Continuous Anhydrous Synthesis of Oxymethylene Dimethyl Ethers by Reaction of Dimethyl Ether with Molecular Formaldehyde.

A novel gaseous synthesis route to oxymethylene dimethyl ethers (OMEn, n = 3-5) starting from CO2 and green H2 by using molecular formaldehyde (FA) and dimethyl ether (DME) is presented. The anhydrous reaction runs in a pressure free, gaseous, and continuous reaction setup. Hetero-geneous cata-lysts including zeolites and ion exchange resins (IER) are investigated, if they catalyze this reaction. While IER is almost inactive, zeolites with a 3D pore structure and an acidity exceeding ρm,H+ (NH3,ads ) = 250 µmol·gcat.-1 proved to be catalytically active. DME conversions of up to 2.76 mol-% are observed. The observed product gas stream compositions confirm thermo-dynamic considerations with back reactions / OMEn decomposition occurring as part of the equilibria under the investigated reaction conditions (90…180 °C). However, feed gas ratio variations (FA:DME = 1:2 to 1:9.5) highlighted the possibility to shift the product selectivity in favor of OMEn and suppress FA disproportionation to methyl formate. FA trimerization to trioxane is almost completely suppressed by running the reaction at 120 °C. The results presented here provide an important and unprecedented contribution to understand the complex reaction network in the OMEn synthesis reaction necessary to establish an energy efficient sustainable OMEn production process.

An engineered toluene dioxygenase for a single step biocatalytical production of (-)-(1S,2R)-cis-1,2-dihydro-1,2-naphthalenediol.

cis-1,2-Dihydro-1,2-naphthalenediol (DHND) is a valuable molecule employed for the pharmaceutical synthesis of bioactive compounds, such as bicyclic conduritol analogues. Enantiopure (+)-(1R,2S)-DHND (>98 % ee) is easily biosynthesized through the dearomatizing dihydroxylation of naphthalene, catalyzed by toluene dioxygenase (TDO) from Pseudomonas putida F1. However, the opposite enantiomer (-)-(1S,2R)-DHND could not be directly accessed, neither by chemical synthesis nor via biocatalytic approaches. Herein, we report a one-step biosynthesis of the opposite enantiomer (-)-(1S,2R)-DHND in a recombinant TDO E. coli BW25113 platform. We based on a semi-rational approach to generate a set of TDO variants, targeting exclusively the hotspot position F366, in order to enable an enantiomeric switch in the generated product. Eight out of nine single point variants were active and showed not only an alteration in enantioselectivity, but also generated an enantiomeric excess of the pursued product. Variant TDOF366V outperformed above the rest of the set, enabling the synthesis of (-)-(1S,2R)-DHND not only with an excellent enantiomeric excess of 90 %, but also with an advantageous product formation. A comparative semi-preparative biosynthesis yielded, 287 mg of (+)-(1R,2S)-DHND (>98 % ee) and 101 mg of (-)-(1S,2R)-DHND (90 % ee), when performed in a total volume of 100 mL with TDO wild-type and TDOF366V resting cells, respectively.

Optimization of Alcohol Dehydrogenase for Industrial Scale Oxidation of Lactols.

Alcohol dehydrogenases (ADH) are widely used to enantioselectively reduce ketones to chiral alcohols, but their application in industrial scale oxidations is rare. Reasons are the need for an NAD(P)+ cofactor regeneration system, often low performance in oxidative reactions and the limited substrate scope of ADHs. ADHA from Candida magnoliae DSMZ 70638 is identified to efficiently catalyze the regio-selective hydroxy-lactone oxidations to hydroxy-lactones. Hydroxy-lactones are common intermediates in industrial processes to cholesterol lowering (va)statin drugs. A biocatalytic aliphatic hydroxy-lactone oxidation process is developed using pure oxygen as oxidant reaching volumetric productivities of up to 12 g L-1 h-1 , product concentrations of almost 50 g L-1 and 95% reaction yield. For co-factor recycling a previously engineered, water-forming NAD(P)H-oxidase from Streptococcus mutans is used. The process is scaled up to industrial pilot plant scale and it could be demonstrated that ADH catalyzed oxidations can be developed to efficient and safe processes. However, the ADHA wild-type enzyme is not productive enough in chlorolactol oxidation. Therefore, enzyme engineering and multi-parameter screening is successfully applied to optimize the enzyme for the target reaction. The optimized ADHA variant shows a 17-fold higher oxidative activity, a 26°C increased stability and is applied to develop an efficient chlorolactol oxidation process.

