Mario Carlos Nazareno Saparrat

Induction, Isolation, and Characterization of Two Laccases from the White Rot Basidiomycete Coriolopsis rigida

ABSTRACT Previous work has shown that the white rot fungus Coriolopsis rigida degraded wheat straw lignin and both the aliphatic and aromatic fractions of crude oil from contaminated soils. To better understand these processes, we studied the enzymatic composition of the ligninolytic system of this fungus. Since laccase was the sole ligninolytic enzyme found, we paid attention to the oxidative capabilities of this enzyme that would allow its participation in the mentioned degradative processes. We purified two laccase isoenzymes to electrophoretic homogeneity from copper-induced cultures. Both enzymes are monomeric proteins, with the same molecular mass (66 kDa), isoelectric point (3.9), N-linked carbohydrate content (9%), pH optima of 3.0 on 2,6-dimethoxyphenol (DMP) and 2.5 on 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), absorption spectrum, and N-terminal amino acid sequence. They oxidized 4-anisidine and numerous phenolic compounds, including methoxyphenols, hydroquinones, and lignin-derived aldehydes and acids. Phenol red, an unusual substrate of laccase due to its high redox potential, was also oxidized. The highest enzyme affinity and efficiency were obtained with ABTS and, among phenolic compounds, with 2,6-dimethoxyhydroquinone (DBQH2). The presence of ABTS in the laccase reaction expanded the substrate range of C. rigida laccases to nonphenolic compounds and that of MBQH2 extended the reactions catalyzed by these enzymes to the production of H2O2, the oxidation of Mn2+, the reduction of Fe3+, and the generation of hydroxyl radicals. These results confirm the participation of laccase in the production of oxygen free radicals, suggesting novel uses of this enzyme in degradative processes.

Differential regulation of laccase gene expression in Coriolopsis rigida LPSC No. 232.

Two laccase isoenzyme genes (lcc2 and lcc3) from the white-rot fungus Coriolopsis rigida were cloned, and together with the previously described lcc1, their transcript levels were analysed by Quantitative RT-PCR in order to study their expression patterns under a range of putative inducers (Cu(2+), Mn(2+), Fe(3+), 2,6-dimethoxy-1,4-benzoquinone, H(2)O(2,) caffeine, amphotericin B and syringic acid). The highest induction was observed for lcc1 in presence of copper, and thus, a kinetic study was performed to analyze its effect on temporary lcc1 gene expression. Our results showed that upregulation due to copper was linked to growth stage, being highest during the trophophase and decreasing during the idiophase. Amphotericin B increased levels of transcripts of lcc1 and lcc2, syringic acid upregulated lcc1 and lcc3 and 2,6-dimethoxy-1,4-benzoquinone induced lcc2 and lcc3. Possible reasons for why laccase genes from C. rigida are differentially regulated at the transcriptional level are discussed.

Production of ligninolytic enzymes by Fusarium solani strains isolated from different substrata

A comparative study on the extracellular ligninolytic enzymatic activity of five strains of Fusarium solani in a carbon-limited medium under shaking, revealed a differential production of these enzymes. Aryl alcohol oxidase (AAO) activity was observed only in the supernatant of strain CLPS no. 568 with levels higher than 57 mU ml−1. Free extracellular laccase activity was detected in strains CLPS nos. 493, 568 and 570, strain no. 568 being the one which showed the highest activity (over 8.6 mU ml−1). Free extracellular lignin peroxidase (LiP) activity was not detected in any isolate tested, whereas low levels of manganese-dependent peroxidase (MnP) and manganese-independent peroxidase (MIP) activities were detected in certain isolates used. The AAO activity of F. solani on primary α-alcohols such as veratryl alcohol, is reported for the first time; this enzyme activity is hydrogen-peroxide independent. This is also the first report for extracellular MnP and MIP activities of F. solani.

Ligninolytic ability and potential biotechnology applications of the South American fungus Pleurotus laciniatocrenatus.

The extracellular ligninolytic enzyme system ofPleurotus laciniatocrenatus, grown under different culture conditions, was characterized and the ability of this strain to degrade different components ofEucalyptus globulus wood was determined. In shaken liquid cultures grown on a C-limited medium supplemented with yeast extract (0.1 %) and peptone (0.5 %), the fungus produced extracellular aryl-alcohol oxidase (Aao), laccase (Lac), manganese-dependent peroxidase (MnP) and manganese-independent peroxidase (MiP) activities, their maximum levels being, respectively, about 600, 50, 1360, and 920 pkat/mL. The supplementation of 1 mmol/L vanillic acid and 150 µmol/L CuSO4 produced an increase of Lac activity levels up to 4-fold and 68.3-fold, respectively. No significant differences were found in the levels of the other ligninolytic enzyme activities when compared to the basal medium. Solid-state fermentation cultures onE. globulus wood chips revealed Lac and MiP activities. These cultures showed degradative activity on lignin and lipophilic wood extractives.

Coriolopsis rigida, a potential model of white-rot fungi that produce extracellular laccases.

