Angewandte Naturwissenschaften (F11)
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AbstractThe triplet‐sensitized (by the solvent acetone) as well as the direct (λex=300–320 nm) photochemical decarboxylation of N‐phthaloylated γ‐aminobutyric acid (GABA) derivatives are versatile and high‐yielding routes to benzopyrrolizidines via intramolecular electron transfer initiated decarboxylation followed by radical coupling. The ß‐mono‐ and ß,ß'‐disubstituted N‐phthaloyl GABA derivatives 7 a–7 g, respectively, were applied as substrates. Decarboxylative photocyclization yielded hydroxy benzopyrrolizidines 8 a–8 g in high chemical yields and with moderate diastereoselectivities from the ß‐monosubstituted substrates. The analogous α‐substituted GABA derivatives 11 a–11 c were also applied as potential substrates for memory of chirality effects. The reaction quantum yields of the photodecarboxylation reactions for the parent GABA derivative 13 and for the new substrates 7 h and 11 a were determined by the quantum yield determination system (QYDS) and showed a remarkable concentration dependency indicating aggregation at higher substrate concentrations. Inhibition studies on the atherogenic human serine hydrolase cholesterol esterase showed derivatives 8 a and 8 d to exhibit a hyperbolic mode of inhibition with moderate IC50 values of about 60–80 μM.
The aldol reaction of bio acetone in presence of a strongly basic ion exchange resin was carried out with and without the addition of water in a temperature range between − 30 °C and 45 °C. The conversion, selectivity and service time of the ion exchange resins were investigated in a stirred batch reactor and a continuous fixed bed reactor. For the batch experiments, both conversion and selectivity increased with decreasing temperature. Furthermore, the addition of water to the reaction medium has a positive effect on selectivity and catalyst service time of the resins. For the continuous flow experiments carried out in a fixed bed reactor, the selectivity towards diacetone alcohol is higher than in a batch reactor. This high selectivity is favored by a short contact time which inhibits as expected most of the consecutive reactions.
Hydroperoxide lyases (HPLs) catalyze the splitting of 13S-hydroperoxyoctadecadienoic acid (13S-HPODE) into the green note flavor hexanal and 12-oxo-9(Z)-dodecenoic acid, which is not yet used industrially. Here, HPL from Carica papaya (HPLCP) was cloned and functionally expressed in Escherichia coli to investigate synthesis of 12-oxo-9(Z)-dodecenoic acid in detail. To improve the low catalytic activity of full-length HPLCP, the hydrophobic, non-conserved N-terminal sequence was deleted. This enhanced enzyme activity from initial 10 to 40 U/l. With optimization of solubilization buffer, expression media enzyme activity was increased to 2700 U/l. The tetrameric enzyme was produced in a 1.5 l fermenter and enriched by affinity chromatography. The enzyme preparation possesses a slightly acidic pH optimum and a catalytic efficiency (kcat/KM) of 2.73 × 106 s−1·M−1 towards 13S-HPODE. Interestingly, HPLCP-N could be applied for the synthesis of 12-oxo-9(Z)-dodecenoic acid, and 1 mM of 13S-HPODE was transformed in just 10 s with a yield of 90%. At protein concentrations of 10 mg/ml, the slow formation of the 10(E)-isomer traumatin was observed, pointing to a non-enzymatic isomerization process. Bearing this in mind, a one-pot enzyme cascade starting from safflower oil was developed with consecutive addition of Pseudomonas fluorescens lipase, Glycine max lipoxygenase (LOX-1), and HPLCP-N. A yield of 43% was obtained upon fast extraction of the reaction mixtures after 1 min of HPLCP-N reaction. This work provides first insights into an enzyme cascade synthesis of 12-oxo-9(Z)-dodecenoic acid, which may serve as a bifunctional precursor for bio-based polymer synthesis.
