Rapid identification of methylase specificity (RIMS-seq) jointly identifies methylated motifs and generates shotgun sequencing of bacterial genomes.

DNA methylation is widespread amongst eukaryotes and prokaryotes to modulate gene expression and confer viral resistance. 5-Methylcytosine (m5C) methylation has been described in genomes of a large fraction of bacterial species as part of restriction-modification systems, each composed of a methyltransferase and cognate restriction enzyme. Methylases are site-specific and target sequences vary across organisms. High-throughput methods, such as bisulfite-sequencing can identify m5C at base resolution but require specialized library preparations and single molecule, real-time (SMRT) sequencing usually misses m5C. Here, we present a new method called RIMS-seq (rapid identification of methylase specificity) to simultaneously sequence bacterial genomes and determine m5C methylase specificities using a simple experimental protocol that closely resembles the DNA-seq protocol for Illumina. Importantly, the resulting sequencing quality is identical to DNA-seq, enabling RIMS-seq to substitute standard sequencing of bacterial genomes. Applied to bacteria and synthetic mixed communities, RIMS-seq reveals new methylase specificities, supporting routine study of m5C methylation while sequencing new genomes.

Identification two key residues at the intersection of domains of a thioether monooxygenase for improving its sulfoxidation performance.

AcCHMO, a cyclohexanone monooxygenase from Acinetobacter calcoaceticus, is a typical Type I Baeyer-Villiger monooxygenase (BVMO). We previously obtained the AcCHMOM6 mutant, which oxidizes omeprazole sulfide (OPS) to the chiral sulfoxide drug esomeprazole. To further improve the catalytic efficiency of the AcCHMOM6 mutant, a focused mutagenesis strategy was adopted at the intersections of the FAD-binding domain, NADPH-binding domain, and α-helical domain based on structural characteristics of AcCHMO. By using focused mutagenesis and subsequent global evolution two key residues (L55 and P497) at the intersections of the domains were identified. Mutant of L55Y improved catalytic efficiency significantly, whereas the P497S mutant alleviated substrate inhibition remarkably. AcCHMOM7 (L55Y/P497S) was obtained by combining the two mutations, which increased the specific activity from 18.5 (M6) to 108 U/g, and an increase in the Ki of the substrate OPS from 34 to 265 µM. The results indicate that catalytic performance can be elevated by modification of the sensitive sites at the intersection of the domains of AcCHMO. The results also provided some insights for the engineering of other Type I BVMOs or other multidomain proteins.

Characterization of the novel OXA-213-like ß-lactamase OXA-822 from Acinetobacter calcoaceticus.

OBJECTIVES: This study analysed the novel carbapenem-hydrolysing class D ß-lactamase OXA-822 identified in the clinical Acinetobacter calcoaceticus isolate AC_2117. METHODS: WGS was employed for identification of ß-lactamases. Micro-broth dilution was used for evaluation of antibiotic susceptibility of AC_2117 and transformants containing blaOXA-822. After heterologous purification of OXA-822, OXA-359 and OXA-213, enzyme kinetics were determined using spectrometry. The effect of OXA-822 upon meropenem treatment was analysed in the Galleria mellonella in vivo infection model. RESULTS: OXA-822 is a member of the intrinsic OXA-213-like family found in A. calcoaceticus and Acinetobacter pittii. Amino acid sequence similarity to the nearest related OXA-359 was 97%. Production of OXA-822, OXA-359 and OXA-213 in Acinetobacter baumannii ATCC® 19606T resulted in elevated MICs for carbapenems (up to 16-fold). Penicillinase activity of the purified OXA-822 revealed high KM values, in the millimolar range, combined with high turnover numbers. OXA-822 showed the highest affinity to carbapenems, but affinity to imipenem was ∼10-fold lower compared with other carbapenems. Molecular modelling revealed that imipenem does not interact with a negatively charged side chain of OXA-822, as doripenem does, leading to the lower affinity. Presence of OXA-822 decreased survival of infected Galleria mellonella larvae after treatment with meropenem. Only 52.7% ±â€Š7.7% of the larvae survived after 24 h compared with 90.9% ±â€Š3.7% survival in the control group. CONCLUSIONS: The novel OXA-822 from a clinical A. calcoaceticus isolate displayed penicillinase and carbapenemase activity in vitro, elevated MICs in different species and decreased carbapenem susceptibility in A. baumannii in vivo.

