Biocatalytic potential of laccase-like multicopper oxidases from Aspergillus niger
© Tamayo-Ramos et al.; licensee BioMed Central Ltd. 2012
Received: 4 July 2012
Accepted: 19 December 2012
Published: 27 December 2012
Laccase-like multicopper oxidases have been reported in several Aspergillus species but they remain uncharacterized. The biocatalytic potential of the Aspergillus niger fungal pigment multicopper oxidases McoA and McoB and ascomycete laccase McoG was investigated.
The laccase-like multicopper oxidases McoA, McoB and McoG from the commonly used cell factory Aspergillus niger were homologously expressed, purified and analyzed for their biocatalytic potential. All three recombinant enzymes were monomers with apparent molecular masses ranging from 80 to 110 kDa. McoA and McoG resulted to be blue, whereas McoB was yellow. The newly obtained oxidases displayed strongly different activities towards aromatic compounds and synthetic dyes. McoB exhibited high catalytic efficiency with N,N-dimethyl-p-phenylenediamine (DMPPDA) and 2,2-azino-di(3-ethylbenzthiazoline) sulfonic acid (ABTS), and appeared to be a promising biocatalyst. Besides oxidizing a variety of phenolic compounds, McoB catalyzed successfully the decolorization and detoxification of the widely used textile dye malachite green.
The A. niger McoA, McoB, and McoG enzymes showed clearly different catalytic properties. Yellow McoB showed broad substrate specificity, catalyzing the oxidation of several phenolic compounds commonly present in different industrial effluents. It also harbored high decolorization and detoxification activity with the synthetic dye malachite green, showing to have an interesting potential as a new industrial biocatalyst.
Multicopper oxidases (MCOs) form a family of redox enzymes that catalyze the reduction of molecular oxygen into water by a four-electron transfer process. It includes laccases (EC 184.108.40.206), ascorbate oxidases (EC 220.127.116.11), bilirubin oxidases (EC 18.104.22.168) and ferroxidases (EC 22.214.171.124), which are key enzymes in many biological processes of prokaryotic and eukaryotic organisms[1, 2]. In fungi, complex MCO gene families exist, possibly due to the variety of functions they accomplish. Fungal MCOs are involved in delignification, morphogenesis, pigment formation, pathogenesis, competitor interactions and transport of metal ions[2, 3]. Their ability to react with a variety of aromatic compounds, by producing just water as a by-product, makes them interesting green biocatalysts[2, 3]. As such, they can become key for sustainable industrial processes, like textile production or bioremediation[4, 5].
The majority of fungal MCOs are distributed, according to Hoegger et al., within the basidiomycete laccases, the ascomycete laccases, the fungal pigment MCOs and the fungal ferroxidases clusters. Laccases form the largest subgroup within the MCO family and they have received most of the attention in biochemical and biotechnological studies. In particular, basidiomycete laccases of several Trametes and Pleurotus species, amongst others, have been well characterized.
Fungal pigment MCOs, mainly found in ascomycetes, have been reported in several Aspergillus species: A. nidulans LccD, TilA and YA[8–10]; A. fumigatus Abr2; and A. niger McoA, McoB and McoC. Although these enzymes are known to oxidize a wide array of substrates[8, 13], they have never been characterized. Therefore no information is available about their molecular properties or substrate specificities. Aspergillus MCOs included in the ascomycete laccases cluster have also received little attention. A significant number of these enzymes, including: A. nidulans LccA, LccB and LccC; and A. niger McoD, McoF, McoG, McoI, McoJ and McoM, remain uncharacterized. Interestingly, A. niger MCOs (both, the ones that belong to the fungal pigment MCO cluster, and to the ascomycete laccase cluster) have a low similarity to laccases included in the basidiomycete laccases cluster (around 25% identical). They also differ from the few deeply characterized ascomycete laccases (i.e. around 25-30% identical to Melanocarpus albomyces laccase, MaL). Thus, to obtain insight into the possible biotechnological potential of this particular group of MCOs, more knowledge about their catalytic properties is required.
The activity patterns observed in plate assays of ten A. niger laccase-like MCOs that were recently homologously overexpressed, indicated that remarkable biochemical differences exist between them. Here we address the biocatalytic potential of three A. niger laccase-like MCOs: two fungal pigment MCOs (McoA and McoB), and one MCO belonging to the ascomycete laccase subfamily (McoG). Their ability to oxidize an array of aromatic compounds and decolorize different dyes was evaluated.
