BackgroundFungal laccases are multicopper oxidases with huge applicability in different sectors. Here, we describe the development of a set of high-throughput colorimetric assays for screening laccase libraries in directed evolution studies.ResultsFirstly, we designed three colorimetric assays based on the oxidation of sinapic acid, acetosyringone and syringaldehyde with λmax of 512, 520 and 370 nm, respectively. These syringyl-type phenolic compounds are released during the degradation of lignocellulose and can act as laccase redox mediators. The oxidation of the three compounds by low and high-redox potential laccases evolved in Saccharomyces cerevisiae produced quantifiable and linear responses, with detection limits around 1 mU/mL and CV values below 16%. The phenolic substrates were also suitable for pre-screening mutant libraries on solid phase format. Intense colored-halos were developed around the yeast colonies secreting laccase. Furthermore, the oxidation of violuric acid to its iminoxyl radical (λmax of 515 nm and CV below 15%) was devised as reporter assay for laccase redox potential during the screening of mutant libraries from high-redox potential laccases. Finally, we developed three dye-decolorizing assays based on the enzymatic oxidation of Methyl Orange (470 nm), Evans Blue (605 nm) and Remazol Brilliant Blue (640 nm) giving up to 40% decolorization yields and CV values below 18%. The assays were reliable for direct measurement of laccase activity or to indirectly explore the oxidation of mediators that do not render colored products (but promote dye decolorization). Every single assay reported in this work was tested by exploring mutant libraries created by error prone PCR of fungal laccases secreted by yeast.ConclusionsThe high-throughput screening methods reported in this work could be useful for engineering laccases for different purposes. The assays based on the oxidation of syringyl-compounds might be valuable tools for tailoring laccases precisely enhanced to aid biomass conversion processes. The violuric assay might be useful to preserve the redox potential of laccase whilst evolving towards new functions. The dye-decolorizing assays are useful for engineering ad hoc laccases for detoxification of textile wastewaters, or as indirect assays to explore laccase activity on other natural mediators.

Background: Wastewaters from the textile industry are an environmental problem for the well-known Colombian textile industry. Ligninolytic fungi and their enzymes are an option for the treatment of these wastewaters; however, the Colombian biodiversity has not been deeply evaluated for fungal strains with ligninolytic activities. In this research, 92 Colombian fungal isolates were collected from four locations around the Aburrá valley, Antioquia, Colombia. Their decolorizing activities were evaluated using Novacron Red, Remazol Black and Turquoise Blue in solid and liquid media at different culture conditions. The best fungal isolate was evaluated in the bioremediation of two real effluents and its enzymatic extracts were used in the decolorization of the three dyes.Results: From 92 Colombian fungal isolates, Leptosphaerulina sp. exhibited the best decolorization percentage (>90 %) in solid and liquid cultures, and in agitated and un-agitated conditions. Leptosphaerulina sp. effectively decolorized the three dyes and two real effluents from textile industries. This decolorization was catalyzed by the production of significant quantities of laccase (650 U/L) and manganese peroxidase (100 U/L). Leptosphaerulina sp. enzymatic extracts exhibited decolorizing activity when ABTS as mediator was added. Leptosphaerulina sp. decolorized two real effluents from textile industries (>90 %) under conditions of low pH and glucose supplementation. Enzymatic degradation and decolorization products' innocuity was demonstrated by cytotoxic and chromatographic analyses.Conclusion: Leptosphaerulina sp. was the best Colombian isolate. This fungal strain achieved a decolorization above 90 % for the three dyes and two real effluents from a textile industry. This decolorization was performed by producing significant amounts of laccase and manganese peroxidase. Leptosphaerulina sp. is an interesting prospect to treat waters polluted with dyes without the production of compounds dangerous for the environment.

BackgroundFungal laccases are multicopper oxidases (MCOs) with high biotechnological potential due to their capability to oxidize a wide range of aromatic contaminants using oxygen from the air. Albeit the numerous laccase-like genes described in ascomycete fungi, ascomycete laccases have been less thoroughly studied than white-rot basidiomycetous laccases. A variety of MCO genes has recently been discovered in plant pathogenic ascomycete fungi, however little is known about the presence and function of laccases in these fungi or their potential use as biocatalysts. We aim here to identify the laccase-type oxidoreductases that might be involved in the decolorization of dyes by Leptosphaerulina sp. and to characterize them as potential biotechnological tools.ResultsA Leptosphaerulina fungal strain, isolated from lignocellulosic material in Colombia, produces laccase as the main ligninolytic oxidoreductase activity during decolorization of synthetic organic dyes. Four laccase-type MCO genes were partially amplified from the genomic DNA using degenerate primers based on laccase-specific signature sequences. The phylogenetic analysis showed the clustering of Lac1, Lac4 and Lac3 with ascomycete laccases, whereas Lac2 grouped with fungal ferroxidases (together with other hypothetical laccases). Lac3, the main laccase produced by Leptosphaerulina sp. in dye decolorizing and laccase-induced cultures (according to the shotgun analysis of both secretomes) was purified and characterized in this study. It is a sensu-stricto laccase able to decolorize synthetic organic dyes with high efficiency particularly in the presence of natural mediator compounds.ConclusionsThe searching for laccase-type MCOs in ascomycetous families where their presence is poorly known, might provide a source of biocatalysts with potential biotechnological interest and shed light on their role in the fungus. The information provided by the use of genomic and proteomic tools must be combined with the biochemical evaluation of the enzyme to prove its catalytic activity and applicability potential.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0192-2) contains supplementary material, which is available to authorized users.