Selective Butyrate Esterase Probe for the Rapid Colorimetric and Fluorogenic Identification of Moraxella catarrhalis.

Clinical identification of the pathogenic bacterium Moraxella catarrhalis in cultures relies on the detection of bacterial butyrate esterase (C4-esterase) using a coumarin-based fluorogenic substrate, 4-methylumbelliferyl butyrate. However, this classical probe may give false-positive responses because of its poor stability and lack of specificity. Here, we report a new colorimetric and fluorogenic probe design employing a meso-ester-substituted boron dipyrromethene (BODIPY) dye for the specific detection of C4-esterase activity expressed by M. catarrhalis. This new probe has resistance to nonspecific hydrolysis that is far superior to the classical probe and also selectively responds to esterase with rapid colorimetric and fluorescence signal changes and large "turn-on" ratios. The probe was successfully applied to the specific detection of M. catarrhalis with high sensitivity.

Cascade Synthesis from Cyclohexane to ϵ-Caprolactone by Visible-Light-Driven Photocatalysis Combined with Whole-Cell Biological Oxidation.

Cyclohexane was directly oxy-functionalised to ϵ-caprolactone through a cascade reaction sequence combining visible-light-driven photocatalysis with cyclohexanone monooxygenase (CHMO) whole-cell biocatalysis. Two available photocatalysts, Au-doped TiO2 (Au-TiO2 ) and graphitic carbonitride (g-C3 N4 ), were evaluated in the experiment and some optimisations to the cascade reaction were applied. In stepwise mode, the highest degree of conversion from cyclohexanol to ϵ-caprolactone can be up to 41 %, with use of g-C3 N4 . The cascade reaction from cyclohexane to ϵ-caprolactone is achievable under a light intensity of 149 µW cm-2 .

Enzyme cocktail containing NADH regeneration system for efficient bioremediation of oil sludge contamination.

Oil sludge is one kind of toxic and persistent contamination to ecology system from petroleum industry. In order to recycle contaminated sands and reduce environmental impacts at a lower operating cost, enzyme cocktail 21/CbFDH including NADH regeneration system for oily sludge bioremediation was constructed for the first time. The intracellular enzymes of oil-degrading strain Acinetobacter calcoaceticus 21 were prepared and the formate dehydrogenase gene Cbfdh from Candida boidinii was cloned and functionally expressed in E.coli BL21 induced by lactose. The activity and stability of CbFDH was enhanced through self-induction medium optimization using Box-Behnken design. The CbFDH activity was 12.2 times increased and was only decreased 3.9% upon storage at 30 °C for 5 d. The CbFDH increased the degradation rate of oil in high concentration. For the sludge with 10% oil (w/w), the degradation rate achieved 35.6% after 12 h using enzyme 21/CbFDH with the protein ratio of 1:4. The results will provide novel perspectives for creation and operation of petroleum-degrading enzymes involving formate dehydrogenase with higher efficiency and lower cost comparing to current microbial strains or consortium.

Generalizable Protein Biosensors Based on Synthetic Switch Modules.

Allosteric protein switches are key controllers of information and energy processing in living organisms and are desirable engineered control tools in synthetic systems. Here we present a generally applicable strategy for construction of allosteric signaling systems with inputs and outputs of choice. We demonstrate conversion of constitutively active enzymes into peptide-operated synthetic allosteric ON switches by insertion of a calmodulin domain into rationally selected sites. Switches based on EGFP, glucose dehydrogenase, NanoLuciferase, and dehydrofolate reductase required minimal optimization and demonstrated a dynamic response ranging from 1.8-fold in the former case to over 200-fold in the latter case. The peptidic nature of the calmodulin ligand enables incorporation of such synthetic switch modules into higher order sensory architectures. Here, a ligand-mediated increase in proximity of the allosteric switch and the engineered activator peptide modulates biosensor's activity. Created biosensors were used to measure concentrations of clinically relevant drugs and biomarkers in plasma, saliva, and urine with accuracy comparable to that of the currently used clinical diagnostic assays. The approach presented is generalizable as it allows rapid construction of efficient protein switches that convert binding of a broad range of analytes into a biochemical activity of choice enabling construction of artificial signaling and metabolic circuits of potentially unlimited complexity.

Xanthine oxidase of Acinetobacter calcoaceticus RL2-M4: Production, purification and characterization.

