Ligninases Production and Partial Purification of Mnp from Brazilian Fungal Isolate in Submerged Fermentation

The potential of ligninases as a green tool for effective valorization of lignin can be shown through enzymatic cocktails containing different lignin degrading enzymes. The present study deals with the screening of potential fungal strains useful for the liquefaction of bark containing lignin. Three different local isolates (Pleurotus ostreatus POS97/14, Pycnoporus sanguineus and the local isolated fungal strain) were selected out of ten different strains for ligninases production. Maximum production of enzymes was observed in the local isolated fungal strain after ten days in submerged fermentation. The isolated fungal strain produces ligninases mainly for manganese peroxidase (MnP). The enzyme oxidized a variety of the usual MnP substrates, including lignin related phenols. Furthermore, the partial purification for MnP was determined by FPLC and the molecular weight was evaluated by SDS-PAGE.


Introduction
White rot fungi are the most significant lignin degraders among the wood inhabiting microorganisms. They degrade lignin by secreting extracellular enzymes, that is, MnP, LiP and laccase which play a key role in lignin biodegradation [1]. P. ostreatus is a white rot fungus which belongs to Basidiomycetes [2]. Some genera of Basidiomycetes, such as Pleurotus spp., were found to lack lignin peroxidases [3], indicating that different enzymes are probably involved in lignin biodegradation and that among these enzymes, laccases play a key role. Studies on the enzymes secreted by the fungus P. ostreatus have shown that the rigorous action of laccase and aryl alcohol oxidase produces significant reduction in the molecular mass of soluble lignosulphonates [3]. The composition of the ligninolytic system is thus very complex and species specific [4]. The MnPs, LiPs and Lcc are very closely related enzymes of white-rot Basidiomycetes and are likely contributors to fungal ligninolysis. Many of them cleave lignin model compounds to give products consistent with those found in residual white rotted lignin depolymerized synthetic lignins [5].
MnP belongs to the family of oxidoreductases, to be specific of those acting on peroxide as acceptor (peroxidases). The systematic name of this enzyme class is Mn (II):hydrogen-peroxide oxidoreductase. Other names in common use includes peroxidase-M2, and Mn-dependent (NADH-oxidizing) peroxidase. This enzyme needs Ca 2+ for activity. They reduce molecular oxygen to water and oxidize phenolic compounds [1]. These ligninases oxidize various environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs), cholorophenols and aromatic dyes. The peroxidases (MnP and LiP) are heme containing enzymes having typical catalytic cycles that are characteristic of other peroxidases. LiP is able to oxidize various aromatic compounds while MnP almost exclusively Mn (II) to Mn (III), that it chelates, acting as a diffusing oxidizer [6,7]. Laccases (benzenediol: oxygen oxidoreductase, EC (1. 10. 3. 2) are multi-copper blue oxidases widely distributed in higher plants, in some insects and in a few bacteria. Laccase production occurs in various fungi [6]. Due to their interesting catalytic properties, laccases have gained considerable interest in various industrial areas.
The most intensively studied applications have included development of oxygen cathodes in biofuel cells, biosensors, labeling in immunoassays and organic synthesis through biocatalysis. Due to their broad substrate specificity, laccases might have great potential in varied environmental applications including pulp delignification, textile dye bleaching and xenobiotics degradation [6,8]. These applications stimulate new fundamental research for which this enzyme can be used.
The current research activity of ligninases includes by utilizing the local lignin sources (eucalyptus and sugarcane bagasse) and checked their delignification pattern. The characterization of ligninases (MnPs, LiPs and Lcc,) from the Brazilian fungal isolates with respect to production, partial purification and time course studies, is reported in this study.

Strain isolation
The unknown fungal strain was isolated from the northeast part of Brazil called Caatinga. Caatinga covers about 735,000 km². It is the most degraded vegetation type in the semi-arid, and has less than 1% of it protected in permanent reserves [9].

Substrate collection and preparation
Ligniocellulosic materials like sugarcane bagasse, eucalyptus were used for delignification. Sugarcane bagasse and eucalyptus were collected LWART Química, Brazil. All the substrates were dried in an oven at 80°C to constant weight and were grounded in an electric grinder to powder form and stored in airtight plastic jars to keep the substrate free of moisture.

Fermentative organism and culturing conditions
Cultures of P. ostreatus and P. sanguineus were collected from Mycology Collection Lab, Department of Plant Protection, UNESP, Botucatu, SP while local fungal isolate fungal strain was isolated from Northeast part of the country named Caatinga forest. Each fungus were plated on the center of a petri dish containing malt extract (malt extract 25 g; agar 20 g; distilled water 1l), supplemented or not with 0.05% Remazol Brilliant Blue R (RBBR). The cultures were incubated in the dark for 14 days at 25°C. Then, the strains cultured in the presence of the dye were evaluated with regard to their ligninolytic potential by decolorization of the dye.

