Decolorization of two synthetic dyes using the purified laccase of Paraconiothyrium variabile immobilized on porous silica beads
© Mirzadeh et al.; licensee BioMed Central Ltd. 2014
Received: 20 June 2013
Accepted: 6 October 2013
Published: 6 January 2014
Decolorization of hazardous synthetic dyes using laccases in both free and immobilized form has gained attention during the last decades. The present study was designed to prepare immobilized laccase (purified from Paraconiothyrium variabile) on porous silica beads followed by evaluation of both free and immobilized laccases for decolorization of two synthetic dyes of Acid Blue 25 and Acid Orange 7. Effects of laccase concentration, pH and temperature alteration, and presence of 1-hydroxybenzotriazole (HBT) as laccase mediator on decolorization pattern were also studied. In addition, the kinetic parameters (K m and V max ) of the free and immobilized laccases for each synthetic dye were calculated.
Immobilized laccase represented higher temperature and pH stability compare to free one. 39% and 35% of Acid Blue 25 and Acid Orange 7 was decolorized, respectively after 65 min incubation in presence of the free laccase. In the case of immobilized laccase decolorization percent was found to be 76% and 64% for Acid Blue 25 and Acid Orange 7, respectively at the same time. Increasing of laccase activity enhanced decolorization percent using free and immobilized laccases. Relative decolorization of both applied dyes was increased after treatment by laccase-HBT system. After nine cycles of decolorization by immobilized laccase, 26% and 31% of relative activity were lost in the case of Acid Blue 25 and Acid Orange 7, respectively.
To sum up, the present investigation introduced the immobilized laccase of P. variabile on porous beads as an efficient biocatalyst for decolorization of synthetic dyes.
Discharge of a wide range of synthetic dyes produced and being increasingly used by different industries to ecosystems made this subject as a major environmental problem [1, 2]. In aquatic ecosystems, entrance of dyes decreases the oxygen level due to reducing the amount of sunlight for photosynthetic organisms . Some of these dyes are toxic and carcinogen in nature and found to be recalcitrant . Time and cost consuming characteristic of physicochemical methods like coagulation, flocculation, adsorption, ion-exchange, oxidation and electrochemical methods applied for removal of synthetic dyes [4, 5] together with generating undesirable by-products prompts researchers to study on biological or enzymatic techniques for degradation of hazardous dyes [6, 7]. Supplying much more eco-friendly process due to production of non-toxic metabolites, requiring mild conditions (temperature, pressure and pH) and less water consumption in comparison with physicochemical techniques and being cost-competitive are some advantages of enzymatic methods for decolorization and removal of synthetic dyes [1, 3, 5].
Laccases (benzenediol: oxygen oxidoreductases, EC 18.104.22.168) are multi-copper oxidases produced mainly by fungal strains, specially white-rot fungi [8, 9]. The ability of laccases for oxidation of a broad range of substrates like chlorinated phenol, polycyclic aromatic hydrocarbons (PAHs), benzenethiols and textile dyes in presence of molecular oxygen as co-substrate introduced this biotechnologically important enzyme as the first choice for xenobiotic removal investigations [8, 10]. However, low stability and low production yield are two main constraints for application of laccases in industrial processes [11, 12]. Such mentioned problems could be overcome by development of immobilized laccases from different resources on solid supports which allow reusing of immobilized enzyme and may improve its stability [12, 13]. Entrapment of enzyme in polymers, enzyme encapsulation in membranes, self-immobilization and covalent binding of enzymes to solid support are techniques used for fixation of enzymes  among which covalent biding supply more stable enzymes by increasing the rigidity of its structure and reducing protein unfolding . During the recent years, wide range of supports like chitosan, chitosan/poly(vinyl alcohol) composite nanofibrous membranes, magnetic mesoporous silica nanoparticles and mesostructured siliceous cellular foams have been used for development of immobilized laccases . Mesoporous silica materials are one of the most applied matrices for immobilization of enzymes due to specific properties such as hydrothermal resistance, well order porous structure and high surface area .
