Band 66, Heft 9

The surface activity of Cryptococcus laurentii α-galactosidase can be significantly (up to 80 times) influenced by a carbon source present in a cultivation medium. Induction of this enzyme is stimulated by the presence of saccharides containing bound galactose. The highest activity observed when the cells grew in a lactose medium was probably a consequence of the absence of cleavage products serving as repressors (glucose). This idea is supported by the poor growth of cells in the medium with lactose as the carbon source. The induction of surface α-galactosidase was accompanied by increased activities in cytosole. The membrane fraction also contained this enzyme, but the influence of the carbon source was not proportional. The induction of α-galactosidase may play an important role in galactose metabolism of the genus Cryptococcus with a direct influence on the virulence of these capsular yeasts.

Protein extract of germinating nasturtium (Tropaeolum majus) seeds containing xyloglucan endotransglycosylase (xyloglucan xyloglucosyl transferase, EC 2.4.1.207, abbreviated XET) exhibited the heterotransglycosylating activity with donor/acceptor substrate pair xyloglucan/sulphorhodamine labelled pustulooligosaccharides (XG/PUOS-SR) in a dot blot assay. The heterotransglycosylating activity was confirmed by the substrate-product changes during transglycosylation by HPLC size-exclusion chromatography. Another donor substrate capable of being coupled with PUOS-SR was cellulose, probably owing to its structural similarity to xyloglucan. Surprisingly, microscopic comparison of the incorporation of the labelled xyloglucan nonasaccharide XGO9-SR (specific substrate for XET) and PUOS-SR into the cell wall structures clearly showed differences in their binding to specific cell structures: the primary cell wall and the plasma membrane. These findings indicate the existence in nasturtium of XETs with different localisation, substrate specificity and, probably, function.

The microalga Chlorella sorokiniana has been used to accumulate selenium and iodine from culture media enriched with these elements as a first stage in the production of supplemented foods. The microalgal colony was grown in a conventional culture medium containing iodine (KI) at concentrations in the range of 150–4000 μg mL−1. Similar experiments were performed with selenium (SeO42−) at concentrations in the range of 20–500 μg mL−1. The concentration of iodine and selenium in the culture medium was analytically monitored daily and the viability of the colony was checked by biomass concentration measurement and by evaluation of the total content of chlorophyll and carotenoids. In addition, photosynthetic activity and the number of cells were also monitored. Iodine accumulation in the algal biomass increased rapidly with time and reached a steady state after 4 h of exposure. With Se exposure the colony viability decreased, although the culture grew well with concentrations of the element of 50 μg mL−1 in the culture medium; this experiment produced Se-enrichment in the alga (3 μg g−1) within 100 h. Sequential extraction of an algal pellet was performed in order to separate Se compounds according to their affinity with the following solvents: hot water to recover low molecular mass Se species, enzymatic extraction with driselase for species associated with the cell wall, sodium dodecyl sulphate (SDS) for water insoluble selenoproteins and, finally, enzymolysis with lipase and pronase that release and fragment residual selenoproteinsproducing compounds with low molecular mass. Size-exclusion chromatography (SEC) coupled with an ICP-MS detector showed the preponderance of Se-containing molecules with low molecular mass, possibly seleno-amino acids. Only a peak of low intensity located at 10 min was observed in the SDS extract that could be associated with a protein with molecular mass of 67 kDa. Finally, analysis of the aqueous extract of alga by reverse-phase chromatography with inductively-coupled plasma mass-spectrometry (RPC-ICP-MS) detection revealed the presence of selenocysteine (SeCys2), selenomethylselenocysteine (SeMetSeCys), selenomethionine (SeMet), and Se(VI), particularly the last two species.

Alzheimer’s disease is the most common neurodegenerative disease, but there is still no cure and early diagnosis remains very difficult. For this reason, the discovery of new biomarkers is of great importance. The application of metabolomics is emerging in this field, based on the use of mass spectrometry as a technique of analysis. In this work, blood serum samples (from Alzheimer’s disease patients and healthy controls) were analysed by mass spectrometry in order to search for potential metabolomic biomarkers. The application of multivariate statistical tools (PLS-DA) enabled us to discriminate between groups. In addition, some phosphatidylcholine compounds were identified as markers of the disease.

