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Molinnus, Denise ; Muschallik, Lukas ; Gonzalez, Laura Osorio ; Bongaerts, Johannes ; Wagner, Torsten ; Selmer, Thorsten ; Siegert, Petra ; Keusgen, Michael ; Schöning, Michael Josef
A capacitive electrolyte-insulator-semiconductor (EIS) field-effect biosensor for acetoin detection has been presented for the first time. The EIS sensor consists of a layer structure of Al/p-Si/SiO₂/Ta₂O₅/enzyme acetoin reductase. The enzyme, also referred to as butane-2,3-diol dehydrogenase from B. clausii DSM 8716T, has been recently characterized. The enzyme catalyzes the (R)-specific reduction of racemic acetoin to (R,R)- and meso-butane-2,3-diol, respectively. Two different enzyme immobilization strategies (cross-linking by using glutaraldehyde and adsorption) have been studied. Typical biosensor parameters such as optimal pH working range, sensitivity, hysteresis, linear concentration range and long-term stability have been examined by means of constant-capacitance (ConCap) mode measurements. Furthermore, preliminary experiments have been successfully carried out for the detection of acetoin in diluted white wine samples.
The integration of biomolecular logic principles with electronic transducers allows designing novel digital biosensors with direct electrical output, logically triggered drug-release, and closed-loop sense/act/treat systems. This opens new opportunities for advanced personalized medicine in the context of theranostics. In the present work, we will discuss selected examples of recent developments in the field of interfacing enzyme logic gates with electrodes and semiconductor field-effect devices. Special attention is given to an enzyme OR/Reset logic gate based on a capacitive field-effect electrolyte-insulator-semiconductor sensor modified with a multi-enzyme membrane. Further examples are a digital adrenaline biosensor based on an AND logic gate with binary YES/NO output and an integrated closed-loop sense/act/treat system comprising an amperometric glucose sensor, a hydrogel actuator, and an insulin (drug) sensor.
Molinnus, Denise ; Sorich, Maren ; Bartz, Alexander ; Siegert, Petra ; Willenberg, Holger S. ; Lisdat, Fred ; Poghossian, Arshak ; Keusgen, Michael ; Schöning, Michael Josef
An amperometric biosensor using a substrate recycling principle was realized for the detection of low adrenaline concentrations (1 nM) by measurements in phosphate buffer and Ringer’s solution at pH 6.5 and pH 7.4, respectively. In proof-of-concept experiments, a Boolean logic-gate principle has been applied to develop a digital adrenaline biosensor based on an enzyme AND logic gate. The obtained results demonstrate that the developed digital biosensor is capable for a rapid qualitative determination of the presence/absence of adrenaline in a YES/NO statement. Such digital biosensor could be used in clinical diagnostics for the control of a correct insertion of a catheter in the adrenal veins during adrenal venous-sampling procedure.
Monti, Elena ; Waldvogel, Janice ; Ritzmann, Ramona ; Freyler, Kathrin ; Albracht, Kirsten ; Helm, Michael ; De Cesare, Niccolò ; Pavan, Piero ; Reggiani, Carlo ; Gollhofer, Albert ; Narici, Marco Vincenzo
Performing tasks, such as running and jumping, requires activation of the agonist and antagonist muscles before (motor unit pre-activation) and during movement performance (Santello and Mcdonagh, 1998). A well-timed and regulated muscle activation elicits a stretch-shortening cycle (SSC) response, naturally occurring in bouncing movements (Ishikawa and Komi, 2004; Taube et al., 2012). By definition, the SSC describes the stretching of a pre-activated muscle-tendon complex immediately followed by a muscle shortening in the concentric push-off phase (Komi, 1984).
Given the importance of SSC actions for human movement, it is not surprising that many studies investigated the biomechanics of this phenomenon; in particular, drop jumps (DJs) represent a good paradigm to study muscle fascicle and tendon behavior in ballistic movements involving the SSC.
Within a DJ, three main phases [pre-activation, braking, and push-off (PO; Komi, 2000)] have been recognized and extensively studied in common and challenging conditions, such as changes in load, falling height, or simulated hypo-gravity (Avela et al., 1994; Arampatzis et al., 2001; Fukashiro et al., 2005; Ishikawa et al., 2005; Sousa et al., 2007; Ritzmann et al., 2016; Helm et al., 2020).
