STD‐NMR‐Based Protein Engineering of the Unique Arylpropionate‐Racemase AMDase G74C

Structure‐guided protein engineering achieved a variant of the unique racemase AMDase G74C, with 40‐fold increased activity in the racemisation of several arylaliphatic carboxylic acids. Substrate binding during catalysis was investigated by saturation‐transfer‐difference NMR (STD‐NMR) spectroscopy. All atoms of the substrate showed interactions with the enzyme. STD‐NMR measurements revealed distinct nuclear Overhauser effects in experiments with and without molecular conversion. The spectroscopic analysis led to the identification of several amino acid residues whose substitutions increased the activity of G74C. Single amino acid exchanges increased the activity moderately; structure‐guided saturation mutagenesis yielded a quadruple mutant with a 40 times higher reaction rate. This study presents STD‐NMR as versatile tool for the analysis of enzyme–substrate interactions in catalytically competent systems and for the guidance of protein engineering.     

Tagging Glycoproteins with Fluorescently Labeled GDP‐Fucoses by Using α1,3‐Fucosyltransferases

Fucose‐containing glycans mediate a variety of biological processes, but there is little information on reaction processes and mechanisms mediated by fucosyltransferases. We recently reported on fluorescently labeled GDP‐β‐L ‐fucose‐ATTO 550, which enabled monitoring of α1,3‐fucosyltransferase activity. Here we present an extension to the previously described results, based on the synthesis of a fluorescein‐isothiocyanate (FITC)‐labeled and two carboxyfluorescein‐labeled (FAM‐labeled) NDP‐β‐L ‐fucose derivatives, and applied all four compounds in labeling of different glycoproteins with the aid of four different fucosyltransferases. The labeling processes were analyzed by in‐gel fluorescence and fluorescence polarization measurements. Comparison with the ATTO‐labeled sugar revealed that the FITC‐labeled fucose was the best of these substrates, and that the bacterial enzyme HP‐FucT tolerated the fluorescent substrates better than human fucosyltransferases.     

P450 BM3 Monooxygenase as an Efficient NAD(P)H‐Oxidase for Regeneration of Nicotinamide Cofactors in ADH‐Catalysed Preparative Scale Biotransformations

Enzymatic oxidations of primary and secondary alcohols catalysed by nicotinamide dependent alcohol dehydrogenases on the preparative scale require cofactor regeneration systems. Of critical value from an economic and ecological perspective is the application of NAD(P)H‐oxidases, which utilise molecular oxygen as a cost‐effective, atom‐efficient and environmentally benign oxidant to regenerate the cofactor NAD(P)⁺. Herein, the P450 BM3 monooxygenase from Bacillus megaterium is presented as an NAD(P)H‐oxidase for the successful regeneration of both NADP⁺ and NAD⁺ on the preparative scale. This enzyme was exemplarily applied for ADH‐catalysed oxidative kinetic resolutions of racemic secondary alcohols and the desymmetrisation of a meso‐diol leading to enantiomerically enriched secondary alcohols in both cases. Furthermore, the ADH‐catalysed oxidation of a primary alcohol targeting the corresponding aldehyde was performed. The obtained results significantly broaden the scope of feasible oxidative biotransformations, thereby increasing the number of synthetic reactions complying with key challenges of a modern and sustainable chemistry such as mild reaction conditions, environmentally benign solvents, and biodegradable non‐toxic catalysts.

     

Systematische Untersuchung der Azikularferritbildung in Stählen

The formation of acicular ferrite is mainly influenced by the steel composition, cooling rate, austenite grain size, and inclusion landscape. However, their precise effects have not been clarified in literature yet. Within the FWF project TRP 266-N19, a systematic methodology to study the influencing factors of the acicular ferrite formation as well as their interactions on laboratory scale was developed. The present work exemplarily demonstrates the application of this methodology for studying the effect of cooling rate and boron on the formation of acicular ferrite in a low-carbon HSLA steel.     

Trans octadecenoic fatty acid (TFA) isomers in German foods and bakery goods

In the present study, 122 food samples from the German food market were analysed for their C18:1 trans fatty acid (TFA) content and profile. A particular focus of the survey were baked and fried foods. TFA analysis was performed by means of silver ion SPE (Ag⁺‐SPE) in combination with high‐resolution GC (HRGC‐FID). Overall, 51 bakery product samples were analysed of which 25 samples were prepacked bakery products purchased from local retail stores and 26 samples of unpacked bakery products purchased from local bakery shops. In addition, 14 French fries samples obtained from small local fast food restaurants as well as from internationally operating fast food chains, 27 potato and tortillas chips, 15 instant soups as well as 15 dry culinary sauces were analysed. The highest amounts of C18:1 TFA isomers were found in deep‐fried bakery products. Prepacked branded cookies and biscuits on the other hand contained only negligible C18:1 TFA amounts. Regarding their C18:1 trans isomer profile most deep‐fried bakery products exhibited a Gaussian‐distributed isomer profile. The analysed prepacked croissants, cookies and biscuits contained predominantly ruminant TFA (TFA) as suggested by the presence of vaccenic acid (C18:1 trans 11), which was the major C18:1 TFA isomer in these products. All non‐bakery samples (n = 71) contained less than 3 g C18:1 TFA per 100 g fat. In conclusion, TFA still occur in considerable amounts in a few German food products, especially in some deep‐fried bakery products (‘Berliner’ type of doughnuts). Practical applications: Trans fatty acids, in particular the trans octadecenoic fatty acid isomers (C18:1), are generally considered from the nutritional point of view as undesirable food components due to their negative health effects. Tremendous efforts have been made by major food processors in order to decrease or even eliminate the presence of TFA in some foodstuffs (e.g. in margarines in European countries). However, some food processors of other food sectors are still applying oils and fats containing partially hydrogenated vegetable oils, whereas others within the same food category have already switched their processing conditions and/or raw materials towards TFA alternatives. Therefore, actual TFA data of foodstuffs determined by means of state‐of‐the‐art analytical procedures (Ag⁺‐SPE in combination with GC‐FID) is necessary to detect areas of further improvement in the food supply chain and to provide data for an update of dietary TFA intake.     

