Determination of Lactose in Lactose-Free and Low-Lactose Milk, Milk Products, and Products Containing Dairy Ingredients by the LactoSens®R Amperometry Method: Final Action 2020.01.

BACKGROUND: The LactoSens®R method was previously shown to have acceptable accuracy and repeatability precision as required by AOAC Standard Method Performance Requirements (SMPR®) 2018.009 for determination of lactose in low-lactose or lactose-free milk, milk products, and products containing dairy ingredients and was awarded Official Method of AnalysisSM (OMA) First Action status in 2020. OBJECTIVE: The method was subjected to a multilaboratory validation (MLV) study to evaluate the reproducibility precision of the method. METHODS: Fourteen validation materials were provided to 15 laboratories in seven countries as blind duplicates. The materials ranged from 0 to 173 mg/100 g lactose. Each laboratory analyzed the blind duplicates according to OMA 2020.01. The data were analyzed for repeatability and reproducibility precision. RESULTS: RSDr values varied from 2.81 to 8.76%, and RSDR values varied from 4.25 to 12.5%. When sorted by category and concentration range, these results met the repeatability and reproducibility criteria required by SMPR 2018.009. CONCLUSIONS: The data generated in the MLV support the adoption of OMA 2020.01 as Final Action status. HIGHLIGHTS: The LactoSensR method, as described by OMA 2020.01, provides an accurate and precise determination of lactose in a variety of low-lactose and lactose-free milk, milk products, and products containing dairy ingredients in minutes.

Interface engineering of cellobiose dehydrogenase improves interdomain electron transfer.

Cellobiose dehydrogenase (CDH) is a bioelectrocatalyst that enables direct electron transfer (DET) in biosensors and biofuel cells. The application of this bidomain hemoflavoenzyme for physiological glucose measurements is limited by its acidic pH optimum and slow interdomain electron transfer (IET) at pH 7.5. The reason for this rate-limiting electron transfer step is electrostatic repulsion at the interface between the catalytic dehydrogenase domain and the electron mediating cytochrome domain (CYT). We applied rational interface engineering to accelerate the IET for the pH prevailing in blood or interstitial fluid. Phylogenetic and structural analyses guided the design of 17 variants in which acidic amino acids were mutated at the CYT domain. Five mutations (G71K, D160K, Q174K, D177K, M180K) increased the pH optimum and IET rate. Structure-based analysis of the variants suggested two mechanisms explaining the improvements: electrostatic steering and stabilization of the closed state by hydrogen bonding. Combining the mutations into six combinatorial variants with up to five mutations shifted the pH optimum from 4.5 to 7.0 and increased the IET at pH 7.5 over 12-fold from 0.1 to 1.24 s-1 . While the mutants sustained a high enzymatic activity and even surpassed the IET of the wild-type enzyme, the accumulated positive charges on the CYT domain decreased DET, highlighting the importance of CYT for IET and DET. This study shows that interface engineering is an effective strategy to shift the pH optimum and improve the IET of CDH, but future work needs to maintain the DET of the CYT domain for bioelectronic applications.

Chimeric Cellobiose Dehydrogenases Reveal the Function of Cytochrome Domain Mobility for the Electron Transfer to Lytic Polysaccharide Monooxygenase.

