Fusarium oxysporum laccase was functionally expressed in Saccharomyces cerevisiae and engineered towards higher expression levels and higher reactivity towards 2,6-dimethoxyphenol, that could be used as a mediator for lignin modification. A combination of classical culture optimization and protein engineering led to around 30 times higher activity in the culture supernatant. The winner mutant exhibited three times lower Km, four times higher kcat and ten times higher catalytic efficiency than the parental enzyme. The strategy for laccase engineering was composed of a combination of random methods with a rational approach based on QM/MM MD studies of the enzyme complex with 2,6-dimethoxyphenol. Laccase mediator system with 2,6-dimethoxyphenol caused fulvic acids release from biosolubilized coal. Laccases are oxidoreductases that catalyze the 4-electron reduction of O 2 to water with simultaneous oxidation of organic substrates. Laccases are able to catalyze oxidation of a substrate of interest directly or indirectly by the formation of a radical, which then may take part in a non-enzymatic event that effects in the oxidation of a substrate of interest. Laccases oxidize phenolic substrates but are also able to oxidize non-phenolic or bigger substrates by Laccase Mediator System (LMS), where a small phenolic compound acts as a mediator 1. Laccases contain 4 copper atoms buried in their 3D structure, which are located in two separate Cu centers. T1 copper ion is a mononuclear center, whereas T2 copper ion and two T3 copper ions are positioned in T2/T3 copper center. The substrate is oxidized closed to T1 copper ion and then the electrons are transferred to the tri-nuclear center where O 2 is reduced to water. The coppers are coordinated by nearby located residues: the T1 copper ion by one Cys and two His residues, the T2 copper by two His residues and a solvent molecule and T3 copper to three His residues 2,3. Biosolubilization of brown coal is a clean coal technology that aims at the conversion of the lignite to its cleaner form or to change its structure to gain new features 4,5. Such solubilized material could be used as a source of value-added products 4. The liquid form of coal-microorganisms, such as Fusarium oxysporum reported before as an excellent coal solubilizer 6 , uses their metabolites, alkaline substances, biosurfactants and enzymes to turn the solid polymer to black liquid. Laccases, from bacteria and fungi, for example from species Pleurotus 7 or Streptomyces 8 , are known to take part in the degradation of lignin and lignite. These two polymers are similar in structure, thus, the mechanism of their degradation is expected to be the same. Lignin comprises only 10-20% of phenolic subunits. In theory, laccase could act on those subunits present on the lignin surface and modify the polymer. However, the possibility of lignin subunits entering the laccase active site is very limited. Moreover, it is not known to what extent lignin could be modified in this way 9. According to literature data, the treatme...

Xylanases are used in the recycling of biomass and have other industrial applications including pulp bleaching. These enzymes are also applied in the baking industry and for the manufacture of animal feed. Such technologies as, for example, pulp bleaching entail high temperatures and high pHs. As a result, there is great demand from industry for thermostable and halostable forms of xylanase. Due to the relatively high variation in the thermo-and halo-stability of xylanases, feature selection was employed as a model to discover the important attributes of their amino acid sequences affecting the thermo-and halo-stability of the enzyme. A data set containing the amino acid sequences of xylanases with different thermo-and halostabilities was collected. Seventy-four amino acid attributes were obtained for each enzyme sequence. After running a feature selection algorithm for each of the thermo-and halostablity variables, features were classified as either important, unimportant or marginal. The results showed a significant correlation between structural amino acid attitudes and stability in harsh temperatures or alkaline conditions. Features such as lysine, glutamic acid, and positively/negatively charged residues showed a positive correlation with both the thermostability and alkalophilicity attributes of the protein. For the first time, we found attributes which were important for both stability at high temperatures as well as in alkaline conditions by mining sequence-derived amino acid attributes using data mining.

Laccases are enzymes of the family of the multicopper oxidases, being widely used for biotechnological applications. The enzyme catalytic cycle consists in the oxidation of the substrate with the concomitant reduction of molecular oxygen to water. In the process the substrate is converted to a free radical, that can oxidize larger substrates acting as a mediator or it can undergo polymerization. Substrate binding is not specific and there is a large diversity of substrates for laccases. Moreover, the binding site shows important differences among diverse species. The goal of the present work is to characterize the laccase binding pocket of different species in order to establish their common pharmacophoric characteristics. For this purpose we have carried out docking studies with a subset of substrates, covering the diversity of substrates using the Glide program. We have also analyze the characteristics of the binding site using diverse probes. We further have rationalized the differential values of Km found among diverse species for a specific substrate. Finally, special attention has been devoted to the binding of the mediator 2,2'-azido-di-(3-ethylbenzothiazoline)-6sulfonic acid (ABTS), commonly used in industrial processes.

