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.

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.

Integrated Mechanical, Thermal, Data, and Power Transfer Interfaces for Future Space Robotics.

In-situ connectability among modules of a space system can provide significantly enhanced flexibility, adaptability, and robustness for space exploration and servicing missions. Connection of modules in extra-terrestrial environment is hence a topic of rising importance in modern orbital or planetary missions. As an example, the increasing number of satellites sent to space have introduced a large set of connections of various type, for transferring mechanical loads, data, electrical power and heat from one module to another. This paper provides a comprehensive review of published work in space robotic connections and presents the different transfer types developed and used to date in robotic applications for orbital and extra-terrestrial planetary missions. The aims of this paper are to present a detailed analysis of the state of the art available technologies, to make an analysis of and comparison among different solutions to common problems, to synthesize and identify future connectability research, and to lay the foundation for future European space robotic connectability effort and work for a complex and growing important future space missions. All types are described in their base characteristics and evaluated for orbital and planetary environments. This analysis shows that despite the large number of connectors developed for each of the four functionalities (mechanical, thermal, data, and electrical power) here considered, the trend is that researchers are integrating more than one functionalizes into a single equipment or device, to reduce costs and improve standardization. The outcomes of this literature review have contributed toward the design of a future multifunctional, standard and scalable interface at the early stage of the Standard Interface for Robotic Manipulation of Payloads in Future Space Missions (SIROM) project, a European Commission funded Horizon 2020 project. SIROM interfaces will be employed by European prime contractors in future extra-terrestrial missions.

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.

Enzymatic network for production of ether amines from alcohols.

We constructed an enzymatic network composed of three different enzymes for the synthesis of valuable ether amines. The enzymatic reactions are interconnected to catalyze the oxidation and subsequent transamination of the substrate and to provide cofactor recycling. This allows production of the desired ether amines from the corresponding ether alcohols with inorganic ammonium as the only additional substrate. To examine conversion, individual and overall reaction equilibria were established. Using these data, it was found that the experimentally observed conversions of up to 60% observed for reactions containing 10 mM alcohol and up to 280 mM ammonia corresponded well to predicted conversions. The results indicate that efficient amination can be driven by high concentrations of ammonia and may require improving enzyme robustness for scale-up. Biotechnol. Bioeng. 2016;113: 1853-1861. © 2016 Wiley Periodicals, Inc.

Wafer-level hysteresis-free resonant carbon nanotube transistors.

We report wafer-level fabrication of resonant-body carbon nanotube (CNT) field-effect transistors (FETs) in a dual-gate configuration. An integration density of >10(6) CNTFETs/cm(2), an assembly yield of >80%, and nanoprecision have been simultaneously obtained. Through combined chemical and thermal treatments, hysteresis-free (in vacuum) suspended-body CNTFETs have been demonstrated. Electrostatic actuation by lateral gate and FET-based readout of mechanical resonance have been achieved at room temperature. Both upward and downward in situ frequency tuning has been experimentally demonstrated in the dual-gate architecture. The minuscule mass, high resonance frequency, and in situ tunability of the resonant CNTFETs offer promising features for applications in radio frequency signal processing and ultrasensitive sensing.

Ironing out their differences: dissecting the structural determinants of a phenylalanine aminomutase and ammonia lyase.

Deciphering the structural features that functionally separate ammonia lyases from aminomutases is of interest because it may allow for the engineering of more efficient aminomutases for the synthesis of unnatural amino acids (e.g., ß-amino acids). However, this has proved to be a major challenge that involves understanding the factors that influence their activity and regioselectivity differences. Herein, we report evidence of a structural determinant that dictates the activity differences between a phenylalanine ammonia lyase (PAL) and aminomutase (PAM). An inner loop region that closes the active sites of both PAM and PAL was mutated within PAM (PAM residues 77-97) in a stepwise approach to study the effects when the equivalent residue(s) found in the PAL loop were introduced into the PAM loop. Almost all of the single loop mutations triggered a lyase phenotype in PAM. Experimental and computational evidence suggest that the induced lyase features result from inner loop mobility enhancements, which are possibly caused by a 310-helix cluster, flanking α-helices, and hydrophobic interactions. These findings pinpoint the inner loop as a structural determinant of the lyase and mutase activities of PAM.

Priming ammonia lyases and aminomutases for industrial and therapeutic applications.

