Purification, partial characterization, and reactivity with aromatic compounds of two laccases from Marasmius quercophilus strain 17.

Two isozymes of laccase were obtained from an induced liquid culture of Marasmius quercophilus with p-hydroxybenzoic acid as the inducer. Both the constitutive and the induced isozyme have a molecular mass of 60 kDa as determined by polyacrylamide gel electrophoresis. Using isoelectric focusing, we found three isozymes with the constitutive enzyme (pI 4, 4.2, 4.4) and four of the induced form (pI 4.75, 4.85, 4.95, 5.1). We observed certain differences between these two isozymes; the specific activity of the induced isozyme was twice as high, and two optimum pH levels (5 and 6) were observed with the induced isozyme (only one, pH 5, for the constitutive isozyme). However, both of these enzymes have the same thermal stability and the same temperature for their highest activity (80 degrees C). Furthermore, the reactivity of both these enzymes with aromatic compounds was similar. The use of mediators extended the oxidized substrate range of the laccases studied. Various products of degradation were observed, depending on the mediator used. When laccase was used alone, the decrease of the signal corresponding to the aromatic cycle, without any formations of other peaks at different wavelengths, suggested polymerisation of aromatic compounds.

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.

Preliminary study on relationships among strains forming a bacterial community selected on naphthalene from a marine sediment.

Two bacterial strains were isolated from a bacterial community formed of nine strains, selected from a marine sediment on a seawater medium with naphthalene as sole carbon source. The two strains studied in the present work were the only strains of this community able to grow in pure culture on naphthalene; therefore, they were called "primary" strains. The seven other strains were maintained in the community by using metabolic intermediates of the two primary strains; they were called "auxiliary" strains. Regulation of naphthalene metabolism was studied for the two primary strains. They oxidized naphthalene into catechol, which was degraded only by the meta pathway. For Pseudomonas Lav. 4, naphthalene oxygenase and salicylate hydroxylase were inducible; catechol 2,3-dioxygenase was constitutive. For Moraxella Lav. 7, naphthalene oxygenase was constitutive; salicylate hydroxylase and catechol 2,3-oxygenase were inducible. The Moraxella strain carries two cryptic plasmids, about 63- and 85-kb in molecular size. In the bacterial community culture medium, Moraxella Lav. 7 prevented accumulation of 2-hydroxymuconate semialdehyde formed by Pseudomonas Lav. 4. The auxiliary strains take up formic, acetic, pyruvic, propionic, and succinic acids released by the two primary strains.

[Degradation of hydrocarbons in the presence of other organic substances by bacteria isolated from seawater]. / Dégradation des hydrocarbures en présence d'autres substances organiques par des bactéries isolées de l'eau de mer

Three bacterial strains, isolated on gas-oil from seawater, have a variously changed growth on hexadecane with supply of two organic substances. Acetate reduces growth of all tested strains and particularly the hexadecane degradation by Acinetobacter sp. On the contrary, trypticase-phytone promotes the degradation by the three strains.

[On the taxonomy and physiology of bacteria utilizing hydrocarbons in the sea (author's transl)]. / Etude taxonomique et physiologique de bactéries se développant sur hydrocarbures en milieu marin

Using mineral media with gas oil as sole carbon source, 191 bacterial strains were isolated from the costal area of Marseille. These strains were attributed to Achromobacter, Alcaligenes, Pseudomonas, Acinetobacter and Arthrobacter genera. Amongst isolated strains there was a predominance of the Alcaligenes-Achromobacter group over others genera. Growth and respiratory activity of 5 strains were studied on hexadecane and acetate. Respiratory activity on hexadecane of 32 strains cultured on acetate has been measured. These strains could be placed in two groups. The group 1 shows an immediate respiratory activity which is not abolished by chloramphenicol. The group 2 presents either an immediate or a delayed respiratory activity which is always abolished by chloramphenicol. Maintenance or suppression of respiration by chloramphenicol is a character which is homogenously distributed amongst the species.