Increased yield of enzymatic synthesis by chromatographic selection of different N-glycoforms of yeast invertase

Invertases are glycosidases applied for synthesis of alkyl glycosides that are important and effective surfactants. Stability of invertases in the environment with increased content of organic solvent is crucial for increase of productivity of glycosidases. Their stability is significantly influenced by N-glycosylation. However, yeast N-glycosylation pathways may synthesize plethora of N-glycan structures. A total natural crude mixture of invertase glycoforms (EINV) extracted from Saccharomyces cerevisiae was subfractionated by anion-exchange chromatography on industrial monolithic supports to obtain different glycoforms (EINV1–EINV3). Separated glycoforms exhibited different stabilities in water-alcohol solutions that are in direct correlation with the amount of phosphate bound to N-glycans. Observed differences in stability of different invertase glycoforms were used to improve productivity of methyl β-d -fructofuranoside (MF) synthesis. The efficiency and yield of MF synthesis were improved more than 50% when the most stabile glycoform bearing the lowest amount of phosphorylated N-glycans is selected and utilized. These data underline the importance of analysis of glycan structures attached to glycoproteins, demonstrate different impact of N-glycans on the surface charge and enzyme stability in regard to particular reaction environment, and provide a platform for improvement of yield of industrial enzymatic synthesis by chromatographic selection of glycoforms on monolithic supports.     

An Integrated approach (thermodynamic, structural, and computational) to the study of complexation of alkali-metal cations by a lower-rim calix[4]arene amide derivative in acetonitrile

The calix[4]arene secondary-amide derivative L was synthesized, and its complexation with alkali-metal cations in acetonitrile (MeCN) was studied by means of spectrophotometric, NMR, conductometric, and microcalorimetric titrations at 25 °C. The stability constants of the 1:1 (metal/ligand) complexes determined by different methods were in excellent agreement. For the complexation of M(+) (M = Li, Na, K) with L, both enthalpic and entropic contributions were favorable, with their values and mutual relations being quite strongly dependent on the cation. The enthalpic and overall stability was the largest in the case of the sodium complex. Molecular and crystal structures of free L, its methanol and MeCN solvates, the sodium complex, and its MeCN solvate were determined by single-crystal X-ray diffraction. The inclusion of a MeCN molecule in the calixarene hydrophobic cavity was observed both in solution and in the solid state. This specific interaction was found to be stronger in the case of metal complexes compared to the free ligand because of the better preorganization of the hydrophobic cone to accept the solvent molecule. Density functional theory calculations showed that the flattened cone conformation (C(2) point group) of L was generally more favorable than the square cone conformation (C(4) point group). In the complex with Na(+), L was in square cone conformation, whereas in its adduct with MeCN, the conformation was slightly distorted from the full symmetry. These conformations were in agreement with those observed in the solid state. The classical molecular dynamics simulations indicated that the MeCN molecule enters the L hydrophobic cavity of both the free ligand and its alkali-metal complexes. The inclusion of MeCN in the cone of free L was accompanied by the conformational change from C(2) to C(4) symmetry. As in solution studies, in the case of ML(+) complexes, an allosteric effect was observed: the ligand was already in the appropriate square cone conformation to bind the solvent molecule, allowing it to more easily and faster enter the calixarene cavity.     

Isomorphism Checking of I-graphs

We consider the class of I-graphs, which is a generalization of the class of the generalized Petersen graphs. We show that two I-graphs I(n, j, k) and I(n, j ₁, k ₁) are isomorphic if and only if there exists an integer a relatively prime to n such that either {j ₁, k ₁} =? {a j mod n, a k mod n } or {j ₁, k ₁} =? {a j mod n, ? a k mod n }. This result has an application in the enumeration of non-isomorphic I-graphs and unit-distance representations of generalized Petersen graphs.     

Comparative studies of methylmercury determination in biological and environmental samples

Some parameters affecting the accuracy of various approaches to methylmercury (MeHg) determination in biological and environmental samples were studied. Different isolation techniques (ion-exchange, extraction, volatilization, distillation) and final measurement via cold vapour atomic absorption spectroscopy (CV AA) or gas chromatography (GC) were compared. Results obtained by the various isolation techniques are comparable for almost all biological and environmental samples, except for soils and some sediments, where disagreement between the results obtained by GC and CV AA was found. In order to resolve these problems, a new separation technique based on distillation of MeHg from the sample followed either by CV AA or GC was developed. The new method results in very good recovery and reproducibility (95 ± 2%) for all samples examined (fish, mussel, shrimp, blood, hair, algae, sediment, etc.), is specific for MeHg and provides for its differentiation from other species by an indirect CV AA determination. Gas-chromatographic measurement of the isolated MeHg using different packings and conditioning of the columns is also discussed. The distillation method with GC detection is advantageous in producing cleaner chromatograms and in prolonging the life-time of the packing and the intervals between reconditioning.     

