULTS: PcCDHTr was recombinantly produced with a yield of 600 U L-1 after 4 days, which is fast compared to the secretion of the enzyme by P. chrysosporium. PcCDHTr and PcCDH were purified to homogeneity by two chromatographic steps. Both enzymes were comparatively characterized in terms of molecular and catalytic properties. The pH optima for electron acceptors are identical for PcCDHTr and PcCDH. The determined FAD cofactor occupancy of 70% for PcCDHTr is higher than for other recombinantly produced CDHs and its catalytic constants are in good accordance with those of PcCDH. Mass spectrometry showed high mannose-type N-glycans on PcCDH, but only single N-acetyl-D-glucosamine additions at the six potential N-glycosylation sites of PcCDHTr, which indicates the presence of an endo-N-acetyl-ß-D-glucosaminidase in the supernatant. CONCLUSIONS: Heterologous production of PcCDHTr is faster and the yield higher than secretion by P. chrysosporium. It also does not need a cellulose-based medium that impedes efficient production and purification of CDH by binding to the polysaccharide. The obtained high uniformity of PcCDHTr glycoforms will be very useful to investigate electron transfer characteristics in biosensors and biofuel cells, which are depending on the spatial restrictions inflicted by high-mannose N-glycan trees. The determined catalytic and electrochemical properties of PcCDHTr are very similar to those of PcCDH and the FAD cofactor occupancy is good, which advocates T. reesei as expression host for engineered PcCDH for biosensors and biofuel cells.

The H2O2-dependent activity of a fungal lytic polysaccharide monooxygenase investigated with a turbidimetric assay.

BACKGROUND: Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent redox enzymes that cleave recalcitrant biopolymers such as cellulose, chitin, starch and hemicelluloses. Although LPMOs receive ample interest in industry and academia, their reaction mechanism is not yet fully understood. Recent studies showed that H2O2 is a more efficient cosubstrate for the enzyme than O2, which could greatly affect the utilization of LPMOs in industrial settings. RESULTS: We probe the reactivity of LPMO9C from the cellulose-degrading fungus Neurospora crassa with a turbidimetric assay using phosphoric acid-swollen cellulose (PASC) as substrate and H2O2 as a cosubstrate. The measurements were also followed by continuous electrochemical H2O2 detection and LPMO reaction products were analysed by mass spectrometry. Different systems for the in situ generation of H2O2 and for the reduction of LPMO's active-site copper were employed, including glucose oxidase, cellobiose dehydrogenase, and the routinely used reductant ascorbate. CONCLUSIONS: We found for all systems that the supply of H2O2 limited LPMO's cellulose depolymerization activity, which supports the function of H2O2 as the relevant cosubstrate. The turbidimetric assay allowed rapid determination of LPMO activity on a cellulosic substrate without the need for time-consuming and instrumentally elaborate analysis methods.

Characterization of pyranose oxidase variants for bioelectrocatalytic applications.

Pyranose oxidase (POx) catalyzes the oxidation of d-glucose to 2-ketoglucose with concurrent reduction of oxygen to H2O2. POx from Trametes ochracea (ToPOx) is known to react with alternative electron acceptors including 1,4-benzoquinone (1,4-BQ), 2,6-dichlorophenol indophenol (DCPIP), and the ferrocenium ion. In this study, enzyme variants with improved electron acceptor turnover and reduced oxygen turnover were characterized as potential anode biocatalysts. Pre-steady-state kinetics of the oxidative half-reaction of ToPOx variants T166R, Q448H, L545C, and L547R with these alternative electron acceptors were evaluated using stopped-flow spectrophotometry. Higher kinetic constants were observed as compared to the wild-type ToPOx for some of the variants. Subsequently, the variants were immobilized on glassy carbon electrodes. Cyclic voltammetry measurements were performed to measure the electrochemical responses of these variants with glucose as substrate in the presence of 1,4-BQ, DCPIP, or ferrocene methanol as redox mediators. High catalytic efficiencies (Imaxapp/KMapp) compared to the wild-type POx proved the potential of these variants for future bioelectrocatalytic applications, in biosensors or biofuel cells. Among the variants, L545C showed the most desirable properties as determined kinetically and electrochemically.