These studies show that the timing and amount of triceps-surae muscle-tendon unit pre-activation in DJs are differentially regulated based on the load applied to the muscle, being optimal in normal “Earth” gravity conditions (Avela et al., 1994), but decreased in simulated hypo-gravity, hyper-gravity (Avela et al., 1994; Ritzmann et al., 2016), or unknown conditions (i.e., unknown falling heights; Helm et al., 2020). Some authors indicated that, when falling from heights different from the optimal one [defined as the drop height giving a maximum DJ performance indicated as peak ground reaction force (GRF) or jump high], electromyographic (EMG) activity of the plantar flexors increases from lower than optimal to higher than optimal heights (Ishikawa and Komi, 2004; Sousa et al., 2007).
These findings highlight the ability of the central nervous system to regulate the timing and amount of pre-activation according to different jumping conditions, thus regulating muscle fascicle length, tendon and joint stiffness as well as position, in order to safely land on the ground and quickly re-bounce.
Similarly, to pre-activation, also in the braking phase, the plantar flexors are differentially regulated. In optimal height (i.e., load) jumping conditions, gastrocnemius medialis (GM) fascicles shorten at early ground contact (possibly due to the intervention of the stretch reflex; Gollhofer et al., 1992) and behave quasi-isometrically in the late braking phase, enabling tendon elongation, and storage of elastic energy (Gollhofer et al., 1992; Fukashiro et al., 2005; Sousa et al., 2007). When increasing the falling height (augmenting the impact GRF), the quasi-isometric behavior of fascicles disappears, and fast fascicle lengthening occurs (Ishikawa et al., 2005; Sousa et al., 2007).
In the third and last PO phase, fascicles shorten and the tendon releases the elastic energy previously stored. Bobbert et al. (1987) reported no influence of jumping height on the work done and on the net vertical impulse assessed during PO; this observation suggests that, despite an optimal DJ performance might be achieved only in specific conditions (falling heights, loads), the central nervous system seems to be able to regulate muscle behavior in order to effectively perform the required task also in challenging situations.
Although the regulation of triceps-surae muscle-tendon unit in DJs has been extensively investigated, very few studies focused on sarcomeres behavior during the performance of this SSC movement (Kurokawa et al., 2003; Fukashiro et al., 2005, 2006). Sarcomeres represent muscle contractile units and are known to express different amounts of force depending on their length (Gordon et al., 1966; Walker and Schrodt, 1974); thus, understanding the time course of their responses during DJs is fundamental to gain further insights into muscle force-generating capacity. In vivo measurement of sarcomere length in humans has been so far been performed only in static positions and under highly controlled experimental conditions (Llewellyn et al., 2008; Sanchez et al., 2015). Instead, human sarcomere length estimation (achieved by dividing GM measured fascicle length for a fixed sarcomere number) in dynamic contractions provided an indirect measure of sarcomere operating range during squat jump, countermovement jump, and DJ (Fukashiro et al., 2005, 2006; Kurokawa et al., 2003). The results of these studies showed that sarcomeres operate in the ascending limb of their length-tension (L-T) relationship in all types of jumps, and particularly so in DJ.
However, most of the available observations on sarcomere and muscle fascicle behavior were made in condition of constant gravity. Thus, in order to understand how sarcomere and muscle fascicle length are regulated in variable gravity conditions, we performed experiments in a parabolic flight, involving variable gravity levels, ranging from about zero-g to about double the Earth’s gravity (1 g; Waldvogel et al., 2021).
Specifically, the aims of the present study were as follows:
1. To investigate the ability of the neuromuscular system in regulating fascicle length in response to conditions of variable gravity.
2. To estimate sarcomere operative length in the different DJ phases, in order to calculate its theoretical force production and its possible modulation in conditions of variable gravity.
We hypothesized that muscle fascicles would be differentially regulated in different gravity conditions compared to 1 g, particularly in anticipation of landing and re-bouncing in unknown gravity levels. In addition, we hypothesized that sarcomeres would operate in the upper part of the ascending limb of their L-T relationship, possibly lengthening during the braking phase (especially in hyper-gravity) while operating quasi-isometrically in 1 g.
Morais, Paulo V. ; Gomes, Vanderley F., Jr. ; Silva, Anielle C. A. ; Dantas, Noelio O. ; Schöning, Michael Josef ; Siqueira, José R., Jr.