In the present study, the purified laccase from a soil isolated ascomycete, P. variabile, was immobilized on the porous silica particles and applied for decolorization of two synthetic dyes of Acid Orange 7 and Acid Blue 25 compared to free enzyme. Furthermore, the influences of conditional parameters such as enzyme activity, temperature, and pH on decolorization were investigated. The kinetic parameters (Km and Vmax) of free and immobilized laccases through the applied dyes were also calculated.
Materials and methods
Enzyme, chemicals and dyes
Chemical type, maximum absorbances, and kinetic parameters of two synthetic dyes
K m (mM)
Vmax(mmol min-1 mg-1)
Acid orange 7
Acid blue 25
The Pv L was immobilized on the porous silica beads based on the method of Champagne and Ramsay . Briefly, 4 g of pre-silanized CPC-silica beads (355–600 mm in diameter, an average surface area of 42.1 m2/g, and a pore size of 37.5 nm) was added to 2.5% glutaraldehyde solution (degassed under 2.0 bar vacuum pressure for 2 h) in 0.1 M KH2PO4 at pH 5.0 and stirred for 2 h. The enzyme solution (prepared in citrate buffer 0.1 M, pH 5) was then introduced to the activated silica beads and incubated at 4°C for 36 h. Consequently, the prepared beads was filtered through paper filter and washed three times with distilled water and twice with citrate buffer (0.1 M, pH 5.0). The SEM image of CPC-silica beads and immobilized Pv L on porous silica beads were then recorded using a scanning electron microscope (Philips XL30) operated at 5 kV.
Determination of thermal and pH stability of free and immobilized Pv L
Stability of the prepared immobilized Pv L and the free purified laccase through temperature was studied by pre-incubating the free or immobilized Pv L at different temperatures between 20 and 60°C for 1 h, followed by determination of the residual activity. The influence of pH on the laccase stability was evaluated by incubating the free or immobilized Pv L at 4°C in different pH levels (3–8) for 24 h and determining the residual activity.
Laccase activity determination
The laccase activity was measured by oxidizing the ABTS as a substrate according to the method previously described [17, 18]. Briefly, 0.5 mL of the purified Pv L solution or 0.1 g of immobilized Pv L was added to 1.5 mL of the ABTS solution (5 mM in citrate buffer 0.1 M, pH 5) and incubated at 40°C and 120 rpm for 10 min. Change in the absorbance at 420 nm was then determined using a UV-vis spectrophotometer (UVD 2950, Labomed, Culver City, USA). The laccase activity was then calculated using the molar extinction coefficient of ABTS (ϵ420 = 36,000 M-1 cm-1). One unit enzyme activity was defined as the amount of enzyme requires oxidizing 1 μmol of ABTS per minute under the assay condition .
Dye decolorization experiments
where Ai is the initial absorbance of the reaction mixture and At is the absorbance after incubation time [5, 18]. In the case of free Pv L, the negative control was prepared by adding the heat-inactivated enzyme to the dye solution and incubating the reaction mixture as described above. In the case of immobilized Pv L, activated glass beads without enzyme applied as negative control. All experiments were performed in triplicate and means of decolorization percentages were reported.
The effect of laccase activity on dye decolorization
In order to determine the effect of laccase activity on decolorization pattern, the purified or immobilized Pv L was added to the reaction mixture (as said above) to reach the enzyme activity of 0.5–8 U/mL followed by incubation at 40°C and 50 rpm for 60 min. The reaction mixture was then monitored for decolorization percentage. The final dye concentration in all experiments was 400 mg/L.
The influence of pH on laccase-mediated decolorization
By adjusting the initial pH of each dye solution (final concentration of 400 mg/L) using 0.1 M citrate-phosphate buffer between 3 - 8, 2 U/mL free or immobilized Pv L was added to the reaction mixture and incubated at 40°C and 50 rpm for 60 min. The decolorization percentage was then monitored as previously described.