Improved spectrophotometric assay for lytic polysaccharide monooxygenase.

BACKGROUND: The availability of a sensitive and robust activity assay is a prerequisite for efficient enzyme production, purification, and characterization. Here we report on a spectrophotometric assay for lytic polysaccharide monooxygenase (LPMO), which is an advancement of the previously published 2,6-dimethoxyphenol (2,6-DMP)-based LPMO assay. The new assay is based on hydrocoerulignone as substrate and hydrogen peroxide as cosubstrate and aims toward a higher sensitivity at acidic pH and a more reliable detection of LPMO in complex matrices like culture media. RESULTS: An LPMO activity assay following the colorimetric oxidation of hydrocoerulignone to coerulignone was developed. This peroxidase activity of LPMO in the presence of hydrogen peroxide can be detected in various buffers between pH 4-8. By reducing the substrate and cosubstrate concentration, the assay has been optimized for minimal autoxidation and enzyme deactivation while maintaining sensitivity. Finally, the optimized and validated LPMO assay was used to follow the recombinant expression of an LPMO in Pichia pastoris and to screen for interfering substances in fermentation media suppressing the assayed reaction. CONCLUSIONS: The biphenol hydrocoerulignone is a better substrate for LPMO than the monophenol 2,6-DMP, because of a ~ 30 times lower apparent K M value and a 160 mV lower oxidation potential. This greatly increases the measured LPMO activity when using hydrocoerulignone instead of 2,6-DMP under otherwise similar assay conditions. The improved activity allows the adaptation of the LPMO assay toward a higher sensitivity, different buffers and pH values, more stable assay conditions or to overcome low concentrations of inhibiting substances. The developed assay protocol and optimization guidelines increase the adaptability and applicability of the hydrocoerulignone assay for the production, purification, and characterization of LPMOs.

Arsenic mitigation in paddy soils by using microbial fuel cells.

Arsenic (As) behavior in paddy soils couples with the redox process of iron (Fe) minerals. When soil is flooded, Fe oxides are transformed to soluble ferrous ions by accepting the electrons from Fe reducers. This process can significantly affect the fate of As in paddy fields. In this study, we show a novel technique to manipulate the Fe redox processes in paddy soils by deploying soil microbial fuel cells (sMFC). The results showed that the sMFC bioanode can significantly decrease the release of Fe and As into soil porewater. Iron and As contents around sMFC anode were 65.0% and 47.0% of the control respectively at day 50. The observed phenomenon would be explained by a competition for organic substrate between sMFC bioanode and the iron- and arsenic-reducing bacteria in the soils. In the vicinity of bioanode, organic matter removal efficiencies were 10.3% and 14.0% higher than the control for lost on ignition carbon and total organic carbon respectively. Sequencing of the 16S rRNA genes suggested that the influence of bioanodes on bulk soil bacterial community structure was minimal. Moreover, during the experiment a maximum current and power density of 0.31 mA and 12.0 mWm-2 were obtained, respectively. This study shows a novel way to limit the release of Fe and As in soils porewater and simultaneously generate electricity.

High performance Cu/Cu2O nanohybrid electrocatalyst for nonenzymatic glucose detection.

Here we report Cu/Cu2O nanocomposites prepared via potential oscillation. High resolution transmission electron microscopy images reveal a homogeneous distribution of Cu and Cu2O in the composite. Due to the synergistic effect between Cu and Cu2O, the nanohybrids show much lower resistivity and higher glucose oxidation activity (a lower over-potential and higher current-output) compared to the Cu2O counterpart. As an example of application, a nanocomposite-based electrode is employed for a nonenzymatic glucose biosensor application, and exhibits a linear detection upper limit of 40 mM, a sensitivity of 1434.12 µA cm-2 mM-1, and good selectivity.

Rhodium nanoparticle-mesoporous silicon nanowire nanohybrids for hydrogen peroxide detection with high selectivity.

