Screening of marine fungal strains for laccase-like activities amenable to biotechnological applications

Background Environmental pollution is one of the major problems that world is facing to date. Several approaches are been studied and oxidative enzymes from microbial organisms represent an eco-friendly and cost–effective processes, amenable to biotechnological applications, as for instance industrial dye decolorization. The aim of this study was to screen marine-derived fungal strains isolated from three coastal areas in Tunisia, to identify laccase-like activities, and to produce and characterize active fungal secretomes of interest for dye decolorization. Results Following the screening of twenty fungal strains isolated from the harbours of Sfax and Monastir (Tunisia), five strains were identified displaying laccase-like activities. Molecular based taxonomic approaches allowed us to identify these strains as belonging to the species Trichoderma asperellum , Stemphylium lucomagnoense and Aspergillus nidulans . Among these five isolates, one T. asperellum strain ( T. asperellum 1) gave the highest level of secreted oxidative activities, and as such it was chosen for further studies. Optimization of the growth medium for liquid cultures was first studied to improve the level of laccase-like activity in culture supernatants. Finally, T. asperellum 1 secretome allowed decolorizing different synthetic dyes belonging to diverse dye families, in the presence or absence of 1-hydroxybenzotriazole (HBT) as a mediator. Conclusions The optimal growth conditions to produce laccase-like active secretomes from T. asperellum 1 were 1.8 mM CuSO 4 as an inducer, 1% NaCl to mimic seawater environment and 3% sucrose as a carbon source. T. asperellum 1 secretome was effective to decolorize different synthetic dyes belonging to diverse chemical classes, and the presence of HBT as a mediator improved the decolorization process.

no real effect on laccase-like activity was found, with a 160 U L -1 maximum at day 4, against 170 U L -1 at day 3 with 0% NaCl. Interestingly, however, with sea salt laccase-like activity did not decrease after 72 h, remaining stable up to 200 h growth.

Influence of CuSO 4 and of different carbon sources on laccase-like activity in Trichoderma asperellum 1
To study the effect of CuSO 4 on secreted laccase-like activity, different concentrations of CuSO 4 (800 µM, 1000 µM, 1800 µM and 2000 µM) were supplemented to the M7 medium used for T. asperellum 1 cultures. The results, reported in fig. 6, indicate that laccase-like activity increased significantly in the supernatant when cultures were supplemented with CuSO 4 . These increments were dose-dependent and significantly higher at around 2000 µM CuSO 4 , as clearly visible at 72 h, when activity was (170 U L -1 ) was more than 3 times higher than in cultures without CuSO 4 (50 U L -1 ).
Carbon sources are also known to strongly affect the levels of secreted fungal laccase-like activities.
For this reason, we tested adding 3% of either sucrose, glucose or starch to the M7 production medium ( Fig. S1 Supplementary data). Our results indicate that 3% sucrose resulted in higher levels of laccase-like activity (270 U L -1 ) in the resulting supernatant.
Decolorization of synthetic dyes asperellum 1 secretome was prepared in the optimized production medium (M7 containing 1% NaCl, 3% sucrose and 1.8 mM CuSO 4 ). The decolorization ability of the culture supernatant was tested on five different dyes, belonging to three different dye families (reactive, azo and anthraquinone). The secretome was incubated in the presence of five dyes (50 μg mL -1 each) namely Remazol Brilliant Blue R (RBBR), Reactive Black 5 (RB5), Direct Red 75 (DR75), Acid Orange 51 (AO51) and Turquoise Blue (TB) for 48 h. Results showed that the presence of HBT, as observed for most laccases and LMCOs [16], improves the decolorization process, probably by facilitating electron transfert between oxidative enzymes from the secretome and the substrate dye molecules. Fig. 7 shows that in all cases HBT improves the decolorization efficiency of the T. asperellum 1 secretome, but only with RB5 it was necessary. Laccase activity differences on different substrates may depend on substrate redox potential and steric match between the substrate and the enzyme active site structures, as observed for most laccases [17]. RB5 was hardly decolorized in the absence of mediator (only 9% decolorization) whereas in 24 h after addition of HBT the decolorization was increased from 9% to 90%. With RBBR, DR75 and TB, the decolorization was increased with the use of HBT from 60 to 80%, while for AO51 5% only of additional decolorization was achieved (from 75 to 80%). Finally, our study shows that, as observed for laccases, the addition of HBT enhances decolorization at different extents depending on the dye to oxidize.