Abiotic stress responses are regulated critically at the transcriptional level. Clarifying the intricate mechanisms that regulate gene expression in response to abiotic stress is crucial and challenging. For this purpose, the factors that regulate gene expression and their binding sites in DNA should be determined. By using bioinformatics tools, the differentially expressed probe sets were studied. A meta-analysis of transcriptomic responses to several abiotic stresses in barley was performed. Motif enrichments revealed that AP2/ERF (APETALA2/Ethylene-Responsive Factor) has the most frequent binding sites. We found that the bHLH transcription factor family has the highest number of transcription factor members. Moreover, network construction revealed that AP2 has the highest number of connections with other genes, which indicates its critical role in abiotic stress responses. The present research further predicted 49 miRNAs belonging to 23 miRNA families. This study identified the probable conserved and enriched motifs, which might have a role in the regulation of differentially expressed genes under abiotic stresses. In addition to shedding light on gene expression regulation, a toolbox of available promoters for genetic engineering of crop plants under such abiotic stresses was developed.

Laccases belong to multi copper oxidase enzyme family (EC 1.10.3.2). Their capacity to oxidíze a wide range of substrates makes them very attractive for the industry and are growing in importance for environmentally-friendly synthesis. Laccases have three different copper sites including, type 1 (T1), type 2 (T2) and type 3 (T3). The function of the T1 site is shuttling electrons from the substrate to the trinuclear copper cluster. During the catalytic cycle of laccase, four electrons are removed from four substrate molecules, which are finally transferred to reduce oxygen to two water molecules .Comparison of the kinetic parameters using several laccases and several substrates reveals that the reaction rate of laccase correlates with the redox potential difference between the T1 copper and the substrate. In recent years, the demonstrated potential of laccases in a range of applications has motivated the progress of laccase engineering efforts. Computational simulations can reveal targets for protein engineering to be explored by site-directed mutagenesis (or semi-rational approaches). In this work we used computational methods for studying interaction of different substrates with laccases and structural activity of the enzyme. The goal of the present study was to characterize the laccase binding pocket of fungal and bacterial laccases in order to establish their common pharmacophoríc characteristics. For this purpose, we first performed molecular docking studies to identify those residues involved in the interaction with diverse substrates. Our results indicate that bacterial laccase {1UVW) has less hydrophobic and aromatic residues in the activity site in comparison to other fugal structures of this study, as a result, find a pose that interacts with residues needs more energy. Subsequently, we evaluated the effect of protonation state of a conserved residue in fungal laccase, Asp/Glu, through molecular dynamics simulation. In a subsequent step, we applied QMMM-2QM-MD approach for one of the fungal laccase structure (3FU8) for calculating redox potential value. The result indicates that the difference in redox potentials changes from 7-17 to 74-92 kJ/mol if the redox state of T1Cu and DMP in the other subunit change and we correctly predict that CuT1ox/DMPred state is more stable than the CuT1red/DMPox state. After the insight gathered from computational studies we started site directed mutagenesis studies on two residues of the binding pocket in order to find their effect on the redox potential value. We made a combinatorial library for position 192 and 296 in MtlL T2. The clone contained A192P and L296W (3H12) mutation and clone contained A192P and 296L {19G8) showed activity with violuríc acid 1.23 and 1.33 fold higher than parental type, respectively. Moreover, the clone contained A192R and L296W (15H11) and clone with mutation A192R and L296L {5B4) showed higher activity with molybdenum compound in comparison to parental type. After experimental characterization of the 19G8 and 5B4 mutants, we studied the structural changes produced in the binding pocket. For this purpose we generated a three-dimensional structure of the two mutants using