The incorporation of nanomaterials that are biocompatible with different types of biological compounds has allowed the development of a new generation of biosensors applied especially in the biomedical field. In particular, the integration of film-based nanomaterials employed in field-effect devices can be interesting to develop biosensors with enhanced properties. In this paper, we studied the fabrication of sensitive nanofilms combining ZnO nanocrystals and carbon nanotubes (CNTs), prepared by means of the layer-by-layer (LbL) technique, in a capacitive electrolyte-insulator-semiconductor (EIS) structure for detecting glucose and urea. The ZnO nanocrystals were incorporated in a polymeric matrix of poly(allylamine) hydrochloride (PAH), and arranged with multi-walled CNTs in a LbL PAH-ZnO/CNTs film architecture onto EIS chips. The electrochemical characterizations were performed by capacitance–voltage and constant capacitance measurements, while the morphology of the films was characterized by atomic force microscopy. The enzymes glucose oxidase and urease were immobilized on film’s surface for detection of glucose and urea, respectively. In order to obtain glucose and urea biosensors with optimized amount of sensitive films, we investigated the ideal number of bilayers for each detection system. The glucose biosensor showed better sensitivity and output signal for an LbL PAH-ZnO/CNTs nanofilm with 10 bilayers. On the other hand, the urea biosensor presented enhanced properties even for the first bilayer, exhibiting high sensitivity and output signal. The presence of the LbL PAH-ZnO/CNTs films led to biosensors with better sensitivity and enhanced response signal, demonstrating that the adequate use of nanostructured films is feasible for proof-of-concept biosensors with improved properties that may be employed for biomedical applications.
Morais, Paulo V. ; Silva, Anielle C. A. ; Dantas, Noelio O. ; Schöning, Michael Josef ; Siqueira, José R., Jr.
Morais, Paulo V. ; Suman, Pedro H. ; Schöning, Michael Josef ; Siqueira Junior, José R. ; Orlandi, Marcelo O.
Lead and nickel, as heavy metals, are still used in industrial processes, and are classified as “environmental health hazards” due to their toxicity and polluting potential. The detection of heavy metals can prevent environmental pollution at toxic levels that are critical to human health. In this sense, the electrolyte–insulator–semiconductor (EIS) field-effect sensor is an attractive sensing platform concerning the fabrication of reusable and robust sensors to detect such substances. This study is aimed to fabricate a sensing unit on an EIS device based on Sn₃O₄ nanobelts embedded in a polyelectrolyte matrix of polyvinylpyrrolidone (PVP) and polyacrylic acid (PAA) using the layer-by-layer (LbL) technique. The EIS-Sn₃O₄ sensor exhibited enhanced electrochemical performance for detecting Pb²⁺ and Ni²⁺ ions, revealing a higher affinity for Pb²⁺ ions, with sensitivities of ca. 25.8 mV/decade and 2.4 mV/decade, respectively. Such results indicate that Sn₃O₄ nanobelts can contemplate a feasible proof-of-concept capacitive field-effect sensor for heavy metal detection, envisaging other future studies focusing on environmental monitoring.
Morat, Mareike ; Faude, Oliver ; Hanssen, Henner ; Ludyga, Sebastian ; Zacher, Jonas ; Eibl, Angi ; Albracht, Kirsten ; Donath, Lars
Exercise training effectively mitigates aging-induced health and fitness impairments. Traditional training recommendations for the elderly focus separately on relevant physiological fitness domains, such as balance, flexibility, strength and endurance. Thus, a more holistic and functional training framework is needed. The proposed agility training concept integratively tackles spatial orientation, stop and go, balance and strength. The presented protocol aims at introducing a two-armed, one-year randomized controlled trial, evaluating the effects of this concept on neuromuscular, cardiovascular, cognitive and psychosocial health outcomes in healthy older adults. Eighty-five participants were enrolled in this ongoing trial. Seventy-nine participants completed baseline testing and were block-randomized to the agility training group or the inactive control group. All participants undergo pre- and post-testing with interim assessment after six months. The intervention group currently receives supervised, group-based agility training twice a week over one year, with progressively demanding perceptual, cognitive and physical exercises. Knee extension strength, reactive balance, dual task gait speed and the Agility Challenge for the Elderly (ACE) serve as primary endpoints and neuromuscular, cognitive, cardiovascular, and psychosocial meassures serve as surrogate secondary outcomes. Our protocol promotes a comprehensive exercise training concept for older adults, that might facilitate stakeholders in health and exercise to stimulate relevant health outcomes without relying on excessively time-consuming physical activity recommendations.
