Degradation of pesticides by the ligninolytic enzyme Laccase : optimisation of in vitro conditions, immobilisation and screening for natural mediators

Pesticides are widely used in many industries but the majority reaches non-target organisms or locations through point or diffuse sources. Understanding conditions for their degradation is therefore important. The degradation of glyphosate, its metabolite AMPA and isoproturon using the ligninolytic enzyme laccase was studied. Optimisation of in vitro conditions were tested with findings indicating that factors such as altering pH and the concentrations of both manganese and redox mediators can impact degradation giving insight into optimal conditions. A method of encapsulation was used showing it is possible to immobilise laccase suggesting a possibility of its suitability as a co-formulation agent in pesticide applications. The immobilised laccase was applied in a laboratory scale experiment to investigate degradation of glyphosate and AMPA in soil and sand. The findings showed an apparent ability of the encapsulated laccase to be liberated and have an effect on glyphosate degradation, although much work still remains in this area. In the final part of this project ligninolytic substrates were screened for natural and easily extractable mediators. Extracts were used to check enzymatic activity and degradation potential. Candidates that showed promising results included extracts from hemp and wheat

Re-designing the substrate binding pocket of laccase for enhanced oxidation of sinapic acid

Iterative saturation mutagenesis was performed over six residues delimiting the substrate binding pocket of a high-redox potential chimeric laccase with the aim of enhancing its activity over sinapic acid, a lignin-related phenol of industrial interest. In total, more than 15000 clones were screened and two selected variants, together with the parent-type laccase, were purified and characterized. The new variants presented shifted pH activity profiles and enhanced turnover rates on sinapic acid and its methyl ester, whereas the oxidation of related phenols was not significantly enhanced. Neither the enzyme's redox potential nor the mechanism of the reaction were affected. Thus, quantum mechanics and molecular dynamics calculations were done to rationalize the effect of the selected mutations, revealing the critical role of the residues of the enzyme pocket to provide the precise binding of the substrate that enables an efficient electron transfer to the T1 copper. The results presented highlight the power of combining directed evolution and computational approaches to give novel solutions in enzyme engineering and to understand the mechanistic reasons behind them, offering new insights for further rational design towards specific targets

Synthetic dye decolorization by three sources of fungal laccase

Decolorization of six synthetic dyes using three sources of fungal laccase with the origin of Aspergillus oryzae, Trametes versicolor, and Paraconiothyrium variabile was investigated. Among them, the enzyme from P. variabile was the most efficient which decolorized bromophenol blue (100%), commassie brilliant blue (91%), panseu-S (56%), Rimazol brilliant blue R (RBBR; 47%), Congo red (18.5%), and methylene blue (21.3%) after 3 h incubation in presence of hydroxybenzotriazole (HBT; 5 mM) as the laccase mediator. It was also observed that decolorization efficiency of all dyes was enhanced by increasing of HBT concentration from 0.1 mM to 5 mM. Laccase from A. oryzae was able to remove 53% of methylene blue and 26% of RBBR after 30 min incubation in absence of HBT, but the enzyme could not efficiently decolorize other dyes even in presence of 5 mM of HBT. In the case of laccase from T. versicolor, only RBBR was decolorized (93%) in absence of HBT after 3 h incubation. © 2012 Forootanfar et al.; licensee BioMed Central Ltd

Enzymatic pretreatment of microalgae using fungal broth from Trametes versicolor and commercial laccase for improved biogas production

Coupling microalgae production to wastewater treatment can reduce the costs of microalgae production for non-food bioproducts and energy consumption for wastewater treatment. Furthermore, microalgae anaerobic digestion can be enhanced by applying pretreatment techniques. The aim of this study is to improve the biogas production from microalgal biomass grown in urban wastewater treatment systems by applying an enzymatic pretreatment with crude fungal broth and commercial laccase. To this end, the fungus Trametes versicolor was cultured, and the enzymatic activity of the culture broth analysed by measuring laccase concentration. The results showed that both the fungal broth and commercial laccase pretreatment (100 U L- 1) over an exposure time of 20 min increased the methane yield in batch tests. Indeed, the fungal broth pretreatment increased the methane yield by 74%, while commercial laccase increased the methane yield by 20% as compared to non-pretreated microalgal biomass. In this manner, laccase addition enhanced microalgal biomass anaerobic biodegradability, and addition of T. versicolor broth further improved the results. This fact may be attributed to the presence of other molecules excreted by the fungus.Peer ReviewedPostprint (author's final draft

Relevamiento de la producción de lacasa en aislamientos de Colletotrichum (Ascomycota), agente causal de antracnosis de la Soja

