Increasing the thermostability of sucrose phosphorylase by a combination of sequence- and structure-based mutagenesis

Sucrose phosphorylase is a promising biocatalyst for the glycosylation of a wide variety of acceptor molecules, but its low thermostability is a serious drawback for industrial applications. In this work, the stability of the enzyme from Bifidobacterium adolescentis has been significantly improved by a combination of smart and rational mutagenesis. The former consists of substituting the most flexible residues with amino acids that occur more frequently at the corresponding positions in related sequences, while the latter is based on a careful inspection of the enzyme's crystal structure to promote electrostatic interactions. In this way, a variant enzyme could be created that contains six mutations and whose half-life at the industrially relevant temperature of 60 °C has more than doubled compared with the wild-type enzyme. An increased stability in the presence of organic co-solvents could also be observed, although these effects were most noticeable at low temperatures.     

Ferroceneboronic acid for the electrochemical probing of interactions involving sugars

Ferroceneboronic acid (FcBA) was used as a redox-active probe suitable for monitoring of diol–boronate interactions. Voltammetric and amperometric measurements allowed to detect FcBA forms – free and bound in the boronate complex. In this way, the complexation interaction was studied for a set of saccharide molecules as model diols and the corresponding affinity equilibrium constants were determined. A shift of the peak potential on voltammograms accompanying formation of the boronate complex with FcBA was proposed as a probe for electrochemical characterization of surface-confined diol-containing structures. The model experiments were carried out using sorbitol- and 1,6-hexandiol-modified polyepichlorhydrin conjugates deposited on the electrodes; the former compound was able to form the boronate complex while no change of the peak potential for the latter conjugate was observed. This approach seems promising for artificial bioelectronic affinity receptors and technology of reagentless biosensors where the binding interaction directly stimulates a measurable electrochemical event.     

<Emphasis Type="Italic">N</Emphasis>-Acylated Bacteriohopanehexol-Mannosamides from the Thermophilic Bacterium <Emphasis Type="Italic">Alicyclobacillus acidoterrestris</Emphasis>

Identification of molecular species of various N-acylated bacteriohopanehexol-mannosamides from the thermophilic bacterium Alicyclobacillus acidoterrestris by semipreparative HPLC and by RP-HPLC with ESI is described. We used triple-quadrupole type mass spectrometer, ¹H and ¹³C NMR for analyzing this complex lipid. CD spectra of two compounds (model compound—7-deoxy-d-glycero-d-allo-heptitol obtained by stereospecific synthesis, and an isolated derivative of hopane) were also measured and the absolute configuration of both compounds was determined. On the basis of all the above methods, we identified the full structure of a new class of bacteriohopanes, represented by various N-acylated bacteriohopanehexol-mannosamides.     

Polymer chain mobility in polyelectrolyte multilayers

Free-standing polyelectrolyte multilayer membranes have been formed by the layer-by-layer technique using a dip-coating apparatus. The polymer-chain mobility has been studied by ¹H relaxation in the rotating frame T _1rho NMR with ¹³C chemical shift resolution. For each of the individual polymers a single relaxation component has been observed for all resolved signals. In the multilayer a significantly different relaxation time T _1rho has been observed with a minor second component. The interaction between the oppositely charged polyelectrolytes influences the molecular mobility.     

Lipidomic Study of Precursors of Endocannabinoids in Freshwater Bryozoan Pectinatella magnifica

