Generation of intra- and interspecific Saccharomyces hybrids with improved oenological and aromatic properties

Non-wine yeasts could enhance the aroma and organoleptic profile of wines. However, compared to wine strains, they have specific intolerances to winemaking conditions. To solve this problem, we generated intra- and interspecific hybrids using a non-GMO technique (rare-mating) in which non-wine strains of S. uvarum, S. kudriavzevii and S. cerevisiae species were crossed with a wine S. cerevisiae yeast. The hybrid that inherited the wine yeast mitochondrial showed better fermentation capacities, whereas hybrids carrying the non-wine strain mitotype reduced ethanol levels and increased glycerol, 2,3-butanediol and organic acid production. Moreover, all the hybrids produced several fruity and floral aromas compared to the wine yeast: β-phenylethyl acetate, isobutyl acetate, γ-octalactone, ethyl cinnamate in both varietal wines. Sc × Sk crosses produced three- to sixfold higher polyfunctional mercaptans, 4-mercapto-4-methylpentan-2-one (4MMP) and 3-mercaptohexanol (3MH). We proposed that the exceptional 3MH release observed in an S. cerevisiae × S. kudriavzevii hybrid was due to the cleavage of the non-volatile glutathione precursor (Glt-3MH) to detoxify the cell from the presence of methylglyoxal, a compound related to the high glycerol yield reached by this hybrid. In conclusion, hybrid generation allows us to obtain aromatically improved yeasts concerning their wine parent. In addition, they reduced ethanol and increased organic acids yields, which counteracts climate change effect on grapes.     

A biomimetic chitosan composite with improved mechanical properties in wet conditions

Chitosan is one of the most widely used structural polymers for biomedical applications because it has many favorable properties. However, one of the most critical drawbacks regarding the use of chitosan as a biomedical material is its poor mechanical properties in wet conditions. Here, we designed a method to improve the mechanical properties of chitosan in wet conditions and minimized the swelling behavior of chitosan film due to water adsorption by mimicking the sclerotization of insect cuticles and squid beaks, that is, catechol‐meditated crosslinking. The biomimetic chitosan composite film was prepared by mixing chitosan with l ‐3,4‐dihydroxyphenylalanine (DOPA) as a catecholic crosslinker and sodium periodate as an oxidant. The catechol‐meditated crosslinking provided a sevenfold enhancement in the stiffness in wet conditions compared to pure chitosan films and reduced the swelling behavior of the chitosan film. This strategy expands the possible applications for the use of chitosan composites as load‐bearing biomaterials. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 505–512, 2013     

Protein engineering of a bacterial N-acyl-d-glucosamine 2-epimerase for improved stability under process conditions

Enzymatic cascade reactions, i.e. the combination of several enzyme reactions in one pot without isolation of intermediates, have great potential for the establishment of sustainable chemical processes. However, many cascade reactions suffer from cross-inhibitions and enzyme inactivation by components of the reaction system. This study focuses on the two-step enzymatic synthesis of N-acetylneuraminic acid (Neu5Ac) using an N-acyl-d-glucosamine 2-epimerase from Anabaena variabilis ATCC 29413 (AvaAGE) in combination with an N-acetylneuraminate lyase (NAL) from Escherichia coli. AvaAGE epimerizes N-acetyl-d-glucosamine (GlcNAc) to N-acetyl-d-mannosamine (ManNAc), which then reacts with pyruvate in a NAL-catalyzed aldol condensation to form Neu5Ac. However, AvaAGE is inactivated by high pyruvate concentrations, which are used to push the NAL reaction toward the product side. A biphasic inactivation was observed in the presence of 50–800 mM pyruvate resulting in activity losses of the AvaAGE of up to 60% within the first hour. Site-directed mutagenesis revealed that pyruvate modifies one of the four lysine residues in the ATP-binding site of AvaAGE. Because ATP is an allosteric activator of the epimerase and the binding of the nucleotide is crucial for its catalytic properties, saturation mutagenesis at position K160 was performed to identify the most compatible amino acid exchanges. The best variants, K160I, K160N and K160L, showed no inactivation by pyruvate, but significantly impaired kinetic parameters. For example, depending on the mutant, the turnover number k_cat was reduced by 51–68% compared with the wild-type enzyme. A mechanistic model of the Neu5Ac synthesis was established, which can be used to select the AvaAGE variant that is most favorable for a given process condition. The results show that mechanistic models can greatly facilitate the choice of the right enzyme for an enzymatic cascade reaction with multiple cross-inhibitions and inactivation phenomena.     

