ABTS, syringaldazine, DMP, starch and yeast extract, Remazol brilliant blue (RBBR), Poly R-478 and Congo red were obtained from Sigma-Aldrich, USA. Methyl violet was obtained from Merck and Brilliant Blue Reafix BFNG (Reactive Blue 268), a dye obtained from a laundry textile in Maringá, Paraná, Brazil. All other reagents were of analytical grade.

P. pulmonarius CCB-20 was obtained from the Culture Collection of the Botany Institute of São Paulo. The stock culture of the fungus was maintained at 4 ºC through periodical transfer in Petri dish containing wheat bran (20 g L^-1), agar (18 g L^-1) and mineral medium Vogel (Vogel, 1956). Orange (Citrussinensis (L.) Osb.) residue was obtained at the local businesses and consisted of orange waste, including membrane tissue as well as peel, after extracting the juice (solid waste). The material was dried at 40 ºC and milled (2 mm). Wheat bran was also obtained in the local businesses.

A central composite design (CCD), 2 ** (5-1), was employed to evaluate the influence of different variables in the production of laccase and to obtain the best conditions in citrus residue (Table 1). Five independent variables were evaluated: orange waste (X1), wheat bran (X2), starch (X3), moisture (X4) and yeast extract (X5). The experiment was carried out with a total of 30 combinations (Table 2), including 16 cubic points, 10 axial points and four central points. The results were adjusted by a second order polynomial equation. The substrates were added in Erlenmeyers flasks of 125 mL and moistened with mineral medium Vogel (Vogel, 1956) to obtain the necessary moistures. The Erlenmeyers flasks were autoclaved at 120°C for 15 min. Two disks with mycelium (7 days and 15 mm of diameter) of P. pulmonarius were inoculated in each Erlenmeyer flask, which were incubated at 28°C in the dark, for 12 days. Cultivation was interrupted with the addition of 20 mL of distilled water, followed by agitation for 20 min. at 8 ºC, filtration and centrifugation (10 min.; 10,000g; 4 ºC). The supernatant was used as crude enzymatic solution.

Table 1.
Experimental range of the five variables studied using CCD in terms of actual and coded factors.

The ligninolytic enzyme activities, laccase (EC 1.10.3.2), Mn peroxidase (EC 1.11.1.13) and lignin peroxidase (EC 1.11.1.4) were measured as described previously by Freitas et al. (2017). The aryl-alcohol oxidase (EC 1.1.3.7) was estimated as Guillén, Martínez, and Martínez (1990). The enzymatic activities were expressed in international unities per milliliter (U L^-1). One unit of enzyme activity was defined as the amount of enzyme required to oxidize 1 µmol of substrate per minute.

The culture filtrates were precipitated by drop-wise addition of chilled acetone and the acetone-precipitated enzymes were used in the following experiments. The molecular mass of the laccase was determined by SDS/PAGE (Laemmli, 1970). Protein bands were visualized by silver staining. The following standards (MW-70 kit-Sigma) were used to estimate the molecular weight of the enzyme: bovine serum albumin (66 kDa), ovalbumin (45 kDa), glucose-6-phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), soybean trypsin inhibitor (20 kDa) and α-lactalbumin (14. 2 kDa). The laccase activity was visualized in the gel after washing with 0.05 mol L^-1 sodium acetate buffer (pH 5.0) and incubation with 1 and 10 mmol L^-1 ABTS for 30 min. at 4°C.

For determination of the pH optimum, the enzyme was assayed with malonate (pH 3.0, 50 mmol L^-1), citrate (pH 4.0–6.0, 50 mmol L^-1), phosphate (pH 6.0–7.5, 50 mmol L-1), Tris-HCl (pH 8.0–9.5, 50 mmol L^-1) and glycine buffer (pH 10.0-11.0, 50 mmo L^-1) at 40. The effect of pH on stability of the crude laccase was determined after 24 hours incubation of laccase in 50 mmol L^-1 sodium acetate buffer (pH 4; 4.5 and 5.0) without substrate at room temperature (25°C). The remaining activities were measured under standard conditions.

