Study of catalytic site structure and diffusion of radicals in porous heterogenous systems with ESR,ENDOR and ESE

The generation of paramagnetic products by adsorption of quinones on activated catalysts has been used for the diagnostic of Lewis acid sites. It has been shown that the application of ENDOR, ESE, and 2 mm-band ESR are extremely effective methods for studying the nature of observed radical species and their environment. Two applications of the ESE method for studying the diffusion of spin probes in porous media are considered. The measurement of effective diffusion coefficients of radical probes in specimens of various heterogeneous systems is described. It has been found that effective diffusion coefficients depend strongly on the mean value of silica gel pore sizes and the mobility of the probe inside the pore.     

Novel laccase—producing ascomycetes

Screening of ascomycetes producing laccases during growth on agar medium or submerged cultivation in the presence of various natural sources of carbon and energy (grain crops and potato) was carried out. The conditions of submerged cultivation of the most active strains (Myrothecium roridum VKM F-3565, Stachybotrys cylindrospora VKM F-3049, and Ulocladium atrum VKM F-4302) were optimized for the purpose of increasing laccase activity. The pH-optima and substrate selectivity of laccases in the culture liquid of the strains in relation to ABTS and phenolic compounds (2,6-dimethoxyphenol, syringaldazine, ferulic acid, p-coumaryl alcohol, and coniferyl alcohol) were investigated. High laccase activity at neutral pH was shown for the culture liquids of M. roridum VKM F-3565 and S. cylindrospora VKM F-3049 strains that provides prospects for using laccases of these strains in various cell biotechnologies.     

Characterization of the Semiquinone Radical Stabilized by the Cytochrome aa3-600 Menaquinol Oxidase of Bacillus subtilis

Cytochrome aa₃-600 is one of the principle respiratory oxidases from Bacillus subtilis and is a member of the heme-copper superfamily of oxygen reductases. This enzyme catalyzes the two-electron oxidation of menaquinol and the four-electron reduction of O₂ to 2H₂O. Cytochrome aa₃-600 is of interest because it is a very close homologue of the cytochrome bo₃ ubiquinol oxidase from Escherichia coli, except that it uses menaquinol instead of ubiquinol as a substrate. One question of interest is how the proteins differ in response to the differences in structure and electrochemical properties between ubiquinol and menaquinol. Cytochrome bo₃ has a high affinity binding site for ubiquinol that stabilizes a ubi-semiquinone. This has permitted the use of pulsed EPR techniques to investigate the protein interaction with the ubiquinone. The current work initiates studies to characterize the equivalent site in cytochrome aa₃-600. Cytochrome aa₃-600 has been cloned and expressed in a His-tagged form in B. subtilis. After isolation of the enzyme in dodecylmaltoside, it is shown that the pure enzyme contains 1 eq of menaquinone-7 and that the enzyme stabilizes a mena-semiquinone. Pulsed EPR studies have shown that there are both similarities as well as significant differences in the interactions of the mena-semiquinone with cytochrome aa₃-600 in comparison with the ubi-semiquinone in cytochrome bo₃. Our data indicate weaker hydrogen bonds of the menaquinone in cytochrome aa₃-600 in comparison with ubiquinone in cytochrome bo₃. In addition, the electronic structure of the semiquinone cyt aa₃-600 is more shifted toward the anionic form from the neutral state in cyt bo₃.     

SO2-dependent cation competition and compartmentalization in Norway spruce needles