Variation of vector potential in laboratory conditions changes biological properties of water

It was found that water preliminary activated with vector potential changes mobility of infusoria brought into it, the rate of sugar fermentation in yeast cells and the drinking attractivity of water for mice. It was also shown that water physical parameters changed after water activation: UV absorption spectrum and state of a silica admixture. It was supposed that water is the initial target when weak electromagnetic fields have an influence on biological objects.     

Variation of vector potential in laboratory conditions changes biological properties of water

It was found that water preliminary activated with vector potential changes mobility of infusoria, bringing into it, the rate of sugar fermentation in yeast cells and a drinking attractivity of water for mice. It was also shown that water physical parameters changed after water activation: UV absorptions spectrum and a state of a silica admixture. It was supposed that water is the initial target when weak electromagnetic fields have an influence on biological objects.     

An improved microindentation technique to measure changes in properties of arterial intima during atherogenesis

A fully automated instrument was developed that measured the amount and time-course of indentation of arterial intima by a spherical tipped probe 25 micrometers in diameter loaded routinely by forces of 10 microN. The relationship of depth of indentation to time was non-exponential but reached an apparent asymptotic value in about 6 s. Routine indentations were recorded 10 s after loading, the relationship of indentation and force being essentially linear between 0-100 microN. The instrument is an order of magnitude more sensitive than one previously described (Gow and Vaishnav, 1975, J. Appl. Physiol. 38, 344-350) and produces indentations of 1-4 micrometers in the intima of rabbit thoracic aorta with a reproducibility of 1-2%. Preliminary usage of the new microindentor has shown increases in the compliance of the thoracic aorta intima in rabbits during cholesterol feeding.     

Synthesis and anti-inflammatory properties of some aromatic and heterocyclic aromatic curcuminoids

A variety of novel aromatic and heterocyclic aromatic curcuminoids were synthesised, characterised and their anti-inflammatory activities (AIA) determined in vivo. Some of these compounds also were tested for inflammatory mediator production. The AIA of the main representatives of these compounds were assessed by oral administration to female Wistar rats using (a) acute carrageenan-induced paw oedema, (b) chronic adjuvant arthritis (therapeutic mode), and (c) anti-pyretic activity assessed in the yeast pyrexia. Gastric ulceration was determined in pre-inflamed rats. Natural curcumin showed modest aspirin-like anti-inflammatory activity which was enhanced when co-administered with the PGE(1) analogue misoprostol as a synergist. In contrast, four novel curcuminoids (RK-97, RK-103, RK-104 and RK-106) in which the bis-methoxy-phenyl group of curcumin was replaced with bis-dimethoxybutenolidyl-(ascorbate), bis-naphthyl, and bis-furanyl derivatives, respectively, had potent activity in the anti-arthritic assay with little gastric or systemic toxicity, compared with the vehicle-treated controls. Of the curcuminoids the furan RK-106 was the only compound to inhibit production of TNFα and IL-1β in a monocytic cell-line THP-1 in vitro. The inactivity of RK-106 on the production of PGE(2) may be related to its absence of gastrotoxicity. None of the curcuminoids exhibited anti-pyretic activity and this may also be related to its insensitivity to PGE(2). Thus, these novel curcuminoids, such as RK-106, may warrant the development of new low gastro-toxic anti-inflammatory agents with selective inhibitory activity of cytokine inflammatory mediators.     

Antimicrobiai properties of aromatic compounds of plant origin

The antimicrobial action of 11 compounds involving guaiacyl- and syringyl-like structures (low-molecular-weight part of lignin), gallic acid and its derivatives, cinnamic acid and its derivatives, veratric acid, anisic acid and crotonic acid (a total of 25 compounds) against bacteria, yeast-like organisms and protozoa was examined. Aromatic compounds modified in the C-side chain and aldehydes were effective preferentially againstTrichomonas vaginalis, whereas against bacteria and yeast-like organisms eugenol was the most effective inhibitor. Dedicated to Academician A. Blažej on the occasion of his 60th birthday.     

Livestock grazing affects microclimate conditions for decomposition process through changes in vegetation structure in mountain grasslands

It is often assumed that a change in litter quality is the main driver of alterations in the decomposition process when grazers modify vegetation structure. Soil microclimate is also modified, but this driver of decomposition has been far less studied than litter quality. We analyzed the relationships among vegetation structure, microclimate and decomposition in different mountain grassland types, across a fence-line separating paddocks with different grazing intensity. Along the fence, we selected nine pairs of contrasting grassland types including lawns and tall tussock grasslands, which are associated with high and low local grazing pressure, respectively. At each site (N = 18) we estimated growth form composition and vegetation height. During the growing season we recorded soil temperature, soil moisture and the photosynthetically active radiation. Within the same period, we measured the decomposition rate of two common litter substrates. We analyzed the relationships among those variables at the landscape and at the local scale. At the landscape scale we considered the variation across all sites (N = 18). At the local scale we considered each pair as a sample (N = 9) and the differences between both sides of the fence as the variables to correlate. Our results indicate that when short grasslands are released from grazing and tall grasslands became dominant, temperature and light at the soil level are reduced, while soil moisture tends to increase, enhancing decomposition. Furthermore, these results show that the microclimatic conditions effect can counteract the litter quality effect (reported in previous studies) on decomposition, resulting in increased decomposition rates when grazing is reduced.     

