INB - Institut für Nano- und Biotechnologien
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Chiral, vicinal diols are of high interest for academic research and industrial applications. For synthesizing chiral diols, enzymes are important catalysts due to their high selectivity and ability to work under tolerable temperature and no pressure. In this study, two consecutive enzyme-catalyzed steps were used for the asymmetric synthesis of aliphatic, vicinal diols with high product concentrations and chiral purity. The reaction comprised a ligation step employing lyases and a subsequent reduction step using oxidoreductases. Either in an aqueous buffer or an organic solvent, the potentially biobased aldehydes acetaldehyde, propanal, butanal, and pentanal were used as substrates. Here, all possible stereoisomers of 2,3-butanediol, 3,4-hexanediol, 4,5-octanediol, and 5,6-decanediol were produced with isomeric content values between 72% and > 99%, and concentrations and conversions between 4.1 and 60 mM. This work shows how four symmetric, chiral, vicinal diols can be synthesized by combining enzymes in a modular way, including exemplarily scaling.
The present invention relates to an isolated polypeptide having aminoacylase activity and comprising an amino acid sequence having over its entire length at least 87 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. The invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding such an aminoacylase, to a plasmid vector comprising said nucleic acid molecule, to a recombinant host cell comprising the isolated nucleic acid molecule or the vector and to methods to produce said aminoacylase. The invention also covers the use of the aminoacylase according to the invention for the N-acylation of amino acids or salts thereof as well as methods to produce N-acyl amino acids or salts thereof using the aminoacylase. Furthermore, the invention relates to the obtained N-acyl amino acids, compositions comprising them and the use of the produced N-acyl amino acids in a cosmetic product, home care product or an industrial and/or institutional product.
Many industrial processes are performed using harmful chemicals. The current technical synthesis of N-acyl-amino acids relies on acyl chlorides, which are typically obtained from phosgene chemistry. A greener alternative is the application of whole cells or enzymes to carry out synthesis in an environmentally friendly manner. Aminoacylases belong to the hydrolase family and the resolution of racemic mixtures of N-acetyl-amino acids is a well-known industrial process. Several new enzymes accepting long-chain fatty acids as substrates were discovered in recent years. This article reviews the synthetic potential of aminoacylases to produce biobased N-acyl-amino acid surfactants. The focus lays on a survey of the different types of aminoacylases available for synthesis and their reaction products. The enzymes are categorized according to their protein family classification and their biochemical characteristics including substrate spectra, reaction optima and process stability, both in hydrolysis and under process conditions suitable for synthesis. Finally, the benefits and future challenges of enzymatic N-acyl-amino acid synthesis with aminoacylases will be discussed.
Magnetic nanoparticles (MNP) are widely investigated for biomedical applications in diagnostics (e.g. imaging), therapeutics (e.g. hyperthermia) and general biosensing. For all these applications, the MNPs’ unique magnetic relaxation mechanism in an alternating magnetic field (AFM) is stimulated to induce desired effects. Whereas magnetic fluid hyperthermia (MFH) and magnetic particle imaging (MPI) are the most prominent examples for biomedical application, we investigate the relatively new biosensing application of frequency mixing magnetic detection (FMMD) from a fundamental perspective. Generally, we ask how specific MNP parameters (core size, magnetic anisotropy) influence the signal, specifically we predict the most effective MNP core size for signal generation. In FMMD, simultaneously two AFM are applied: a low-frequency magnetic driving field, driving MNP close to saturation, and a high-frequency excitation field that probes MNP susceptibility: . Resulting from the nonlinear magnetization of the MNP, harmonics of both individual incident frequencies as well as intermodulation products of these frequencies are generated. In this work, we present numerical Monte-Carlo(MC)-based simulations of the MNP relaxation process, solving the Landau-Lifshitz-Gilbert (LLG) equation to predict FMMD signals: As Figure 1 shows for the first four intermodulation signals , with , we can clearly see that larger core sizes generally increase the signal intensity. Same trend is predicted by a simple Langevin-function based thermal equilibrium model. Both predictions include a lognormal size distribution. The effect of core size distribution presumably dominates the effect of magnetic anisotropy. The findings are supported by comparison with experimental data and help to identify which MNP are best suited for magnetic biosensing applications using FMMD.
Frequency mixing magneticdetection(FMMD) has been widely utilized as a measurement technique in magnetic immunoassays. It can also be used for characterization[1]and distinction[2](also known as “colorization”) ofdifferent types of magnetic nanoparticlesaccording totheircore sizes.It is well known that the large particles contribute most of the FMMD signal. Typically, 90% of the signal stems from the largest 10% of the particles [1]. This leads to ambiguities in core size fitting since thecontribution of thesmall sized particles is almostundetectable among the strong responses from the large ones. In this work, we report on how this ambiguity can be overcome. Magnetic nanoparticle samples from Micromod (Rostock, Germany) were prepared in liquid and filterbound state. Their FMMD response at mixing frequencies f1 ± nf2 to magnetic excitation H(t)=H0+H1sin(2 f1t)+H2sin(2 f2t),with H1=1.3mT/μ0 at f1=40.5kHzandH2=16mT/μ0 at f2=63Hz,was measured as a function ofoffset field strength H0= (0,…,24) mT/μ0.The signal calculated fromLangevin model in thermodynamic equilibrium[1]with a lognormal core size distribution fL(dc,d0, ,A) = Aexp(–ln²(dc/d0)/(2 ²))/(dc (2 )1/2)was fitted to the experimental data. For each choice of median diameter d0, pairs of parameters ( ,A) are found which yield excellent fit results with R²>0.99.All the lognormal core size distributions shown in Figure (a) are compatible with the measurements because their large-size tails are almost equal. However, all distributions have different number of particles and different total iron content. We determined the samples’ total iron mass with inductively coupled plasma optical emission spectrometry(ICP-OES) and, out of all possible lognormal distributions, determined the one with the same amount of iron. With this additional externally measured parameter, we resolved the ambiguity in core size distribution and determined the parameters (d0, ,A).
