Isolation and characterisation of 8-hydroxy-3Z,5Z-tetradecadienoic acid, a putative intermediate in Pichia guilliermondii gamma-decalactone biosynthesis from ricinoleic acid.

During a screening procedure for the discovery of a strong gamma-decalactone producer from ricinoleic acid, we observed that the yeast Pichia guilliermondii accumulated transiently 8-hydroxy-3Z,5Z-tetradecadienoic acid 1 during gamma-decalactone biosynthesis in the stationary phase of growth. The structural elucidation of 1 was based on nuclear magnetic resonance, infrared, ultraviolet and gas chromatography-mass spectrometry experiments. The occurrence of 1 is discussed in relation with previously proposed gamma-decalactone biosynthetic pathways.

Response of electrically stimulated cells of Pseudomonas oleovorans strain ATCC 29347 suspended in silicone oil.

A high intensity direct current was applied for more than 10 min onto a bacterial suspension of Pseudomonas oleovorans ATCC 29347 suspended in silicone oil. The application of a gradually increased electric field from 0 to 2500 V x cm(-1) resulted in a decrease of the optical density of the bacterial suspension and the occurrence of a peak current of several hundred microA for living cells instead of a linear increase (few microA) for killed or lyophilised cells. This procedure is not only a rapid way of investigating the living state of cell cultures but also an efficient experimental tool to study the cellular effects of a controlled electrical stress.

A new tannase substrate for spectrophotometric assay.

A new tannase substrate, protocatechuic acid p-nitrophenyl ester, 5, was synthesized using modern synthetic methods. The synthesis was designed to be performed by non-specialized chemists. It only involves four steps, three of which are protection-deprotection, and uses standard methods of separation and purification, such as recrystallization and column chromatography over silica. Under tannase action, protocatechuic acid p-nitrophenyl ester, 5, releases p-nitrophenol, which is easily measured spectrophotometrically either at 350 nm for pH values<6 or at 400 nm for pH values of 6-7 (yellow). The pH-response and the catalytic parameters of a crude Penicillium sp. tannase preparation were determined using 5 as substrate, thus showing the usefulness of this substrate in determining tannase activity.

Biochemical and molecular characterization of a laccase from Marasmius quercophilus.

The basidiomycete Marasmius quercophilus is commonly found during autumn on the decaying litter of the evergreen oak (Quercus ilex L.), a plant characteristic of Mediterranean forest. This white-rot fungus colonizes the leaf surface with rhizomorphs, causing a total bleaching of the leaf. In synthetic liquid media, this white-rot fungus has strong laccase activity. From a three-step chromatographic procedure, we purified a major isoform to homogeneity. The gene encodes a monomeric glycoprotein of approximately 63 kDa, with a 3.6 isoelectric point, that contains 12% carbohydrate. Spectroscopic analysis of the purified enzyme (UV/visible and electron paramagnetic resonance, atomic absorption) confirmed that it belongs to the "blue copper oxidase" family. With syringaldazine as the substrate, the enzyme's pH optimum was 4.5, the optimal temperature was 75 degrees C, and the K(m) was 7.1 microM. The structural gene, lac1, was cloned and sequenced. This gene encodes a 517-amino-acid protein 99% identical to a laccase produced by PM1, an unidentified basidiomycete previously isolated from wastewater from a paper factory in Spain. This similarity may be explained by the ecological distribution of the evergreen oak in Mediterranean forest.

Measurement of thermally produced volatile alkanes: an assay for plant hydroperoxy fatty acid evaluation.

A new method designed to monitor lipid peroxidation in plants has been set up with soybean hypocotyl/radicles. The hydroperoxy fatty acids present in situ are converted by rapid thermal treatment (80 s and 210 J g-1) of the biological sample into ethane and n-pentane, which are analyzed by gas chromatography. The method has been directly calibrated by quantification of the hydroperoxy fatty acids by silica-phase HPLC analysis of their reduced hydroxy derivatives. Hypocotyl/radicles from the two soybean cultivars Argenta and Soriano were submitted to various chemical oxidative treatments and were analyzed for both thermally produced volatile alkanes and hydroperoxy fatty acid levels. Our results showed that ethane and n-pentane production are in both cases closely correlated with linolenic as well as linoleic acid hydroperoxide levels (P < 0.001). Within a given plant material, thermal conversion of both hydroperoxides into alkanes occurred with yields which were not dependent on the oxidative treatment. These yields are however functions of the biological material since in Soriano and Argenta cultivars they were around 6 and 25%, respectively. Taking into account the last point, the alkane test cannot be used to directly quantify the absolute lipid hydroperoxide levels of plant tissues but it is convenient to monitor the peroxidative phenomenon as it occurs. The assay is easy and rapid to perform (analysis of 50 samples per day) since no sample preparation is needed, and the low detection limit (20 pmol of alkane g-1) permits the analysis of small samples.