Significant efforts have been made on filamentous fungal biotechnology in recent years in order to obtain value added products such as enzymes, chemicals, liquid biofuel, secondary metabolites and spores (Reina et al., 2013). White-rot basidiomycetes are the principal organisms that secrete a unique set of extracellular oxidoreductases comprising   lignin    peroxidase    (LiP; Ec1.11.1.14), manganese peroxidase (MnP; EC 1.11.1.13) and laccase (Lac; EC 1.10.3.2). These catalysts are known with remarkable potential to depolymerize lignin and various environmental pollutants such as polycyclic aromatic hydrocarbons, synthetic dyes and cholorophenols (Du et al., 2015). Different white rot fungi (WRF)  secrete  different  set  of  lignin  mineralizing enzymes (LMEs) and each fungus secretes one or more of the three enzymes essential for lignin degradation (Levin et al., 2008). Besides, some versatile peroxidases (VP; EC.1.11.1.16) with combined LiP and MnP catalytic properties have also been reported (Morgenstern et al., 2008).

 

Due to their interesting catalytic properties, MnPs have gained considerable interest in various industrial areas. The most intensively studied applications have included bioremediation, biomass delignification, oxidation of organic pollutants, bio sensing, textile, animal feed, cosmetics, detergent manufacturing, paper and pulp, transformation of antibiotics and steroids etc (Sylvia et al., 2015). MnP (EC 1.11.1.7) is a lignin-modifying glycoprotein synthesized by wood-colonizing basidiomycetes during secondary metabolism. It catalyzes phenolic compounds to phenoxy radicals by oxidation of Mn²⁺ to reactive Mn³⁺ in H₂O₂ dependent enzymatic reaction (Ferhan et al., 2012). However, some low molecular weight mediators can increase the substrate range of MnPs to non-phenolic structures (Giardina et al., 2000). Further, many proteins acting synergistically with MnPs, has expanded the role of these enzymes in fungal lignolysis (Hilden et al., 2000).

 

Among WRF, Ganoderma lucidum is considered as the most commonly used organism in biodegradation studies due to its good ligninolytic properties, fast growth potentials, and environmental-friendly nature (Batool et al., 2013). Keeping in view the extensive industrial applications of MnP, the present study was accomplished with an objective to purify and characterize the extracellular MnP from an indigenous WRF strain G. lucidum IBL-05 and then tested it for its ability to decolorize different textile dyes.

Fungal culture and inoculum development

 

For inoculum development, the indigenously isolated G. lucidum IBL-05 strain was grown in Kirk’s basal nutrient medium (Tien and Kirk, 1988) in Erlenmeyer flask (250 ml) that was supplemented with Millipore filtered 1% glucose. Prior to sterilization, the medium was adjusted at pH 4.5 with 1 M NaOH/1 M HCl and inoculated with spores of G. lucidum IBL-05 from slant culture. The inoculated flask was incubated (120 rpm) at 30°C for 5 to 7 days to get homogenous spore suspension of the fungus (1×10⁶-10⁸ spores/ml) and used as inoculum.

 

Production and extraction of MnP

 

Triplicate conical flasks containing 5 g semi-solid wheat bran were autoclaved and inoculated with 5 ml homogenous inoculum (Ramzan et al., 2013). The inoculated flasks were allowed to ferment at 30°C in a temperature controlled incubator for 5 to 7 days at pH 4.5. After growth, sterile samples were taken after every 24 h and MnP activity was monitored. When MnP activity was peaked to a maximum level, the fermented biomass was harvested by  adding  100 ml  distilled  water,  filtered,  centrifuged   (Ependorf 5415C, Germany) and clear supernatant was assayed for MnP enzyme and stored at 4°C in refrigerator for further characterization. All experiments were carried in triplicate to avoid the discrepancy in results.

 

Determination of MnP activity and protein contents

 

The MnP activity was determined by monitoring the formation of Mn³⁺-malonate complexes at 270 nm (ε₂₇₀ = 11570 M cm^-1, Wariishi et al., 1992). Assay mixture contained 1 ml of 1x10^-3M MnSO₄, 1 ml of 50 x 10^-3M sodium malonate buffer (pH 4.5) and 0.5 ml of H₂O₂ in combination with 0.1 ml enzyme solution. Absorbance of each sample was measured Spectrophotometrically (HALO DB-20). The protein concentration was estimated according to Bradford method (1976) using bovine serum albumin (BSA) as a standard.

