TY - JOUR A1 - Tadjoung Waffo, Armel Franklin A1 - Mitrova, Biljana A1 - Tiedemann, Kim A1 - Iobbi-Nivol, Chantal A1 - Leimkühler, Silke A1 - Wollenberger, Ulla T1 - Electrochemical trimethylamine n-oxide biosensor with enzyme-based oxygen-scavenging membrane for long-term operation under ambient air JF - Biosensors : open access journal N2 - An amperometric trimethylamine N-oxide (TMAO) biosensor is reported, where TMAO reductase (TorA) and glucose oxidase (GOD) and catalase (Cat) were immobilized on the electrode surface, enabling measurements of mediated enzymatic TMAO reduction at low potential under ambient air conditions. The oxygen anti-interference membrane composed of GOD, Cat and polyvinyl alcohol (PVA) hydrogel, together with glucose concentration, was optimized until the O-2 reduction current of a Clark-type electrode was completely suppressed for at least 3 h. For the preparation of the TMAO biosensor, Escherichia coli TorA was purified under anaerobic conditions and immobilized on the surface of a carbon electrode and covered by the optimized O-2 scavenging membrane. The TMAO sensor operates at a potential of -0.8 V vs. Ag/AgCl (1 M KCl), where the reduction of methylviologen (MV) is recorded. The sensor signal depends linearly on TMAO concentrations between 2 mu M and 15 mM, with a sensitivity of 2.75 +/- 1.7 mu A/mM. The developed biosensor is characterized by a response time of about 33 s and an operational stability over 3 weeks. Furthermore, measurements of TMAO concentration were performed in 10% human serum, where the lowest detectable concentration is of 10 mu M TMAO. KW - trimethylamine N-oxide KW - biosensor KW - TMAO-reductase KW - oxygen scavenger KW - immobilized enzyme KW - multienzyme electrode KW - viologen Y1 - 2021 U6 - https://doi.org/10.3390/bios11040098 SN - 2079-6374 VL - 11 IS - 4 PB - MDPI CY - Basel ER - TY - JOUR A1 - Kaufmann, Hans Paul A1 - Duffus, Benjamin R. A1 - Mitrova, Biljana A1 - Iobbi-Nivol, Chantal A1 - Teutloff, Christian A1 - Nimtz, Manfred A1 - Jaensch, Lothar A1 - Wollenberger, Ulla A1 - Leimkühler, Silke T1 - Modulating the Molybdenum Coordination Sphere of Escherichia coli Trimethylamie N-Oxide Reductase JF - Biochemistry N2 - The well-studied enterobacterium Escherichia coli present in the human gut can reduce trimethylamine N-oxide (TMAO) to trimethylamine during anaerobic respiration. The TMAO reductase TorA is a monomeric, bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor-containing enzyme that belongs to the dimethyl sulfoxide reductase family of molybdoenzymes. We report on a system for the in vitro reconstitution of TorA with molybdenum cofactors (Moco) from different sources. Higher TMAO reductase activities for TorA were obtained when using Moco sources containing a sulfido ligand at the molybdenum atom. For the first time, we were able to isolate functional bis-MGD from Rhodobacter capsulatus formate dehydrogenase (FDH), which remained intact in its isolated state and after insertion into apo-TorA yielded a highly active enzyme. Combined characterizations of the reconstituted TorA enzymes by electron paramagnetic resonance spectroscopy and direct electrochemistry emphasize that TorA activity can be modified by changes in the Mo coordination sphere. The combination of these results together with studies of amino acid exchanges at the active site led us to propose a novel model for binding of the substrate to the molybdenum atom of TorA. Y1 - 2018 U6 - https://doi.org/10.1021/acs.biochem.7b01108 SN - 0006-2960 VL - 57 IS - 7 SP - 1130 EP - 1143 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Mitrova, Biljana A1 - Tadjoung Waffo, Armel Franklin A1 - Kaufmann, Paul A1 - Iobbi-Nivol, Chantal A1 - Leimkühler, Silke A1 - Wollenberger, Ulla T1 - Trimethylamine N-Oxide Electrochemical Biosensor with a Chimeric Enzyme JF - ChemElectroChem N2 - For the first time, an enzyme-based electrochemical biosensor system for determination of trimethylamine N-oxide (TMAO) is described. It employs an active chimeric variant of TorA in combination with an enzymatically deoxygenating system and a low-potential mediator for effective regeneration of the enzyme and cathodic current generation. TMAO reductase (TorA) is a molybdoenzyme found in marine and most enterobacteria that specifically catalyzes the reduction of TMAO to trimethylamine (TMA). The chimeric TorA, named TorA-FDH, corresponds to the apoform of TorA from Escherichia coli reconstituted with the molybdenum cofactor from formate dehydrogenase (FDH). Each enzyme, TorA and TorA-FDH, was immobilized on the surface of a carbon electrode and protected with a dialysis membrane. The biosensor operates at an applied potential of -0.8V [vs. Ag/AgCl (1M KCl)] under ambient air conditions thanks to an additional enzymatic O-2-scavenger system. A comparison between the two enzymatic sensors revealed a much higher sensitivity for the biosensor with immobilized TorA-FDH. This biosensor exhibits a sensitivity of 14.16nA/M TMAO in a useful measuring range of 2-110M with a detection limit of LOD=2.96nM (S/N=3), and was similar for TMAO in buffer and in spiked serum samples. With a response time of 16 +/- 2 s, the biosensor is stable over prolonged daily measurements (n=20). This electrochemical biosensor provides suitable applications in detecting TMAO levels in human serum. KW - trimethylamine N-oxide (TMAO) KW - TMAO reductase KW - chimeric enzyme KW - molybdoenzyme KW - electrochemical biosensor Y1 - 2018 U6 - https://doi.org/10.1002/celc.201801422 SN - 2196-0216 VL - 6 IS - 6 SP - 1732 EP - 1737 PB - Wiley-VCH CY - Weinheim ER - TY - THES A1 - Mitrova, Biljana T1 - Bioelectrochemical investigation of E. coli TMAO reductase and R. capsulatus formate dehydrogenase BT - From small to complex molybdoenzymes Y1 - ER -