Organocatalytic Enantioselective Strecker Synthesis of α‐Quaternary α‐Trifluoromethyl Amino Acids

The first organocatalytic enantioselective Strecker synthesis of α‐quaternary α‐trifluoromethylated amino acids has been developed. Employing Takemoto’s thiourea catalyst the nucleophilic addition of trimethylsilyl cyanide to trifluoromethyl ketimines affords α‐amino nitriles in good to excellent yields (50–99%) and very good enantioselectivities (ee=83–95%). The enantiopure amino nitriles can be obtained by recrystallization. Deprotection and hydrolysis leads to the title amino acids.     

GC–MS analysis of oxysterols and their formation in cultivated liver cells (HepG2)

Oxysterols play a key role in many (patho)physiological processes and they are potential biomarkers for oxidative stress in several diseases. Here we developed a rapid gas chromatographic-mass spectrometry-based method for the separation and quantification of 11 biologically relevant oxysterols bearing hydroxy, epoxy, and dihydroxy groups. Efficient chromatographic separation (resolution ≥ 1.9) was achieved using a medium polarity 35%-diphenyl/65%-dimethyl polysiloxane stationary phase material (30 m × 0.25 mm inner diameter and 0.25 μm film thickness). Based on thorough analysis of the fragmentation during electron ionization we developed a strategy to deduce structural information of the oxysterols. Optimized sample preparation includes (i) extraction with a mixture of n-hexane/iso-propanol, (ii) removal of cholesterol by solid phase extraction with unmodified silica, and (iii) trimethylsilylation. The method was successfully applied on the analysis of brain samples, showing consistent results with previous studies and a good intra- and interday precision of ≤20%. Finally, we used the method for the investigation of oxysterol formation during oxidative stress in HepG2 cells. Incubation with tert-butyl hydroperoxide led to a massive increase in free radical formed oxysterols (7-keto-chol > 7β-OH-chol >> 7α-OH-chol), while 24 h incubation with the glutathione peroxidase 4 inhibitor RSL3 showed no increase in oxidative stress based on the oxysterol pattern. Overall, the new method described here enables the robust analysis of a biologically meaningful pattern of oxysterols with high sensitivity and precision allowing us to gain new insights in the biological formation and role of oxysterols.     

Leakage of Metallic Ball Seat Valves with Anisotropic Surfaces

Metallic ball seat valves are an essential component of many hydraulic systems. They are used for many different purposes such as pressure relief valves or check valves. Despite their universal usage, their sealing mechanism is not yet fully understood. In previous works, a successful method for the simulation of the fluid leakage of metallic ball seat valves has been developed and confirmed experimentally. The method is based on Persson's contact mechanics theory, which is based on surface roughness power spectrum C(q). This theory takes a wide range of roughness values at different length scales into account. The previous method has been restricted to isotropic surfaces, but most surfaces of practical interest are highly anisotropic. A method for the calculation of pressure flow factors using Persson's method is presented. Based on these, a model for the leakage calculation can be developed. The simulation results are validated using an experiment.     

Phase separation of a hafnium alkoxide-modified polysilazane upon polymer-to-ceramic transformation—A case study

The polymer-to-ceramic transformation of a hafnium alkoxide-modified polysilazane was investigated via thermogravimetric analysis coupled with in situ mass spectrometry (TG/MS), nuclear magnetic resonance (MAS NMR) and transmission electron microscopy (TEM). The results indicate that the structural evolution of the polysilazane upon ceramization is strongly affected by the modification with hafnium alkoxide. Thus, the content of carbon in the ceramic backbone was relatively low, whereas a large amount of SiN₄ sites and a segregated carbon phase was present in the sample. Furthermore, this study revealed the formation of a SiHfCNO amorphous single phase ceramic via pyrolysis of the polymer at 700 °C, whereas at higher pyrolysis temperatures precipitation of hafnia was observed, leading to an amorphous hafnia/silicon carbonitride ceramic nanocomposite. The precipitation of hafnia was shown to not rely on decomposition processes, but to be a result of rearrangement reactions occurring within the ceramic material.     

Mechanical robustness of Pseudomonas aeruginosa biofilms

Biofilms grow on various surfaces and in many different environments, a phenomenon that constitutes major problems in industry and medicine. Despite their importance little is known about the viscoelastic properties of biofilms and how these depend on the chemical microenvironment. Here, we find that the mechanical properties of Pseudomonas aeruginosa (P.a.) biofilms are highly robust towards chemical perturbations. Specifically, we observe that P.a. biofilms are able to fully regain their initial stiffness after yielding is enforced, even in the presence of chemicals. Moreover, only trivalent ions and citric acid significantly affect the biofilm elasticity, the first of which also alter the texture of the material. Finally, our results indicate that biofilm mechanics and bacteria viability inside the biofilm are not necessarily linked which suggests that targeting bacteria alone might not be sufficient for biofilm removal strategies.