Identification and characterization of laccase-type multicopper oxidases involved in dye-decolorization by the fungus Leptosphaerulina sp.

13 p.-4 fig.-4 tab.[Background] Fungal 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.[Results] A 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.[Conclusions] The 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.This research was supported by the Program for Interuniversity Cooperation and Scientific Reasearch (PCI) from the Spanish Agency for International Cooperation and Development (AECID), Project AP/033932/11, and the Spanish Project NOESIS BIO2014-56388-R.Peer reviewe

Hongos nativos con potencial degradador de tintes industriales en el valle de aburrá, colombia

Los colorantes industriales poseen estructuras químicas estables que dificultan su tratamiento mediante procesos fisicoquímicos convencionales. En los últimos años, como una alternativa biotecnológica para la degradación de compuestos recalcitrantes, se han utilizado hongos ligninolíticos de diferentes grupos taxonómicos, que producen enzimas oxidantes de dichas moléculas. El aislamiento e identificación de especies fúngicas nativas con potencial decolorante, resulta promisorio para biorremediar efluentes provenientes de industrias textiles. En esta investigación se identificaron, con base en análisis de secuencias de las regiones ITS1 e ITS2 y 28S del ADNr, y por sus características morfológicas, cuatro hongos nativos aislados de material lignocelulósico colectado en el Valle de Aburrá (Antioquia, Colombia). Los aislamientos fueron identificados como el ascomycete Leptosphaerulina sp., y los hongos anamórficos Trichoderma viride (dos cepas) y Aspergillus niger

New colorimetric screening assays for the directed evolution of fungal laccases to improve the conversion of plant biomass

Abstract Background Fungal 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. Results Firstly, 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. Conclusions The 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.This work has been funded by the Spanish National Project EVOFACEL, BIO2010-19697. The internship of X. Chanagá at CIB-CSIC, Madrid, was supported by a grant from the UNAL-Medellín, Colombia. I. Pardo thanks Agencia Estatal CSIC for her JAE-Predoc Fellowship and A.I. Vicente thanks MINECO for her FPI Fellowship.Peer Reviewe

MOESM1 of Degradation and detoxification of synthetic dyes and textile industry effluents by newly isolated Leptosphaerulina sp. from Colombia

Additional file 1. Statistical analysis

Additional file 1: Table S1. of Identification and characterization of laccase-type multicopper oxidases involved in dye-decolorization by the fungus Leptosphaerulina sp.

Oxidoreductases identified from the secretome of Leptosphaerulina sp. grown in dye-supplemented culture (7d). Only significant hits identified from the shotgun nLC/MS-MS analysis of the entire EPP after search against Uniprot Ascomycota database are shown. Protein identities provided on the basis of a single matching peptide, were considered as tentative. Table S2. Oxidoreductases identified from the secretome of Leptosphaerulina sp. grown in the laccase-induced culture (3d). Only significant hits identified from the shotgun nLC/MS-MS analysis of the entire EPP after search against Uniprot Ascomycota database are shown. Protein identities provided on the basis of a single matching peptide, were considered as tentative. Figure S1. Laccase (-♦-) and peroxidase (-■-) activities with ABTS detected in Leptosphaerulina. sp. standard liquid culture (black lines), dye-supplemented culture (red lines) and laccase-induced culture with CuSO4 and ethanol (blue lines). Figure S2. Biochemical characterization of the purified laccase from Leptosphaerulina sp. MALDI-TOF spectrometry analysis of the native and deglycosylated protein (A); SDS-PAGE and Coomassie Blue staining (B); UV-Vis absorbance spectrum (C). Figure S3. Optimum pH for oxidation of ABTS (A) and 2,6-dimethoxyphenol (B) by Leptosphaerulina sp. laccase. (PDF 190 kb