Moreno i Codinachs, L. ; Birkenstock, C. ; Garma, T. ; Zierold, R. ; Bachmann, J. ; Nielsch, K. ; Schöning, Michael Josef ; Fontcuberta i Morral, A.
Moreno i Codinachs, Lia ; Kloock, Joachim P. ; Schöning, Michael Josef ; Baldi, Antoni ; Ipatov, Andrey ; Bratov, Andrey ; Jimenez-Jorquera, Cecilia
Moritz, W. ; Yoshinobu, T. ; Finger, F. ; Krause, S. ; Martin-Fernandez, M. ; Schöning, Michael Josef
Moseley, Fiona ; Halamek, Jan ; Kramer, Friederike ; Poghossian, Arshak ; Schöning, Michael Josef ; Katz, Evgeny
An enzyme system organized in a flow device was used to mimic a reversible Controlled NOT (CNOT) gate with two input and two output signals. Reversible conversion of NAD⁺ and NADH cofactors was used to perform a XOR logic operation, while biocatalytic hydrolysis of p-nitrophenyl phosphate resulted in an Identity operation working in parallel. The first biomolecular realization of a CNOT gate is promising for integration into complex biomolecular networks and future biosensor/biomedical applications.
Development of an optimized LSO/LuYAP phoswich detector head for the Lausanne ClearPET demonstrator
(2006)
Mosset, Jean-Baptiste ; Devroede, Olivier ; Krieguer, Magalie ; Rey, M. ; Vieira, J.-M. ; Jung, J. H. ; Kuntner, Claudia ; Streun, Matthias ; Ziemons, Karl ; Auffray, Etiennette ; Sempere-Roldan, P. ; Lecoq, Paul ; Bruyndonckx, Peter ; Loude, Jean-François ; Tavernier, Stefaan ; Morel, Christian
This paper describes the LSO/LuYAP phoswich detector head developed for the ClearPET small animal PET scanner demonstrator that is under construction in Lausanne within the Crystal Clear Collaboration. The detector head consists of a dual layer of 8×8 LSO and LuYAP crystal arrays coupled to a multi-anode photomultiplier tube (Hamamatsu R7600-M64). Equalistion of the LSO/LuYAP light collection is obtained through partial attenuation of the LSO scintillation light using a thin aluminum deposit of 20-35 nm on LSO and appropriate temperature regulation of the phoswich head between 30°C to 60°C. At 511keV, typical FWHM energy resolutions of the pixels of a phoswich head amounts to (28±2)% for LSO and (25±2)% for LuYAP. The LSO versus LuYAP crystal identification efficiency is better than 98%. Six detector modules have been mounted on a rotating gantry. Axial and tangential spatial resolutions were measured up to 4 cm from the scanner axis and compared to Monte Carlo simulations using GATE. FWHM spatial resolution ranges from 1.3 mm on axis to 2.6 mm at 4 cm from the axis.
Mourzina, I.G. ; Yoshinobu, T. ; Ermolenko, Y.E. ; Vlasov, Y. G. ; Schöning, Michael Josef ; Iwasaki, H.
Mourzina, Ioulia G. ; Yoshinobu, Tatsuo ; Ermolenko, Yuri E. ; Vlasov, Yuri G. ; Schöning, Michael Josef ; Iwasaki, H.
Mourzina, Y. G. ; Ermelenko, Y. E. ; Yoshinobu, T. ; Vlasov, Y. ; Iwasaki, H. ; Schöning, Michael Josef
Thin film chemical sensors based on chalcogenide glasses for „electronic tongue“ applications
(2001)
Mourzina, Y. G. ; Legin, A. ; Vlasov, Y. G. ; Schöning, Michael Josef ; Schubert, J. ; Zander, W. ; Lüth, H.
Mourzina, Y. G. ; Schöning, Michael Josef ; Schubert, J. ; Zander, W. ; Legin, A. V. ; Vlasov, Y. G. ; Lüth, H.
