Modeling and experimental validation of covalent immobilization of Trametes maxima laccase on glyoxyl and MANA-Sepharose CL 4B supports, for the use in bioconversion of residual colorants.

Our novel strategy for the rational design of immobilized derivatives (RDID) is directed to predict the behavior of the protein immobilized derivative before its synthesis, by the usage of mathematic algorithms and bioinformatics tools. However, this approach needs to be validated for each target enzyme. The objective of this work was to validate the RDID strategy for covalent immobilization of the enzyme laccase from Trametes maxima MUCL 44155 on glyoxyl- and monoaminoethyl-N-aminoethyl (MANA)-Sepharose CL 4B supports. Protein surface clusters, more probable configurations of the protein-supports systems at immobilization pHs, immobilized enzyme activity, and protein load were predicted by RDID1.0 software. Afterward, immobilization was performed and predictions were experimentally confirmed. As a result, the laccase-MANA-Sepharose CL 4B immobilized derivative is better than laccase-glyoxyl-Sepharose CL 4B in predicted immobilized derivative activity (63.6% vs. 29.5%). Activity prediction was confirmed by an experimentally expressed enzymatic activity of 68%, using 2,6-dimethoxyphenol as substrate. Experimental maximum protein load matches the estimated value (11.2 ± 1.3 vs. 12.1 protein mg/support mL). The laccase-MANA-Sepharose CL 4B biocatalyst has a high specificity for the acid blue 62 colorant. The results obtained in this work suggest the possibility of using this biocatalyst for wastewater treatment.

Correction to: Metagenomic approaches: effective tools for monitoring the structure and functionality of microbiomes in anaerobic digestion systems.

The original version of this article was revised: After publication of this article, the publisher was notified that Michael Klocke has been listed as an author without his consent.

Metagenomic approaches: effective tools for monitoring the structure and functionality of microbiomes in anaerobic digestion systems.

Microbial metagenome analysis has proven its usefulness to investigate the microbiomes present in technical engineered ecosystems such as anaerobic digestion systems. The analysis of the total microbial genomic DNA allows the detailed determination of both the microbial community structure and its functionality. In addition, it enables to study the response of the microbiome to alterations in technical process parameters. Strategies of functional microbial networks to face abiotic stressors, e.g., resistance, resilience, and reorganization, can be evaluated with respect to overall process optimization. The objective of this paper is to review the main metagenomic tools used for effective studies on anaerobic digestion systems in monitoring the dynamic of the microbiomes, as well as the factors that have been identified so far as limiting the metagenomic studies in this ecosystems.

Biodegradation of polycyclic aromatic hydrocarbons by native Ganoderma sp. strains: identification of metabolites and proposed degradation pathways.

Since polycyclic aromatic hydrocarbons (PAHs) are mutagenic, teratogenic, and carcinogenic, they are of considerable environmental concern. A biotechnological approach to remove such compounds from polluted ecosystems could be based on the use of white-rot fungi (WRF). The potential of well-adapted indigenous Ganoderma strains to degrade PAHs remains underexplored. Seven native Ganoderma sp. strains with capacity to produce high levels of laccase enzymes and to degrade synthetic dyes were investigated for their degradation potential of PAHs. The crude enzymatic extracts produced by Ganoderma strains differentially degraded the PAHs assayed (naphthalene 34-73%, phenanthrene 9-67%, fluorene 11-64%). Ganoderma sp. UH-M was the most promising strain for the degradation of PAHs without the addition of redox mediators. The PAH oxidation performed by the extracellular enzymes produced more polar and soluble metabolites such as benzoic acid, catechol, phthalic and protocatechuic acids, allowing us to propose degradation pathways of these PAHs. This is the first study in which breakdown intermediates and degradation pathways of PAHs by a native strain of Ganoderma genus were determined. The treatment of PAHs with the biomass of this fungal strain enhanced the degradation of the three PAHs. The laccase enzymes played an important role in the degradation of these compounds; however, the role of peroxidases cannot be excluded. Ganoderma sp. UH-M is a promising candidate for the bioremediation of ecosystems polluted with PAHs.

Diversity of Ligninolytic Enzymes and Their Genes in Strains of the Genus Ganoderma: Applicable for Biodegradation of Xenobiotic Compounds?

