Identification of a Hotspot Residue for Improving the Thermostability of a Flavin-Dependent Monooxygenase

HadA is a flavin-dependent monooxygenase that can catalyze the denitration and dehalogenation of a wide variety of toxicants such as pesticides. Although these enzymatic reactions are useful for bioremediation or biocatalysis, the application of HadA for these purposes is not yet possible because of its low thermostability. In this work we have engineered HadA to be more thermostable through the use of structural, in silico, and rational approaches. The X-ray structure of HadA was solved to obtain a reliable three-dimensional protein model for further prediction of thermostable variants. In silico analysis by using two bioinformatic tools—FireProt and Disulfide by Design—suggested 102 variants that we then further refined by applying rational criteria including the location of a particular residue and its nearby interactions, as well as other biophysical parameters to narrow down the list to six candidates. The G513Y variant was found to be an optimal engineered candidate because it has significantly improved stability relative to the wild-type enzyme and equivalent activity. G513Y has an activity half-life 72 (50 °C) and 160 times (45 °C) longer than that of the wild-type enzyme. Coupled together with thermostable reactions of reduced flavin and NADH-regenerating systems, the G513Y variant can be used to catalyze denitration of 4nitrophenol at 45 °C. Structure/sequence alignments of HadA and its homologues indicate that several flavin-dependent monooxygenases also contain amino acid residues homologous to the G513 of HadA, hence opening up the possibility of applying this engineering approach to improving their thermostabilities as well. Molecular dynamics (MD) simulations confirmed that the improved thermostability of the G513Y variant was due to aromatic hydrocarbon interactions between Y513 and N359, L347, G348, and F349.     

Functional characterization of a thermostable methionine adenosyltransferase from <Emphasis Type="Italic">Thermus thermophilus</Emphasis> HB27

MATTt (a thermostable methionine adenosyltransferase from Thermus thermophilus HB27) was overexpressed in Escherchia coli and purified using Ni-NTA affinity column. The enzymatic activity of MATTt was investigated in a temperature range from 30 °C to 90 °C, showing that MATTt exhibited a high enzymatic activity and good thermostability at 80 °C. Circular dichroism spectra reveals that MATTt contains high portion of β-sheet structures contributing to the thermostability of MATTt. The kinetic parameter, K _m is 4.19 mmol/L and 1.2 mmol/L for ATP and methionine, respectively. MATTt exhibits the highest enzymatic activity at pH 8. Cobalt (Co²⁺) and zinc ion (Zn²⁺) enhances remarkably the activity of MATTt compared to the magnesium ion (Mg²⁺). All these results indicated that the thermostable MATTt has great potential for industry applications.

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Stabilization of the Reductase Domain in the Catalytically Self‐Sufficient Cytochrome P450BM3 by Consensus‐Guided Mutagenesis

The multidomain, catalytically self‐sufficient cytochrome P450 BM‐3 from Bacillus megaterium (P450_BM3) constitutes a versatile enzyme for the oxyfunctionalization of organic molecules and natural products. However, the limited stability of the diflavin reductase domain limits the utility of this enzyme for synthetic applications. In this work, a consensus‐guided mutagenesis approach was applied to enhance the thermal stability of the reductase domain of P450_BM3. Upon phylogenetic analysis of a set of distantly related P450s (>38 % identity), a total of 14 amino acid substitutions were identified and evaluated in terms of their stabilizing effects relative to the wild‐type reductase domain. Recombination of the six most stabilizing mutations generated two thermostable variants featuring up to tenfold longer half‐lives at 50 °C and increased catalytic performance at elevated temperatures. Further characterization of the engineered P450_BM3 variants indicated that the introduced mutations increased the thermal stability of the FAD‐binding domain and that the optimal temperature (T_opt) of the enzyme had shifted from 25 to 40 °C. This work demonstrates the effectiveness of consensus mutagenesis for enhancing the stability of the reductase component of a multidomain P450. The stabilized P450_BM3 variants developed here could potentially provide more robust scaffolds for the engineering of oxidation biocatalysts.     