 

Purification of MnP

 

All the purification steps were conducted below 4°C. Briefly, crude MnP extract obtained from 5 days old culture of G. lucidum IBL-05 was centrifuged at 3,000 × g for 15 min. The cell-free supernatant was saturated (up to 35%) by gradual addition of ammonium sulphate and kept for overnight at 4°C. The resulting precipitate, thus obtained was recovered (3,000 × g for 20 min at 4°C) and supernatant was again saturated by adding ammonium sulfate (up to 65%), allowed to stand overnight at 4°C, centrifuged and pellets were dissolved in 50 mM Sodium Malonate buffer  (pH 4.5). The solution was kept in dialysis bag and dialyzed against the same buffer, after sealing it securely and finally, dialyzate was freeze dried. The dialyzate obtained was submitted to ion-exchange chromatography using diethyl amino ethyl (DEAE) cellulose column, equilibrated with phosphate buffer (100 mM; pH 6.5) for 24 h and eluted with 0 to 1.0 M linear gradient of NaCl at a flow rate of 0.5 ml/min. The MnP active fractions were pooled and loaded onto Sephadex-G-100 column (10×300 mm). Up to 30 positive fractions (1 ml) with flow rate of 0.3 ml/min were collected and absorbance was measured at 280 nm (Zeng et al., 2013).

 

Gel electrophoresis and staining

 

The MnP purification was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) following the method of Laemmli (1970). The molecular mass of MnP was approximated after gel staining with Coomassie Brilliant Blue G-250 followed by calibration against standard protein markers (Sigma, USA), ranging from 17-170 kDa.

 

Characterization of purified MnP

 

Effect of pH on MnP activity was investigated by incubating enzyme for 15 min at varying pH values (3 to 10). For stability studies the enzyme was pre-incubated at varying pH for 1 h. MnP activities were also determined at various temperatures between 30 and 70°C under optimal pH values. The enzyme was incubated for 15 min at varying temperatures before running the enzyme assay. For thermal-stability the enzyme was incubated at different temperatures for 1 h without substrate before carrying out MnP assay. K_m and V_max for the purified enzyme were calculated using the Lineweaver Burk transformation of Michaelis-Menten equation. The standard quartz cuvettes of 1 mm path length were used to calculate the values of kinetic parameters. The enzyme was incubated for 15 min at 30°C in sodium malonate buffer of pH 4.5 before carrying out standard enzyme assay protocol. Lineweaver?Burk  (Double   reciprocal)   plot   was   generated   with Microsoft Excel Windows updated version 7 via nonlinear regression analysis using different concentrations (0.1 to 1.0 mM) of manganese sulphate as substrate at optimum pH 5 and 40°C temperature. The effects of various modulators (Mn²⁺, Zn²⁺, Co²⁺, Cu²⁺, Fe²⁺, Hg²⁺, K⁺, Ethylene diamine tetra acetic acid (EDTA) and cysteine) on enzyme activities were tested in the concentration range of 5 to 20 mM. Enzyme activities measured without any modulator was considered as 100% (Zeng et al., 2013).

 

Dyes and decolorization studies

 

Three dyes namely Sandal-fix Red C₄BLN (?max: 540 nm), Sandal-fix Turq Blue GWF (?max: 664 nm) and Sandal-fix Black CKF (?max: 598 nm) were used to investigate the decolorization potential of purified MnP. For this, MnP solution was transferred to triplicate Erlenmeyer flasks (500 ml) containing 100 ml of individual dye solution (0.1 mg/ml) in combination with Na-malonate buffer (50 mM; pH 4.5). Flasks were incubated in rotary shaker (Sanyo-Gallenkamp, UK) at 40°C for 12 h. The flasks content were filtered, centrifuged (8,000 × g, 10 min) and dye removal was monitored spectrophotometrically (HALO DB-20) at respective wavelengths. The decolorization efficiency was calculated using relation (Equation 1).

