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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.
Three amperometric biosensors have been developed for the detection of L-malic acid, fumaric acid, and L -aspartic acid, all based on the combination of a malate-specific dehydrogenase (MDH, EC 1.1.1.37) and diaphorase (DIA, EC 1.8.1.4). The stepwise expansion of the malate platform with the enzymes fumarate hydratase (FH, EC 4.2.1.2) and aspartate ammonia-lyase (ASPA, EC 4.3.1.1) resulted in multi-enzyme reaction cascades and, thus, augmentation of the substrate spectrum of the sensors. Electrochemical measurements were carried out in presence of the cofactor β-nicotinamide adenine dinucleotide (NAD+) and the redox mediator hexacyanoferrate (III) (HCFIII). The amperometric detection is mediated by oxidation of hexacyanoferrate (II) (HCFII) at an applied potential of + 0.3 V vs. Ag/AgCl. For each biosensor, optimum working conditions were defined by adjustment of cofactor concentrations, buffer pH, and immobilization procedure. Under these improved conditions, amperometric responses were linear up to 3.0 mM for L-malate and fumarate, respectively, with a corresponding sensitivity of 0.7 μA mM−1 (L-malate biosensor) and 0.4 μA mM−1 (fumarate biosensor). The L-aspartate detection system displayed a linear range of 1.0–10.0 mM with a sensitivity of 0.09 μA mM−1. The sensor characteristics suggest that the developed platform provides a promising method for the detection and differentiation of the three substrates.
The immobilization of NAD+-dependent dehydrogenases, in combination with a diaphorase, enables the facile development of multiparametric sensing devices. In this work, an amperometric biosensor array for simultaneous determination of ethanol, formate, d- and l-lactate is presented. Enzyme immobilization on platinum thin-film electrodes was realized by chemical cross-linking with glutaraldehyde. The optimization of the sensor performance was investigated with regard to enzyme loading, glutaraldehyde concentration, pH, cofactor concentration and temperature. Under optimal working conditions (potassium phosphate buffer with pH 7.5, 2.5 mmol L-1 NAD+, 2.0 mmol L-1 ferricyanide, 25 °C and 0.4% glutaraldehyde) the linear working range and sensitivity of the four sensor elements was improved. Simultaneous and cross-talk free measurements of four different metabolic parameters were performed successfully. The reliable analytical performance of the biosensor array was demonstrated by application in a clarified sample of inoculum sludge. Thereby, a promising approach for on-site monitoring of fermentation processes is provided.
Modulation of muscle-tendon interaction in the human triceps surae during an energy dissipation task
(2017)
Abstractauthoren Graphene oxide (GO) nanoparticles were incorporated in temperature-sensitive Poly(N-isopropylacrylamide) (PNIPAAm) hydrogels. The nanoparticles increase the light absorption and convert light energy into heat efficiently. Thus, the hydrogels with GO can be stimulated spatially resolved by illumination as it was demonstrated by IR thermography. The temporal progression of the temperature maximum was detected for different concentrations of GO within the polymer network. Furthermore, the compatibility of PNIPAAm hydrogels with GO and cell cultures was investigated. For this purpose, culture medium was incubated with hydrogels containing GO and the viability and morphology of chinese hamster ovary (CHO) cells was examined after several days of culturing in presence of this medium.
Four members of a homologous series of chlorinated poly(vinyl ester) oligomers CCl₃–(CH₂CH (OCO(CH₂)ₘCH₃))ₙ–Cl with degrees of polymerization of 10 and 20 were prepared by telomerisation using carbon tetrachloride. The number of side chain carbon atoms ranges from 2 (poly(vinyl acetate) to 18 (poly(vinyl stearate)). The effect of the n-alkyl side chain length and of the degree of polymerization on the thermal stability and crystallization behaviour of the synthesized compounds was investigated.
All oligomers degrade in two major steps by first losing HCl and side chains with subsequent breakdown of the backbone. The members with short side chains, up to poly(vinyl octanoate), are amorphous and show internal plasticization, whereas those with high number of side chain carbon atoms are semi-crystalline due to side-chain crystallization. A better packing for poly(vinyl stearate) is also noticeable. The glass transition and melting temperatures as well as the onset temperature of decomposition are influenced to a larger extent by the side chain length than by the degree of polymerization. Thermal stability is improved if both the size and number of side chains increase, but only a long side chain causes a significant increase of the resistance to degradation. This results in a stabilization of PVAc so that oligomers from poly(vinyl octanoate) on are stable under atmospheric conditions. Thus, the way to design stable, chlorinated PVEs oligomers is to use a long n-alkyl side chain.
Regardless of size or destination, synthetic biology starts with com-parably small information units, which need to be combined and properly arranged in order to achieve a certain goal. This may be the de novo synthesis of individual genes from oligonucleotides, a shuffling of protein domains in order to create novel biocatalysts, the assembly of multiple enzyme encoding genes in metabolic pathway design, or strain development at the production stage. The CoLibry concept has been designed in order to close the gap between recombinant production of individual genes and genome editing.
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.