Engineering residues on C interface to improve thermostability of nitrilase for biosynthesis of Pregabalin precursor

Nitrilase-mediated bioprocesses exhibited great potential in the production of value-added carboxylic acids. However, poor thermostability of nitrilases usually restricts their industrial applications. Herein, the thermostability of nitrilase BaNIT_M0 was significantly improved by engineering the amino acid residues on the intersection of two dimers (C interface). Except for simultaneous enhancement of enantioselectivity and activity, the best variant V82L/M127I/L159M/F166Q/C237S/Q260H (BaNIT_M4) showed a 10.8-fold increase in half-life at 30°C compared with BaNIT_M0. Structural analysis demonstrated that additional hydrogen bonds were formed between the residues on the C interface, which strengthened the interactions of two symmetrical regions and spirals. Subsequently, the engineered nitrilase was immobilized onto epoxy resins LXTE-603 and the immobilized nitrilase exhibited excellent stability over 12 repeated cycles, which indicated a great industrial potential for biosynthesis of Pregabalin precursor.     

Modeling-Assisted Design of Thermostable Benzaldehyde Lyases from Rhodococcus erythropolis for Continuous Production of α-Hydroxy Ketones

Enantiopure α-hydroxy ketones are important building blocks of active pharmaceutical ingredients (APIs), which can be produced by thiamine-diphosphate-dependent lyases, such as benzaldehyde lyase. Here we report the discovery of a novel thermostable benzaldehyde lyase from Rhodococcus erythropolis R138 (ReBAL). While the overall sequence identity to the only experimentally confirmed benzaldehyde lyase from Pseudomonas fluorescens Biovar I (PfBAL) was only 65 %, comparison of a structural model of ReBAL with the crystal structure of PfBAL revealed only four divergent amino acids in the substrate binding cavity. Based on rational design, we generated two ReBAL variants, which were characterized along with the wild-type enzyme in terms of their substrate spectrum, thermostability and biocatalytic performance in the presence of different co-solvents. We found that the new enzyme variants have a significantly higher thermostability (up to 22 °C increase in T₅₀) and a different co-solvent-dependent activity. Using the most stable variant immobilized in packed-bed reactors via the SpyCatcher/SpyTag system, (R)-benzoin was synthesized from benzaldehyde over a period of seven days with a stable space-time-yield of 9.3 mmol ⋅ L^-1 ⋅ d⁻¹. Our work expands the important class of benzaldehyde lyases and therefore contributes to the development of continuous biocatalytic processes for the production of α-hydroxy ketones and APIs.     

The Effects of Insect Extracts and Some Insect-Derived Compounds on the Settling Behavior of Liposcelis bostrychophila

Extracts of whole booklice (Liposcelis bostrychophila)—sequentially extracted in hexane and aqueous 80% methanol (80%MeOH)—repel conspecifics. A methanol-soluble fraction (MFr) of the 80% methanol extract was more repellent than either its corresponding water fraction (WFr) or the hexane extract. The repellent effect of the MFr was repeatable across extracts prepared on different occasions over a 1 month period. Gas chromatography, mass-spectrometry (GC-MS) analyses showed that saturated (C₁₆; C₁₈) monoenoic (C_16:1; C_18:1) and a dienoic fatty acid (C_18:2) and the corresponding methyl esters of all but C_16:1 and C₁₈ constituted approximately 95% and 30%, of the detected compounds in the methanol fractions and the hexane extract, respectively. Qualitative thin layer chromatography showed that cholesterol was present in methanol fractions and the hexane extract, and also enabled tentative identification of triacylglycerols and phospholipids in the methanol fractions. Extracts of wheatgerm, dried skimmed milk powder, active yeast, and wholemeal flour—L. bostrychophila dietary components—were analyzed by GC-MS, and C₁₆, C_18:1 and C_18:2 were detected, indicating that C₁₈ and the methyl esters were not directly extractable and/or that they were products of booklice metabolism. A fatty acid amide (stearamide) previously identified in cuticular extracts of L. bostrychophila was not detected, and therefore was not responsible for the observed biological activity. Pure fatty acids and fatty acid methyl esters repelled settling of L. bostrychophila at 10 mM, with the exception of palmitic and stearic acids, indicating, among other things, a difference between the efficacy of saturated and unsaturated fatty acids. The effect of concentrations <10 mM was less significant, although palmiteoleic acid appeared to be attractive to L. bostrychophila at 0.1 mM. Fatty acids and fatty acid methyl esters were at a much lower concentration than 10 mM in the repellent methanol fractions, indicating that an interaction between known and as yet unidentified compounds is likely. The significance of fatty acids in relation to the biology and behavior of L. bostrychophila and their potential for use in traps and monitoring are discussed.     