Mourzina, Y. ; Mai, T. ; Poghossian, Arshak ; Ermolenko, Y. ; Yoshinobu, T. ; Vlasov, Y. ; Iwasaki, H. ; Schöning, Michael Josef
Mourzina, Y. ; Yoshinobu, T. ; Ermelenko, Y. ; Furuichi, K. ; Vlasov, Y. ; Schöning, Michael Josef ; Iwasaki, H.
Mourzina, Y.G. ; Schöning, Michael Josef ; Schubert, J. ; Zander, W. ; Legin, A. V. ; Vlasov, Y. G. ; Kordos, P. ; Lüth, H.
Mourzina, Yu. G. ; Schubert, J ; Zander, W. ; Legin, A. ; Vlasov, Y. G. ; Lüth, H. ; Schöning, Michael Josef
Mourzina, Yu.G. ; Schöning, Michael Josef ; Schubert, J. ; Zander, W. ; Legin, A. ; Vlasov, Y. G. ; Lüth, H.
Muck, A. ; Wang, J. ; Jacobs, M. ; Chen, G. ; Chatrathi, M. P. ; Jurka, V. ; Vyborny, Z. ; Spillmann, S. D. ; Sridharan, G. ; Schöning, Michael Josef
Murgan, Ilina ; Beyer, Sonja ; Kotliar, Konstantin ; Weber, Lutz ; Bechtold-Dalla Pozza, Susanne ; Dalla Pozza, Robert ; Wegner, Aharon ; Sitnikova, Diana ; Stock, Konrad ; Heemann, Uwe ; Schmaderer, Christoph ; Baumann, Marcus
Murib, M. S. ; Grinsven, B. van ; Grieten, L. ; Janssens, S. D. ; Vermeeren, V. ; Eersels, K. ; Broeders, J. ; Ameloot, Marcel ; Michiels, L. ; Ceuninck, W. De ; Haenen, K. ; Schöning, Michael Josef ; Wagner, Patrick
Murib, M. S. ; Yeap, W. S. ; Eurlings, Y. ; Grinsven, B. van ; Boyen, H.-G. ; Conings, B. ; Michiels, L. ; Ameloot, Marcel ; Carleer, R. ; Warmer, J. ; Kaul, P. ; Haenen, K. ; Schöning, Michael Josef ; Ceuninck, W. de ; Wagner, P.
In this study, we show that synthetic sapphire (Al₂O₃), an established implant material, can also serve as a platform material for biosensors comparable to nanocrystalline diamond. Sapphire chips, beads, and powder were first modified with (3-aminopropyl) triethoxysilane (APTES), followed by succinic anhydride (SA), and finally single-stranded probe DNA was EDC coupled to the functionalized layer. The presence of the APTES-SA layer on sapphire powders was confirmed by thermogravimetric analyis and Fourier-transform infrared spectroscopy. Using planar sapphire chips as substrates and X-ray photoelectron spectroscopy (XPS) as surface-sensitive tool, the sequence of individual layers was analyzed with respect to their chemical state, enabling the quantification of areal densities of the involved molecular units. Fluorescence microscopy was used to demonstrate the hybridization of fluorescently tagged target DNA to the probe DNA, including denaturation- and re-hybridization experiments. Due to its high thermal conductivity, synthetic sapphire is especially suitable as a chip material for the heat-transfer method, which was employed to distinguish complementary- and non-complementary DNA duplexes containing single-nucleotide polymorphisms. These results indicate that it is possible to detect mutations electronically with a chemically resilient and electrically insulating chip material.
Murib, M. S. ; Yeap, W. S. ; Martens, D. ; Liu, X. ; Bienstman, P. ; Fahlman, M. ; Schöning, Michael Josef ; Michiels, L. ; Haenen, K. ; Serpengüzel, A. ; Wagner, Patrick
In this study we show an optical biosensor concept, based on elastic light scattering from sapphire microspheres. Transmitted and elastic scattering intensity of the microspheres (radius 500 μm, refractive index 1.77) on an optical fiber half coupler is analyzed at 1510 nm. The 0.43 nm angular mode spacing of the resonances is comparable to the angular mode spacing value estimated using the optical size of the microsphere. The spectral linewidths of the resonances are in the order of 0.01 nm, which corresponds to quality factors of approximately 105. A polydopamine layer is used as a functionalizing agent on sapphire microspherical resonators in view of biosensor implementation. The varying layer thickness on the microsphere is determined as a function of the resonance wavelength shift. It is shown that polymer functionalization has a minor effect on the quality factor. This is a promising step toward the development of an optical biosensor.