The optimum temperature of laccase was determined between 35°C and 70°C. Thermal stability was investigated by incubating the enzyme in 50 mmol L^-1 sodium acetate buffer (pH 5.0) at 40, 50 and 60°C for different times. Immediately afterwards, the enzyme was immersed in an ice bath and then activity was tested at 40°C.

The kinetic constants was calculated using syringaldazine (ε₅₂₅= 65 L mmol^–1 cm^–1) in 100 mmol L^-1 sodium phosphate buffer, pH 6.5, ABTS (ε₄₂₀= 36 mmol^–1 cm^–1) in 100 mmol acetate buffer, pH 5.0 and DMP (ε477= 14.8 mmol^–1cm^–1) in 50 mmol L^-1 malonate buffer, pH 4.5 from a Lineweaver-Burk plot. One unit of enzymatic activity was defined as the amount of enzyme required to oxidize 1 μmol of substrate per min. at 40°C. The influence of chemicals reagents CuSO₄, NaF, HgCl, NaCl, Na₂SO₄, NaN₃, β-mercaptoethanol were also tested in different concentrations in laccase activity.

To study of crude laccase capacity (after optimization) in decolorization the following dyes were used: Remazol brilliant blue (RBBR), Ethyl violet, Methyl violet, Methyl green, Methylene blue, Poly R-478, Congo red, Bromophenol blue, Green bromocresol, Methyl red and Brilliant Blue Reafix BFNG. A reaction mixture containing 2.0 mL of dye in 0.05 mol L^-1 sodium acetate buffer (pH 5.0) and 2.0 mL crude laccase (diluted to give a final activity of 3 U) was incubated at 28°C in the dark. After 24 hours, the decrease in absorbance was measured at the maximum absorbance of each dye in a UV and VIS Shimadzu Spectrophotometer and expressed in terms of percentage. In parallel, a boiled crude laccase preparation was used as negative control.

All the analysis was carried out in triplicate. The data was expressed as the means ± standard deviations. Differences of 5% (p < 0.05) among the means were considered significant according to Dunnett test. The results were analyzed using the Statistica 7.0 software (StatSoft, Tulsa, Oklahoma) and GraphPad Prism® 5.0 (San Diego, USA).

A factorial design was developed in the pursuit of studying the influence of five variables and also to verify the interactions between them on the production of laccase by P. pulmonarius. The selected variables were a certain amount of orange waste (g), wheat bran (g), starch (g), yeast extract (g) and moisture (%). The experimental results are shown in Table 2.

The data for the dependent variable were also analyzed and converted into natural logarithm (Lambda value equal to 0) according to the Box-Cox transformation system. The adjustment of the quadratic model to the data with second order interactions resulted in a coefficient of determination (R2) of 0.84, indicating that 84 % of the variability in response can be explained by the model. To validate the regression coefficient, an analysis of the laccase production variance (ANOVA) was performed (Table 3). The maximum laccase activity of 25,447 U L^-1 was observed in the experiment under the following conditions: orange waste, 3.5 g; wheat bran, 1.0 g; starch, 2.0 g; yeast extract 0.2 g, and 80% moisture. The treatment validation confirmed the maximum activity. The variables considered significant by the model were moisture and starch, which provided a positive effect (p < 0.05) in the laccase production and are highlighted in Table 3. The optimal values (predicted by the model) for these variables were starch, 2.5 g and 90% moisture. Therefore, the increased amount of starch and the initial moisture content of the medium may result in higher laccase titers. However, the combination of starch with lower amount of orange waste resulted in lower colonization and low enzymatic activity (Table 2 - treatment Nº 4). Figure 1 represents the response surface showing the effect of interaction between starch and orange waste in the laccase production.

Table 2
Results of experimental range of variables studied in the optimization of laccase production using CCD in terms of actual and coded factors.