The natural function of cellobiose dehydrogenase (CDH) to donate electrons from its catalytic flavodehydrogenase (DH) domain via its cytochrome (CYT) domain to lytic polysaccharide monooxygenase (LPMO) is an example of a highly efficient extracellular electron transfer chain. To investigate the function of the CYT domain movement in the two occurring electron transfer steps, two CDHs from the ascomycete Neurospora crassa (NcCDHIIA and NcCDHIIB) and five chimeric CDH enzymes created by domain swapping were studied in combination with the fungus' own LPMOs (NcLPMO9C and NcLPMO9F). Kinetic and electrochemical methods and hydrogen/deuterium exchange mass spectrometry were used to study the domain movement, interaction, and electron transfer kinetics. Molecular docking provided insights into the protein-protein interface, the orientation of domains, and binding energies. We find that the first, interdomain electron transfer step from the catalytic site in the DH domain to the CYT domain depends on steric and electrostatic interface complementarity and the length of the protein linker between both domains but not on the redox potential difference between the FAD and heme b cofactors. After CYT reduction, a conformational change of CDH from its closed state to an open state allows the second, interprotein electron transfer (IPET) step from CYT to LPMO to occur by direct interaction of the b-type heme and the type-2 copper center. Chimeric CDH enzymes favor the open state and achieve higher IPET rates by exposing the heme b cofactor to LPMO. The IPET, which is influenced by interface complementarity and the heme b redox potential, is very efficient with bimolecular rates between 2.9 × 105 and 1.1 × 106 M-1 s-1.

Determination of Lactose in Lactose-Free and Low-Lactose Milk, Milk Products, and Products Containing Dairy Ingredients by the LactoSens®R Amperometry Method: First Action 2020.01.

BACKGROUND: The AOAC Stakeholder Panel on Strategic Food Analytical Methods approved Standard Method Performance Requirements (SMPR®) 2018.009 for lactose in low-lactose or lactose-free milk, milk products, and products containing dairy ingredients. The LactoSens®R Method is a biosensor assay kit developed for the determination of lactose in a variety of lactose-free or low-lactose milk, dairy, and infant formula products produced with yeast-neutral lactases. OBJECTIVE: In response to a call for methods, the LactoSensR method was validated in a single laboratory study with comparison to SMPR 2018.009. METHOD: The LactoSensR method was evaluated for calibration, interference, repeatability, recovery, and robustness. In a method comparison study samples naturally containing low levels of lactose were evaluated using LactoSensR and an accredited high-performance anion-exchange chromatography with pulsed amperometric detection. RESULTS: Calibration with lactose standard solutions was shown to be linear and the method was shown to be free of interference from a variety of sugars, vitamins, alcohols, flavorings, and other compounds. Matrix studies, including 85 spiked materials, 55 products naturally containing lactose, and 13 reference materials, resulted in RSDr of 0-10.5% at 8-100 mg lactose/100 g and 0.2-5.4% at >100 mg lactose/100 g for milk and dairy products and 1.0-6.8% for infant formula, in compliance with SMPR 2018.009 with few exceptions. Recovery was 85.0-110.3% at 8-100 mg lactose/100 g and 85.6-109.7% at >100 mg lactose/100 g for milk and dairy products and 91.1-97.0% for infant formula, also meeting the performance requirements with few exceptions. The method was shown to be robust to changes in ambient temperature, sample temperature, and sample volume. CONCLUSIONS: The LactoSensR method compares favorably with the requirements of SMPR 2018.009 and should be adopted as a First Action AOAC Official MethodSM. HIGHLIGHTS: The LactoSensR method is a fast, easy-to-use method that meets the requirements of SMPR 2018.009.

Polysaccharide oxidation by lytic polysaccharide monooxygenase is enhanced by engineered cellobiose dehydrogenase.

The catalytic function of lytic polysaccharide monooxygenases (LPMOs) to cleave and decrystallize recalcitrant polysaccharides put these enzymes in the spotlight of fundamental and applied research. Here we demonstrate that the demand of LPMO for an electron donor and an oxygen species as cosubstrate can be fulfilled by a single auxiliary enzyme: an engineered fungal cellobiose dehydrogenase (CDH) with increased oxidase activity. The engineered CDH was about 30 times more efficient in driving the LPMO reaction due to its 27 time increased production of H2 O2 acting as a cosubstrate for LPMO. Transient kinetic measurements confirmed that intra- and intermolecular electron transfer rates of the engineered CDH were similar to the wild-type CDH, meaning that the mutations had not compromised CDH's role as an electron donor. These results support the notion of H2 O2 -driven LPMO activity and shed new light on the role of CDH in activating LPMOs. Importantly, the results also demonstrate that the use of the engineered CDH results in fast and steady LPMO reactions with CDH-generated H2 O2 as a cosubstrate, which may provide new opportunities to employ LPMOs in biomass hydrolysis to generate fuels and chemicals.