Regioselective biocatalytic self-sufficient Tishchenko-type reaction via formal intramolecular hydride transfer.

A self-sufficient nicotinamide-dependent intramolecular bio-Tishchenko-type reaction was developed. The reaction is catalyzed by alcohol dehydrogenases and proceeds through formal intramolecular hydride transfer on dialdehydes to deliver lactones. Regioselectivity on [1,1'-biphenyl]-2,2'-dicarbaldehyde substrates could be controlled via the electronic properties of the substituents. Preparative scale synthesis provided access to substituted dibenzo[c,e]oxepin-5(7H)-ones.

Approaching boiling point stability of an alcohol dehydrogenase through computationally-guided enzyme engineering.

Enzyme instability is an important limitation for the investigation and application of enzymes. Therefore, methods to rapidly and effectively improve enzyme stability are highly appealing. In this study we applied a computational method (FRESCO) to guide the engineering of an alcohol dehydrogenase. Of the 177 selected mutations, 25 mutations brought about a significant increase in apparent melting temperature (ΔTm ≥ +3 °C). By combining mutations, a 10-fold mutant was generated with a Tm of 94 °C (+51 °C relative to wild type), almost reaching water's boiling point, and the highest increase with FRESCO to date. The 10-fold mutant's structure was elucidated, which enabled the identification of an activity-impairing mutation. After reverting this mutation, the enzyme showed no loss in activity compared to wild type, while displaying a Tm of 88 °C (+45 °C relative to wild type). This work demonstrates the value of enzyme stabilization through computational library design.

Exploring the sequence variability of polymerization-involved residues in the production of levan- and inulin-type fructooligosaccharides with a levansucrase.

The connection between the gut microbiome composition and human health has long been recognized, such that the host-microbiome interplay is at present the subject of the so-called "precision medicine". Non-digestible fructooligosaccharides (FOS) can modulate the microbial composition and therefore their consumption occupies a central place in a strategy seeking to reverse microbiome-linked diseases. We created a small library of Bacillus megaterium levansucrase variants with focus on the synthesis of levan- and inulin-type FOS. Modifications were introduced at positions R370, K373 and F419, which are either part of the oligosaccharide elongation pathway or are located in the vicinity of residues that modulate polymerization. These amino acids were exchanged by residues of different characteristics, some of them being extremely low- or non-represented in enzymes of the levansucrase family (Glycoside Hydrolase 68, GH68). F419 seemed to play a minor role in FOS binding. However, changes at R370 abated the levansucrase capacity to synthesize levan-type oligosaccharides, with some mutations turning the product specificity towards neo-FOS and the inulin-like sugar 1-kestose. Although variants retaining the native R370 produced efficiently levan-type tri-, tetra- and pentasaccharides, their capacity to elongate these FOS was hampered by including the mutation K373H or K373L. Mutant K373H, for instance, generated 37- and 5.6-fold higher yields of 6-kestose and 6-nystose, respectively, than the wild-type enzyme, while maintaining a similar catalytic activity. The effect of mutations on the levansucrase product specificity is discussed.

A novel cytochrome P450 mono-oxygenase from Streptomyces platensis resembles activities of human drug metabolizing P450s.