Autoradiography is a well-established method of nuclear imaging. When different radionuclides are present simultaneously, additional processing is needed to distinguish distributions of radionuclides. In this work, a method is presented where aluminium absorbers of different thickness are used to produce images with different cut-off energies. By subtracting images pixel-by-pixel one can generate images representing certain ranges of β-particle energies. The method is applied to the measurement of irradiated reactor graphite samples containing several radionuclides to determine the spatial distribution of these radionuclides within pre-defined energy windows. The process was repeated under fixed parameters after thermal treatment of the samples. The greyscale images of the distribution after treatment were subtracted from the corresponding pre-treatment images. Significant changes in the intensity and distribution of radionuclides could be observed in some samples. Due to the thermal treatment parameters the most significant differences were observed in the ³H and ¹⁴C inventory and distribution.
NVS123 is a poorly water-soluble protease 56 inhibitor in clinical development. Data from in vitro hepatocyte studies suggested that NVS123 is mainly metabolized by CYP3A4. As a consequence of limited solubility, NVS123 therapeutic plasma exposures could not be achieved even with high doses and optimized formulations. One approach to overcome NVS123 developability issues was to increase plasma exposure by coadministrating it with an inhibitor of CYP3A4 such as ritonavir. A clinical boost effect was predicted by using physiologically based pharmacokinetic (PBPK) modeling. However, initial boost predictions lacked sufficient confidence because a key parameter, fraction of drug metabolized by CYP3A4 (ƒₘCYP3A4), could not be estimated with accuracy on account of disconnects between in vitro and in vivo preclinical data. To accurately estimate ƒₘCYP3A4 in human, an in vivo boost effect study was conducted using CYP3A4-humanized mouse model which showed a 33- to 56-fold exposure boost effect. Using a top-down approach, human ƒₘCYP3A4 for NVS123 was estimated to be very high and included in the human PBPK modeling to support subsequent clinical study design. The combined use of the in vivo boost study in CYP3A4-humanized mouse model mice along with PBPK modeling accurately predicted the clinical outcome and identified a significant NVS123 exposure boost (∼42-fold increase) with ritonavir.
Breast cancer resistance protein (BCRP) is expressed in various tissues, such as the gut, liver, kidney and blood brain barrier (BBB), where it mediates the unidirectional transport of substrates to the apical/luminal side of polarized cells. Thereby BCRP acts as an efflux pump, mediating the elimination or restricting the entry of endogenous compounds or xenobiotics into tissues and it plays important roles in drug disposition, efficacy and safety. Bcrp knockout mice (Bcrp−/−) have been used widely to study the role of this transporter in limiting intestinal absorption and brain penetration of substrate compounds. Here we describe the first generation and characterization of a mouse line humanized for BCRP (hBCRP), in which the mouse coding sequence from the start to stop codon was replaced with the corresponding human genomic region, such that the human transporter is expressed under control of the murine Bcrp promoter. We demonstrate robust human and loss of mouse BCRP/Bcrp mRNA and protein expression in the hBCRP mice and the absence of major compensatory changes in the expression of other genes involved in drug metabolism and disposition. Pharmacokinetic and brain distribution studies with several BCRP probe substrates confirmed the functional activity of the human transporter in these mice. Furthermore, we provide practical examples for the use of hBCRP mice to study drug-drug interactions (DDIs). The hBCRP mouse is a promising model to study the in vivo role of human BCRP in limiting absorption and BBB penetration of substrate compounds and to investigate clinically relevant DDIs involving BCRP.
Mice that have been genetically humanized for proteins involved in drug metabolism and toxicity and mice engrafted with human hepatocytes are emerging and promising in vivo models for an improved prediction of the pharmacokinetic, drug–drug interaction and safety characteristics of compounds in humans. The specific advantages and disadvantages of these models should be carefully considered when using them for studies in drug discovery and development. Here, an overview on the corresponding genetically humanized and chimeric liver humanized mouse models described to date is provided and illustrated with examples of their utility in drug metabolism and toxicity studies. We compare the strength and weaknesses of the two different approaches, give guidance for the selection of the appropriate model for various applications and discuss future trends and perspectives.
The interplay of albumin (BSA) and lysozyme (LYZ) adsorbed simultaneously on titanium was analyzed by gel electrophoresis and BCA assay. It was found that BSA and lysozyme adsorb cooperatively. Additionally, the isoelectric point of the respective protein influences the adsorption. Also, the enzymatic activity of lysozyme and amylase (AMY) in mixtures with BSA was considered with respect to a possible influence of protein-protein interaction on enzyme activity. Indeed, an increase of lysozyme activity in the presence of BSA could be observed. In contrast, BSA does not influence the activity of amylase.
Evaluation of lignocellulosic material for butanol production using enzymatic hydrolysate medium
(2016)
Butanol is a promising gasoline additive and platform chemical that can be readily produced via acetone-butanolethanol (ABE) fermentation from pretreated lignocellulosic materials. This article examines lignocellulosic material from beech wood for ABE fermentation, using Clostridium acetobutylicum. First, the utilization of both C₅₋ (xylose) and C₆₋ (glucose) sugars as sole carbon source was investigated in static cultivation, using serum bottles and synthetic medium. The utilization of pentose sugar resulted in a solvent yield of 0.231 g·g_sugar⁻¹, compared to 0.262 g·g_sugar⁻¹ using hexose. Then, the Organosolv pretreated crude cellulose fibers (CF) were enzymatically decomposed, and the resulting hydrolysate medium was analyzed for inhibiting compounds (furans, organic acids, phenolics) and treated with ionexchangers for detoxification. Batch fermentation in a bioreactor using CF hydrolysate medium resulted in a total solvent yield of 0.20 gABE·g_sugar⁻¹.