Results and discussion
Homologous expression, purification and molecular properties of A. niger MCOs
Steady-state kinetic parameters of McoA, McoB and McoG with DMPPDA and ABTS
K m (mM)
V max (Δ Α 550 /min mg)
K i (mM)
K m (mM)
V max (Δ Α 420 /min mg)
K i (mM)
3.6 ± 0.2
4.6 × 102 ± 8
0.4 ± 0.1
7.5 × 103 ± 6.0 × 102
22.4 ± 6.4
0.5 ± 0.1
2.9 × 102 ± 4
1.8 ± 0.3
4.4 × 102 ± 40
4.6 ± 0.7
5.6 ± 1.0
2.5 × 102 ± 16
Effect of pH and temperature on A. niger MCOs activity
Effect of potential inhibitors on A. niger MCOs activity
Effect of metal salts and inhibitors on McoA, McoB and McoG activity
Residual activity (%)
NaN3 (1 mM)
5.0 ± 0.3
20.0 ± 1.3
16.6 ± 4.2
EDTA (50 mM)
93.8 ± 1.8
82.9 ± 9.8
202.8 ± 33.9
CaCl2 (50 mM)
63.7 ± 0.4
76.4 ± 4.2
53.3 ± 8.6
MgCl2 (50 mM)
81.3 ± 0.1
91.0 ± 0.3
78.2 ± 0.5
NaCl (100 mM)
72.5 ± 4.1
83.8 ± 4.2
62.0 ± 9.5
CuSO4 (0.1 mM)
83.0 ± 10.2
73.1 ± 10.9
14.2 ± 6.5
CuSO4 (0.5 mM)
51.3 ± 8.1
41.9 ± 1.6
2.9 ± 2.0
CuSO4 (1 mM)
36.2 ± 6.7
43.0 ± 3.1
CuSO4 (2.5 mM)
31.6 ± 6.4
36.2 ± 3.8
SDS (1 mM)
109.0 ± 1.0
139.0 ± 10.1
143.7 ± 27.0
46.1 ± 4.4
29.8 ± 1.3
21.9 ± 5.5
10.8 ± 1.2
34.6 ± 1.1
13.9 ± 4.0
28.8 ± 0.4
46.1 ± 4.2
24.1 ± 1.6
Biocatalytic potential with natural and synthetic substrates
Substrate specificity of McoA, McoB and McoG
Substrate (1 mM)
7.1 × 103 ± 1.6 × 102
13.0 × 103 ± 5.6 × 102
75.6 × 103 ± 2.3 × 103
1.7 × 103 ± 4.1 × 102
3.1 × 103 ± 7.9 × 102
2.9 × 103 ± 3.1 × 102
7.0 × 103 ± 4.1 × 102
4.9 × 103 ± 2.9 × 103
6.6 × 102 ± 3.2 × 102
6.1 × 103 ± 4.4 × 102
4.5 × 103 ± 1.2 × 102
7.3 × 102 ± 5.2 × 102
2.3 × 102 ± 20
3.9 × 102 ± 1.1 × 102
3.1 × 103 ± 1.1 × 102
1.6 × 103 ± 1.1 × 102
The inability of McoA to oxidize 2,6-dimethoxyphenol (as observed before) nor ABTS is a remarkable result, as a significant number of reported laccases react with these two common substrates. Nevertheless, as discussed in the “Kinetic parameters” section, other laccases from eukaryotic and prokaryotic sources are not active with ABTS as well. Also, other laccases have been reported to be inactive with laccase model substrates. For instance, the Agaricus bisporus laccase is unable to convert syringaldazine and EpoA, from Streptomyces griseus, does not oxidize syringaldazine and guaiacol. The range of substrates oxidized varies from one laccase to another. It has been suggested that differences in substrate access to the T1 copper site of laccases could imply different substrate affinities. In order to study and understand if this factor could have influence in the narrow range of substrate specificity of McoA, the availability of its three-dimensional structure would be desirable.
The MG decolorization ability of McoB, when the dye is present at a final concentration of 50 mg/L, is similar to the one reported recently from a new laccase from Trametes sp. 48424, and better than that of the laccase from the white-rot fungus Ganoderma lucidum. The direct decolorization of MG during the cultivation of several basidiomycete and non-basidiomycete fungi has been also investigated, but the reported efficiencies in similar conditions (e.g. incubation time, MG concentration) were lower than the ones achieved by means of using the purified enzymes[39–42]. In addition, McoB was also able to oxidize higher concentrations of MG. Similar results, have not been reported for other fungal laccases, although recently the Pseudomonas sp. strain DY1, grown in the presence of the dye, has been shown to be a very efficient tool for MG degradation, in concentration ranges from 100 mg/L to 1000 mg/L. MG successful degradation (50 mg/L) by other bacterial species has been also reported[43, 44]. However, in none of the cases the enzymes involved in the biodegradation process were identified. The results obtained in the present study suggest that McoB could be used in bioremediation processes of this compound. To confirm this, the toxicity of MG transformation compounds should be assessed, as its biodegradation pathway varies depending on the biological treatment, and thus the generated intermediates and final products[35, 38]. The fact that decolorization of MG was achieved with no need of mediators would mean an additional advantage for the use of McoB, as synthetic mediators may be expensive, toxic, and inhibit the enzyme activity at higher concentrations[45, 46]. Nevertheless, natural mediators have also been shown to effectively enhance the transformation of MG in combination with a fungal laccase.