Determination of dye Remazol Brilliant Blue R (RBBR) oxidation in liquid culture medium
Seven strains that showed the best ligninolytic potential were grown in liquid culture medium (2.5% malt extract; 0.05% RBBR and water 100%). The cultures were incubated at 30°C, in the dark, under constant agitation. Decolorization, which is associated with dye oxidation, was determined at 10 days by monitoring the decrease in the absorbance peak at 595 nm, using a Shimadzu brand UV-1601PC spectrophotometer. The mycelial biomass was separated by filtration and weighed; quantification was performed through gravimetry, by oven-drying at 70°C until constant weight was achieved. The experimental design was completely randomized and consisted of three replicates.
The laccase activity was also measured by spectrophotometry, as o-dianisidine oxidation at 525 nm (ε 525 =65,000 M -1 cm -1 ). The reactive mixture contained per 1 mL: citrate-phosphate buffer, 0.5 M with pH 5.0 (200 µl); 1 mM o-dianisidine solution (100 µl), culture medium (600 µl) and hydrogen peroxide (100 µl). The boiled culture medium was used as a control. For all enzymes under evaluation, one activity unit was defined as the amount of enzyme necessary to oxidize 1 µmol of substrate per minute. The specific activity was expressed as units per microgram of protein [11]. Each value presented here represents the mean of three replicates.

Ligninases cocktail partial purification and characterization
All steps were carried out at 4°C. Proteins in 2 L of culture filtrate were precipitated by addition of ammonium sulfate (80% saturation). The precipitate was collected by centrifugation (5000 g×30 min) and dissolved in 50 mM potassium phosphate buffer, pH 6.5 (buffer A). The enzyme solution was dialyzed overnight against buffer A, then loaded onto a DEAE Sepharose Fast Flow column (10×300 mm) previously equilibrated with buffer A. The column was washed with buffer A and eluted with 220 ml of 0 to 1.0 M linear gradient of NaCI in buffer A at a flow rate of 0.5 ml/min. Fractions containing MnP activity were collected. Fractions with MnP activity were pooled and dialyzed overnight against buffer A, then loaded onto a Superdex 75 (10×300 mm), previously equilibrated with buffer A.
The active fractions (30 ml) were collected and concentrated to 5 ml by ultrafiltration with a Centriprep-3 (3 kDa cut-off, Amicon). The concentrated supernatant was loaded onto a Sephadex G-100 column equilibrated with buffer A, containing 100 mM NaCl in a fast protein liquid chromatography (FPLC) system (Pharmacia, AKTA purifier). The samples were detected by UV detector by using Unicorn 5.11 software of Pharmacia. At this step, MnP activity was eluted as a single peak corresponding to a peak of absorbance at 280 nm. The purified and concentrated enzyme was kept frozen at -20°C without significant loss of activity for several months [12].

Scanning electron microscopy (SEM)
Samples of sugarcane bagasse and black liquor of eucalyptus were oven-dried at 50°C for 1 h and thick layer of samples were supported in the sample-holder fixed on a carbon ribbon. This assembly was maintained in a vacuum-desiccator until the analysis. The SEM with a Jeol model JSM-6360LV microscope was used for observing the lignin samples before and after fungal treatment [13].

Determination of total proteins
The quantification of total proteins was performed by means of a standard curve obtained from solutions containing bovine serum albumin (BSA) at known concentrations (0; 0.005; 0.010; 0.015; 0.020; and 0.025 mg ml −1 ), Bradford's reagent (0.20 mL), and sufficient water to complete a final volume of 1 ml. The samples to be analyzed contained 800 μl of the enzymatic mixture and 200 μl of Bradford's reagent. After agitation, the absorbance was measured at 595 nm.

Gel electrophoresis and staining
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Höfer [14]. Gels were stained with Coomassie brilliant blue R-250. Molecular weights of proteins were determined with commercial molecular weight markers (Bio-Rad, Munich, Germany).

MS Analysis to determine the lignin monomers
MALDI-TOF-MS spectra were acquired on Autoflex III Series

Statistical analysis
Treatment effects were compared by the protected least significant difference methods with ANOVA using Statistical Assistance version 7.6 beta software.