Discussion
Fungi are recognized for their aptitude to produce a large variety of extra-cellular enzymes [18].
However, most of the fungi studied to date are isolated from forests and other terrestrial environments, while very few studies have focused on the exploration of marine fungal diversity. A large proportion of the diversity of marine-derived fungi would have originated from their terrestrial counterparts, with the appearal of strains able to live in marine harsh environments (high pressure, low temperature, oligotrophic nutrient, high salinity, etc.) [19,20]. These specific conditions are responsible for the significant differences between the enzymes generated by marine microorganisms and their homologues from terrestrial counterparts [21]. Finally, marine-derived microorganisms have been studied to exploit their potential to generate new natural products and to degrade plant biomass [22].
In this study, twenty marine derived fungi were isolated from Tunisian marine biotopes and five of them were selected for their oxidative profile on DMP and ABTS. These five strains were identified as ascomycetes belonging to the species Aspergillus nidulans, Stemphylium lucomagnoense and Trichoderma asperellum (three strains belonging to the latter species). Among these marine strains, Aspergillus nidulans, anamorph of Emericella nidulans, is an important model ascomycete for Previously, a number of molecular markers have successfully been used for the taxonomic identification of fungal genera and species, and ITS rDNA region has been often considered as a marker of choice for the fungal kingdom [27]. However, sequencing of the TEF-1α region is considered as a sensitive tool for identification in mycology with superior resolution then ITS, e.g. when studying the genus Trichoderma [28]. In this study, TEF-1α sequence-based phylogeny suggests that the most phylogenetically related species to our three isolates Trichoderma sp 1, 2 and 3 is Trichoderma asperellum, a fungus which is naturally found in soils [29]. Even if Trichoderma species are usually found in terrestrial habitats, some isolates were collected from marine environments, where they live in association to algae [ [38]. Moreover, a terrestrial T. asperellum secretome producing oxidases including LMCOs was applied to degrade polycyclic aromatic hydrocarbons in soil [39]. In our study, the secretomes of five fungal isolates showed different amounts of laccase-like activities, in liquid cultures and eventually under saline conditions. The highest laccase-like activity was observed with the strain T. asperellum 1, in cultures with as well as without 1% NaCl. For comparison, while marine-derived A. sclerotiorum produced 9.26 U L − 1 laccase-like activity after 7 day-culture in 3% (w/v) NaCl, for T. asperellum 1 about 190 U L − 1 were obtained. In another study [40] optimization of laccase-like activity levels from Trichoderma sp. grown in 0.5% NaCl yielded approximately 2000 U L − 1 , but activity was assayed using o-tolidine instead of ABTS as a substrate, and as such those results are not directly comparable with ours. The finding of laccase-like activities from fungal cultures grown in NaCl-containing media could be benifical for industrial and biotechnological processes in which saline conditions are high [41]. In our study, we show that high levels of salt-tolerant laccase-like activity could be spot out using synthetic dyes as substrates. These findings pave the way to the discovery of novel biocatalysts for the textile industry, whose effluents contain not only dyes, but also high salt concentrations.
Secretome and enzyme characterization will then be the next step of our research.
To maximize the levels of laccase-like activity in T. asperellum 1 cultures, we evaluated the effect of different concentrations of NaCl and known inducers, such as CuSO 4 and three carbon sources. These parameters in fact can affect the productivity of various oxidases secreted in the culture medium, due to an inhibition of fungal growth or to effects on enzyme stability and activity, possibly in relationship to protein surface charges and to perturbation of global or local protein folding [42]. In our study, higher levels of laccase-like secreted activity were found when 1% NaCl was added to T. asperellum 1 cultures. Above this concentration, activity gradually decreased with increasing NaCl concentration.  [50] reported that the best CuSO 4 concentration for LMCO production in Polyporus brumalis was 0.25 mM. CuSO 4 induction of LMCOs is related to the active site architecture of these enzymes, which contain generally 4 copper atoms per polypeptide. Copper addition to the culture medium was also reported to induce laccase gene transcription [51]. In addition, it has been reported that copper could be toxic as it interacts with nucleic acids, proteins, enzymes and metabolites associated with major cell functions, explaining why CuSO 4 concentration should be tested case by case [51]. Several studies have proved that the choice of carbon sources affects the production of ligninolytic enzymes [52]. The purpose of glucose supplementation to lignocellulose for fungal cultures has two reasons. First, it promotes the growth and rapid establishment of the fungus within the solid raw material. Second, the fungus needs an additional, easily metabolizable carbon source to sustain lignin degradation from lignocellulosic substrates [53].