Mechanism of levulinic acid formation

Based on the intermediates revealed by ¹³C-NMR spectroscopy, the mechanism of levulinic acid ($\text{6}$) formation in acid catalysed hydrolysis of 2-hydroxymethylfurane ($\text{1}$) and 5-hydroxymethylfuran-2-carbaldehyde ($\text{14}$) is proposed.     

The early glycation products of the Maillard reaction: mass spectrometric characterization of novel imidazolidinones derived from an opioid pentapeptide and glucose

Glucose-substituted imidazolidinones related to the endogenous opioid peptide leucine-enkephalin have been investigated using fast atom bombardment tandem mass spectrometry (FAB-MS/MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). In addition to Amadori compounds, the studied imidazolidinones represent a novel type of the early glycation products formed in the Maillard reaction. To obtain insight into the fragmentation behavior of these carbohydrate-peptide adducts, we also studied synthetic precursors of the glucose-substituted imidazolidinones as well as the corresponding isopropylidene derivatives. The collision-induced dissociation (CID) spectra of [M + H](+) ions of all these imidazolidinones have been compared. Detailed analysis showed that fragmentation of each compound generates two ions at m/z 566 and m/z 598 which are characteristic and undoubtedly confirm the imidazolidinone-type structure. These two significant ions were identified as the M + 10 and M + 42 modifications of the N-terminus of the parent opioid pentapeptide effected by the carbohydrate moiety. Furthermore, the ion at m/z 178 is identified as the M + 42 modification of the immonium ion of the N-terminal amino acid (tyrosine) also effected by the carbohydrate moiety. They can be used as diagnostic ions for imidazolidinone-type compounds in studying the Maillard reaction. Thus, we have demonstrated the utility of FAB-MS/MS and ESI-MS/MS in the structural determination and identification of such novel peptide-carbohydrate adducts, useful in understanding the details of the mechanism of non-enzymatic glycation in vivo.     

Nonleptonic kaon decays and nonperturbative QCD effects in chiral-bag model

The instanton induced-term, proposed by Konishi and Ranfone [1] was introduced in the chiral-bag model based analysis ofK→2π decays. Almost perfect fit of the experimental data was possible with the induced-term coefficient \(\tilde C_1 \cong 1,4\) . However, this conclusion, which is appreciably larger than the rough theoretical estimate [1] (which was \(\tilde C_1 = 0.15\) m _s /μ) depends on the approximation used in the theoretical approach.     

Thermal behaviour of diclofenac sodium: decomposition and melting characteristics

The thermal behaviour and melting characteristics of diclofenac sodium were investigated using various instrumental techniques--differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), Fourier-transform infrared (FT-IR) spectroscopy and thin layer chromatography (TLC). DSC analysis of diclofenac sodium performed under dynamic flow of either synthetic air or helium or nitrogen did not produce any sharp endothermic peak characteristic of melting peak of a pure substance. Both the rate of scanning of the sample and the environmental atmospheric condition significantly affected the thermographic profile of diclofenac sodium. An exothermic peak prior to an endothermic peak corresponding to melting of the substance appeared when heated under dynamic flow of synthetic air suggesting oxidation (decomposition) of diclofenac sodium before reaching its melting point. In fact, at a scanning rate of 1 degree C/min only the exothermic peak appeared in the thermogram, suggesting complete decomposition prior to melting under the dynamic flow of synthetic air. DSC, FT-IR and TLC data obtained from samples heated under the dynamic flow of either helium or nitrogen revealed formation of a related compound, 1-(2,6-dichlorophenyl)-indolin-2-one, an indol-cyclic amide, as a result of an intramolecular cyclization reaction during the heating process. TGA data demonstrated a loss of 11.4-20.2% of the mass of diclofenac sodium when heated under various environmental conditions, and also supported the oxidative nature of degraded product(s) when the thermal process occurred slowly under a dynamic flow of synthetic air.