Xanthine oxidase (EC 1.17.3.2) is a key enzyme of purine metabolism and has potential applications in food and pharmaceutical industries. In the present study, a new bacterial source of xanthine oxidase i.e. Acinetobacter calcoaceticus RL2-M4 with high oxidase activity was isolated from soil. The culture conditions were optimized with one variable at a time (OVAT) and response surface methodology (RSM) approaches included: a minimal salt medium (MSM) of pH 7.0 supplemented with 0.8% yeast extract, 8.5 mM xanthine and incubation at 30 °C for 24 h. Under these culture conditions 11.57 fold increase in the production of this enzyme was achieved. The enzyme was purified from A. calcoaceticus RL2-M4 using anion exchange chromatography to 8.18 fold with 31% yield and specific activity of 4.58 U/mg protein. SDS-PAGE analysis of the purified enzyme revealed that it was homodimer of 95 kDa and its native molecular mass was estimated to be 190 kDa. This enzyme was found to be stable at 35 °C for 5 h. The purified xanthine oxidase of A. calcoaceticus RL2-M4 had Km 0.3 mM and Vmax 5.8 U/mg protein using xanthine as substrate. The activity and stability characteristic of xanthine oxidase of A. calcoaceticus RL2-M4 makes it a potentially good enzyme for industrial applications.

Identification of the novel class D ß-lactamase OXA-679 involved in carbapenem resistance in Acinetobacter calcoaceticus.

OBJECTIVES: The aim of this study was to characterize the Acinetobacter calcoaceticus clinical isolate AC_2117 with the novel carbapenem-hydrolysing class D ß-lactamase (CHDL) OXA-679. METHODS: Identification of the species and ß-lactamases was verified by genome sequencing (PacBio) and phylogenetic analyses. Antibiotic susceptibility of AC_2117 and transformants harbouring cloned blaOXA-679 was evaluated using antibiotic gradient strips and microbroth dilution. OXA-679 was purified heterologously and kinetic parameters were determined using spectrometry or isothermal titration calorimetry. The impact of OXA-679 production during imipenem therapy was evaluated in the Galleria mellonella infection model. RESULTS: Sequencing of the complete genome of the clinical A. calcoaceticus isolate AC_2117 identified a novel CHDL, termed OXA-679. This enzyme shared sequence similarity of 71% to each of the families OXA-143 and OXA-24/40. Phylogenetic analyses revealed that OXA-679 represents a member of a new OXA family. Cloning and expression of blaOXA-679 as well as measurement of kinetic parameters revealed the effective hydrolysis of carbapenems which resulted in reduced susceptibility to carbapenems in Escherichia coli and A. calcoaceticus, and high-level carbapenem resistance in Acinetobacter baumannii. Infection of larvae of G. mellonella with a sublethal dose of blaOXA-679-expressing A. baumannii could not be cured by high-dose imipenem therapy, indicating carbapenem resistance in vivo. CONCLUSIONS: We identified blaOXA-679 in a clinical A. calcoaceticus isolate that represents a member of the new OXA-679 family and that conferred high-level carbapenem resistance in vitro and in vivo.

Prevalence and antimicrobial susceptibilities of Acinetobacter baumannii and non-baumannii Acinetobacters from Terengganu, Malaysia and their carriage of carbapenemase genes.

A total of 153 non-repeat Acinetobacter spp. clinical isolates obtained in 2015 from Hospital Sultanah Nur Zahirah (HSNZ) in Terengganu, Malaysia, were characterized. Identification of the isolates at species level was performed by ribosomal DNA restriction analysis (ARDRA) followed by sequencing of the rpoB gene. The majority of the isolates (n=128; 83.7 %) were A. baumannii while the rest were identified as A. nosocomialis (n=16), A. calcoaceticus (n=5), A. soli (n=2), A. berezeniae (n=1) and A. variabilis (n=1). Multidrug resistance (MDR) was most prevalent in A. baumannnii (66.4 %) whereas only one non-baumannii isolate (A. nosocomialis) was MDR. The blaOXA-23 gene was the predominant acquired carbapenemase gene (56.2 %) and was significantly associated (P<0.001) with carbapenem resistance. However, no significant association was found for carbapenem resistance and isolates that contained the ISAba1-blaOXA-51 configuration.