Behaviour of pectinases under process conditions

Hydrolysis of pectic substances by pectinases were carried out under process conditions. Polymethylgalacturonase, polygalacturonase and pectinlyase are three enzymes studies in this regard. Pectin is used as the substrate for polymethylgalacturonase and pectinlyase whereas polygalacturonic acid was used as the substrate for polygalacturonase. The initial concentration was varied between 1.0 and 5.0 kg/m³ for polymethylgalacturonase and polygalacturonase and between 4.0 and 8.0 kg/m³ for pectinylase. Hydrolysis experiments were carried out by varying initial substrate to enzyme ratio and reducing groups formed are measured. The behaviour of pectinases during hydrolysis are studied from the plot of (reducing groups formed)/initial substrate vs. initial substrate to enzyme ratio. This study is helpful in predicting the amount of substrate and enzyme required to produce the required amount of reducing groups within a stipulated time.     

Amidosulfobetaines, a family of detergents with improved solubilization properties: application for isoelectric focusing under denaturing conditions

The synthesis of a family of new detergents and their use for isoelectric focusing are described. These detergents differ from the conventional sulfobetaines by the presence of an amido group bridging the hydrophobic linear tail and the polar head, which increases their water solubility and urea tolerance considerably. Four different linear alkyl hydrophobic tails were tested, together with three different polar heads. The solubilization of red blood cell ghosts by the 12 resulting detergent performances was evaluated both in solution and in isoelectric focusing runs. Six chemicals gave performances equal or better than those of conventional detergents and could replace them for general use in focusing experiments. In addition, the synthesis of a whole range of compounds allowed valuable insights in the empirical understanding of protein-detergent interactions, which are extensively discussed.     

Degradation of aromatic compounds in plants grown under aseptic conditions

The aim of the work is to investigate the ability of higher plants to absorb and detoxify environmental pollutants - aromatic compounds via aromatic ring cleavage. Transformation of 14C specifically labelled benzene derivatives, [1-6-14C]-nitrobenzene, [1-6-(14)C]-aniline, [1-(14)C]- and [7-(14)C]-benzoic acid, in axenic seedlings of maize (Zea mays L.), kidney bean (Phaseolus vulgaris L.), pea (Pisum sativum L.) and pumpkin (Cucurbita pepo L.) were studied. After penetration in plants, the above xenobiotics are transformed by oxidative or reductive reactions, conjugation with cell endogenous compounds, and binding to biopolymers. The initial stage of oxidative degradation consists in hydroxylation reactions. The aromatic ring can then be cleaved and degraded into organic acids of the Krebs cycle. Ring cleavage is accompanied by 14CO2 evolution. Aromatic ring cleavage in plants has thus been demonstrated for different xenobiotics carrying different substitutions on their benzene ring. Conjugation with low molecular peptides is the main pathway of aromatic xenobiotics detoxification. Peptide conjugates are formed both by the initial xenobiotics (except nitrobenzene) and by intermediate transformation products. The chemical nature of the radioactive fragment and the amino acid composition of peptides participating in conjugation were identified.     

Drosophila wing-spot test: improved detectability of genotoxicity of polycyclic aromatic hydrocarbons

The somatic mutation and recombination tests (SMART) using eyes or wings in the fruit fly Drosophila melanogaster are flexible and sensitive systems for the detection of genotoxicity of individual chemical compounds and complex mixtures. It is of special interest that adults and larvae possess cytochrome P-450-dependent activation systems able to metabolize most promutagens, e.g., nitrosamines, aflatoxins, pyrrolizidine alkaloids, safrole, etc. The polycyclic aromatic hydrocarbons (PAHs) represent a class of promutagens poorly detectable in Drosophila genotoxicity systems. Therefore, new tester strains for the wing-spot test were constructed by introducing chromosomes 1 and 2 from a wild-type strain with increased cytochrome P-450-dependent metabolism linked to a gene on chromosome 2. Previous investigations with the new strains showed their increased detection capability for diethylnitrosamine. Comparative tests with the 3 PAHs benzo[a]pyrene, benz[a]anthracene and 7,12-dimethylbenz[a]anthracene demonstrate, in a reproducible way, that with the new strains all 3 can be detected as active genotoxic compounds. The dose-response curves for all compounds show a plateau with higher exposures. This is interpreted as indicative of a saturation of the cytochrome P-450-dependent activation systems.     