 

 

Where, Ai and At are representing absorbance at zero and time t.

 

Statistical analysis

 

Mean and standard deviation (SD) of the results based on three independent experiments were calculated using Microsoft Excel-software (Microsoft) and the standard error (SE) values were displayed as Y?error bars in figures.

Production of MnP

 

In  this study, a locally isolated fungal strain, G. lucidum IBL -5was exploited for ligninolytic enzyme (Lip, MnP and laccase) production potential in solid state medium of wheat bran under pre-optimized growth conditions such as moisture, 50%; substrate, 5 g; pH, 5.5; temperature, 30°C; carbon source, 2% glucose; nitrogen source, 0.02% yeast extract; C:N ratio, 25:1; fungal spore suspension, 5 ml and fermentation time period, 5 days (Ramzan et al., 2013). The enzyme extract contained 576.3, 717.7 and 323.2 UmL^-1 of LiP, MnP and laccase, respectively.

 

Purification of MnP

 

A sequential four-step purification procedure involving ammonium sulphate fractionation, dialysis, DEAE-cellulose ion exchange and G-100 sephadex gel permeation chromatography was employed for the purification of MnP. A mixture of crude ligninolytic extract obtained from five days incubated culture (300 ml) of G. lucidum on wheat bran was purified to homogeneity as summarized in Table 1. The MnP was completely salted out at 65% saturation with (NH₄)₂SO₄ to 1.73 fold purification with specific activity of 273.01 U/mg. After ammonium sulphate precipitation, the crude extract was applied to ion exchange chromatography. The elution pattern on the DEAE-cellulose column showed two protein peaks at 280 nm (Figure 1). The fractions with high MnP activities were pooled, concentrated and loaded onto a Sephadex G-100 column. In Figure 2 it was clearly indicated that the MnP eluted in a single prominent peak on gel filtration chromatography. At the end of fourth purification step, the enzyme had been 3.43-fold purified with corresponding specific activity of 539.59 U/mg. The purified MnP appeared as a single band in 43 KDa region on SDS-PAGE analysis (Figure 3), suggesting that the enzyme was a monomeric protein. The MnPs also secreted in a multiple isoform with distinct structural configuration and molecular mass. The molecular masses of MnP vary from 32 to75kDa (Asgher et al., 2013).

 

 

 

Characterization of purified MnP

 

Effect of pH on MnP activity and stability

 

The pH profile for purified MnP has been illustrated in Figure 4. The MnP displayed maximum activity at optimum pH 5; beyond this pH value a marked decreasing trend in the activity was observed. Moreover, the purified MnP was fairly stable over a wide range of pH (4-6) at incubated time of 1 h, pH above 6 caused inactivation of the enzyme irrespective of incubation time. In previous studies, maximum activity of MnP from different WRF has been reported in the pH range of 4.5 to 6.5 (de Oliveira et al., 2009). MnP isolated from solid-state culture of corncobs by Lentinula edodes exhibited optimum activity at pH 4.5 (Boer et al., 2006); whereas, MnP from S. commune IBL-06 was optimally active at pH 5 (Asgher et al., 2013).

 

 

Effect of temperature on MnP activity and stability

 

The MnP activity against temperature curve shown in Figure 5, the plot indicated that initial rise of temperature up to 40°C increased the activity as well as stability of the MnP. Further rise of temperature caused decrease of activity and stability as well due to denaturation of enzyme at elevated heat.  The optimum temperature for purified MnP was found to be 40°C. The purified MnP showed fascinating thermal-stability up to 50°C without dropping much of its activity, which would be an attractive and desirable feature for a variety of industrial processes. However,  MnP  lost  almost  50% of its activity when incubated at 55°C for 1 h. The MnP isolated from different WRF demonstrated optimum temperature around 40 to 60°C (Hakala et al., 2005). The MnP from WRF strain, Irpex lacteus was stable in the temperature range of 30 to 40°C (Shin et al., 2005), while Rhizoctonia sp. isolated MnP was deactivated over 55°C (Cai et al., 2010). The MnP2 isozyme from Lentinula edodes showed thermal-stability up to 40°C (Boer et al., 2006).