A Dual-Enzyme Hydrogen Peroxide Generation Machinery in Hydrogels Supports Antimicrobial Wound Treatment.

The aging population and accompanying diseases like diabetes resulted in an increased occurrence of chronic wounds. Topical wound treatment with antimicrobial agents to inhibit bacterial invasion and promote wound healing is often associated with difficulties. Here, we investigated the potential of succinyl chitosan (SC)-carboxymethyl cellulose (CMC) hydrogels which constantly release clinically relevant levels of hydrogen peroxide (H2O2). CMC hydrogel matrix was in situ converted by limited hydrolysis by a cellulase into substrates accepted by cellobiose dehydrogenase (CDH) for continuous production of H2O2 (30 µM over 24 h). This dual-enzyme catalyzed in situ H2O2 generation system proved its antimicrobial activity in a zone of inhibition (ZOI) assay best simulating the application as wound dressing and was found to be biocompatible toward mouse fibroblasts (95% viability). The hydrogels were thoroughly characterized regarding their rheological properties indicating fast gel formation (<3 min) and moderate cross-linking (1.5% strain, G' = 10 Pa). Cooling (fridge conditions) was found to be the simple on/off switch of the enzymatic machinery which is of great importance regarding storage and applicability of the bioactive hydrogel. This robust and bioactive antimicrobial hydrogel system overcomes dosing issues of common topical wound treatments and constitutes a promising wound healing approach for the future.

Characterization of three pyranose dehydrogenase isoforms from the litter-decomposing basidiomycete Leucoagaricus meleagris (syn. Agaricus meleagris).

Multigenicity is commonly found in fungal enzyme systems, with the purpose of functional compensation upon deficiency of one of its members or leading to enzyme isoforms with new functionalities through gene diversification. Three genes of the flavin-dependent glucose-methanol-choline (GMC) oxidoreductase pyranose dehydrogenase (AmPDH) were previously identified in the litter-degrading fungus Agaricus (Leucoagaricus) meleagris, of which only AmPDH1 was successfully expressed and characterized. The aim of this work was to study the biophysical and biochemical properties of AmPDH2 and AmPDH3 and compare them with those of AmPDH1. AmPDH1, AmPDH2 and AmPDH3 showed negligible oxygen reactivity and possess a covalently tethered FAD cofactor. All three isoforms can oxidise a range of different monosaccarides and oligosaccharides including glucose, mannose, galactose and xylose, which are the main constituent sugars of cellulose and hemicelluloses, and judging from the apparent steady-state kinetics determined for these sugars, the three isoforms do not show significant differences pertaining to their reaction with sugar substrates. They oxidize glucose both at C2 and C3 and upon prolonged reaction C2 and C3 double-oxidized glucose is obtained, confirming that the A. meleagris genes pdh2 (AY753308.1) and pdh3 (DQ117577.1) indeed encode CAZy class AA3_2 pyranose dehydrogenases. While reactivity with electron donor substrates was comparable for the three AmPDH isoforms, their kinetic properties differed significantly for the model electron acceptor substrates tested, a radical (the 2,2'-azino-bis[3-ethylbenzothiazoline-6-sulphonic acid] cation radical), a quinone (benzoquinone) and a complexed iron ion (the ferricenium ion). Thus, a possible explanation for this PDH multiplicity in A. meleagris could be that different isoforms react preferentially with structurally different electron acceptors in vivo.

Cellobiose dehydrogenase and chitosan-based lysozyme responsive materials for antimicrobial wound treatment.