We studied Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) content and activity in juvenile tree species — broadleaved Fagus sylvatica L. and coniferous Picea abies (L.) Karsten exposed for three growing seasons to ambient (A = 385 μmol mol−1) and elevated (E = 700 μmol mol−1) CO2 concentrations. Rubisco content was determined by the SDS-PAGE method, Rubisco activity was assayed spectrophotometrically. The highest content of Rubisco enzyme in F. sylvatica was measured immediately after full leaf development followed by a gradual decrease throughout the growing season. By contrast, Rubisco content in P. abies increased markedly during the growing season. In both tree species, down-regulation of Rubisco content in trees cultivated under elevated CO2 concentration was observed. Rubisco activity was stimulated in F. sylvatica by E treatment but not in P. abies. Because no significant differences were found in Rubisco activation state between A and E, we assume that stimulation of Rubisco activity in E is not a consequence of higher carbamylation but could be caused by the release of inhibitors from active sites of Rubisco under elevated CO2.

Several DNA amplification-based methods were used for identification and evaluation of the relation between lactobacilli isolated from breastfed full-term infant faeces (31 strains), dairy products (5 strains) and silage (1 strain). Twenty-seven strains isolated from infant faeces were identified as Lactobacillus rhamnosus (9), Lactobacillus gasseri (6), Lactobacillus paracasei (4), Lactobacillus fermentum (4), Lactobacillus salivarius (2), Lactobacillus plantarum (1), and Lactobacillus helveticus (1) using 10 species-specific polymerase chain reactions (PCRs), multiplex PCR for the Lactobacillus casei group, and sequencing of 16S rDNA. Four strains were not identified. Six strains isolated from dairy products and silage were identified as Lactobacillus rhamnosus. A repetitive extragenic palindromic polymerase chain reaction (rep-PCR) with primer (GTG)5 and a randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) with primer M13 were used for confirmation of species identification. Fingerprints were used for evaluation of the relatedness of lactobacilli. Differences between strains from infant faeces, dairy products, and silage were not detected.

Geotrichum candidum CCY 16-1-29 (teleomorph Galactomyces geotrichum) is able to grow and produce polygalacturonase of remarkable activities on pectin or grape pomace as a sole carbon source. The highest activities of extracellular enzymes were found on the third and the seventh day of cultivation. After extraction and precipitation, polygalacturonases produced in these cultivation periods were characterized. Production of multiple forms of polygalacturonase was observed in both cultivation periods. Two major forms, polygalacturonase with random action pattern (endo-PGase, EC 3.2.1.15) and oligogalacturonate hydrolase (exoPGase, exopolygalacturonase preferring oligogalacturonides as substrates), as well as numerous minor forms were detected by IEF-PAGE using the print technique detection. EndoPGase was identified by mass spectrometry. The major forms have similar isoelectric points (below pH 6.0) and pH optima (4.6 and 4.8, respectively). pH optimum of 4.6 was associated with exoPGase and that of 4.8 with endoPGase. Both enzymes were stable after freeze-drying and storage at 4°C. EndoPGase had molecular mass of about 29 kDa (36 kDa by SDS-PAGE) as determined by gel filtration, temperature optimum of about 45°C and it was stable only below 35°C. Molecular mass of exoPGase was about 50 kDa, its temperature optimum was about 60°C, and it was stable to 60°C. Optimal substrate for exoPGase was a pentamer, for endoPGase it was a pectate. Values of K m for optimal substrate reached the values of 11.4 × 10−5 M for for exoPGase and 6.6 × 10−5 M for endoPGase. Pectin methylesterase as another pectolytic enzyme was also identified by mass spectrometry.