White-rot fungi (WRF) and their ligninolytic enzymes (laccases and peroxidases) are considered promising biotechnological tools to remove lignin related Persistent Organic Pollutants from industrial wastewaters and contaminated ecosystems. A high diversity of the genus Ganoderma has been reported in Cuba; in spite of this, the diversity of ligninolytic enzymes and their genes remained unexplored. In this study, 13 native WRF strains were isolated from decayed wood in urban ecosystems in Havana (Cuba). All strains were identified as Ganoderma sp. using a multiplex polymerase chain reaction (PCR)-method based on ITS sequences. All Ganoderma sp. strains produced laccase enzymes at higher levels than non-specific peroxidases. Native-PAGE of extracellular enzymatic extracts revealed a high diversity of laccase isozymes patterns between the strains, suggesting the presence of different amino acid sequences in the laccase enzymes produced by these Ganoderma strains. We determined the diversity of genes encoding laccases and peroxidases using a PCR and cloning approach with basidiomycete-specific primers. Between two and five laccase genes were detected in each strain. In contrast, only one gene encoding manganese peroxidase or versatile peroxidase was detected in each strain. The translated laccases and peroxidases amino acid sequences have not been described before. Extracellular crude enzymatic extracts produced by the Ganoderma UH strains, were able to degrade model chromophoric compounds such as anthraquinone and azo dyes. These findings hold promises for the development of a practical application for the treatment of textile industry wastewaters and also for bioremediation of polluted ecosystems by well-adapted native WRF strains.

Obtención de semilla biotecnológica de caña de azúcar (Saccharum spp. híbrido) de alta calidad genética y fitosanitaria en el Ecuador / Obtainment of sugarcane (Saccharum spp. hybrid) biotechnological seed of high genetic and phytosanitary quality in Ecuador

Los métodos de saneamiento de plantas se basan fundamentalmente en el empleo combinado de cultivo in vitro de meristemos con tratamientos mediante hidrotermoterapia. Este trabajo se realizó con el objetivo de obtener plantas para usar como semilla libre de patógenos sistémicos, con énfasis en la enfermedad raquitismo de los retoños de la caña de azúcar (RSD). El trabajo se llevó a cabo con diferentes cultivares de caña de azúcar, los cuales fueron previamente identificados mediante marcadores bioquímicos basados en los patrones electroforéticos de isoenzimas peroxidasas. El saneamiento se realizó en tres etapas sucesivas mediante tratamiento hidrotérmico a 52 ºC durante 2h y uso del fungicida vitavax, un segundo tratamiento hidrotérmico a los meristemos a 51 ºC durante 10 min y una tercera etapa donde las vitroplántulas se propagaron en presencia del antibiótico gentamicina. La detección de RSD se efectuó mediante el empleo de la técnica de tinción de haces vasculares funcionales. Los resultados permitieron la creación de un banco de germoplasma de plantas libres de los principales patógenos bacterianos sistémicos y con alta calidad genética, lo que aportó semilla básica de excelente calidad para establecer semilleros. La conservación incluyó el mantenimiento de plántulas in vitro y la creación de un "banco de ADN" que permite conservar el genofondo en condiciones de laboratorio. Se propone finalmente una metodología para la obtención de plantas saneadas a partir de yemas y meristemos.

Methods for obtaining healthy plants are mainly based on the combined use of meristem in vitro culture and hydrothermotherapy. This work was carried out in order to obtain free- pathogen plants for using as seeds, emphasizing ratoon stunting disease (RSD) of sugarcane. The work was performed using a group of sugarcane cultivars, which was confirmed the variety identification by means of peroxidases isozymes electrophoretic patterns. Plant sanitation was performed in three successive steps by means of hydrothermal treatment at 52 ºC during 2h and using the fungicide vitavax, a second hydrothermal treatment at 51 °C during 10 min to the meristem and a third step in which vitroplantlets were propagated in presence of the antibiotic gentamicin. RSD detection was done by using the staining transpiration methods (STM) of functional bundles. Results allowed the creation of a germoplasm bank of sugarcane plants free of the most important systemic bacterial pathogens, with high genetic quality, which provided basic seed of excellent quality for establishing seedlings. Conservation included an in vitro-plantlets bank and the creation of a "DNA bank" which allows preserving the genetic fond in laboratory conditions. Finally a methodology for obtaining free-pathogens plants from buds and meristems is included.