Co-expression of an alcohol dehydrogenase and a cyclohexanone monooxygenase for cascade reactions facilitates the regeneration of the NADPH cofactor.

The introduction of a three-enzyme cascade (comprising a cyclohexanone monooxygenase (CHMO), an alcohol dehydrogenase (ADH) and a lipase (CAL-A)) for the production of oligo-ε-caprolactone provided self-sufficiency with respect to NADPH-cofactor regeneration and reduced inhibiting effects on the central CHMO enzyme. For further optimization of cofactor regeneration, now a co-expression of CHMO and ADH in E. coli using a Duet™ vector was performed. This led to higher conversion values of the substrate cyclohexanol in whole-cell biocatalysis compared to an expression of both enzymes from two separate plasmids. Furthermore, a more advantageous balance of expression levels between the partial cascade enzymes was achieved via engineering of the ribosome binding site. This contributed to an even faster cofactor regeneration rate.

Rechargeable, flexible and mediator-free biosupercapacitor based on transparent ITO nanoparticle modified electrodes acting in µM glucose containing buffers.

We present a transparent and flexible self-charging biosupercapacitor based on an optimised mediator- and membrane-free enzymatic glucose/oxygen biofuel cell. Indium tin oxide (ITO) nanoparticles were spray-coated on transparent conducting ITO supports resulting in a flocculent, porous and nanostructured electrode surface. By this, high capacitive currents caused by an increased electrochemical double layer as well as enhanced catalytic currents due to a higher number of immobilised enzyme molecules were obtained. After a chemical pre-treatment with a silane derivative, bilirubin oxidase from Myrothecium verrucaria was immobilized onto the ITO nanostructured electrode surface under formation of a biocathode, while bioanodes were obtained by either immobilisation of cellobiose dehydrogenase from Corynascus thermophilus or soluble PQQ-dependent glucose dehydrogenase from Acinetobacter calcoaceticus. The latter showed a lower apparent KM value for glucose conversion and higher catalytic currents at µM glucose concentrations. Applying the optimised device as a biosupercapacitor in a discontinuous charge/discharge mode led to a generated power output of 0.030mW/cm2 at 50µM glucose, simulating the glucose concentration in human tears. This represents an enhancement by a factor of 350 compared to the power density obtained from the continuously operating biofuel cell with a maximum power output of 0.086µW/cm2 under the same conditions. After 17h of charging/discharging cycles a remarkable current enhancement was still measured. The entire device was transferred to flexible materials and applied for powering a flexible display showing its potential applicability as an intermittent power source in smart contact lenses.

Emergence of New Delhi metallo-beta-lactamase 1 and other carbapenemase-producing Acinetobacter calcoaceticus-baumannii complex among patients in hospitals in Ha Noi, Viet Nam.

Acinetobacter baumannii is an important cause of multidrug-resistant hospital acquired infections in the world. Here, we investigate the presence of NDM-1 and other carbapenemases among carbapenem-resistant A. baumannii isolated between August 2010 and December 2014 from three large hospitals in Hanoi, Vietnam. We identified 23/582 isolates (4 %) (11 from hospital A, five from hospital B, and seven from hospital C) that were NDM-1 positive, and among them 18 carried additional carbapenemase genes, including seven isolates carrying NDM-1, IMP-1, and OXA-58 with high MICs for carbapenems. Genotyping indicated that NDM-1 carrying A. baumannii have expanded clonally in these hospitals. Five new STs (ST1135, ST1136, ST1137, ST1138, and ST1139) were identified. One isolate carried NDM-1 on a plasmid belonging to the N-repA replicon type; no NDM-1-positive plasmids were identified in the other isolates. We have shown the extent of the carbapenem resistance and the local clonal spread of A. baumannii carrying NDM-1 in these hospitals; coexistence of NDM-1 and IMP-1 is reported for the first time from Vietnam here, and this will further seriously limit future therapeutic options.

Engineering Cyclohexanone Monooxygenase for the Production of Methyl Propanoate.