In enology, yeasts play an important role in the characteristics of the final product. They are predominant in the biochemical interaction with components of must. Rapid identification of the yeast population is necessary for fermentation process monitoring and for obtaining a good quality wine. The main goal of this study was the isolation and characterisation of the yeast microbial community naturally present on grape berries, leaves and occurring during the spontaneous fermentation process of the white wine Veltlin green from the South Moravian region, Czech Republic. The results, based on PCR-RFLP of the 5.8S-ITS region of rDNA, PCR-fingerprinting using microsatellite oligonucleotide primers (GAG)5, (GTG)5, (GAC)5, and M13 primer, showed great diversity of the yeast population. Including grape berries and fermented must, the following yeast species were identified: Hanseniaspora uvarum, Aureobasidium pullulans, Metschnikowia pulcherrima, Torulaspora delbrueckii, a number of Pichia species such as P. fermentans, P. membranifaciens, P. kluyveri, also Sporidiobolus salmonicolor, Rhodosporidium toruloides, Rhodotorula mucilaginosa, Rhodotorula glutinis as well as Saccharomyces cerevisiae and Saccharomyces bayanus. Monitoring of the yeast strains during the wine fermentation process of traditional Moravian wine can contribute to the improvement of wine quality.

In the proposed work, new elastomeric bio-polyol based polyurethanes (bio-PUs) with specific mechanical properties were prepared by a one-shot process without the presence of a solvent. Commercial non-degradable polyether polyol derived from petrochemical feed stock was partly (in the amount of 1 mass %, 5 mass %, and 10 mass %) substituted by the biodegradable polyhydroxybutyrate (PHB). Morphology of elastomeric PU composites was evaluated by scanning electron microscopy and mechanical properties of the prepared samples were obtained by both tensile measurements and prediction via the Mooney-Rivlin equation. Electron microscopy proved that the prepared materials have the character of a particle filled composite material, where PHB particles are regular with their size of about 1–2 μm in diameter. Tensile measurements demonstrated that the Young’s modulus, tensile stress at break, and tensile strain at break of each sample increase with the increase of the volume fraction of the filler. From the measured stress-strain data, the first and the second term of the Mooney-Rivlin equation were calculated. The obtained constants were applied to recalculate the stress-strain curves. It was found that the Mooney-Rivlin equation corresponds well with the stress-strain behavior of the prepared specimens.

Conformational changes in humic acids in two different aqueous solutions (NaCl and NaOH) are studied by means of high resolution ultrasound spectrometry. The method is based on the measurement of parameters of ultrasonic waves propagating through the sample. The attenuation describes the decay of the amplitude of the ultrasonic wave with the distance travelled. The velocity is the speed of this wave and is related to the wavelength and the frequency of oscillation of the deformation. It is determined by the density and elasticity of the sample, which is strongly influenced by the molecular arrangement. The minimal velocity of ultrasound was observed at 1 g dm−3 for lignitic humic acids and at 0.5 g dm−3 for IHSS Leonardite standard. The values of compressibility as computed are almost constant up to humic acids’ content corresponding to the minimum velocity of ultrasound and then decrease with the increase in concentration. This shows that the organisation of particles in diluted and concentrated humic acids sols is different. The decrease in compressibility points to the formation of a more rigid structure, which could lead to the decrease in humic acids’ binding ability. It was confirmed that the method employed was very sensitive and could be utilised as an indicator of conformational changes in humic acids in solutions with varying concentrations.

Three fiber-reinforced cement composites were prepared and cured in an autoclave for up to 168 h at 2 MPa of steam pressure in order to investigate the effect of hydrothermal curing on the alteration of pore structure, density, and formation and stability of hydrated products with time. Compressive strength was reviewed in connection with sample porosity. It was found that the time of autoclaving plays a crucial role in objective assessment of the durability of composites as potential candidates for geothermal applications. A mercury intrusion porosimeter Quantachrome Poremaster 60GT was used for the estimation of the pore structure parameters of composites. The thermal analysis method was used to identify different temperature ranges of cured samples’ thermal decomposition and to characterize the nature of hydrated products. Two kinds of products were formed. The first group consisted of calcium-silicate-hydrate (C-S-H), calcium-silicate-aluminate-hydrate (C-S-A-H), calcium-aluminate-hydrate (C-A-H), and calcium-carbonate (C-C) as a product of carbonation. The second group are chemically bond products, e.g. hydroxyapatite (Ca5(PO4)3(OH)) and gibbsite (Al(OH)3). These two hydroceramic products formed under hydrothermal conditions act also as binders and they can be useful as geothermal cement binders.