Fungicide activity of MG and its transformation products
The toxicity of higher concentrations (6 mg/L, 10 mg/L and 20 mg/L) of MG and DMG, obtained by incubating MG with McoB for 20 h was also tested. As expected, MG completely inhibited A. niger growth, whereas a negative effect of DMG in the radial growth of A. niger was hardly observed (Figure7b). Furthermore, the radial growth and the mycelium appeareance 72 h after inoculation in complete medium with DMG was not different to the one observed in the control condition, where no dye was added (Figure7c). These results show that the compounds generated after MG decolorization by McoB have a lower toxic effect in A. niger when compared with the one produced by the untreated dye. Therefore, McoB could be a good candidate to be used in detoxification processes of MG. The ability of McoB to detoxify MG without the need of redox mediators is an advantage over other MG detoxification processes reported[38, 46].
The different biocatalytic properties of the A. niger MCOs determined in this study are remarkable. McoA and McoB, showing 56% of sequence identity, are severely divergent in their biochemical features. The potential applications in wastewaters pre-treatment processes and dye decolorization, along with the fact that yellow MCOs are far less well characterized than their blue counterparts, makes more challenging to continue with a deeper characterization of McoB. In this sense, it would be interesting to test its suitability for applications related to the food industry, as A. niger is a safe production organism and many of its enzymes are considered generally recognized as safe (GRAS) by the United States Food and Drug Administration. Also, the possibility to use industrial A. niger strains to overproduce the native form of McoB could overcome issues related to production yields and stability, that can occur during heterologous expression of recombinant proteins, and are less expected in homologous expression systems. Interestingly, the production yields of this enzyme have been recently optimized, together with those of other A. niger MCOs. By improving the performance of the glucoamylase expression system, A. niger strains able to accumulate extracellular McoB up to 42% of the total secreted protein were generated.
This study reports the first biochemical characterization of A. niger MCOs. The purified McoA, McoB, and McoG enzymes showed clearly different substrate specificities. Yellow McoB turned out to be the most efficient biocatalyst, showing broad substrate specificity and high decolorization activity with the synthetic dye malachite green. The lower fungicide activity of decolorized MG suggests that McoB can be an interesting biocatalyst for bioremediation processes involving this dye.
Strains, media and culture conditions
A. niger N593 strains expressing recombinant McoA, McoB and McoG, were used as cell factories to produce the three enzymes. Complete medium plates were used for spores preparation and fungicide activity tests. Minimal medium liquid cultures (containing 50 mM of maltose and 0.1 mM of CuSO4) were used for MCOs production. Liquid cultures were performed at 30°C and 250 rpm in an orbital shaker, in 2 L erlenmeyer flasks containing 800 mL of minimal medium inoculated with 1 × 106 spores/mL.
Purification of A. niger MCOs
All MCO proteins were purified from filtered supernatants of 24 h cultures. Ammonium sulfate was applied at 100% saturation in 3 L of culture supernatant containing McoA. After centrifugation, the recovered pellet was dissolved in 50 mL of 20 mM Tris-HCl buffer (pH 7.5). The resulting solution was dialyzed against 20 mM Tris-HCl buffer (pH 7.5) and then concentrated using an Amicon Ultra-15 Centrifugal Filter device. The resulting solution was applied to a Resource Q 1mL column (GE Healthcare) installed in an Äkta purifier FPLC system (GE Healthcare). A linear gradient of NaCl from 0 to 1 M in 20 mM Tris-HCl buffer (pH 7.5) was performed in a total volume of 200 mL, at a flow rate of 1 mL/min.
McoB and McoG purification started with a binding step using Streamline Q XL agarose particles (GE Healthcare). Prior to the binding step, 1.5 L of culture supernatants containing McoB and McoG were diluted five times in de-mineralized water. 50 mL of Streamline Q XL resin was added to each solution, and stirred during 3 h at 4°C. Proteins were eluted from the resin with 50 mL of a 1 M NaCl solution in 20 mM Tris-HCl buffer (pH 7.5). McoB and McoG solutions were concentrated in an Amicon Ultra-15 Centrifugal Filter device and the NaCl concentration was severely reduced through several washing steps with 20 mM Tris-HCl buffer (pH 7.5). McoB and McoG were further purified on Resource Q, using the same protocol as described for McoA. Purified enzymes were stored at -80°C in 10 mM HEPES buffer (pH 7.0).
Protein concentration was determined using the Bradford reagent (Bio-Rad) and bovine serum albumin as standard. SDS-PAGE was carried out using Precast Polyacrylamide (12%) Mini Gels (Thermo Scientific). Page Blue Protein Stain (Fermentas) was used for SDS-PAGE gels staining.