Ion contents in needles from Norway spruce trees [Picea abies (L.) Karst.] growing in Würzburg and in the SO₂-polluted Erzgebirge mountains were analysed to quantify cations which accumulate together with sulphate. In Würzburg there was a positive correlation of potassium (0.680 ± 0.300 Eq Eq^?1 SO₄^?2), magnesium (0.415 ± 0.111 Eq Eq^?1 SO₄^?2) and zinc (0.059 ± 0.006 Eq Eq^?1 SO₄^2?). In the Erzgebirge, potassium was also the stoichiometrically most important cation (0–887 ± 0–180 Eq K⁺ Eq^?1 SO₄^2?). All other correlations examined were weak or statistically non-significant. At both sites the calcium content of spruce needles did not depend on the sulphate content. The lack of a role for Ca²⁺ in neutralizing sulphate is a consequence of the presence of free oxalic acid in needles. Soluble oxalic acid precipitates Ca²⁺, which thereby becomes unavailable as a counterion for SO₄^2?. The activity coefficients of Ca²⁺ and oxalate^2?, and the solubility product of Ca-oxalate, were determined from in vivo data. It is concluded that the chronic accumulation of atmospheric sulphate in spruce needle vacuoles depletes available potassium and thereby strongly interferes with spruce growth and canopy turnover. This leads to impaired spruce vitality, even at sites where acute SO₂ disease symptoms are absent.     

Characterization of mutants that change the hydrogen bonding of the semiquinone radical at the QH site of the cytochrome bo3 from Escherichia coli

The cytochrome bo3 ubiquinol oxidase catalyzes the two-electron oxidation of ubiquinol in the cytoplasmic membrane of Escherichia coli, and reduces O2 to water. This enzyme has a high affinity quinone binding site (QH), and the quinone bound to this site acts as a cofactor, necessary for rapid electron transfer from substrate ubiquinol, which binds at a separate site (QL), to heme b. Previous pulsed EPR studies have shown that a semiquinone at the QH site formed during the catalytic cycle is a neutral species, with two strong hydrogen bonds to Asp-75 and either Arg-71 or Gln-101. In the current work, pulsed EPR studies have been extended to two mutants at the QH site. The D75E mutation has little influence on the catalytic activity, and the pattern of hydrogen bonding is similar to the wild type. In contrast, the D75H mutant is virtually inactive. Pulsed EPR revealed significant structural changes in this mutant. The hydrogen bond to Arg-71 or Gln-101 that is present in both the wild type and D75E mutant oxidases is missing in the D75H mutant. Instead, the D75H has a single, strong hydrogen bond to a histidine, likely His-75. The D75H mutant stabilizes an anionic form of the semiquinone as a result of the altered hydrogen bond network. Either the redistribution of charge density in the semiquinone species, or the altered hydrogen bonding network is responsible for the loss of catalytic function.     

Effectivity of bacterial repair systems in near UV radiation-induced damage

Inactivation of seven strains derived fromEscherichia coli B differing in their capacity to repair damage to their DNA (exc, pol, rec) after irradiation with far (254 nm) and middle and near (300 to 380 and 320–400 nm) UV light was investigated. The same bacterial strains were also used as hosts for the UV-irradiated pliage T7. The damage induced in bacteria and the phage by the near UV radiation was repaired only to a lesser extent by the investigated repair mechanisms or was not repaired at all.     

ODOR PERCEPTION OVER LIQUID EMULSIONS CONTAINING SINGLE AROMA COMPOUNDS: EFFECTS OF AROMA CONCENTRATION AND OIL VOLUME FRACTION

This study aimed to check the hypothesis that aroma concentration in the aqueous phase of an oil-in-water emulsion controlled the odor intensity of single aroma compounds. A set of flavored oil-in-water emulsions, prepared according to a 2² experimental design (aroma concentration, oil volume fraction) with two central points, was assessed for odor intensity by a 24-member panel during four sessions. In each session, three of the four-studied aroma molecules (benzaldehyde, ethyl butyrate, linalool and acetophenone) were investigated. Whatever the aroma, the experimental data showed that the oil volume fraction of the emulsion (from 0.12 to 0.48) did not influence the odor intensity. For each emulsion composition, aroma concentrations at equilibrium in both phases were calculated using the oil-water partition coefficient of the compound. Odor intensities, estimated from aroma concentration in the aqueous phase using previously reported modeling of odor intensity above water solutions, were then compared to experimental data. It is confirmed that the perceived odor intensity is governed by the aroma concentration in the aqueous phase at the time of the trial and not by the averaged apparent concentration in the emulsion.