The treatment of wound infection still constitutes a major threat in health care due to the increasing number of bacterial resistances and the difficulty of timely infection detection. Here, we present a smart antimicrobial system that is activated in case of infection based on elevated lysozyme activities. N-acetyl chitosan (degree of N-acetylation: 40%) was synthesized and hydrolysis by lysozyme in artificial wound fluid (AWF) was demonstrated. This resulted in the formation of N-acetylated chito oligosaccharides (COS) with a degree of polymerization of 2-5 units. The COS were shown to serve as substrate for cellobiose dehydrogenase (CDH) leading to the production of 1 mM antimicrobial hydrogen peroxide (H2 O2 ) after 24 h incubation at 37°C in AWF. Growth inhibition was seen upon incubation of Escherichia coli and Staphylococcus aureus with this chitosan-CDH system over 8 h. This approach represents the first self-regulating system for the infection responsive inhibition of bacterial growth in response to lysozyme as infection biomarker. Biotechnol. Bioeng. 2017;114: 416-422. © 2016 Wiley Periodicals, Inc.

Transcription analysis of pyranose dehydrogenase from the basidiomycete Agaricus bisporus and characterization of the recombinantly expressed enzyme.

Agaricus bisporus is a litter degrading basidiomycete commonly found in humic-rich environments. It is used as model organism and cultivated in large scale for food industry. Due to its ecological niche it produces a variety of enzymes for detoxification and degradation of humified plant litter. One of these, pyranose dehydrogenase, is thought to play a role in detoxification and lignocellulose degradation. It is a member of the glucose-methanol-choline family of flavin-dependent enzymes and oxidizes a wide range of sugars with concomitant reduction of electron acceptors like quinones. In this work, transcription of pdh in A. bisporus was investigated with real-time PCR revealing influence of the carbon source on pdh expression levels. The gene was isolated and heterologously expressed in Pichia pastoris. Characterization of the recombinant enzyme showed a higher affinity towards disaccharides compared to other tested pyranose dehydrogenases from related Agariceae. Homology modeling and sequence alignments indicated that two loops of high sequence variability at substrate access site could play an important role in modulating these substrate specificities.

Antimicrobial Cellobiose Dehydrogenase-Chitosan Particles.

Increasing prevalence of chronic wounds and microbial infection constitute a severe health challenge. The situation is further complicated by emerging multidrug resistance making the treatment of infections increasingly difficult. Here, a novel antimicrobial system based on in situ release of hydrogen peroxide (H2O2) by cellobiose dehydrogenase (CDH) immobilized on chitosan (CTS) particles is described. Covalent immobilization using carbodiimide coupling lead to a higher amount of protein immobilized on CTS (104 µg CDH/mg CTS) when compared to noncovalent immobilization, which, however, showed highest recovery of CDH activity (0.01 U/mg CTS). The CDH-CTS in situ generated H2O2 completely inhibited growth of Escherichia coli and Staphylococcus aureus over a period of 24 h. This resilient antimicrobial system represents a novel strategy for preventing infection with potential application in counteracting microbial colonization of chronic wounds.

Cellobiose dehydrogenase functionalized urinary catheter as novel antibiofilm system.

Urinary catheters expose patients to a high risk of acquiring nosocomial infections. To prevent this risk of infection, cellobiose dehydrogenase (CDH), an antimicrobial enzyme able to use various oligosaccharides as electron donors to produce hydrogen peroxide using oxygen as an electron acceptor, was covalently grafted onto plasma-activated urinary polydimethylsiloxane (PDMS) catheter surfaces. Successful immobilization of CDH on PDMS was confirmed by Fourier transformed-infrared spectrometry and production of H2 O2 . The CDH functionalized PDMS surfaces reduced the amount of viable Staphylococcus aureus by 60%, total biomass deposited on the surface by 30% and 70% of biofilm formation. The immobilized CDH was relatively stable in artificial urine over 16 days, retaining 20% of its initial activity. The CDH coated PDMS surface did not affect the growth and physiology of HEK 239 and RAW 264,7 mammalian cells. Therefore this new CDH functionalized catheter system shows great potential for solving the current problems associated with urinary catheters. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1448-1456, 2016.

Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation.