Engineering Leifsonia Alcohol Dehydrogenase for Thermostability and Catalytic Efficiency by Enhancing Subunit Interactions

Leifsonia alcohol dehydrogenase (LnADH) is a promising biocatalyst for the synthesis of chiral alcohols. However, limitations of wild-type LnADH observed for practical application include low activity and poor stability. In this work, protein engineering was employed to improve its thermostability and catalytic efficiency by altering the subunit interfaces. Residues T100 and S148 were identified to be significant for thermostability and activity, and the melting temperature (ΔT_m) and catalytic efficiency of the mutant T100R/S148I toward ketone substrates was improved by 18.7 °C and 1.8–5.5-fold. Solving the crystal structures of the wild-type enzyme and T100R/S148L revealed beneficial effects of mutations on stability and catalytic activity. The most robust mutant T100R/S148I is promising for industrial applications and can produce 200 g liter⁻¹ day⁻¹ chiral alcohols at 50 °C by only a 1 : 500 ratio of enzyme to substrate.     

Reactions of ethylene oxide and ethanol in the presence of amberlyst 15: Products and product rate modeling

The reactions of ethylene oxide and ethanol catalyzed by Amberlyst 15 (H⁺) were studied at 60°, 90°, and 120°C. The concentration ranges investigated at 90°C were 0.9–5 and 0.3–6 mol/1 for the oxide and ethanol, respectively, while the mass of resin taken was in the range of 60 to 28 g. It was found that ethylene oxide reacts with the sulfonic acid groups of the resin to form polymeric ester species which cause catalyst deactivation. Despite the deactivation several reactions occur at 90°C yielding 2-ethoxyethanol, 2-(2-ethoxyethoxy)-ethanol and 1,4-dioxane. Two parallel reaction paths and the overall rate model r = r = C₁C₂C₃/(β₁C + β₂C₂) are proposed for the formation of 2-ethoxyethanol. A back-biting mechanism is tentatively proposed for the formation of 1,4-dioxane from the polymer ester species and the model r = βC₃ for its rate of formation.     

Synthesis and study of the oligo(ether–ester)s based on butylene oxide

Unsaturated oligo(ether–ester) was prepared by ring‐opening polymerization of butylene oxide (BO) with glycidyl methacrylate (GMA) in the presence of cationic catalyst BF₃^?O(C₂H₅)₂. The effects of mol ratio of the initial components, amount of catalyst, temperature, and reaction times on the copolymerization reaction were examined. Oligo(ether–ester) were achieved in highest yield of 82% after reaction times of 4 h at 0°C. Synthesized copolymer was characterized using spectroscopic (¹H NMR and IR) and chemical analysis methods. The thermal degradation and softing points of oligo(ether–ester)‐styrene composites were studied. It is shown that crosslinking of the unsaturated oligo(ether–ester) with styrene takes place through the use of thermostable materials. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008.     

Graphite intercalation compound of magnesium fluoride and fluorine

A graphite intercalation compound of C_xF(MgF₂)_y was prepared under a fluorine atmosphere of 1 atm at temperatures of 20–350°C. The 1st stage compound has the identity period of 9.3–9.4Å. ESCA and ¹⁹F NMR spectra indicate that the chemical interaction of intercalated fluorine with carbon is similar to that for graphite fluoride, however, with C_xF(MgF₂)_y having slightly mobile fluorine atoms chemically adsorbed on carbon atoms of graphite layers.     