Murib, Mohammed S. ; Tran, Anh Quang ; Ceuninck, Ward de ; Schöning, Michael Josef ; Nesladek, Milos ; Serpengüzel, Ali ; Wagner, Patrick
Deoxyribonucleic acid (DNA) and protein recognition are now standard tools in biology. In addition, the special optical properties of microsphere resonators expressed by the high quality factor (Q-factor) of whispering gallery modes (WGMs) or morphology dependent resonances (MDRs) have attracted the attention of the biophotonic community. Microsphere-based biosensors are considered as powerful candidates to achieve label-free recognition of single molecules due to the high sensitivity of their WGMs. When the microsphere surface is modified with biomolecules, the effective refractive index and the effective size of the microsphere change resulting in a resonant wavelength shift. The transverse electric (TE) and the transverse magnetic (TM) elastic light scattering intensity of electromagnetic waves at 600 and 1400 nm are numerically calculated for DNA and unspecific binding of proteins to the microsphere surface. The effect of changing the optical properties was studied for diamond (refractive index 2.34), glass (refractive index 1.50), and sapphire (refractive index 1.75) microspheres with a 50 µm radius. The mode spacing, the linewidth of WGMs, and the shift of resonant wavelength due to the change in radius and refractive index, were analyzed by numerical simulations. Preliminary results of unspecific binding of biomolecules are presented. The calculated shift in WGMs can be used for biomolecules detection.
Murib, Mohammed Sharif ; Yeap, Weng-Siang ; Martens, Daan ; Bienstman, Peter ; Ceuninck, Ward de ; Grinsven, Bart van ; Schöning, Michael Josef ; Michiels, Luc ; Haenen, Ken ; Ameloot, Marcel ; Serpengüzel, Ali ; Wagner, Patrick
A microcavity-based deoxyribonucleic acid (DNA) optical biosensor is demonstrated for the first time using synthetic sapphire for the optical cavity. Transmitted and elastic scattering intensity at 1510 nm are analyzed from a sapphire microsphere (radius 500 μm, refractive index 1.77) on an optical fiber half coupler. The 0.43 nm angular mode spacing of the resonances correlates well with the optical size of the sapphire sphere. Probe DNA consisting of a 36-mer fragment was covalently immobilized on a sapphire microsphere and hybridized with a 29-mer target DNA. Whispering gallery modes (WGMs) were monitored before the sapphire was functionalized with DNA and after it was functionalized with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). The shift in WGMs from the surface modification with DNA was measured and correlated well with the estimated thickness of the add-on DNA layer. It is shown that ssDNA is more uniformly oriented on the sapphire surface than dsDNA. In addition, it is shown that functionalization of the sapphire spherical surface with DNA does not affect the quality factor (Q≈104) of the sapphire microspheres. The use of sapphire is especially interesting because this material is chemically resilient, biocompatible, and widely used for medical implants.
Muschallik, Lukas ; Kipp, Carina Ronja ; Recker, Inga ; Bongaerts, Johannes ; Pohl, Martina ; Gelissen, Melanie ; Schöning, Michael Josef ; Selmer, Thorsten ; Siegert, Petra
The enantioselective synthesis of α-hydroxy ketones and vicinal diols is an intriguing field because of the broad applicability of these molecules. Although, butandiol dehydrogenases are known to play a key role in the production of 2,3-butandiol, their potential as biocatalysts is still not well studied. Here, we investigate the biocatalytic properties of the meso-butanediol dehydrogenase from Bacillus licheniformis DSM 13T (BlBDH). The encoding gene was cloned with an N-terminal StrepII-tag and recombinantly overexpressed in E. coli. BlBDH is highly active towards several non-physiological diketones and α-hydroxyketones with varying aliphatic chain lengths or even containing phenyl moieties. By adjusting the reaction parameters in biotransformations the formation of either the α-hydroxyketone intermediate or the diol can be controlled.