A new paradigm for cellulose depolymerization by fungi focuses on an oxidative mechanism involving cellobiose dehydrogenases (CDH) and copper-dependent lytic polysaccharide monooxygenases (LPMO); however, mechanistic studies have been hampered by the lack of structural information regarding CDH. CDH contains a haem-binding cytochrome (CYT) connected via a flexible linker to a flavin-dependent dehydrogenase (DH). Electrons are generated from cellobiose oxidation catalysed by DH and shuttled via CYT to LPMO. Here we present structural analyses that provide a comprehensive picture of CDH conformers, which govern the electron transfer between redox centres. Using structure-based site-directed mutagenesis, rapid kinetics analysis and molecular docking, we demonstrate that flavin-to-haem interdomain electron transfer (IET) is enabled by a haem propionate group and that rapid IET requires a closed CDH state in which the propionate is tightly enfolded by DH. Following haem reduction, CYT reduces LPMO to initiate oxygen activation at the copper centre and subsequent cellulose depolymerization.

Inter-domain electron transfer in cellobiose dehydrogenase: modulation by pH and divalent cations.

The flavocytochrome cellobiose dehydrogenase (CDH) is secreted by wood-decomposing fungi, and is the only known extracellular enzyme with the characteristics of an electron transfer protein. Its proposed function is reduction of lytic polysaccharide mono-oxygenase for subsequent cellulose depolymerization. Electrons are transferred from FADH2 in the catalytic flavodehydrogenase domain of CDH to haem b in a mobile cytochrome domain, which acts as a mediator and transfers electrons towards the active site of lytic polysaccharide mono-oxygenase to activate oxygen. This vital role of the cytochrome domain is little understood, e.g. why do CDHs exhibit different pH optima and rates for inter-domain electron transfer (IET)? This study uses kinetic techniques and docking to assess the interaction of both domains and the resulting IET with regard to pH and ions. The results show that the reported elimination of IET at neutral or alkaline pH is caused by electrostatic repulsion, which prevents adoption of the closed conformation of CDH. Divalent alkali earth metal cations are shown to exert a bridging effect between the domains at concentrations of > 3 mm, thereby neutralizing electrostatic repulsion and increasing IET rates. The necessary high ion concentration, together with the docking results, show that this effect is not caused by specific cation binding sites, but by various clusters of Asp, Glu, Asn, Gln and the haem b propionate group at the domain interface. The results show that a closed conformation of both CDH domains is necessary for IET, but the closed conformation also increases the FAD reduction rate by an electron pulling effect.

Ultrasound coating of polydimethylsiloxanes with antimicrobial enzymes.

There is an urgent need for antimicrobial functionalization of urinary catheters to prevent microbial colonization and biofilm formation on them. Here, the antimicrobial hydrogen peroxide (H2O2) producing enzyme cellobiose dehydrogenase (CDH) was for the first time grafted onto polydimethylsiloxanes (PDMS) using an ultrasound assisted coating method. This resulted in the development of an effective in situ continous H2O2 producing system able to continuously prevent microbial colonization and biofilm formation on catheters. This enzyme has an added advantage that it uses various oligosaccharides including expolysaccharides (an important part of the bioflim produced by the microbes while colonizing biomaterials) as electron donors to produce H2O2. Successful immobilization of active CDH nanoparticles on PDMS was confirmed by ESEM and AFM analysis as well as quantification of H2O2. Depending on the initial enzyme concentration, CDH-nanoparticles of varying sizes from 65 ± 17 nm to 93 ± 17 nm were created by the ultrasonic waves and subsequently deposited on the PDMS surface. PDMS sheets treated for 3 min produced 18 µM of H2O2 within 2 hours which was sufficient to significantly reduce the amount of viable S. aureus cells as well as the total amount of biomass deposited on the surface. The ultrasound assisted coating of antimicrobial enzymes therefore provides an easy approach to immobilize enzymes and create a surface with antimicrobial properties.

Preventing microbial colonisation of catheters: antimicrobial and antibiofilm activities of cellobiose dehydrogenase.