Preparation and characterisation of BaTiO3 based PTC ceramics with high performance

Abstract

The nanocrystalline BaTiO₃ based PTC powders had been prepared by a simple sol–gel method starting from comparatively cheap raw materials. The powders and ceramics were characterised by thermogravimetry–differential scanning calorimetry, X-ray diffraction and scanning electron microscopy, while electronic properties of the ceramics were also studied. The good material electronic properties were obtained, including a Curie temperature (T _C) of 100°C, a resistivity at room temperature (ρ _25°C) of 18 Ω cm, a high resistivity step ratio (ρ _max/ρ _min) of 1·2×10⁶, a temperature coefficient of resistivity (α ₃₀) of 17% °C^?1 and a withstand voltage intensity (V _b) of 196 V mm^?1.     

Preparation and characterization of Pb(Lu1/2Nb1/2)O3–Pb(In1/2Nb1/2)O3–PbTiO3 ternary ferroelectric ceramics with high phase transition temperatures

To explore new relaxor‐PbTiO₃ systems for high‐power and high‐temperature electromechanical applications, a ternary ferroelectric ceramic system of Pb(Lu_1/2Nb_1/2)O₃–Pb(In_1/2Nb_1/2)O₃–PbTiO₃ (PLN–PIN–PT) have been investigated. The phase structure, dielectric, piezoelectric, and ferroelectric properties of the as‐prepared PLN–PIN–PT ceramics near the morphotropic phase boundary (MPB) were characterized. A high rhombohedral‐tetragonal phase transition temperature T_R‐T of 165°C and a high Curie temperature T_C of 345°C, together with a good piezoelectric coefficient d₃₃ of 420 pC/N, were obtained in 0.38PLN–0.20PIN–0.42PT ceramics. Furthermore, for (0.8?x)PLN–0.2PIN–xPT ceramics, the temperature‐dependent piezoelectric coefficients, coercive fields and electric‐field‐induced strains were further studied. At 175°C, their coercive fields were found to be above 9.5 kV/cm, which is higher than that of PMN–PT and soft P5H ceramics at room temperature, indicating PLN–PIN–PT ceramics to be one of the promising candidates in piezoelectric applications under high‐driven fields. The results presented here could benefit the development of relaxor‐PbTiO₃ with enhanced phase transition temperatures and coercive fields.     

α‐Thiophene end‐capped styrene copolymer containing fullerene pendant moieties: Synthesis,characterization, and gas sensing properties

We report the synthesis, characterization, and gas sensing properties of a styrene copolymer bearing α‐thiophene end group and fullerene (C₆₀) pendant moieties P(S‐co‐CMS‐C₆₀). First, the copolymer of styrene (S) and chloromethylstyrene (CMS) monomers was prepared in bulk via a bimolecular nitroxide‐mediated radical polymerization (NMP) technique using benzoyl peroxide (BPO) as the radical initiator and nitroxy‐functional thiophene compound (Thi‐TEMPO) as the co‐radical and this gave α‐thiophene end‐capped copolymer P(S‐co‐CMS). The chloromethylstyrene units of P(S‐co‐CMS) allowed further side‐chain functionalization onto P(S‐co‐CMS). The obtained P(S‐co‐CMS) was then reacted with sodium azide (NaN₃) and this led to the copolymer with pendant azide groups, P(S‐co‐CMS‐N₃), and then grafted with electron‐acceptor C₆₀ via the reaction between N₃ and C₆₀. The final product was characterized by using NMR, FTIR, and UV–vis methods. Electrical characterization of P(S‐co‐CMS‐C₆₀) thin film was also investigated at between 30 and 100 °C as the ramps of 10 °C. Temperature dependent electrical characterization results showed that P(S‐co‐CMS‐C₆₀) thin film behaves like a semiconductor. Furthermore, P(S‐co‐CMS‐C₆₀) was employed as the sensing layer to investigate triethylamine (TEA), hydrogen (H₂), acetone, and ethanol sensing properties at 100 °C. The results revealed that P(S‐co‐CMS‐C₆₀) thin film has a sensing ability to H₂. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43641.     