Glycosylation sites in McoA, McoB and McoG amino acid sequences were predicted using NetNGlyc 1.0 (http://www.cbs.dtu.dk/services/NetNGlyc/) and GPP Prediction Servers (http://comp.chem.nottingham.ac.uk/glyco/index.html).
Analytical gel filtration was performed on a Superdex 200 HR 10/30 column (GE Healthcare), using a similar protocol to the one previously described.
Enzyme activity determination
All chemicals were purchased from Sigma and Invitrogen. McoA, McoB and McoG activity was determined using similar conditions as previously described, by measuring the initial oxidation rate (Δ Α), during a period of 6 min, of 6.0 mM ABTS at 420 nm and 2.5 mM DMPPDA at 550 nm (pH 5.0). Enzyme kinetics were determined measuring the oxidation rate of, at least, 10 different solutions of ABTS and DMPPDA, in concentrations ranging from 0.125 to 15 mM. The kinetic parameters of the three enzymes were determined by Michaelis-Menten analysis using the Sigma Plot 8.0 Software for Enzyme Kinetics. V max values were expressed in Δ absorbance/min mg protein.
Effect of pH and temperature on MCO activity
The pH-dependent activity of McoA, McoB and McoG with ABTS (6.0 mM) or DMPPDA (2.5 mM) was measured in McIlvaine's buffer ranging from pH 2.2 to 8.0 as mentioned in the “Enzyme activity determination” section. The temperature-dependent activity of McoB and McoG was measured from 30 to 100°C by end point determination. Reaction mixtures (in closed microcentrifuge tubes) were incubated at different temperatures for 6 min with ABTS (6.0 mM) in 100 mM sodium acetate buffer (pH 5.0). After an incubation step on ice of 5 min, the absorbance of the samples was measured at 420 nm.
Effect of inhibitors on MCO activity
The activity of McoA, McoB and McoG with 2.5 mM DMPPDA in 100 mM sodium acetate buffer (pH 5.0) was measured at 23°C in the absence and presence of 1.0 mM NaN3; 1.0 mM SDS; 50 mM EDTA, CaCl2, and MgCl2; 100 mM NaCl; different concentrations of CuSO4 (0.1, 0.5, 1.0 and 2.5 mM) and 50% methanol, ethanol and acetone.
The activity of McoA, McoB and McoG with the following aromatic compounds was investigated: ADBP, phenol, 2,6-dimethoxyphenol, hydroquinone, vanillin, p-coumaric acid, vanillic acid, ferulic acid, syringic acid, and cinnamic acid. Quantification of substrate specificity was achieved through direct measurement of oxygen consumption. For this purpose an Oxytherm (Hansatech Instruments) was used. All reaction mixtures contained 1 mM of substrate and 100 mM sodium acetate buffer (pH 5.0), and reactions were performed at 23°C. One unit of enzyme activity (U) was defined as the amount of enzyme that oxidizes 1 μmol of substrate per min.
Bromocresol purple (440 nm), amido black 10B (600 nm), crystal violet (560 nm), malachite green (600 nm), blue dextran (600 nm) and bromothymol blue (440 nm) were incubated with 100 μg/mL MCO in 100 mM sodium acetate buffer (pH 5.0) at 55°C for 20 h. As a negative control, the different dyes were incubated with an equal volume of 10 mM HEPES buffer (pH 7.0). The degree of dye decolorization was measured 3 and 20 h after the incubation started.
Fungicide activity of MG and its transformation products
A malachite green solution (100 mg/L) was incubated with McoB or an equal volume of 10 mM HEPES buffer (pH 7.0), for 3 and 20 h, following the conditions mentioned in the “Dye decolorization” methods section. After the 3 h incubation, DMG and MG were mixed with Aspergillus complete medium in 24 well agar plates to reach the following concentrations: 0.25 mg/L, 0.5 mg/L, 1 mg/L, 2 mg/L and 4 mg/L. Similarly, after 20 h incubation, complete medium agar plates were prepared with DMG and MG at the following concentrations: 6 mg/L, 10 mg/L and 20 mg/L. DMG and MG toxicity was measured by their ability to inhibit or reduce A. niger mycelium radial growth after an incubation period of 24 h. In order to monitor toxicity effects and major growth delays produced by DMG or MG, the mycelium growth was monitored for two weeks.
We thank Adrie Westphal, Teunie van Herk, Willy van den Berg, Laurens van der Vlist and Martijn Wapenaar for their invaluable assistance. We also thank the reviewers for their valuable comments. This work has been carried out under the IBOS SME research project 053.63.322 granted by the ACTS national research program from the Netherlands. JATR was recipient of a postdoctoral contract within research project 053.63.322.
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