Table 3
ANOVA for response surface quadratic model. R2 = 0.84; Adjusted R2= 0.70.

^aSS – sum of squares; ^bDF – degrees of freedom; ^cSQM – sum squares mean; ^dF – F value; ^ep– significance probability (p < 0.05).

Figure 1
In a: Response surface plots showing the effect of interaction between orange waste (X1) and starch (X3) on the yield of laccase. In b: Response surface plots showing the effect of interaction between moisture (X4) and orange waste (X1) on the yield of laccase.

As a feature of the RSM, the interaction between the variables in the laccase production was easily noticed, because even in experiments where there were large pools of carbon, the enzyme activities were low when the initial moisture of the culture was at or below 70%, for example, in treatments numbers 11, 18 and 22 in Table 2.

No aryl-alcohol oxidase, Mn peroxidase and lignin peroxidase activity was detected in this crude extract. A small range of optimum pH for the oxidation of ABTS was observed, pH 4.5–5.0, whereas a relative activity of 33.1% and 8.5% at pH 6.0 and 8.0, respectively, were found (Figure 2a). The crude laccase was more stable in pH 5.0 and maintained about 60% of its initial activity after 24 hours (Figure 2b). The effect of temperature on the activity of the crude laccase is shown in Figure 2c and 2d. The temperature optimum of the laccase was 60°C with ABTS as a substrate in acetate buffer, pH 5.0, and relative activities at 70°C were 77%. The enzyme retained about 68%, 35% and 18% of its initial activity when heated for 2 hours at 40, 50 and 60°C, respectively (Figure 2d). Crude laccase retaining 100% activity after 15 min. incubation at 40°C and presented a half-life of 100 min. and 40 min. at 50 and 60°C, respectively (Figure 2d). At 4°C, the enzyme was stable for several days.

Figure 2
Effect of pH (a and b) and temperature (c and d) on the crude laccase Pleurotus pulmonarius

The SDS-PAGE indicates a main band in the crude laccase with molecular weight of 23 kDa and 46.5 kDa, confirmed by the zymogram. The kinetic studies were carried out using ABTS, syringaldazine and DMP as substrate. The kinetic parameters suggest that the order of affinity toward the tested substrates was syringaldazine > ABTS > DMP. The apparent K_M value of the enzyme for syringaldazine determined from the Lineweaver-Burk plot was estimated to be 0.16 mmol L-1, and the corresponding V_max value was 8.1 mmol L^-1min^-1. The K_M value of the enzyme for ABTS was estimated to be 0.43 mmol L mmol L^-1, and the corresponding Vmax value was 15.9 mmol L^-1min^-1. For DMP, the values of K_M and V_max were 2.10 mmol L^-1 and 89.84 mol L^-1min^-1, respectively.

The effects of several chemicals in the laccase activity were determined with ABTS as substrate (Table 4). Crude laccase was strongly inhibited by HgCl and slightly inhibited by CuSO₄. NaF and the reducing agent 2-mercaptoethanol caused complete inhibition of laccase activity. The potential laccase inhibitor NaN₃ stimulated laccase activity in the concentrations studied: 0.375; 1.5 and 3.0 mmol L^-1.

Table 4.
Effect of several reagents on crude Pleurotus pulmonarius laccase.

*Final concentration.

The enzyme was activated by NaCl and Na2SO4at 0.375 mmol L^-1, but at higher concentrations there was inhibition.

The crude extract free peroxidase and aryl-alcohol oxidase activity, prepared from optimizated culture, was evaluated for enzymatic decolorization activity using ten different groups of reactive dyes (Table 5). Great decolorization occurred to bromophenol blue, green bromocresol, methyl green and RBBR at 0.05%. Methyl violet, congo red and methyl red were partially decolorized. Low decolorization was observed to methylene blue and Poly R-478. Ethyl Violet showed better decolorization in lower concentrations.

Table 5.
Decolorization of some synthetic dyes by the laccase of P. pulmonarius