Bi(Zn2/3Nb1/3)O3–(K0.5Na0.5)NbO3 High‐Temperature Lead‐FreeFerroelectric Ceramics with Low Capacitance Variation in a Broad Temperature Usage Range

The xBi(Zn_2/3Nb_1/3)O₃–(1?x)(K_0.5Na_0.5)NbO₃ (abbreviated as xBZN–(1?x)KNN) ceramics have been synthesized using the conventional solid‐state sintering method. The phase structure, dielectric properties and “relaxorlike” behavior of the ceramics were investigated. The 0.03BZN–0.97KNN ceramics show a broad and stable permittivity maximum near 2000 and lower dielectric loss (≤5%) at a broad temperature usage range (100°C–400°C) and the capacitance variation (ΔC/C_150°C) is maintained smaller than ±15%. The 0.03BZN–0.97KNN ceramics only possess the diffuse phase transition and no frequency dispersion of dielectric permittivity, which indicates that 0.03BZN–0.97KNN ceramics is a high temperature “relaxorlike” ferroelectric ceramics. These results indicate that 0.03BZN–0.97KNN ceramics are excellent promising candidates for preparing high‐temperature multilayer ceramics capacitors.     

Antibacterial efficiency of alkali-free bio-glasses incorporating ZnO and/or SrO as therapeutic agents

A series of seven alkali-free silica-based bioactive glasses (SBG) with ZnO and/or SrO additives (in concentrations of 0–12?mol%) were synthesized by melt-quenching, aiming to delineate a candidate formulation possessing (i) a coefficient of thermal expansion (CTE) similar to the one of titanium (Ti) and its medical grade super-alloys (crucial for the future development of mechanically adherent implant-type SBG coatings) and (ii) antibacterial efficiency, while (iii) conserving a good cytocompatibility. The SBGs powders were multi-parametrically evaluated by X-ray diffraction, Fourier transform infrared and micro-Raman spectroscopy, dilatometry, inductively coupled plasma mass spectrometry, antibacterial (against Staphylococcus aureus and Escherichia coli strains) suspension inhibition and agar diffusion tests, and human mesenchymal stem cells cytocompatibility assays. The results showed that the coupled incorporation of zinc and strontium ions into the parent glass composition has a combinatorial and additive benefit. In particular, the “Z6S4” formulation (mol%: SiO₂—38.49, CaO—32.07, P₂O₅—5.61, MgO—13.24, CaF₂—0.59, ZnO—6.0, SrO—4.0) conferred strong antimicrobial activity against both types of strains, minimal cytotoxicity combined with good stem cells viability and proliferation, and a CTE (~?8.7?×?10^?6 ×?°C^?1) matching well those of the Ti-based implant materials.     

Preparation of Rh-containing cellulose acetate films and the chemistry of Rh in cellulose acetate

RhCl [P(C₆H₅)₃]₃ complexes have been incorporated in cellulose acetate as a dispersion medium using cosolvent (tetrahydrofuran). The interactions between Rh (I) complexes and cellulose acetate (CA) are examined by infrared spectroscopy and thermal analysis. The chemical reactivities of Rh–CA films have been investigated by reacting Rh sites with CO, H₂, O₂, and C₂H₄ in the temperature range 90–150°C and at a pressure of less than 1 atm. Three different Rh-carbonyls and a Rh-hydride species formed in CA are characterized by their infrared spectra. Treatment of 10 or 20 wt % Rh–CA films with hydrogen (600 torr) at 150°C produces small Rh metal particles of ca. 10 Å or less in diameter in CA, which show catalytic activities under mild conditions in various reactions such as hydrogenation of C₂H₄, oxidation of CO, and Fischer–Tropsch type reactions.     

Thermal and thermo‐oxidative stability of thermoplastic polymer nanocomposites with arylated [60]fullerene derivatives

Nanocomposite films of polystyrene (PS) and poly(methyl methacrylate) (PMMA) were prepared by loading four variations of fullerenes such as pristine C₆₀, multiarylated [60]fullerenes with tolyl (tolyl‐C₆₀) and phenol groups (phenol‐C₆₀), and [6,6]‐phenyl‐C61‐butyric acid methyl ester (PCBM). The TGA analysis showed no appreciable change in their thermal and thermo‐oxidative stabilities for PS/tolyl‐C₆₀ and PS/phenol‐C₆₀ films, but significant improvement up to +45°C for PS/C₆₀ and PS/PCBM films even under air. The thermo‐oxidative stability of PMMA/phenol‐C₆₀ and PMMA/PCBM, however, exhibited slightly larger improvements over that of PMMA/C₆₀. We believe that the radical‐scavenging ability of π‐conjugative fullerenes and the dispersibility of fullerene–polymer combinations play key roles in these enhancements. We also found that optimal loading occurred at a relatively low content of fullerenes (0.4–0.8 wt%) probably because larger amounts may interfere with the morphological interaction of polymer chains which is essential for the thermal persistency of polymer. POLYM. COMPOS. 37:1143–1151, 2016. © 2014 Society of Plastics Engineers     