Cytochrome P450 mono-oxygenases (P450) are versatile enzymes which play essential roles in C-source assimilation, secondary metabolism, and in degradations of endo- and exogenous xenobiotics. In humans, several P450 isoforms constitute the largest part of phase I metabolizing enzymes and catalyze oxidation reactions which convert lipophilic xenobiotics, including drugs, to more water soluble species. Recombinant human P450s and microorganisms are applied in the pharmaceutical industry for the synthesis of drug metabolites for pharmacokinetics and toxicity studies. Compared to the membrane-bound eukaryotic P450s, prokaryotic ones exhibit some advantageous features, such as high stability and generally easier heterologous expression. Here, we describe a novel P450 from Streptomyces platensis DSM 40041 classified as CYP107L that efficiently converts several commercial drugs of various size and properties. This P450 was identified by screening of actinobacterial strains for amodiaquine and ritonavir metabolizing activities, followed by genome sequencing and expression of the annotated S. platensis P450s in Escherichia coli. Performance of CYP107L in biotransformations of amodiaquine, ritonavir, amitriptyline, and thioridazine resembles activities of the main human metabolizing P450s, namely CYPs 3A4, 2C8, 2C19, and 2D6. For application in the pharmaceutical industry, an E. coli whole-cell biocatalyst expressing CYP107L was developed and evaluated for preparative amodiaquine metabolite production.

Development of microreactors with surface-immobilized biocatalysts for continuous transamination.

The industrial importance of optically pure compounds has thrown a spotlight on ω-transaminases that have shown a high potential for the synthesis of bioactive compounds with a chiral amine moiety. The implementation of biocatalysts in industrial processes relies strongly on fast and cost effective process development, including selection of a biocatalyst form and the strategy for its immobilization. Here, microscale reactors with selected surface-immobilized amine-transaminase (ATA) in various forms are described as platforms for high-throughput process development. Wild type ATA (ATA-wt) from a crude cell extract, as well as Escherichia coli cells intracellularly overexpressing the enzyme, were immobilized on the surfaces of meander microchannels of disposable plastics by means of reactor surface silanization and glutaraldehyde bonding. In addition, a silicon/glass microchannel reactor was used for immobilization of an ATA-wt, genetically engineered to contain a silica-binding module (SBM) at the N-terminus (N-SBM-ATA-wt), leading to immobilization on the non-modified inner microchannel surface. Microreactors with surface-immobilized biocatalysts were coupled with a quenching system and at-line HPLC analytics and evaluated based on continuous biotransformation, yielding acetophenone and l-alanine. E. coli cells and N-SBM-ATA-wt were efficiently immobilized and yielded a volumetric productivity of up to 14.42 g L-1 h-1, while ATA-wt small load resulted in two orders of magnitude lower productivity. The miniaturized reactors further enabled in-operando characterization of biocatalyst stability, crucial for successful transfer to a production scale.

Quantitative Analysis of Hepatitis C NS5A Viral Protein Dynamics on the ER Surface.

Exploring biophysical properties of virus-encoded components and their requirement for virus replication is an exciting new area of interdisciplinary virological research. To date, spatial resolution has only rarely been analyzed in computational/biophysical descriptions of virus replication dynamics. However, it is widely acknowledged that intracellular spatial dependence is a crucial component of virus life cycles. The hepatitis C virus-encoded NS5A protein is an endoplasmatic reticulum (ER)-anchored viral protein and an essential component of the virus replication machinery. Therefore, we simulate NS5A dynamics on realistic reconstructed, curved ER surfaces by means of surface partial differential equations (sPDE) upon unstructured grids. We match the in silico NS5A diffusion constant such that the NS5A sPDE simulation data reproduce experimental NS5A fluorescence recovery after photobleaching (FRAP) time series data. This parameter estimation yields the NS5A diffusion constant. Such parameters are needed for spatial models of HCV dynamics, which we are developing in parallel but remain qualitative at this stage. Thus, our present study likely provides the first quantitative biophysical description of the movement of a viral component. Our spatio-temporal resolved ansatz paves new ways for understanding intricate spatial-defined processes central to specfic aspects of virus life cycles.

Draft Genome Sequences of Three Actinobacteria Strains Presenting New Candidate Organisms with High Potentials for Specific P450 Cytochromes.

The three Actinobacteria strains Streptomyces platensis DSM 40041, Pseudonocardia autotrophica DSM 535, and Streptomyces fradiae DSM 40063 were described to selectively oxyfunctionalize several drugs. Here, we present their draft genomes to unravel their gene sets encoding promising cytochrome P450 monooxygenases associated with the generation of drug metabolites.

Impaired coordination of nucleophile and increased hydrophobicity in the +1 subsite shift levansucrase activity towards transfructosylation.