Die Erfindung liegt auf dem Gebiet der Enzymtechnologie. Die Erfindung betrifft Proteasen aus Metabacillus indicus, die insbesondere im Hinblick auf den Einsatz in Wasch- und Reinigungsmitteln verwendet werden können, alle hinreichend ähnlichen Proteasen mit einer entsprechend ähnlichen Sequenz zu SEQ ID NO:1 und für sie codierende Nukleinsäuren. Die Erfindung betrifft ferner deren Herstellung sowie Verfahren zur Verwendung dieser Proteasen, deren Verwendung als solche sowie diese enthaltende Mittel, insbesondere Wasch- und Reinigungsmittel.
Die Erfindung liegt auf dem Gebiet der Enzymtechnologie. Die Erfindung betrifft Proteasen aus Fictibacillus arsenicus, die insbesondere im Hinblick auf den Einsatz in Wasch- und Reinigungsmitteln verwendet werden können, alle hinreichend ähnlichen Proteasen mit einer entsprechend ähnlichen Sequenz zu SEQ ID NO:1 und für sie codierende Nukleinsäuren. Die Erfindung betrifft ferner deren Herstellung sowie Verfahren zur Verwendung dieser Proteasen, deren Verwendung als solche sowie diese enthaltende Mittel, insbesondere Wasch- und Reinigungsmittel.
In this work, we present a compact, bifunctional chip-based sensor setup that measures the temperature and electrical conductivity of water samples, including specimens from rivers and channels, aquaculture, and the Atlantic Ocean. For conductivity measurements, we utilize the impedance amplitude recorded via interdigitated electrode structures at a single triggering frequency. The results are well in line with data obtained using a calibrated reference instrument. The new setup holds for conductivity values spanning almost two orders of magnitude (river versus ocean water) without the need for equivalent circuit modelling. Temperature measurements were performed in four-point geometry with an on-chip platinum RTD (resistance temperature detector) in the temperature range between 2 °C and 40 °C, showing no hysteresis effects between warming and cooling cycles. Although the meander was not shielded against the liquid, the temperature calibration provided equivalent results to low conductive Milli-Q and highly conductive ocean water. The sensor is therefore suitable for inline and online monitoring purposes in recirculating aquaculture systems.
As one class of molecular imprinted polymers (MIPs), surface imprinted polymer (SIP)-based biosensors show great potential in direct whole-bacteria detection. Micro-contact imprinting, that involves stamping the template bacteria immobilized on a substrate into a pre-polymerized polymer matrix, is the most straightforward and prominent method to obtain SIP-based biosensors. However, the major drawbacks of the method arise from the requirement for fresh template bacteria and often non-reproducible bacteria distribution on the stamp substrate. Herein, we developed a positive master stamp containing photolithographic mimics of the template bacteria (E. coli) enabling reproducible fabrication of biomimetic SIP-based biosensors without the need for the “real” bacteria cells. By using atomic force and scanning electron microscopy imaging techniques, respectively, the E. coli-capturing ability of the SIP samples was tested, and compared with non-imprinted polymer (NIP)-based samples and control SIP samples, in which the cavity geometry does not match with E. coli cells. It was revealed that the presence of the biomimetic E. coli imprints with a specifically designed geometry increases the sensor E. coli-capturing ability by an “imprinting factor” of about 3. These findings show the importance of geometry-guided physical recognition in bacterial detection using SIP-based biosensors. In addition, this imprinting strategy was employed to interdigitated electrodes and QCM (quartz crystal microbalance) chips. E. coli detection performance of the sensors was demonstrated with electrochemical impedance spectroscopy (EIS) and QCM measurements with dissipation monitoring technique (QCM-D).
Electrolyte-insulator-semiconductor capacitors (EISCAP) belong to field-effect sensors having an attractive transducer architecture for constructing various biochemical sensors. In this study, a capacitive model of enzyme-modified EISCAPs has been developed and the impact of the surface coverage of immobilized enzymes on its capacitance-voltage and constant-capacitance characteristics was studied theoretically and experimentally. The used multicell arrangement enables a multiplexed electrochemical characterization of up to sixteen EISCAPs. Different enzyme coverages have been achieved by means of parallel electrical connection of bare and enzyme-covered single EISCAPs in diverse combinations. As predicted by the model, with increasing the enzyme coverage, both the shift of capacitance-voltage curves and the amplitude of the constant-capacitance signal increase, resulting in an enhancement of analyte sensitivity of the EISCAP biosensor. In addition, the capability of the multicell arrangement with multi-enzyme covered EISCAPs for sequentially detecting multianalytes (penicillin and urea) utilizing the enzymes penicillinase and urease has been experimentally demonstrated and discussed.