Developing nanostructured electrocatalysts, with low overpotential, high selectivity and activity has fundamental and technical importance in many fields. We report here rhodium nanoparticle and mesoporous silicon nanowire (RhNP@mSiNW) hybrids for hydrogen peroxide (H2O2) detection with high electrocatalytic activity and selectivity. By employing electrodes that loaded with RhNP@mSiNW nanohybrids, interference caused from both many electroactive substances and dissolved oxygen were eliminated by electrochemical assaying at an optimal potential of +75 mV. Furthermore, the electrodes exhibited a high detection sensitivity of 0.53 µA/mM and fast response (< 5 s). This high-performance nanohybrid electrocatalyst has great potential for future practical application in various oxidase-base biosensors.

Layer-by-layer assembly of graphene, Au and poly(toluidine blue O) films sensor for evaluation of oxidative stress of tumor cells elicited by hydrogen peroxide.

High level of oxidative stress is involved in formation of incipient tumor and carcinomatous cells. Here in this contribution we have explored a facile strategy to assess the oxidative stress elicited by hydrogen peroxide (H(2)O(2)) in cells with amperometric current-time technique in vitro. An electrochemical biosensor exhibiting high sensitivity and selectivity to H(2)O(2) is fabricated by integration of graphene with gold nanoparticles and poly(toluidine blue O) films. The efflux of H(2)O(2) from several representative tumor cells and normal cells on exposure to ascorbic acid could be detected by using the graphene-based nanocomposite films. The results indicate that tumor cells release much more H(2)O(2) than do the normal cells. The novel sensor raises the possibility for clinical diagnostic application to evaluate the higher level of intracellular oxidative stress of tumor cells in comparison with normal cells.

Apoptosis induction and inhibition of drug resistant tumor growth in vivo involving daunorubicin-loaded graphene-gold composites.

Daunorubicin (DNR) loaded graphene-gold nanocomposites offer a novel strategy for inducing apoptosis in drug resistant leukemia cells (K562/A02; KA). In vitro and in vivo investigations on xenografted tumors in KA nude mice demonstrate that the combination of monoclonal P-glycoprotein (P-gp) antibodies and DNR anticancer drug loaded on graphene-gold nanocomposites (GGN) is an efficient drug delivery vector, with remarkable targeting and binding properties towards drug resistant KA cell lines, and induces apoptosis of KA cells and inhibits tumor growth in KA nude mice. Cellular treatment with DNR-loaded GGN remarkably reduced drug resistant-related P-gp expression and activated apoptosis-related caspase protein expression in KA cells. Cell apoptosis provoked in vitro by such nanocomposites corresponds to a rapid induction of active caspase 8,3 activities and stimulation of poly-(ADP-ribose) polymerase (PARP) proteolytic cleavage. In vivo studies indicate that DNR-loaded GGN nanocomposites effectively overcome the inhibition of drug resistant leukemia cell-induced tumor growth in KA nude mice. This nanocomposite raises the possibility of modulating apoptosis in cancer cells, and of inhibiting tumor growth, showing that nanocomposites of this kind have promising applications in efficient multifunctional therapy.

Catalytic oxidation and determination of ß-NADH using self-assembly hybrid of gold nanoparticles and graphene.

A self-assembly hybrid of gold nanoparticles on graphene modified electrodes for low-potential NADH detection has been achieved. We used the natural polymer chitosan (Chit) to assist the stabilization of graphene in aqueous solution, and immobilize the electronegative Au nanoparticles (NPs) through electrostatic attraction. The synergy of Au NPs with graphene for catalytic oxidation of NADH made the overpotential ca. 220 mV less positive than that on the bare electrode, and remarkably increased the oxidation current. The amperometric sensors based on such modified electrodes for detection of NADH exhibited a good linearity from 1.5 to 320 µM, and showed high sensitivity with a low detection limit of 1.2 µM (S/N = 3). It could also exclude common interfering electroactive compounds like ascorbic acid and possessed good reproducibility and operational stability. Such eminent performance of the Au-RGO/Chit film together with the ability of graphene to significantly enhance the electron transfer between enzymes and the electrode suggested its promise for constructing novel graphene based dehydrogenase biosensors.