In our study, sucrose is the best substrate for secreted laccase-like activity from T. asperellum 1 cultures (290 U L − 1 ), as it has previously been showed for Arthrospira maxima [54].
Industrial dyes usually have a synthetic origin and complex aromatic structures which make them highly resilient and more difficult to biodegrade [55]. Reactive dyes, for exemple, contain chromophoric groups such as azo, anthraquinone and others. Most of these dyes are not toxic by themselves, but after release into aquatic environments they may be converted into potentially carcinogenic amines that have an impact on the ecosystem downstream from the mill [56]. Currently employed physico-chemical methods were showed to have some serious limitations, such as high cost, high salt content utilization, and problems related to disposal of concentrate [57,58]. In this regard, considerable focus has been placed on developing biological processes, because they are more effective compared to more conventional, physico-chemical methods [56]. The production of LMCOs from marine-derived ascomycetes, zygomycetes and basidiomycetes has been poorly investigated [41,59]. Similarly, to our knowledge, only one work reports on the application of LMCOcontaining secretomes from a marine Trichoderma to degrade synthetic dyes [40], one describes the production of laccase from marine-derived Aspergillus sclerotiorum [59] and no work is available on LMCOs derived from Stemphylium species. In this study, the dye decolorization ability of T.  60]. Due to its high molecular weight, for example, sulfonated azo dyes are unable to pass through the cell membrane, and therefore degradation of these dyes must occur extracellularly. The role of redox mediators in an azo bond detoxification has also been shown before [61]. For instance, it has been reported that the addition of the mediator HBT to the LMCO-containing secretome of Paraconiothyrium variabile enhanced the decolorization of RB5, RBBR, DR75 and TB [62].
In a previous study we investigated RBBR decolorization by the culture filtrate of the terrestrial ascomycete Trametes trogii and by the LMCO isolated from it [63]. The purified LMCO decolorized up to 97% of a 100 mg L − 1 dye solution, with only 0.2 U mL − 1 enzyme. In our test conditions, we reached comparable results (60-80% decolorization) with T. asperellum 1 culture supernatant, with or without HBT. In general, different marine strains are able to degrade RBBR to different extents, for exemple Flavodon flavis degraded RBBR to more than 90% [64] while Cerrena unicolor only to 46% [65].
Biodegradation of RB5 was investigated using the secretome of the ascomycete Trichoderma atroviride F03 yielding 91.1% decolorization without mediators [66]. Three products of this biodegradation reaction (1, 2, 4-trimethyl benzene, 2, 4-ditert butylphenol and benzoic acid-TMS derivatives) were identified, confirming the validity of enzymatic treatment without generating aromatic amines, which are higly toxic [66]. In comparison, the T. asperellum 1 secretome allowed attaining only 10% of RB5 decoulorization without HBT, and up to 80% in the presence of mediator. AO51, is a water-soluble anionic azo dye. Typically containing one to three sulfonic groups, it is widely applied to colour wool, silk and polyamide. The nature and level of toxicity of AO51 has not been well established yet [67], but sulfonated azo dyes (including naphthalene sulfonic acids, naphthols, naphthoic acids, benzidines, etc), and particularly benzidines are in the focus of attention because of their carcinogenicity [67]. AO51 degradation by crude LMCO from Trametes trogii grown in solid cultures on sawdust has been investigated [67] and above 88% decolorization in the presence of HBT was achieved. Our results show that with T. asperellum 1 culture supernatant, instead, HBT was not essential for achieving AO51 decolorization. To our knowledge, this is the first report of AO51 decolorization without the need of laccase mediators.
To date, only a few studies have dealt with decolorization of the phthallocinine dye TB. Plácido et al. showed that Leptosphaerulina sp. effectively decolorized TB and two real effluents from textile industries [68]. This decolorization was catalyzed by the production of significant quantities of LMCO (650 U L − 1 ) and manganese peroxidase (100 U L − 1 ). Leptosphaerulina sp. enzymatic extracts exhibited decolorizing activity when ABTS was added as mediator. Similarly, the secretome of T. asperellum 1 showed maximum TB biodegradation capacity when HBT was added.
Remarkably high levels of DR75 degradation (95-100%) were achieved after 120 h incubation with Penicillium oxalicaum culture supernatant [69]. In that study, high levels of manganese peroxidase activity (659.4 ± 20 U L − 1 ) were measured in the P. oxalicaum secretome, indicating the involvement of heme peroxidases in the decolorization process. In our study, instead, no peroxidase activity was detected in T. asperellum 1 secretomes, suggesting for the first time, to our knowledge, that LMCOcatalyzed DR75 degradation takes place instead.
Further studies will be necessary to get further insight into the enzymatic mechanisms deployed by marine-derived fungi to cope with their environment. It will be necessary to identify the key enzymes secreted by T. asperellum 1 growing in saline conditions, as well as to produce and characterize them, numbers : CECT 21166, CECT 21167 and CECT 21168 for Trichoderma asperellum 1, 2 and 3 respectively, CECT 21164 for Stemphylium lucomagnoense and CECT 21165 for Aspergillus nidulans.