Bacterial levansucrases produce ß(2,6)-linked levan-type polysaccharides using sucrose or sucrose analogs as donor/acceptor substrates. However, the dominant reaction of Bacillus megaterium levansucrase (Bm-LS) is hydrolysis. Single domain levansucrases from Gram-positive bacteria display a wide substrate-binding pocket with open access to water, challenging engineering for transfructosylation-efficient enzymes. We pursued a shift in reaction specificity by either modifying the water distribution in the active site or the coordination of the catalytic acid/base (E352) and the nucleophile (D95), thus affecting the fructosyl-transfer rate and allowing acceptors other than water to occupy the active site. Two serine (173/422) and two water-binding tyrosine (421/439) residues located in the first shell of the catalytic pocket were modified. Library variants of S173, Y421 and S422, which coordinate the position of D95 and E352, show increased transfructosylation (30-200%) and modified product spectra. Substitutions at position 422 have a higher impact on sucrose affinity, while changes at position 173 and 421 have a strong effect on the overall catalytic rate. As most retaining glycoside hydrolases (GHs) Bm-LS catalyzes hydrolysis and transglycosylation via a double displacement reaction involving two-transition states (TS1 and TS2). Hydrogen bonds of D95 with the side chains of S173 and S422 contribute a total of 2.4 kcal mol-1 to TS1 stabilization, while hydrogen bonds between invariant Y421, E352 and the glucosyl C-2 hydroxyl-group of sucrose contribute 2.15 kcal mol-1 stabilization. Changes at Y439 render predominantly hydrolytic variants synthesizing shorter oligosaccharides.

Three in One: Temperature, Solvent and Catalytic Stability by Engineering the Cofactor-Binding Element of Amine Transaminase.

Amine transaminase (ATA) catalyse enantioselectively the direct amination of ketones, but insufficient stability during catalysis limits their industrial applicability. Recently, we revealed that ATAs suffer from substrate-induced inactivation mechanism involving dissociation of the enzyme-cofactor intermediate. Here, we report on engineering the cofactor-ring-binding element, which also shapes the active-site entrance. Only two point mutations in this motif improved temperature and catalytic stability in both biphasic media and organic solvent. Thermodynamic analysis revealed a higher melting point for the enzyme-cofactor intermediate. The high cofactor affinity eliminates the need for pyridoxal 5'-phosphate supply, thus making large-scale reactions more cost effective. This is the first report on stabilising a tetrameric ATA by mutating a single structural element. As this structural "hotspot" is a common feature of other transaminases it could serve as a general engineering target.

Direct observation of isolated Damon-Eshbach and backward volume spin-wave packets in ferromagnetic microstripes.

The analysis of isolated spin-wave packets is crucial for the understanding of magnetic transport phenomena and is particularly interesting for applications in spintronic and magnonic devices, where isolated spin-wave packets implement an information processing scheme with negligible residual heat loss. We have captured microscale magnetization dynamics of single spin-wave packets in metallic ferromagnets in space and time. Using an optically driven high-current picosecond pulse source in combination with time-resolved scanning Kerr microscopy probed by femtosecond laser pulses, we demonstrate phase-sensitive real-space observation of spin-wave packets in confined permalloy (Ni80Fe20) microstripes. Impulsive excitation permits extraction of the dynamical parameters, i.e. phase- and group velocities, frequencies and wave vectors. In addition to well-established Damon-Eshbach modes our study reveals waves with counterpropagating group- and phase-velocities. Such unusual spin-wave motion is expected for backward volume modes where the phase fronts approach the excitation volume rather than emerging out of it due to the negative slope of the dispersion relation. These modes are difficult to excite and observe directly but feature analogies to negative refractive index materials, thus enabling model studies of wave propagation inside metamaterials.

Stochastic formation of magnetic vortex structures in asymmetric disks triggered by chaotic dynamics.