Ammonia lyases (AL) and aminomutases (AM) are emerging in green synthetic routes to chiral amines and an AL is being explored as an enzyme therapeutic for treating phenylketonuria and cancer. Although the restricted substrate range of the wild-type enzymes limits their widespread application, the non-reliance on external cofactors and direct functionalization of an olefinic bond make ammonia lyases attractive biocatalysts for use in the synthesis of natural and non-natural amino acids, including ß-amino acids. The approach of combining structure-guided enzyme engineering with efficient mutant library screening has extended the synthetic scope of these enzymes in recent years and has resolved important mechanistic issues for AMs and ALs, including those containing the MIO (4-methylideneimidazole-5-one) internal cofactor.

Biochemical properties and crystal structure of a ß-phenylalanine aminotransferase from Variovorax paradoxus.

By selective enrichment, we isolated a bacterium that can use ß-phenylalanine as a sole nitrogen source. It was identified by 16S rRNA gene sequencing as a strain of Variovorax paradoxus. Enzyme assays revealed an aminotransferase activity. Partial genome sequencing and screening of a cosmid DNA library resulted in the identification of a 1,302-bp aminotransferase gene, which encodes a 46,416-Da protein. The gene was cloned and overexpressed in Escherichia coli. The recombinant enzyme was purified and showed a specific activity of 17.5 U mg(-1) for (S)-ß-phenylalanine at 30°C and 33 U mg(-1) at the optimum temperature of 55°C. The ß-specific aminotransferase exhibits a broad substrate range, accepting ortho-, meta-, and para-substituted ß-phenylalanine derivatives as amino donors and 2-oxoglutarate and pyruvate as amino acceptors. The enzyme is highly enantioselective toward (S)-ß-phenylalanine (enantioselectivity [E], >100) and derivatives thereof with different substituents on the phenyl ring, allowing the kinetic resolution of various racemic ß-amino acids to yield (R)-ß-amino acids with >95% enantiomeric excess (ee). The crystal structures of the holoenzyme and of the enzyme in complex with the inhibitor 2-aminooxyacetate revealed structural similarity to the ß-phenylalanine aminotransferase from Mesorhizobium sp. strain LUK. The crystal structure was used to rationalize the stereo- and regioselectivity of V. paradoxus aminotransferase and to define a sequence motif with which new aromatic ß-amino acid-converting aminotransferases may be identified.

A single active nanoelectromechanical tuning fork front-end radio-frequency receiver.

Nanoelectromechanical systems (NEMS) offer the potential to revolutionize fundamental methods employed for signal processing in today's telecommunication systems, owing to their spectral purity and the prospect of integration with existing technology. In this work we present a novel, front-end receiver topology based on a single device silicon nanoelectromechanical mixer-filter. The operation is demonstrated by using the signal amplification in a field effect transistor (FET) merged into a tuning fork resonator. The combination of both a transistor and a mechanical element into a hybrid unit enables on-chip functionality and performance previously unachievable in silicon. Signal mixing, filtering and demodulation are experimentally demonstrated at very high frequencies ( > 100 MHz), maintaining a high quality factor of Q = 800 and stable operation at near ambient pressure (0.1 atm) and room temperature (T = 300 K). The results show that, ultimately miniaturized, silicon NEMS can be utilized to realize multi-band, single-chip receiver systems based on NEMS mixer-filter arrays with reduced system complexity and power consumption.

Mechanism-inspired engineering of phenylalanine aminomutase for enhanced ß-regioselective asymmetric amination of cinnamates.

Turn to switch: A mutant of phenylalanine aminomutase was engineered that can catalyze the regioselective amination of cinnamate derivatives (see scheme, red) to, for example, ß-amino acids. This regioselectivity, along with the X-ray crystal structures, suggests two distinct carboxylate binding modes differentiated by C(ß)-C(ipso) bond rotation, which determines if ß- (see scheme) or α-addition takes place.

Nanomechanical silicon resonators with intrinsic tunable gain and sub-nW power consumption.