Biobased polymers derived from plant oils are sustainable alternatives to petro based polymers. In recent years, multienzyme cascades have been developed for the synthesis of biobased ω-aminocarboxylic acids, which serve as building blocks for polyamides. In this work, we have developed a novel enzyme cascade for the synthesis of 12-aminododeceneoic acid, a precursor for nylon-12, starting from linoleic acid. Seven bacterial ω-transaminases (ω-TAs) were cloned, expressed in Escherichia coli and successfully purified by affinity chromatography. Activity towards the oxylipin pathway intermediates hexanal and 12-oxododecenoic acid in their 9(Z) and 10(E) isoforms was demonstrated for all seven transaminases in a coupled photometric enzyme assay. The highest specific activities were obtained with ω-TA from Aquitalea denitrificans (TRAD), with 0.62 U mg−1 for 12-oxo-9(Z)-dodecenoic acid, 0.52 U mg−1 for 12-oxo-10(E)-dodecenoic acid and 1.17 U mg−1 for hexanal. A one-pot enzyme cascade was established with TRAD and papaya hydroperoxide lyase (HPLCP-N), reaching conversions of 59% according to LC-ELSD quantification. Starting from linoleic acid, up to 12% conversion to 12-aminododecenoic acid was achieved with a 3-enzyme cascade comprising soybean lipoxygenase (LOX-1), HPLCP-N and TRAD. Higher product concentrations were achieved by the consecutive addition of enzymes compared to simultaneous addition at the beginning.
The use of two-component transposon plasmid vector systems, namely, a transposase construct and a donor vector carrying the gene of interest (GOI) can accelerate the development of recombinant cell lines. However, the undesired stable transfection of the transposase construct and the sustained expression of the enzyme can cause genetic instability due to the re-mobilization of the previously transposed donor vectors. Using a Sleeping Beauty -derived vector system, we established three recombinant cell pools and demonstrate stable integration of the transposase construct and sustained expression of the transposase over a period of 48 days. To provide an alternative approach, transcripts of the transposase gene were generated in vitro and co-transfected with donor vector plasmid at different ratios and mediating high GOI copy number integrations and expression levels. We anticipate that the use of transposase mRNA will foster further improvements in future cell line development processes.
Retroviral vectors derived from murine leukemia virus (MLV) are used in somatic gene therapy applications e.g. for genetic modification of hematopoietic stem cells. Recently, we reported on the establishment of a suspension viral packaging cell line (VPC) for the production of MLV vectors. Human embryonic kidney 293-F (HEK293-F) cells were genetically modified for this purpose using transposon vector technology. Here, we demonstrate the establishment of a continuous high cell density (HCD) process using this cell line. First, we compared different media regarding the maximum achievable viable cell concentration (VCC) in small scale. Next, we transferred this process to a stirred tank bioreactor before we applied intensification strategies. Specifically, we established a perfusion process using an alternating tangential flow filtration system. Here, VCCs up to 27.4E + 06 cells/mL and MLV vector titers up to 8.6E + 06 transducing units/mL were achieved. Finally, we established a continuous HCD process using a tubular membrane for cell retention and continuous viral vector harvesting. Here, the space-time yield was 18-fold higher compared to the respective batch cultivations. Overall, our results clearly demonstrate the feasibility of HCD cultivations for high yield production of viral vectors, especially when combined with continuous viral vector harvesting.
This work aims to address the knowledge gap in the thermal efficiency performance of a locally made cookstove in Mali. Despite the fact that the thermal efficiency of cookstoves is a crucial aspect of cooking, the performance of commercially produced cookstoves in Mali has not been thoroughly studied. In this context, the thermal efficiency of a single-mouth biomass stove has been investigated using a theoretical and experimental approach. First, the fundamental principles of physics for the three forms of heat transfer were applied. Then, the theoretical thermal efficiency of the stove was calculated based on the percentage share of energy gains and losses for the respective heat transfer modes. This analysis shows that the highest energy gain is achieved by radiation heat transfer from the flame and the fuel bed, followed by convection heat transfer to the bottom and sides of the pot, respectively. In order to validate the findings, the theoretical results have been compared with the experimental data at a case study site in Katibougou, Mali. Accordingly, the experimental thermal efficiency is slightly lower than the theoretical value, with a measured value of 27% compared to the theoretical value of 31.45%. The theoretical thermal efficiency can be closer to the experimental efficiency if the combustion losses caused by incomplete combustion of the fuel are taken into account.