Freshwater bryozoan Pectinatella magnifica was collected from a sand pit (South Bohemia). The total lipids after extraction from lyophilized bryozoans were analyzed using high‐performance liquid chromatography/high‐resolution negative tandem electrospray mass spectrometry. A total of 19 lipid classes were identified, including N‐acyl‐substituted phospholipids, that is, N‐acylphosphatidylethanolamine and N‐acylphosphatidylserine in their plasmenyl forms. Based on gas chromatography/mass spectrometry of 3‐pyridylcarbonyl (picolinyl) esters, a very unusual fatty acid was identified, namely 24:7n‐3 (all‐cis‐3,6,9,12,15,18,21‐tetracosaheptaenoic acid). The presence of polyunsaturated fatty acids in individual classes is very specific: arachidonic and eicosapentaenoic acids being predominantly bound as amides in N‐acyl phospholipids, that is, diacyl‐N‐acylphosphatidylethanolamines (NAPtdEtn), plasmenyl‐N‐acylphosphatidyl ethanolamines (PlsNAPtdEtn), diacyl‐N‐acylphosphatidylserines (NAPtdSer), and plasmenyl‐N‐acylphosphatidylserines (PlsNAPtdSer). While 24:6n‐3 was identified in the sn‐2 position of several phospholipids, 24:7n‐3 was identified in only two plasmalogens, that is, PlsNAPtdEtn and PlsNAPtdSer. Thanks to the tandem mass spectrometry, we managed to identify the position of all acyl groups in both diacyl‐ and also in alkenyl‐acyl‐(plasmenyl) molecular species of N‐acylphospholipids. The identification of the molecular species of N‐acyl‐substituted phosphatidylethanolamine and phosphatidylserine, including their plasmalogen forms, in the freshwater bryozoan P. magnifica has enabled the identification of endogenous cannabinoid precursors.     

Biodiesel from rapeseed oil,methanol and KOH. 3. Analysis of composition of actual reaction mixture

Detailed analysis is described of the samples taken after suitable reaction times from the actual reaction mixture during the production of biodiesel fuel using methanolysis of rapeseed oil catalyzed by KOH. Three methods for stoppage of reaction (neutralisation of catalyst, dilution by two suitable solvents) in the sample are used. The contents of mono‐, di‐ and triacylglycerols, methylesters of fatty acids (biodiesel) and potassium salts of fatty acids of rapeseed oil, glycerol (by HPLC method), basicity (by potentiometric titration) and water (by GC and Karl‐Fischer method) in the samples are determined. An example of these determinations is described.     

Polymer anion-selective membranes for electrolytic splitting of water. Part II: Enhancement of ionic conductivity and performance under conditions of alkaline water electrolysis

An attempt was made to increase the ionic conductivity of novel, heterogeneous, anion-selective membranes by increasing the porosity of their surface skin. This was based on the addition of a water-soluble component, namely poly(ethylene-ran-propylene glycol), to an inert polymer matrix, based on low-density polyethylene, while mixing it with the ion-exchange particles. A series of membranes was prepared, consisting of 66 wt% of anion-exchange phase represented by a styrene-divinyl benzene copolymer matrix with quaternary ammonium functional groups and an inert polymer matrix in a mixture with variable amounts of water-soluble component added. The membranes were subsequently tested with respect to their morphology, mechanical properties, apparent ion-exchange capacity, ionic conductivity, and performance under conditions of alkaline water electrolysis. When added in the appropriate amount, the addition of a water-soluble component was found to improve the electrochemical properties of the resulting membrane efficiently, while at the same time not reducing its mechanical properties to below a critical level.     

The influence of electrolyte composition on electrochemical ferrate(VI) synthesis. Part III: anodic dissolution kinetics of a white cast iron anode rich in iron carbide