Nanoelectromechanical systems (NEMS) as integrated components for ultrasensitive sensing, time keeping, or radio frequency applications have driven the search for scalable nanomechanical transduction on-chip. Here, we present a hybrid silicon-on-insulator platform for building NEM oscillators in which fin field effect transistors (FinFETs) are integrated into nanomechanical silicon resonators. We demonstrate transistor amplification and signal mixing, coupled with mechanical motion at very high frequencies (25-80 MHz). By operating the transistor in the subthreshold region, the power consumption of resonators can be reduced to record-low nW levels, opening the way for the parallel operation of hundreds of thousands of NEM oscillators. The electromechanical charge modulation due to the field effect in a resonant transistor body constitutes a scalable nanomechanical motion detection all-on-chip and at room temperature. The new class of tunable NEMS represents a major step toward their integration in resonator arrays for applications in sensing and signal processing.

The crystal structure of an esterase from the hyperthermophilic microorganism Pyrobaculum calidifontis VA1 explains its enantioselectivity.

The highly thermostable esterase from the hyperthermophilic archaeon Pyrobaculum calidifontis VA1 (PestE) shows high enantioselectivity (E > 100) in the kinetic resolution of racemic chiral carboxylic acids, but little selectivity towards acetates of tertiary alcohols (E = 2-4). To explain these unique properties, its crystal structure has been determined at 2.0 Å resolution. The enzyme is a member of the hormone-sensitive lipase group (group H) of the esterase/lipase superfamily on the basis of the amino acid sequence identity. The PestE structure shows a canonical α/ß-hydrolase fold as core domain with a cap structure at the C-terminal end of the ß-sheet. A tetramer in the crystal packing is formed of two dimers; the dimeric form is observed in solution. Conserved dimers and even tetramers are found in other group H proteins. The amino acid residues Ser157, His284, and Asp254 form the catalytic triad, which is typically found in α/ß-hydrolases. The oxyanion hole is composed of Gly85 and Gly86 within the conserved sequence motif HGGG(M,F,W) (amino acid residues 83-87) and Ala158. With the elucidated structure, experimental results about enantioselectivity towards the two model substrate classes (as exemplified for 3-phenylbutanoic acid ethyl ester and 1,1,1-trifluoro-2-phenylbut-3-yn-2-yl acetate) could be explained by molecular modeling. For both enantiomers of the tertiary alcohol, orientations in two binding pockets were obtained without significant energy differences corresponding to the observed low enantioselectivity due to missing steric repulsions. In contrast, for the carboxylic acid ester, two different orientations with significant energy differences for each enantiomer were found matching the high E values.

Mutational analysis of phenylalanine ammonia lyase to improve reactions rates for various substrates.

Phenylalanine ammonia lyases (PAL) catalyze the reversible, non-reductive amination of trans-cinnamic acid to l-phenylalanine in the presence of high ammonia concentrations. Since neither cofactor recycling nor other additives are needed and by this asymmetric synthesis theoretical yields of 100% can be reached, it is an interesting reaction for industrial processes. In this study we demonstrate the superior properties of p-nitro-cinnamic acid (p-n-CA) in the amination reaction using the PAL from Petroselinum crispum (pcPAL). By focused-directed evolution, three mutants were identified showing increased reaction rates and decreased substrate inhibition. Together, the F137V mutant with p-n-CA showed a 15-fold increased reaction rate compared with the pcPAL WT with the natural cinnamic acid. The high reaction rates were also proven in preparative scale experiments. Activities towards other p-substituted cinnamic acids showing different electronic effects of the substituent were analyzed. Focused-directed evolution around the carboxylic acid- and amine-binding site always decreased PAL activity, due to a sensitive H-bond network.

Comparative Multi-Epitope-Ligand-Cartography reveals essential immunological alterations in Barrett's metaplasia and esophageal adenocarcinoma.