Colletotrichum truncatum es el hongo patógeno más comúnmente asociado con la antracnosis de soja. Aunque la enzima ligninolítica lacasa se relaciona con la patogenicidad de un amplio rango de hongos fitopatógenos, su rol biológico en la interacción Colletotrichum -soja aún se desconoce. La extensión de la infección en la Argentina , nos ha llevado a examinar la producción de lacasa en cepas aisladas de plantas enfermas de soja de diferentes regiones de nuestro país. Se evaluó la producción in vitro de lacasa en diez cepas (ocho de ellas identificadas como C. truncatum ). Sólo seis, todas correspondientes a C. truncatum , produjeron lacasa cuando fueron cultivadas en un medio con pectina y asparagina. La máxima actividad en este medio (44 U/L) se detectó en la cepa BAFC 3102 (aislada en la Provincia del Chaco). La electroforesis en SDS PAGE de los sobrenadantes de cultivo reveló una única banda con actividad lacasa (peso molecular 67 kDa). La adición de CuSO 4 al medio incrementó hasta siete veces su producción (280 U/L en medio con glucosa), sin embargo la edad del cultivo o la composición del medio no produjeron variaciones en el patrón isoenzimático. Este es el primer reporte sobre producción de lacasa por C. truncatum.Colletotrichum truncatum is the most common pathogen fungus associated with soybean anthracnose. Although the lignin-degrading enzyme laccase has been implicated in pathogenicity of a wide range of plant pathogenic fungi, its biological role in the Colletotrichum -soybean disease system is unknown. The extent of the infection in our country led us to examine laccase production in Argentinean Colletotrichum strains isolated from diseased soybean plants from different geographic locations. Ten strains (eight of them identified as C. truncatum) , were screened for in vitro laccase production. Only six of the isolates, all of them C. truncatum , produced laccase activity when cultured on a defined medium based on pectin and asparagine as carbon and nitrogen sources, respectively. Strain BAFC 3102 (isolated from Chaco province), yielded the highest laccase titers (44 U/L) in this medium. Denaturing polyacrylamide gel electrophoresis of extracellular culture fluids revealed one band with laccase activity (mol wt 67 kDa). CuSO 4 addition to media with either glucose or pectin as carbon sources increased up to 7-fold laccase production (280 U/L in the glucose medium), but the pattern of isoenzyme was not affected by culture age or medium composition. This is the first report on laccase production by C. truncatum.Fil: Levin, Laura Noemí. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; ArgentinaFil: Ramos, Araceli Marcela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; ArgentinaFil: Parisi, Matías. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; ArgentinaFil: Gally, Marcela Edith. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Producción Vegetal. Cátedra de Fitopatología; Argentin

Effect of fibre treatments on mechanical properties of flax/tannin composites

Due to the inherent environmental benefits of using natural resin (tannin) and natural fibre (flax), flax/tannin composites could be potentially used for vehicle applications. One of the main limitations is the hydrophilic property of flax, resulting in the poor fibre/hydrophobic matrix interface quality. Alkali, acetylation, silane treatment and enzymatic treatment were selected to modify non-woven flax mats to prepare the composites. The fibre morphology was studied through scanning electronic microscopes (SEM). The effects of fibre pre-treatments on dynamic and static mechanical properties of composites were investigated through adequate experiments, such as dynamic mechanical analysis (DMA) and static tensile testing. The modified rougher fibre surface broadened the glass transition peaks of composites due to the improved surface adhesion. However, there is no big improvement of tensile strength after modifications. The pure NaOH (sodium hydroxide) treated composites remain the tensile properties and offer good flax/tannin wettability

Effect of mycoviruses on the virulence of Fusarium circinatum and laccase activity

Producción CientíficaLaccase enzymes (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) play a major role in the degradation of phenolic compounds such as lignin. They are common in fungi and have been suggested to participate in host colonization by pathogenic fungi. Putative mycoviruses have recently been isolated from the causal agent of pine pitch canker disease, Fusarium circinatum Nirenberg & O'Donell. In this study, the effects of single and double mycoviral infections on laccase activity, growth rate and pathogenicity were investigated in fourteen F. circinatum strains. Extracellular laccase activity was analyzed by the Bavendamm test, image processing and a spectrophotometric method. Mycelial growth, in vivo pathogenicity and seedling survival probability were also determined in Monterrey pine (Pinus radiata D. Don) seedlings. The findings showed that (i) mycelial growth of isolates from the same fungal population was homogeneous, (ii) the presence of mycovirus appears to increase the virulence of fungal isolates, (iii) co-infection (with two mycoviruses) caused cryptic effects in fungal isolates, and (iv) laccases embody a possible auxiliary tool in fungal infection. The prospects for biocontrol, the adaptive role of F. circinatum mycoviruses and the importance of laccase enzymes in host colonization are discussed.Ministerio de Economía, Industria y Competitividad (Project AGL2012-39912