A previous study showed that cyclohexanone monooxygenase from Acinetobacter calcoaceticus (AcCHMO) catalyzes the Baeyer-Villiger oxidation of 2-butanone, yielding ethyl acetate and methyl propanoate as products. Methyl propanoate is of industrial interest as a precursor of acrylic plastic. Here, various residues near the substrate and NADP+ binding sites in AcCHMO were subjected to saturation mutagenesis to enhance both the activity on 2-butanone and the regioselectivity toward methyl propanoate. The resulting libraries were screened using whole cell biotransformations, and headspace gas chromatography-mass spectrometry was used to identify improved AcCHMO variants. This revealed that the I491A AcCHMO mutant exhibits a significant improvement over the wild type enzyme in the desired regioselectivity using 2-butanone as a substrate (40% vs 26% methyl propanoate, respectively). Another interesting mutant is the T56S AcCHMO mutant, which exhibits a higher conversion yield (92%) and kcat (0.5 s-1) than wild type AcCHMO (52% and 0.3 s-1, respectively). Interestingly, the uncoupling rate for the T56S AcCHMO mutant is also significantly lower than that for the wild type enzyme. The T56S/I491A double mutant combined the beneficial effects of both mutations leading to higher conversion and improved regioselectivity. This study shows that even for a relatively small aliphatic substrate (2-butanone), catalytic efficiency and regioselectivity can be tuned by structure-inspired enzyme engineering.

Role of Quinones in Electron Transfer of PQQ-Glucose Dehydrogenase Anodes­Mediation or Orientation Effect.

In this study, the influence of two quinones (1,2- and 1,4-benzoquinone) on the operation and mechanism of electron transfer in PQQ-dependent glucose dehydrogenase (PQQ-sGDH) anodes has been determined. Benzoquinones were experimentally explored as mediators present in the electrolyte. The electrochemical performance of the PQQ-sGDH anodes with and without the mediators was examined and for the first time molecular docking simulations were used to gain a fundamental understanding to explain the role of the mediator molecules in the design and operation of the enzymatic electrodes. It was proposed that the higher performance of the PQQ-sGDH anodes in the presence of 1,2- and 1,4-benzoquinones introduced in the solution is due to the shorter distance between these molecules and PQQ in the enzymatic molecule. It was also hypothesized that when 1,4-benzoquinone is adsorbed on a carbon support, it would play the dual role of a mediator and an orienting agent. At the same time, when 1,2-benzoquinone and ubiquinone are adsorbed on the electrode surface, the enzyme would transfer the electrons directly to the support, and these molecules would primarily play the role of an orienting agent.

Epidemiological characteristics of blaNDM-1 in Enterobacteriaceae and the Acinetobacter calcoaceticus-Acinetobacter baumannii complex in China from 2011 to 2012.

OBJECTIVES: The study aimed to investigate the prevalence and epidemiological characteristics of blaNDM-1 (encoding New Delhi metallo-ß-lactamase 1) in Enterobacteriaceae and the Acinetobacter calcoaceticus-Acinetobacter baumannii complex (ABC) in China from July 2011 to June 2012. METHODS: PCR was used to screen for the presence of blaNDM-1 in all organisms studied. For blaNDM-1-positive strains, 16S rRNA analysis and Analytical Profile Index (API) strips were used to identify the bacterial genus and species. The ABCs were reconfirmed by PCR detection of blaOXA-51-like. Antibiotic susceptibilities of the bacteria were assessed by determining minimum inhibitory concentration (MIC) of them using two-fold agar dilution test, as recommended by the Clinical and Laboratory Standards Institute (CLSI). Molecular typing was performed using pulsed-field gel electrophoresis (PFGE). S1 nuclease-pulsed-field gel electrophoresis (S1-PFGE) and Southern blot hybridization were conducted to ascertain the gene location of blaNDM-1. Conjugation experiments were conducted to determine the transmission of blaNDM-1-positive strains. RESULTS: Among 2,170 Enterobacteriaceae and 600 ABCs, seven Enterobacteriaceae strains and two A. calcoaceticus isolates from five different cities carried the blaNDM-1 gene. The seven Enterobacteriaceae strains comprised four Klebsiella pneumoniae, one Enterobacter cloacae, one Enterobacter aerogen and one Citrobacter freundii. All seven were non-susceptible to imipenem, meropenem or ertapenem. Two A. calcoaceticus species were resistant to imipenem and meropenem. Three K. pneumoniae showed the same PFGE profiles. The blaNDM-1 genes of eight strains were localized on plasmids, while one was chromosomal. CONCLUSIONS: Compared with previous reports, the numbers and species containing the blaNDM-1 in Enterobacteriaceae have significantly increased in China. Most of them are able to disseminate the gene, which is cause for concern. Consecutive surveillance should be implemented and should also focus on the dissemination of blaNDM-1 among gram-negative clinical isolates.