The ability of cellobiose dehydrogenase (CDH) to produce hydrogen peroxide (H(2)O(2)) for antimicrobial and antibiofilm functionalisation of urinary catheters was investigated. A recombinantly produced CDH from Myriococcum thermophilum was shown to completely inhibit the growth of Escherichia coli and Staphylococcus aureus both in liquid and solid media when supplemented with either 0.8 mM or 2 mM cellobiose as substrate. Biofilm formation on silicone films was prevented by CDH when supplemented with 1mM cellobiose. The CDH/cellobiose system also successfully inhibited many common urinary catheter-colonising micro-organisms, including multidrug-resistant S. aureus, Staphylococcus epidermidis, Proteus mirabilis, Stenotrophomonas maltophilia, Acinetobacter baumannii and Pseudomonas aeruginosa. Interestingly, CDH was also able to produce H(2)O(2) during oxidation of extracellular polysaccharides (exPS) formed by micro-organisms in the absence of cellobiose. The H(2)O(2) production and consequently antimicrobial and antibiofilm activities on these exPS were enhanced by incorporation of glycoside hydrolases such as amylases. Hydrolysis of polysaccharides by these enzymes increases the number of terminal reducing sugars as substrates for CDH as well as destabilises the biofilm. Furthermore, CDH suspended in catheter lubricants killed bacteria in biofilms colonising catheters. Incorporation of the CDH/cellobiose system in the lubricant therefore makes it an easy strategy for preventing microbial colonisation of catheters.

Galactose oxidase from Fusarium oxysporum--expression in E. coli and P. pastoris and biochemical characterization.

A gene coding for galactose 6-oxidase from Fusarium oxysporum G12 was cloned together with its native preprosequence and a C-terminal His-tag, and successfully expressed both in Escherichia coli and Pichia pastoris. The enzyme was subsequently purified and characterized. Among all tested substrates, the highest catalytic efficiency (kcat/Km) was found with 1-methyl-ß-D-galactopyranoside (2.2 mM(-1) s(-1)). The Michaelis constant (Km) for D-galactose was determined to be 47 mM. Optimal pH and temperature for the enzyme activity were 7.0 and 40°C, respectively, and the enzyme was thermoinactivated at temperatures above 50°C. GalOx contains a unique metalloradical complex consisting of a copper atom and a tyrosine residue covalently attached to the sulphur of a cysteine. The correct formation of this thioether bond during the heterologous expression in E. coli and P. pastoris could be unequivocally confirmed by MALDI mass spectrometry, which offers a convenient alternative to prove this Tyr-Cys crosslink, which is essential for the catalytic activity of GalOx.

Engineering of pyranose dehydrogenase for increased oxygen reactivity.

Pyranose dehydrogenase (PDH), a member of the GMC family of flavoproteins, shows a very broad sugar substrate specificity but is limited to a narrow range of electron acceptors and reacts extremely slowly with dioxygen as acceptor. The use of substituted quinones or (organo)metals as electron acceptors is undesirable for many production processes, especially of food ingredients. To improve the oxygen reactivity, site-saturation mutagenesis libraries of twelve amino acids around the active site of Agaricus meleagris PDH were expressed in Saccharomyces cerevisiae. We established high-throughput screening assays for oxygen reactivity and standard dehydrogenase activity using an indirect Amplex Red/horseradish peroxidase and a DCIP/D-glucose based approach. The low number of active clones confirmed the catalytic role of H512 and H556. Only one position was found to display increased oxygen reactivity. Histidine 103, carrying the covalently linked FAD cofactor in the wild-type, was substituted by tyrosine, phenylalanine, tryptophan and methionine. Variant H103Y was produced in Pichia pastoris and characterized and revealed a five-fold increase of the oxygen reactivity.

Calculation of substrate binding affinities for a bacterial GH78 rhamnosidase through molecular dynamics simulations.