Fungal cultures
Selected marine fungal strains were grown in submerged cultures in 50 mL M7-medium, and culture supernartant was used to retrieve ABTS-oxidizing, laccase-like activity as previously described [75].

Laccase-like activity assay
Laccase-like activity was measured by monitoring the oxidation of 5 mM ABTS (Sigma-Aldrich) in 0.1 M citrate phosphate buffer (pH 5) at 436 nm for 1 min [76]. The reaction mixture (1 mL) contained 0.1 mL supernatant of the culture medium which was centrifuged for 10 min at 12000 rpm. Oxidase activity was determined as the increase in absorbance at 436 nm [(ε 436nm = 29300 M -1 cm -1 ) [77].
One unit of ABTS-oxidizing activity is defined as the amount of enzyme needed to oxidize 1 μmol of ABTS per minute at room temperature. Measurements were also conducted in the presence of either H 2 O 2 (0.5 mM) or catalase (280 unit per ml of assay), in order to confirm that no activity was due to heme-containing peroxidases.

Influence of NaCl, sea salt, CuSO 4 and different carbon sources on laccase-like activity
To compare the effect of NaCl and sea salt on the production of active secretomes, standard M7 medium was supplemented with increasing concentrations of either NaCl or sea salt (1 to 5% w/v). 50 mL cultures were grown in 250 mL erlenmeyer flasks for a period of 7 days at 30 °C, and samples were withdrawn periodically. CuSO 4 was also supplemented to cultures as an inducer of laccase-like activity in case LMCOs were involved. In order to find out the suitable concentration of CuSO 4 for an optimal production of LMCOs, the following concentrations of CuSO 4 were tested: 800 µM, 1000 µM, 1800 µM and 2000 µM. In order to find the suitable carbon source for highest laccase-like activity in culture secretomes, the effect of different carbon sources, such as sucrose, glucose and starch was studied. The carbon sources were tested at a concentration of 3% in M7 production medium. The Erlenmeyer flasks (250 mL) containing 50 mL of the production medium were incubated at 30 °C for a period of 7 days.

Dye decolorization by Trichoderma asperellum 1 secretome
To test the ability of T. asperellum 1 cultures to decolorize industrial dyes, five different dyes used in

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