The anolyte composition and process temperature could improve the kinetics of iron anode dissolution and subsequent ferrate(VI) production significantly. This also holds for the anode composition. Following pure iron and silicon-rich steel (SRS), white cast iron (WCI) was the last representative of anode material tested that is typically used to produce ferrate(VI). Using anolytes 14 M NaOH, 14 M KOH, and mixtures thereof, the systems were studied by potentiodynamic methods, electrochemical impedance spectroscopy, and batch electrolysis experiments. Additionally, metallographic analysis of the material was performed. The dissolution kinetics increases with increasing temperature and also, at 60 °C, with increasing K⁺ content in the anolyte, but less progressively than in the case of SRS. Similar to SRS, WCI also easily dissolves into ferrate(VI) even at 20 °C in pure NaOH, thus indicating the inferior protective properties of oxo-hydroxide surface layers. In general, a maximum current efficiency of approx. 60 % was obtained at 60 °C in pure KOH solution. The authors conclude that, at 60 °C, the high efficiency of the synthesis is caused by the low protective properties of the oxo-hydroxide surface layer caused by the preferential dissolution of cementite and at the same time by the precipitation of the potassium salt of the product in the electrolyte immediately after its formation. This minimizes the effect of its decomposition.     

Evaluation of an Improved Indirect Method for the Analysis of 3-MCPD Esters Based on Acid Transesterification

The quantification of 3-chloropropane-1,2-diol (3-MCPD) esters in fat-based matrices is currently carried out according to a number of methods that involve either the conversion of all the esters to the free form that is then quantified (indirect methods), or the separation and quantification of the individual esters (direct methods). Indirect methods of analysis show a better sensitivity, however, the series of chemical reactions that take place during sample preparation may affect the reliability of the results because of the potential ex-novo formation of 3-MCPD from precursors present in the sample. This study is focused on the evaluation of the selectivity and robustness of an indirect acid-catalysed method of 3-MCPD esters analysis. The interference of chloride ions and glycidyl esters was evaluated. 3-MCPD esters are overestimated only when high levels of chloride ions (>1.7 mmol/kg oil) were added to the samples. The interference by chloride ions can be easily eliminated by a single extraction step of the samples before analysis. In contrast, glycidyl esters did not interfere with the determination of 3-MCPD esters. Further investigation on the robustness of the method showed that the time allowed for the transesterification, a major drawback of the previous version of the method, can be reduced from 16 to 4 h without any significant reduction of the accuracy and repeatability (RSD = 0.7%).     

Silica nanoparticles inhibit the cation channel TRPV4 in airway epithelial cells

Background

Silica nanoparticles (SiNPs) have numerous beneficial properties and are extensively used in cosmetics and food industries as anti-caking, densifying and hydrophobic agents. However, the increasing exposure levels experienced by the general population and the ability of SiNPs to penetrate cells and tissues have raised concerns about possible toxic effects of this material. Although SiNPs are known to affect the function of the airway epithelium, the molecular targets of these particles remain largely unknown. Given that SiNPs interact with the plasma membrane of epithelial cells we hypothesized that they may affect the function of Transient Receptor Potential Vanilloid 4 (TRPV4), a cation-permeable channel that regulates epithelial barrier function. The main aims of this study were to evaluate the effects of SiNPs on the activation of TRPV4 and to determine whether these alter the positive modulatory action of this channel on the ciliary beat frequency in airway epithelial cells.

Results

Using fluorometric measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i) we found that SiNPs inhibit activation of TRPV4 by the synthetic agonist GSK1016790A in cultured human airway epithelial cells 16HBE and in primary cultured mouse tracheobronchial epithelial cells. Inhibition of TRPV4 by SiNPs was confirmed in intracellular Ca²⁺ imaging and whole-cell patch-clamp experiments performed in HEK293T cells over-expressing this channel. In addition to these effects, SiNPs were found to induce a significant increase in basal [Ca²⁺]_i, but in a TRPV4-independent manner. SiNPs enhanced the activation of the capsaicin receptor TRPV1, demonstrating that these particles have a specific inhibitory action on TRPV4 activation. Finally, we found that SiNPs abrogate the increase in ciliary beat frequency induced by TRPV4 activation in mouse airway epithelial cells.

Conclusions

Our results show that SiNPs inhibit TRPV4 activation, and that this effect may impair the positive modulatory action of the stimulation of this channel on the ciliary function in airway epithelial cells. These findings unveil the cation channel TRPV4 as a primary molecular target of SiNPs.