Results and Discussion
The white-rot fungi possess an enzymatic system for lignin degradation, which, due to its wide substrate specificity, has been reported as responsible for the transformation and mineralization of organic pollutants structurally similar to lignin. The enzymes produced by these fungi are manganese peroxidase (MnP), lignin peroxidase (LiP),and laccases, which are frequently referred to as lignin-modifying enzymes (LMEs). Generally, laccases and MnP are more widely distributed among white rot fungi than LiP [16].
Several white-rot ligninolytic fungi have been reported as responsible by the degradation and decolorization of a number of organic pollutants [17]. Regardless of the involvement of MnP and LiP in the degradation of dyes [18], it is expected that white-rot fungi possess differentiated dye decolorization capabilities, due to their qualitative and quantitative differences in ligninolytic enzyme production ( Figure  1). A simple and quick method to select fungi with ligninolytic activity is the use of polymeric dyes, similar to the lignin polymer. The most utilized dye, due to its industrial importance, is Remazol Brilliant Blue R (RBBR), an anthracene derivative that represents an important class of frequently toxic and persistent organic pollutants. Its decolorization has been described by many authors as related mainly to lignin peroxidase, manganese peroxidase, and laccases [18].

Ligninases production during the time course studies
After every 24 h, the triplicate fermentation flasks were harvested and culture supernatants were analyzed for ligninases and dry weight of biomass (residue) was also recorded. The results of isolated fungal strain during time course study showed that maximum production of MnP (64 IUl −1 ), LiP (26.35 IUl −1 ) and laccase (5.44 IUl −1 ) were achieved in 10 days from local isolated fungal strain as compared to other two strains (Pleurotus ostreatus, Pycnoporus sanguineus) The enzymatic pattern for isolated fungal strain was differentiated among the known strains (Table 1); P. ostreatus, P. sanguineus showed ligninolytic activities were distinctive as compared to other strains (Ganoderma spp, Stereum ostrea, and Trametes versicolor) had the least remazol decolorization efficiency and did not show any peroxidase activity. The negative peroxidase test suggests that the fungi did not produce the enzymes at levels significant enough to be detected, or the production of ligninases requires different growing conditions for those strains.
As the incubation time increased, the biomass weight also increased. It was observed that ligninases production steadily increased with an increasing fermentation time and further increase in fermentation time showed a decrease in ligninases activities (Table  1). It was also important to note that local isolate is the best producer of MnP (64 IUl −1 ) as the major enzyme activity, followed by LiP (26.35 IUl −1 ) and laccase (5.44 IUl −1 ) respectively. The isolated fungal strain had the highest percentage of RBBR decolorization (94%) in liquid medium. As a control, the fungi were grown in liquid culture medium without supplementation. During the development period, a followup was conducted for biomass and protein yield in the medium. The concentration of proteins was parallel to the biomass yield, with the exception of the 12 th day, when biomass began to decrease. The specific activities of MnP and LiP however continued to increase [19].

Purification of MnP
The purification of MnP consisted of six steps, including ammonium sulfate precipitation and ion-exchange chromatography. The target protein peak (A 280 ) was then fractionated by the subsequent size exclusion chromatography (Figure 2A). Finally, the activity was eluted as a single peak that corresponded to a peak in the absorbance at 280 nm in the second gel filtration ( Figure 2B). The purified enzyme showed protein band on native-PAGE ( Figure 3A). This band  coincided with the band stained for activity in a gel run simultaneously. The purification procedure is summarized in Table 2. The molecular mass of purified MnP was 37 kDa shown in Figure 3B and the specific activity of purified enzyme was 3.22-fold.
Analysis of the effect of isolated fungal strain treatment on sugarcane bagasse and black liquor of Eucalyptus by scanning electron microscope (SEM) was carried out (Figure 4). When the SEM technique was used, an abundant fungal growth on the bagasse and black liquor fibers were observed which indicates the decay of lignin samples. The fiber surface of bagasse sample clearly shows that modifications resulting from the fungus action on the fiber structure with the presence of bore holes occurred, indicated by arrows in Figure 4 (C and D).

Conclusions
With the increasing global concern on fossil fuel depletion and environmental footprint, there is a strong interest in exploring biorenewable resources as alternative feedstock for making more environmental-friendly biomaterials. The results of the present study indicate the screening of potential fungal strain for ligninases production, partial purification of MnP and the degrading pattern of local lignin resources found in Brazil. It allows a deeper insight into the mechanism of delignification process. The demand for application of ligninases in industry and biotechnology is ever increasing due to their use in a variety of processes. Their capacities to remove xenobiotic substances and produce polymeric products make them a useful tool for bioremediation purposes. The unknown isolated fungal strain has a potential for delignification. Nevertheless, further molecular biology studies are needed to identify the specie by 18S-rDNA.