M.albomices laccase as template by homology modelling. Whereas the former mutant exhibits a similar binding pocket to the template, the latter appears to be smaller. In any case, subsequent docking studies did not show any differential behaviour and ligands could bind to both binding pockets in a similar way. Finally, we calculated the redox potential of the mutant A296L MaL that is similar to the former mutant, yielding a value of 167 kJ/mol. This is higher than the value obtained for MalL supporting the effect of this mutation on the redox potential. Las lacasas pertenecen a la familia de enzimas multícobre oxidadas (EC 1.10.3.2). Su capacidad de oxidar una amplia gama de sustratos las hace muy atractivas para la industria y su utilización está creciendo en importancia para la síntesis respetuosa del medio ambiente. Las lacasas tienen tres tipos diferentes de cobre: tipo 1 (T1), Tipo 2 (T2) y típo 3 (T3). La función del sitio de T1 es la de transportar electrones desde el sustrato al clúster de cobre trinuclear. Durante el ciclo catalítico de la lacasa, cuatro electrones son transferidos desde cuatro moléculas de sustrato para reducir oxígeno a dos moléculas de agua. La comparación de los parámetros cinéticos utilizando varias lacasas y varios sustratos revela que la velocidad de reacción de la lacasa se correlaciona con la diferencia de potencial redox entre el cobre T1 y el sustrato. En los últimos años, el potencial demostrado por las lacasas en una gama de aplicaciones ha motivado el progreso en la ingeniería de lacasas. Las simulaciones computacionales pueden revelar residuos clave que pueden ser cambiados por mutagénesís dirigida (o enfoques semi-racionales). En este trabajo se han utilizado métodos computacionales para el estudio de la interacción de diferentes sustratos con lacasas y ver su efecto sobre la actividad. El objetivo del presente estudio fue caracterizar la unión de lacasa bolsillo de lacasas fúngícas y bacterianas con el fin de establecer sus características farmacofórícas comunes. Para este propósito, hemos realizado estudios de anclaje moleculares para identificar aquellos residuos que participan en la interacción con diversos substratos. Nuestros resultados indican que la lacasa bacteriana (1UVN) tiene un número menor de residuos hidrófobos y aromáticos que las estructuras fúngicas, como consecuencia la unión no es tan fuerte. Posteriormente, se evaluó el efecto del estado de protonación de un residuo Asp / Glu conservado en lacasas fúngicas a través de dinámica molecular. En una etapa posterior, se aplicó enfoque QMMM-2QM-MD para uno de la estructura lacasa fúngica (3FU8) para calcular el valor potencial redox. El resultado índica que la diferencia en los potenciales redox cambios 7-17 a 74-92 kJ/mol sí el estado redox de T1Cu y DMP en la otra subunidad cambio y correctamente predecir qué estado CuT1ox / DMPred es más estable que el CuT1red / estado DMPox. Después de los estudios computacionales se llevó a cabo un estudio de mutagénesis dirigida sobre dos residuos del bolsillo de unión, con el fin de encontrar su efecto sobre el valor potencial redox. Con este objetivo se llevó a cabo una biblioteca combinatoria para la posición 192 y 296 en MtL T2. El clan contenía A192P y L296W (3H 12) y el clan contenía la mutación A192P y L296L (19G8) mostraron una actividad con ácido violurico 1,23 y 1,33 veces mayor que la de tipo parental, respectivamente. Por otra parte, el clon contenía A192R y L296W (15h11) y el clon con A192R mutación y L296L (5B4) mostraron una mayor actividad con el compuesto de molibdeno en comparación con el tipo parental. Después de la caracterización experimental de los mutantes 19G8 y 5B4, estudiamos los cambios estructurales que se producen en el bolsillo de unión. Con este fin generamos una estructura tridimensional de los dos mutantes utilizando la lacasa de M.albomices como plantilla, por medio de la modelización por homología. Mientras que el primer mutante exhibe un bolsillo de unión similar al de la plantilla, éste es más pequeño en el segundo mutante. En cualquier caso, los estudios de anclaje molecular posteriores no mostraron ningún comportamiento diferencial y los ligandos podrían unirse a los dos bolsillos de unión de una manera similar. Finalmente, se calculó el potencial redox de la mutante A296L MaL que es similar al mutante 19G8, obteniéndose un valor de 167 kJ/mol. Este valor es más alto que el obtenido para MaL, apoyando el efecto que tiene esta mutacíón sobre el potencial redox.