Utilization of metabolic energy under saline conditions: changes in properties of ATP dependent enzymes in plant cells grown under saline conditions

The effect of growth in saline medium on the activity of two ATP utilizing enzymes was studied. Hexokinase in carrot (Daucus carota L.) cells grown in suspension culture either in the absence or presence of 150 ml NaCl, and tonoplast H⁺-ATPase in tobacco (Nicotiana tabacum L. cv. Wisconsin 38) cells grown in suspension culture either in the absence of presence of 428 mM NaCl. There was no difference in the pH profiles, NaCl sensitivity and kinetic parameters towards glucose of hexokinase activities from carrot cells grown in the presence or the absence of NaCl, but the activity from cells grown in the presence of NaCl was more resistant to inhibition by N-ethylmaleimide and to inactivation by heat. Two separate apparent Km values toward ATP were delineated in the extract from cells grown in presence of NaCl while extracts from cells grown in the absence of NaCl had only one apparent Km value. The tonoplast H⁺-ATPase from NaCl grown tobacco cells showed changed kinetic compared to this activity from cells grown in the absence of NaCl. These data may indicate that growth in NaCl results in the appearance of isozymic activity that enhances the ability of plant cells to utilize metabolic energy more efficiently.     

Antigenic properties of nitro aromatic derivatives of polyethylene glycol

The immunogenic properties of the dihapten and monohapten derivatives of polyethylene glycol with different nitroaromatic groupings were studied. 2,4-dinitrophenyl, 2, 4, 6-trinitrophenyl and trinitrophenyl-ethyl groupings were used as hapten groups. The injection of monohapten compounds was found to induce the accumulation of antibody-forming cells secreting antibodies to trinitrophenyl in the spleen of normal and athymic nude mice. As early as on day 3 the number of antibody-forming cells considerably exceeded their background level, the process of B-cell activation being, to a certain extent, thymus-independent. Dihapten compounds were not immunologically active. The effect rendered by the nitroaromatic derivatives of polyethylene glycol, revealed in this study, is linked with the known capacity of polyethylene glycol to adsorb on the surface of cell membranes.     

Bidirectional transformation of aromatic aldehydes by Desulfovibrio desulfuricans under nitrate-dissimilating conditions

M. PAREKH, H.L. DRAKE AND S.L. DANIEL 1996. Desulfovibrio desulfuricans ATCC 27774 was screened for reactivity against aromatic compounds during lactate-dependent, nitrate-dissimilating growth. Only aromatic aldehydes (benzaldehyde, 2-hydroxybenzaldehyde, 3-hydroxybenzaldehyde, 4-hydroxybenzaldehyde, vanillin, iso -vanillin and o -vanillin) were reactive and, with the exception of 2-hydroxybenzaldehyde, were stimulatory to lactate-dependent growth. Aromatic aldehydes were transformed to their corresponding benzoate and benzyl alcohol derivatives, with the ratio of benzoate-to-benzyl alcohol derivatives being dependent upon lactate availability. In presence of lactate, aromatic aldehydes were primarily reduced to their corresponding benzyl alcohol derivatives; in the absence of lactate, aromatic aldehydes were mainly oxidized to their corresponding benzoate derivatives. In the absence of nitrate, 3-hydroxybenzaldehyde was neither reduced nor oxidized. These results indicate that D. desulfuricans is competent in the bidirectional transformation of aromatic aldehydes under nitrate-dissimilating conditions and that the direction of transformation (i.e. reduction or oxidation) is regulated by reductant availability.     

Seasonal changes in some photosynthetic properties of Ceratonia siliqua (carob tree) leaves under natural conditions

Rates of photosynthesis and leaf conductance of the leaves of carob trees ( Ceratonia siliqua L.) growing in natural conditions were measured during the course of the seasons to define the effects of the main climatic factors limiting growth in the region: temperature during the winter and water in the summer. The highest photosynthetic rates were measured in spring and autumn and could reach 25 μmol m₋₂ s₋₁ with optimal temperature and available water. Due to lower temperatures (4 to 6°C in the night) these values were frequently around 15 μmol m₋₂ s₋₁ during winter, but the strongest depression was due to prolonged drought in summer. However a reduction in photosynthesis rate down to 5 μmol m₋₂ s₋₁ occurred only after depletion of all the available water in the soil layer up to a depth of 50 cm. In the end of the summer, leaf conductance and water potential were in the order of 20 mmol m₋₂ s₋₁ and −3 MPa respectively. Compared to other trees that make up the Mediterranean sclerophyll forest, the photosynthetic activity of carob is high, and the tree tolerates a considerable depletion of soil water.