In the last two decades, a significant amount of work aimed at studying the ability of the white-rot fungus Coriolopsis rigida strain LPSC no. 232 to degrade lignin, sterols, as well as several hazardous pollutants like dyes and aliphatic and aromatic fractions of crude oil, including polycyclic aromatic hydrocarbons, has been performed. Additionally, C. rigida in association with arbuscular mycorrhizal fungi appears to enhance plant growth, albeit the physiological and molecular bases of this effect remain to be elucidated. C. rigidas ability to degrade lignin and lignin-related compounds and the capacity to transform the aromatic fraction of crude oil in the soil might be partially ascribed to its ligninolytic enzyme system. Two extracellular laccases are the only enzymatic components of its lignin-degrading system. We reviewed the most relevant findings regarding the activity and role of C. rigida LPSC no. 232 and its laccases and discussed the work that remains to be done in order to assess, more precisely, the potential use of this fungus and its extracellular enzymes as a model in several applied processes.

Insights into Hydrocarbon Assimilation by Eurotialean and Hypocrealean Fungi: Roles for CYP52 and CYP53 Clans of Cytochrome P450 Genes

Several filamentous fungi are able to concomitantly assimilate both aliphatic and polycyclic aromatic hydrocarbons that are the biogenic by-products of some industrial processes. Cytochrome P450 monooxygenases catalyze the first oxidation reaction for both types of substrate. Among the cytochrome P450 (CYP) genes, the family CYP52 is implicated in the first hydroxylation step in alkane-assimilation processes, while genes belonging to the family CYP53 have been linked with oxidation of aromatic hydrocarbons. Here, we perform a comparative analysis of CYP genes belonging to clans CYP52 and CYP53 in Aspergillus niger, Beauveria bassiana, Metarhizium robertsii (formerly M. anisopliae var. anisopliae), and Penicillium chrysogenum. These species were able to assimilate n-hexadecane, n-octacosane, and phenanthrene, exhibiting a species-dependent modification in pH of the nutrient medium during this process. Modeling of the molecular docking of the hydrocarbons to the cytochrome P450 active site revealed that both phenanthrene and n-octacosane are energetically favored as substrates for the enzymes codified by genes belonging to both CYP52 and CYP53 clans, and thus appear to be involved in this oxidation step. Analyses of gene expression revealed that CYP53 members were significantly induced by phenanthrene in all species studied, but only CYP52X1 and CYP53A11 from B. bassiana were highly induced with n-alkanes. These findings suggest that the set of P450 enzymes involved in hydrocarbon assimilation by fungi is dependent on phylogeny and reveal distinct substrate and expression specificities.

Draft Genome Sequence and Gene Annotation of Stemphylium lycopersici Strain CIDEFI-216

ABSTRACT Stemphylium lycopersici is a plant-pathogenic fungus that is widely distributed throughout the world. In tomatoes, it is one of the etiological agents of gray leaf spot disease. Here, we report the first draft genome sequence of S. lycopersici, including its gene structure and functional annotation.

Bioremediation of PAH-Contaminated Soil by Fungi

Polycyclic aromatic hydrocarbons (PAHs) are by-products of the incomplete combustion of organic materials. They are considered to be priority pollutants in the environment due to their recalcitrance and mutagenic properties. The principal PAH loss process from soil is through microbial degradation; therefore, the bioremediation is considered as an efficient, financially affordable, and adaptable alternative for the recuperation of PAH-contaminated soil. Several microorganisms, such as bacteria, yeasts, and filamentous fungi, are capable of degrading different types of PAHs. The ability of the fungi to degrade the high-molecular-weight PAHs, together with their physiological versatility, converts the fungal remediation in a promising technique for the cleanup of PAH-contaminated soil. This chapter summarizes the recent information on the metabolic pathway of the fungal transformation of PAHs and provides a critical review of previous work about fungal bioremediation of PAH-contaminated soil. Also, some of the most recently used fungal technology to enhance PAHs bioremediation processes is discussed.

Extracellular laccase activity in Tetraploa aristata

Tetraploa aristata CLPS 419 produced maximum extracellular laccase activity at over 9 mU ml−1 in shaking cultures supplemented with glucose and 3.5 mU ml−1 in sucrose-grown ones. Laccase activity did not exceed 0.7 mU ml−1 in stationary cultures with glucose and was not detected in similar cultures with sucrose or in ones grown on lignin.

Response of the fungus Pseudocercospora griseola f. mesoamericana to Tricyclazole

Pseudocercospora griseola, an anamorph of Mycosphaerella, causes Angular Leaf Spot (ALS). The mycelia and conidia from P. griseola are coloured due to the synthesis of 1,8 dihydroxynaphthalene (DHN)-melanin. The aim of this work was to identify in P. griseola f. mesoamericana isolate T4, a highly pigmented fungus, intermediary compounds as a result of the inhibition of melanin synthesis by tricyclazole, and to analyze at the structural level the localization of these dark pigments. The main metabolites were analyzed using ultraviolet matrix-assisted laser desorption-ionization mass spectrometry (UV–MALDI MS). Tricyclazole affected P. griseola f. mesoamericana in several different ways. The most evident effect was the reduction of melanin synthesis, and therefore diffusible shunt products were found and identified. Flaviolin was the main intermediate metabolite found in cultures supplemented with tricyclazole. This inhibitor, which affected pigmentation and the cell wall structure of mycelium, revealed macroscopically by the reduction in growth, decreased the stratification and deposition of melanin in the hyphal wall. These results suggest a possible role of tricyclazole to control ALS.