Muschallik, Lukas ; Molinnus, Denise ; Bongaerts, Johannes ; Pohl, Martina ; Wagner, Torsten ; Schöning, Michael Josef ; Siegert, Petra ; Selmer, Thorsten
The gene encoding a putative (R,R)-butane-2,3-diol dehydrogenase (bdhA) from Bacillus clausii DSM 8716T was isolated, sequenced and expressed in Escherichia coli. The amino acid sequence of the encoded protein is only distantly related to previously studied enzymes (identity 33–43%) and exhibited some uncharted peculiarities. An N-terminally StrepII-tagged enzyme variant was purified and initially characterized. The isolated enzyme catalyzed the (R)-specific oxidation of (R,R)- and meso-butane-2,3-diol to (R)- and (S)-acetoin with specific activities of 12 U/mg and 23 U/mg, respectively. Likewise, racemic acetoin was reduced with a specific activity of up to 115 U/mg yielding a mixture of (R,R)- and meso-butane-2,3-diol, while the enzyme reduced butane-2,3-dione (Vmax 74 U/mg) solely to (R,R)-butane-2,3-diol via (R)-acetoin. For these reactions only activity with the co-substrates NADH/NAD+ was observed. The enzyme accepted a selection of vicinal diketones, α-hydroxy ketones and vicinal diols as alternative substrates. Although the physiological function of the enzyme in B. clausii remains elusive, the data presented herein clearly demonstrates that the encoded enzyme is a genuine (R,R)-butane-2,3-diol dehydrogenase with potential for applications in biocatalysis and sensor development.
Muschallik, Lukas ; Molinnus, Denise ; Jablonski, Melanie ; Kipp, Carina Ronja ; Bongaerts, Johannes ; Pohl, Martina ; Wagner, Torsten ; Schöning, Michael Josef ; Selmer, Thorsten ; Siegert, Petra
α-hydroxy ketones (HK) and 1,2-diols are important building blocks for fine chemical synthesis. Here, we describe the R-selective 2,3-butanediol dehydrogenase from B. clausii DSM 8716ᵀ (BcBDH) that belongs to the metal-dependent medium chain dehydrogenases/reductases family (MDR) and catalyzes the selective asymmetric reduction of prochiral 1,2-diketones to the corresponding HK and, in some cases, the reduction of the same to the corresponding 1,2-diols. Aliphatic diketones, like 2,3-pentanedione, 2,3-hexanedione, 5-methyl-2,3-hexanedione, 3,4-hexanedione and 2,3-heptanedione are well transformed. In addition, surprisingly alkyl phenyl dicarbonyls, like 2-hydroxy-1-phenylpropan-1-one and phenylglyoxal are accepted, whereas their derivatives with two phenyl groups are not substrates. Supplementation of Mn²⁺ (1 mM) increases BcBDH's activity in biotransformations. Furthermore, the biocatalytic reduction of 5-methyl-2,3-hexanedione to mainly 5-methyl-3-hydroxy-2-hexanone with only small amounts of 5-methyl-2-hydroxy-3-hexanone within an enzyme membrane reactor is demonstrated.
Musholt, Petra B. ; Schöndorf, Thomas ; Pfützner, Andreas ; Hohberg, Cloth ; Kleine, Iris ; Fuchs, Winfried ; Hehenwarter, Silvia ; Dikta, Gerhard ; Kerschgens, Benedikt ; Forst, Thomas
Validation of a novel method for detecting and stabilizing malfunctioning areas in fuel cell stacks
(2014)
Müller, Martin ; Hirschfeld, Julian A. ; Lambertz, Rita ; Schulze Lohoff, Andreas ; Lustfeld, Hans ; Pfeifer, Heinz ; Reißel, Martin
In this paper a setup for detecting malfunctioning areas of MEAs in fuel cell stacks is described. Malfunctioning areas generate electric cross currents inside bipolar plates. To exploit this we suggest bipolar plates consisting not of two but of three layers. The third one is a highly conducting layer and segmented such that the cross currents move along the segments to the surface of the stack where they can be measured by an inductive sensor. With this information a realistic model can be used to detect the malfunctioning area. Furthermore the third layer will prevent any current inhomogeneity of a malfunctioning cell to spread to neighbouring cells in the stack. In this work the results of measurements in a realistic cell setup will be compared with the results obtained in simulation studies with the same configuration. The basis for the comparison is the reliable characterisation of the electrical properties of the cell components and the implication of these results into the simulation model. The experimental studies will also show the limits in the maximum number of segments, which can be used for a reliable detection of cross currents.