The non-trivial spin configuration in a magnetic vortex is a prototype for fundamental studies of nanoscale spin behaviour with potential applications in magnetic information technologies. Arrays of magnetic vortices interfacing with perpendicular thin films have recently been proposed as enabler for skyrmionic structures at room temperature, which has opened exciting perspectives on practical applications of skyrmions. An important milestone for achieving not only such skyrmion materials but also general applications of magnetic vortices is a reliable control of vortex structures. However, controlling magnetic processes is hampered by stochastic behaviour, which is associated with thermal fluctuations in general. Here we show that the dynamics in the initial stages of vortex formation on an ultrafast timescale plays a dominating role for the stochastic behaviour observed at steady state. Our results show that the intrinsic stochastic nature of vortex creation can be controlled by adjusting the interdisk distance in asymmetric disk arrays.

Wave modes of collective vortex gyration in dipolar-coupled-dot-array magnonic crystals.

Lattice vibration modes are collective excitations in periodic arrays of atoms or molecules. These modes determine novel transport properties in solid crystals. Analogously, in periodical arrangements of magnetic vortex-state disks, collective vortex motions have been predicted. Here, we experimentally observe wave modes of collective vortex gyration in one-dimensional (1D) periodic arrays of magnetic disks using time-resolved scanning transmission x-ray microscopy. The observed modes are interpreted based on micromagnetic simulation and numerical calculation of coupled Thiele equations. Dispersion of the modes is found to be strongly affected by both vortex polarization and chirality ordering, as revealed by the explicit analytical form of 1D infinite arrays. A thorough understanding thereof is fundamental both for lattice vibrations and vortex dynamics, which we demonstrate for 1D magnonic crystals. Such magnetic disk arrays with vortex-state ordering, referred to as magnetic metastructure, offer potential implementation into information processing devices.

Enantioselective oxidation of aldehydes catalyzed by alcohol dehydrogenase.

Teaching old dogs new tricks: Alcohol dehydrogenases (ADHs) may be established redox biocatalysts but they still are good for a few surprises. ADHs can be used to oxidize aldehydes, and this was demonstrated by the oxidative dynamic kinetic resolution of profens. In the presence of a suitable cofactor regeneration system, this reaction can occur with high selectivity.

Coupled vortex oscillations in spatially separated permalloy squares.

We experimentally study the magnetization dynamics of pairs of micron-sized permalloy squares coupled via their stray fields. The trajectories of the vortex cores in the Landau-domain patterns of the squares are mapped in real space using time-resolved scanning transmission x-ray microscopy. After excitation of one of the vortex cores with a short magnetic-field pulse, the system behaves like coupled harmonic oscillators. The coupling strength depends on the separation between the squares and the configuration of the vortex-core polarizations. Considering the excitation via a rotating in-plane magnetic field, it can be understood that only a weak response of the second vortex core is observed for equal core polarizations.

Tunable negligible-loss energy transfer between dipolar-coupled magnetic disks by stimulated vortex gyration.

A wide variety of coupled harmonic oscillators exist in nature. Coupling between different oscillators allows for the possibility of mutual energy transfer between them and the information-signal propagation. Low-energy input signals and their transport with negligible energy loss are the key technological factors in the design of information-signal processing devices. Here, utilizing the concept of coupled oscillators, we experimentally demonstrated a robust new mechanism for energy transfer between spatially separated dipolar-coupled magnetic disks - stimulated vortex gyration. Direct experimental evidence was obtained by a state-of-the-art experimental time-resolved soft X-ray microscopy probe. The rate of energy transfer from one disk to the other was deduced from the two normal modes' frequency splitting caused by dipolar interaction. This mechanism provides the advantages of tunable energy transfer rates, low-power input signals and negligible energy loss in the case of negligible intrinsic damping. Coupled vortex-state disks might be implemented in applications for information-signal processing.

Influence of dipolar interaction on vortex dynamics in arrays of ferromagnetic disks.

The influence of the magnetostatic interaction on vortex dynamics in arrays of ferromagnetic disks is investigated by means of a broadband ferromagnetic-resonance setup. Transmission spectra reveal a strong dependence of the resonance frequency of vortex-core motion on the ratio between the center-to-center distance and the element size. For a decreasing ratio, a considerable broadening of the absorption peak is observed following an inverse sixth power law. An analogy between the vortex system and rotating dipoles is confirmed by micromagnetic simulations.