HIV Gag virus-like particles (HIV Gag VLPs) are promising HIV vaccine candidates. In the literature, they are often described as shear-sensitive particles, and authors usually recommend the operation of tangential flow filtration (TFF) gently at shear rates below 4,000 s −1 to 6,000 s −1 . This in turn poses a severe limitation to the performance of TFF-mediated concentration of VLPs, which would be substantially enhanced by working at higher shear rates. To our knowledge, studies examining the shear sensitivity of HIV Gag VLPs and providing detailed information and evidence for the fragility of these particles have not been conducted yet. Thus, we investigated the effect of high shear rates on the colloidal stability of mosaic VLPs (Mos-VLPs) as relevant examples for HIV Gag VLPs. For this purpose, Mos-VLPs were exposed to different shear rates ranging from 3,395 s −1 to 22, 365 s −1 for 2 h. The average hydrodynamic diameter (AHD) and the polydispersity index (PDI) of the associated particle size distribution were used as stability indicators and measured after the treatment and during storage through dynamic light scattering. At high shear rates, we observed an increase in both AHD and PDI during the storage of HIV Mos1.Gag VLPs (bVLP—without envelope proteins) and Mos1.Gag + Mos2S.Env VLPs (eVLP—with envelope proteins). eVLPs exhibited higher colloidal stability than bVLPs, and we discuss the potential stabilizing role of envelope proteins. We finally demonstrated that the dispersion medium also has a considerable impact on the stability of Mos-VLPs.
The rapid increase in the use and development of statistical design of experiments (DoE), particularly in pharmaceutical process development, has become increasingly important over the last decades. This rise aligns with Green Chemistry Principles, seeking reduced resource usage and heightened efficiency. In this study, we employed a comprehensive design of experiments (DoE) approach to optimize the catalytic conversion of 1-decene to n-decanal through direct Wacker-type oxidation using the previously determined efficient PdCl2(MeCN)2 catalytic system. The aim was to maximize selectivity and conversion efficiency. Through systematic variation of seven factors, including substrate amount, catalyst and co-catalyst amount, reaction temperature, reaction time, homogenization temperature, and water content, this study identified critical parameters influencing the process to direct the reaction toward the desired product. The statistical analysis revealed high significance for both selectivity and conversion, with surface diagrams illustrating optimal conditions. Notably, catalyst amount emerged as a pivotal factor influencing conversion, with reaction temperature and co-catalyst amount significantly affecting both conversion efficiency and selectivity. The refined model demonstrated strong correlations between predicted and observed values, highlighting the impact of these factors on both selectivity and conversion.
Bridging the Implementation Gap between Pomace Waste and Large-Scale Baker’s Yeast Production
(2023)
The objectives set in the European Green Deal constitute the starting point of this review, which then focuses on the current implementation gap between agro-industrial wastes as resources for large-scale bioprocesses (e.g., baker’s yeast, bioethanol, citric acid, and amino acids). This review highlights the current lack of sustainability of the post-harvest processing of grapes and apples. In light of the European Green Deal, industrial biotechnology often lacks sustainability as well. We reviewed the recent progress reported in the literature to enhance the valorization of grape and apple pomace and the current failure to implement this research in technical processes. Nevertheless, selected recent papers show new perspectives to bridge this gap by establishing close collaborations between academic teams and industrial partners. As a final outcome, for the first time, we drew a circular flow diagram that connects agriculture post-harvest transformation with the industrial biotechnology and other industries through the substantial valorization of apple and grape pomace into renewable energy (solid biofuels) and sugar extracts as feedstock for large-scale bioprocesses (production of baker’s yeast industry, citric acid, bioethanol and amino acids). Finally, we discussed the requirements needed to achieve the successful bridging of the implementation gap between academic research and industrial innovation.