BACKGROUND: Barrett's esophagus (BE) is caused by gastroesophageal reflux with consecutive mucosal inflammation, predisposing patients to the development of esophageal adenocarcinoma (EAC). We investigated changes in T cell-related mucosal combinatorial molecular protein patterns in both diseases using the novel Multi-Epitope-Ligand-Cartography, a unique robotic whole-cell imaging technology that simultaneously visualizes dozens of proteins in structurally intact tissues and correlates cellular localization of proteins with function. RESULTS: Biopsies were taken during endoscopy from BE, EAC, and normal control tissue, and proteomic microscopy was performed on 32 different epitopes. When the significance level was set to p < 0.0005 and the search depth to five antibody combinations, controls and BE can be differentiated by 63, controls and EAC by 3222, and BE from EAC by 1521 distinct protein combinations.For example, the number of activated apoptotic naïve and memory T cells was significantly increased only in BE, whereas the number of activated apoptotic helper and regulatory T cells was significantly elevated in BE and EAC. In contrast, the number of activated apoptotic cytotoxic T cells was significantly elevated only in EAC. Confirming different pathways in BE and EAC, the number of T lymphocytes with p53 expression and downregulation of bcl2 expression (CD3+p53+Bcl2-NfkB-) was significantly increased in EAC compared to BE and controls. Interestingly, the number of precursor T cells (CD7+) was significantly elevated only in EAC. These cells lack Bax and caspase-8, suggesting impaired apoptosis in the early stages of T cell differentiation. CONCLUSION: Proteomic analysis showed for the first time that proteins, which are critically involved in the mucosal immune system of the esophagus, are distinctly expressed in BE and EAC, whereas others are comparably altered in both diseases, suggesting that many pathogenic events might be shared by both diseases. Topological proteomic analysis, therefore, helps us to understand the different pathogenic events in the underlying disease pathways.

A single residue influences the reaction mechanism of ammonia lyases and mutases.

All ways lead to Rome? Computer modeling and kinetic measurements identified a distinct residue in Phe/Tyr ammonia lyases (PAL/TAL) which controls whether the Friedel-Crafts or an E(1)cB reaction mechanism takes place. Hence, Glu484 in pcPAL favors the Friedel-Crafts reaction (see picture, MIO = 4-methylidene imidazol-5-one) whereas an Asn in TAL gives an elimination reaction. These mechanistic investigations also reveal activity of a PAL mutant and a TAL towards an amino alcohol.

Recovery of choline oxidase activity by in vitro recombination of individual segments.

Initial attempts to express a choline oxidase from Arthrobacter pascens (APChO-syn) in Escherichia coli starting from a synthetic gene only led to inactive protein. However, activity was regained by the systematic exchange of individual segments of the gene with segments from a choline oxidase-encoding gene from Arthrobacter globiformis yielding a functional chimeric enzyme. Next, a sequence alignment of the exchanged segment with other choline oxidases revealed a mutation in the APChO-syn, showing that residue 200 was a threonine instead of an asparagine, which is, thus, crucial for confering enzyme activity and, hence, provides an explanation for the initial lack of activity. The active recombinant APChO-syn-T200N variant was biochemically characterized showing an optimum at pH 8.0 and at 37 degrees C. Furthermore, the substrate specificity was examined using N,N-dimethylethanolamine, N-methylethanolamine and 3,3-dimethyl-1-butanol.

Systematic high-content proteomic analysis reveals substantial immunologic changes in colorectal cancer.

The immune system is a significant determinant of epithelial tumorigenesis, but its role in colorectal cancer pathogenesis is not well understood. The function of the immune system depends upon the integrity of the protein network environment, and thus, we performed MELC immunofluorescence microscopy focusing on the lamina propria. By analyzing structurally intact tissues from colorectal cancer, ulcerative colitis, and healthy colonic mucosa, we used this unique and novel highly multiplexed robotic-imaging technology, which allows visualizing dozens of proteins simultaneously, and explored the toponome in colorectal cancer mucosa for the first time. We identified 1,930 motifs that distinguish control from colorectal cancer tissue. In colorectal cancer, the number of activated T cells is increased, explained by a lack of bax, caspase-3, and caspase-8. Whereas CD4(+)CD25(+) T cells are decreased and are, other than in ulcerative colitis, not activated, cytotoxic T cells are significantly increased in colorectal cancer. Furthermore, the number of activated human lymphocyte antigen (HLA)-DR(+) T-cells is increased in colorectal cancer, pointing to an altered antigen presentation. In colorectal cancer, CD3(+)CD29(+) expression and assembly of the LFA-1 and LFA-3 receptor are differentially changed, indicating a distinct regulation of T-cell adhesion in colorectal cancer. We also identified increased numbers of natural killer and CD44(+) cells in the colorectal cancer mucosa and nuclear factor-kappaB as regulator of apoptosis in these cell populations. High-content proteomic analysis showed that colorectal cancer induces a tremendous modification of protein expression profiles in the lamina propria. Thus, topological proteomic analysis may help to unravel the role of the adaptive immune system in colorectal cancer and aid the development of new antitumor immunotherapy approaches.