 

 

Determination of kinetic constants Km and Vmax

 

The K_m and V_max values were calculated by intercepting line on X-axis and Y-axis of the reciprocal plot, respectively, using different concentration (0.1 to 1.0 mM) of MnSO₄ as assay substrate (Figure 6). At 0.5 mM MnSO₄ concentration, the maximum MnP activity (859 UmL^-1) was furnished with K_m 65.64 µM and V_max 640 UmL^-1 using non-linear regression analysis at optimum pH and temperature. The difference in K_m values of MnP from different reported fungal species might be due to the genetic variations and substrate specificities among various species. The interaction of enzyme with its substrate was indicated through K_m values and a lower K_m value reflect high affinity of enzyme for its substrate and higher V_max indicated that  small  amount  of  enzyme can convert substrate into the product (Asgher et al., 2014).

 

Effect of various modulators on activity of MnP

 

In order to identify the nature of enzyme, the effects of various organic compounds and metal ions as possible inhibitors and activators on MnP activities were studied (Table 2). The results revealed that Mn²⁺ and Cu²⁺ enhanced the activity of MnP at all tested concentrations. The metal ion Mn²⁺ showed the most significant role to activate G. lucidum MnP, which was consistent with the findings of Boer et al. (2006). In addition, low concentrations of Co²⁺ (5 mM) drive up MnP activity but at higher concentration of Co²⁺ (20 mM) a slight inhibition was observed (91%). Elevated concentrations of K⁺ enhanced the MnP activity (117%) but lower K⁺ concentrations (5 mM) did not exert any effect on MnP activity. On the other hand, Zn²⁺and Fe²⁺ partially inhibited the MnP, whereas MnP activity was strongly inhibited by Ethylene diamine tetra acetic acid (EDTA) and cysteine and even Hg²⁺ fully inactivated MnP enzyme. The MnP from P. chrysosporium was inhibited by NaN₃, β-mercaptoethanol and dithreitol, whereas co-oxidants such as glutathione, un-saturated fatty acids and Tween 80, significantly enhanced the MnP  activity  (Urek and Pazarlioglu, 2004). Trichophyton rubrum LSK-27 MnP was entirely inhibited by Hg²⁺, while Fe³⁺, Ca²⁺ and Ni²⁺ did not show any inhibitory effect on enzyme activity (Boer et al., 2006).

 

 

 

Decolorization of different textile (synthetic) dyes by purified MnP

 

The dye-decolorizing potential of purified MnP from G. lucidum was demonstrated for different synthetic dyes (Sandal-reactive dyes) at different time periods. From data in Figure 7 it can be seen that MnP caused maximum decolorization of S.F. Turq Blue dye to 81.3%, followed by S.F. Black CKF to 74.2% and S. F. Red C₄BLN dye to 67.8% within 12 h of incubation period. The MnP was more effective for decolorization of different textile dyes including Remazol brilliant blue R (RBBR), Congo red, methylene blue and ethyl violet (Bazanella et al., 2013). The findings correlated with previous investigations (Cheng et al., 2007), which confirmed that MnP has remarkable catalytic potential to degrade and mineralize dyes and colored effluents. The peroxidases caused dye degradation by generating highly active free radicals such as Mn³⁺, lipid, hydroxyl, and peroxy-radicals (Hofrichter, 2002). However, the dyes are not uniformly susceptible to biodegradation because of the structural diversity (Murugesan et al., 2006).

 

The ion exchange and gel filtration column chromatography techniques were  used  to  purify   MnP enzyme from G. lucidum up to 3.43-fold. The molecular weight of purified MnP was determined to be 43 kDa from SDS-PAGE analysis. Purified MnP showed encouraging activity and stability at their optimal pH and temperature. Further, the purified MnP possesses effective dye decolorization capability, indicating a useful tool for bioremediation purposes. The high level MnP production and its novel catalytic features suggest its suitability for industrial and biotechnological applications. Nevertheless, further molecular approaches are needed for improving its catalytic and thermal-stability characteristics that will be the focus of future research.

The authors have not declared any conflict of interest.

This study was supported by Higher Education Commission, Islamabad, Pakistan.