Evaluation of clonality and carbapenem resistance mechanisms among Acinetobacter baumannii-Acinetobacter calcoaceticus complex and Enterobacteriaceae isolates collected in European and Mediterranean countries and detection of two novel ß-lactamases, GES-22 and VIM-35.

We evaluated doripenem-resistant Acinetobacter baumannii-Acinetobacter calcoaceticus complex (ACB; n = 411) and Enterobacteriaceae (n = 92) isolates collected from patients from 14 European and Mediterranean countries during 2009 to 2011 for the presence of carbapenemase-encoding genes and clonality. Following susceptibility testing, carbapenem-resistant (doripenem MIC, >2 µg/ml) isolates were screened for carbapenemases. New ß-lactamase genes were expressed in a common background and susceptibility was tested. Class 1 integrons were sequenced. Clonality was evaluated by pulsed-field gel electrophoresis and multilocus sequence typing (Pasteur scheme). Relative expression of ß-lactam intrinsic resistance mechanisms was determined for carbapenemase-negative Enterobacteriaceae. ACB and Enterobacteriaceae displayed 58.9 and 0.9% doripenem resistance, respectively. bla(OXA-23), bla(OXA-58), and bla(OXA-24/OXA-40) were detected among 277, 77, and 29 ACB, respectively (in 8, 6, and 5 countries). Ten Turkish isolates carried bla(GES-11) or bla(GES-22). GES-22 (G243A and M169L mutations in GES-1) had an extended-spectrum ß-lactamase profile. A total of 33 clusters of ≥ 2 ACB isolates were observed, and 227 isolates belonged to sequence type 2/international clone II. Other international clones were limited to Turkey and Israel. Doripenem-resistant Enterobacteriaceae increased significantly (0.7 to 1.6%), and 15 blaKPC-2- and 22 blaKPC-3-carrying isolates, mostly belonging to clonal complexes 11 and 258, were observed. Enterobacteriaceae isolates producing OXA-48 (n = 16; in Turkey and Italy), VIM-1 (n = 10; in Greece, Poland, and Spain), VIM-26 (n = 1; in Greece), and IMP-19, VIM-4, and the novel VIM-35 (n = 1 each from Poland) were detected. VIM-35 had one substitution compared to VIM-1 (A235T) and a similar susceptibility profile. One or more resistance mechanisms were identified in 4/6 carbapenemase-negative Enterobacteriaceae. This broad evaluation confirms results from country-specific surveys and shows a highly diverse population of carbapenemase-producing ACB and Enterobacteriaceae in Europe and Mediterranean countries.

Characterization of affinity-purified isoforms of Acinetobacter calcoaceticus Y1 glutathione transferases.

Glutathione transferases (GST) were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) of Acinetobacter calcoaceticus strain PHEA-2, respectively.

Biofuel cells based on direct enzyme-electrode contacts using PQQ-dependent glucose dehydrogenase/bilirubin oxidase and modified carbon nanotube materials.

Two types of carbon nanotube electrodes (1) buckypaper (BP) and (2) vertically aligned carbon nanotubes (vaCNT) have been used for elaboration of glucose/O2 enzymatic fuel cells exploiting direct electron transfer. For the anode pyrroloquinoline quinone dependent glucose dehydrogenase ((PQQ)GDH) has been immobilized on [poly(3-aminobenzoic acid-co-2-methoxyaniline-5-sulfonic acid), PABMSA]-modified electrodes. For the cathode bilirubin oxidase (BOD) has been immobilized on PQQ-modified electrodes. PABMSA and PQQ act as promoter for enzyme bioelectrocatalysis. The voltammetric characterization of each electrode shows current densities in the range of 0.7-1.3 mA/cm(2). The BP-based fuel cell exhibits maximal power density of about 107 µW/cm(2) (at 490 mV). The vaCNT-based fuel cell achieves a maximal power density of 122 µW/cm(2) (at 540 mV). Even after three days and several runs of load a power density over 110 µW/cm(2) is retained with the second system (10mM glucose). Due to a better power exhibition and an enhanced stability of the vaCNT-based fuel cells they have been studied in human serum samples and a maximal power density of 41 µW/cm(2) (390 mV) can be achieved.