Synthesis of C60‐bonded polystyrene initiated with C60Cln/Ni(naph)2/P(Ph)3

A linear‐shaped polystyrene with C₆₀ core was prepared with a novel initiator system, C₆₀Cl_n (n average value is 20)/Ni(naph)₂/P(Ph)_3, and T_p was 130 °C. The results of gel‐permeation chromatography detected by UV detector and fluorescence spectrum of C₆₀‐PSt demonstrated that C₆₀ was chemically bonded to polystyrene. The linear increasing of molecular weights (M_n,GPC) with conversion indicated that this novel initiator system had some characters of living polymerization. A bathochromic shift was found in the UV–vis spectra curves with increasing concentration of C₆₀‐PSt in THF solution. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 98: 1215–1218, 2005     

Extraction,purification and characterization of wax from flax (Linum usitatissimum) straw

The chemical composition and selected physical parameters of wax extracted from flax straw with supercritical CO₂ (SC‐CO₂) and hexane have been determined. From the GC/MS results, clear variations in composition and component distributions were observed between SC‐CO₂‐ and hexane‐extracted samples. The major components of the SC‐CO₂ and hexane extracts from three flax cultivars were: fatty acids (36–49%), fatty alcohols (20–26%), aldehydes (10–14%), wax esters (5–12%), sterols (7–9%) and alkanes (4–5%). Purification of SC‐CO₂‐extracted wax with silica gel chromatography yielded 0.4–0.5% (dry matter) and was composed primarily of wax esters (C₄₄, C₄₆ and C₄₈) and alkanes (C₂₇, C₂₉ and C₃₁). UV‐Vis scans of the purified wax samples exhibited two main peaks indicating the presence of conjugated dienes and carotenoids or related compounds. Fourier transform infrared results showed prominent peaks at 2918 (‐C‐H), 2849 (‐C‐H), 1745 (‐C=O), 1462 (‐C‐H), 1169 (‐C‐O) and 719 cm^–1 (‐(CH₂)_n‐), with NorLin wax showing a slightly deviating pattern compared to the other samples. Thermal analysis by differential scanning calorimetry revealed a mean melting point of 55–56 °C and oxidation temperatures of 146–153 °C for purified wax from flax straw processed using different procedures.     

Mechanical properties of C58 materials and their dependence on thermal treatment

C₅₈ fullerene cages made by electron-impact induced fragmentation of C₆₀ fullerenes have been assembled into several micron thick solid films by low energy cluster beam deposition onto inert substrates held at room temperature under ultrahigh vacuum. The resulting as-prepared material, RT-C₅₈, behaves as an amorphous wide-band semiconductor. Nanoindentation was used to measure its mechanical properties revealing that RT-C₅₈ has a higher elastic modulus E and hardness H than the reference carbon allotropes solid C₆₀ and Highly Ordered Pyrolytic Graphite (HOPG): E(RT-C₅₈) = 14 GPa and H(RT-C₅₈) = 1.2 GPa. This effect can be explained by the unique intrinsic “functionalization” of C₅₈ cages: they comprise reactive surface sites constituted by annelated pentagon rings which give rise to covalently stabilized oligomers, –C₅₈–C₅₈–C₅₈, under our deposition conditions. Annealing, thick RT-C₅₈ films up to 1100 K in ultrahigh vacuum results in HT-C₅₈, a new material with considerably modified electronic and vibrational properties compared to the as-prepared RT-C₅₈ film. The associated molecular transformations, including also partial cage–cage coalescence reactions, raise the overall mechanical hardness of the material: H(HT-C₅₈) = 3.9 GPa.