Ram2 from Pediococcus acidilactici is a rhamnosidase from the glycoside hydrolase family 78. It shows remarkable selectivity for rutinose rather than para-nitrophenyl-alpha-l-rhamnopyranoside (p-NPR). Molecular dynamics simulations were performed using a homology model of this enzyme, in complex with both substrates. Free energy calculations lead to predicted binding affinities of -34.4 and -30.6 kJ mol-1 respectively, agreeing well with an experimentally estimated relative free energy of 5.4 kJ mol-1. Further, the most relevant binding poses could be determined. While p-NPR preferably orients its rhamnose moiety toward the active site, rutinose interacts most strongly with its glucose moiety. A detailed hydrogen bond analysis confirms previously implicated residues in the active site (Asp217, Asp222, Trp226, Asp229 and Glu488) and quantifies the importance of individual residues for the binding. The most important amino acids are Asp229 and Phe339 which are involved in many interactions during the simulations. While Phe339 was observed in more simulations, Asp229 was involved in more persistent interactions (forming an average of at least 2 hydrogen bonds during the simulation). These analyses directly suggest mutations that could be used in a further experimental characterization of the enzyme. This study shows once more the strength of computer simulations to rationalize and guide experiments at an atomic level.

Substrate specificity and interferences of a direct-electron-transfer-based glucose biosensor.

OBJECTIVE: Electrochemical sensors for glucose monitoring employ different signal transduction strategies for electron transfer from the biorecognition element to the electrode surface. We present a biosensor that employs direct electron transfer and evaluate its response to various interfering substances known to affect glucose biosensors. METHODS: The enzyme cellobiose dehydrogenase (CDH) was adsorbed on the surface of a carbon working electrode and covalently bound by cross linking. The response of CDH-modified electrodes to glucose and possible interfering compounds was measured by flow-injection analysis, linear sweep, and chronoamperometry. RESULTS: Chronoamperometry showed initial swelling/wetting of the electrode. After stabilization, the signal was stable and a sensitivity of 0.21 µA mM-1 cm-2 was obtained. To investigate the influence of the interfering substances on the biorecognition element, the simplest possible sensor architecture was used. The biosensor showed little (<5% signal deviation) or no response to various reported electroactive or otherwise interfering substances. CONCLUSIONS: Direct electron transfer from the biorecognition element to the electrode is a new principle applied to glucose biosensors, which can be operated at a low polarization potential of -100 mV versus silver/silver chloride. The reduction of interferences by electrochemically active substances is an attractive feature of this promising technology for the development of continuous glucose biosensors.

Optimization of a membraneless glucose/oxygen enzymatic fuel cell based on a bioanode with high coulombic efficiency and current density.

After initial testing and optimization of anode biocatalysts, a membraneless glucose/oxygen enzymatic biofuel cell possessing high coulombic efficiency and power output was fabricated and characterized. Two sugar oxidizing enzymes, namely, pyranose dehydrogenase from Agaricus meleagris (AmPDH) and flavodehydrogenase domains of various cellobiose dehydrogenases (DH(CDH)) were tested during the pre-screening. The enzymes were mixed, "wired" and entrapped in a low-potential Os-complex-modified redox-polymer hydrogel immobilized on graphite. This anode was used in combination with a cathode based on bilirubin oxidase from Myrothecium verrucaria adsorbed on graphite. Optimization showed that the current density for the mixed enzyme electrode could be further improved by using a genetically engineered variant of the non-glycosylated flavodehydrogenase domain of cellobiose dehydrogenase from Corynascus thermophilus expressed in E. coli (ngDH(CtCDHC310Y)) with a high glucose-turnover rate in combination with an Os-complex-modified redox polymer with a high concentration of Os complexes as well as a low-density graphite electrode. The optimized biofuel cell with the AmPDH/ngDH(CtCDHC310Y) anode showed not only a similar maximum voltage as with the biofuel cell based only on the ngDH(CtCDHC310Y) anode (0.55 V) but also a substantially improved maximum power output (20 µW cm(-2)) at 300 mV cell voltage in air-saturated physiological buffer. Most importantly, the estimated half-life of the mixed biofuel cell can reach up to 12 h, which is apparently longer than that of a biofuel cell in which the bioanode is based on only one single enzyme.