The effect of temperature on enzymatic decolorization
The effect of temperature on enzymatic decolorization was studied by incubating 400 mg/L of the synthetic dyes in the presence of 2 U/mL of free or immobilized Pv L at temperature range of 25 - 70°C and pH 5.
Application of laccase-HBT mediated system for dye decolorization
HBT as a non-phenolic laccase mediator was introduced to the reaction mixture (dye, 400 mg/L and free or immobilized Pv L, 2 U/mL in citrate buffer 0.1 M pH 5) to reach the final concentrations of 0.1, 1, and 5 mM. Decolorization was determined as previously described.
Reusability of immobilized laccase
In order to determine the reusability of immobilized biocatalyst, the applied immobilized Pv L was recovered from the reaction mixture of each dye using filtration and washed three times with citrate buffer (0.1 M, pH 5). It was then introduced again to the reaction mixture and residual activity of the immobilized Pv L was measured as said above. The mentioned experiment was done in triplicate and mean of obtained results were reported.
After determination of velocity in presence of different concentrations (100 - 600 mg/L) of each dye, the Michaelis - Menten curve was drawn by plotting the obtained velocity (V) against the dye concentrations (S). The kinetic parameters [Km (Michaelis constant) and Vmax (maximal velocity)] of the free or immobilized Pv L through each dye were then obtained using the Lineweaver - Burk transformation of the Michaelis - Menten equation.
All above mentioned experiments performed in triplicate and the reported data were presented as mean ± standard deviation. The independent sample t-test and one-way analysis of variance (ANOVA) with Dunnett’s T3 post Hoc test was done using the SPSS 15 software (SPSS Inc) to calculate statistical significance between mean values. Probability values <0.05 were considered to be significant.
Results and discussion
Immobilization of laccase
Compare to free enzymes, immobilization of biocatalysts on solid supports made them more stable for industrial processes and allow them to be reused many times [14, 20]. The obtained results of the present study revealed that immobilized laccase on porous silica beads represented higher temperature and pH stability compared to the free laccase. Same results were reported by Arica et al.  and Bayramoglu et al.  where the purified laccase of Trametes versicolor was immobilized on poly(GMA/EGDMA) and epoxy-functionalized magnetic chitosan beads, respectively. In the study of Wang et al. , it was revealed that immobilization of laccase on magnetic mesoporous silica particles enhanced the optimum catalytic temperature and pH of the enzyme from 20°C and pH 4 to 60°C and pH 5, respectively. Meanwhile, they showed that the immobilized laccase exhibited more than 95% of the maximum activity with a wider pH range between 4.0 and 5.5 .
Decolorization using free and immobilized laccase
Effect of laccase activity on decolorization
Effect of increasing laccase concentration on decolorization of studied dyes was illustrated in Figure 3c and Figure 3d. In the case of free Pv L, the highest decolorization percent (52.2% for AB25 and 46.2% for AO7) was achieved at laccase activity of 4 U/mL (Figure 3c) while in the case of immobilized laccase maximum decolorization percent (75% for AB25 and 62% for AO7) was obtained in presence of 2 U/mL of laccase (Figure 3d). Depends on the sources of laccases (bacteria, fungi, etc.) decolorization pattern even on one special dye has been found to be different [4, 27]. In our previous studies [4, 7–10, 18], the produced laccase of P. varibile was efficiently decolorized wide range of synthetic dyes including diazo dyes (Amido black, Sudan black, Congo red and Ponceau-S), triphenylmethane dyes (Crystal violet, Bromothymol blue, Commassie brilliant blue and Malachite green) and anthraquinone dyes (Rimazol brilliant blue R). The recent study of Ashrafi et al.  showed that increasing of laccase concentration from 0.025 to 0.1 U/mL significantly increased decolorization percent in all thirteen studied synthetic dyes.
Influence of pH on decolorization
Effect of temperature on laccase decolorization
As shown in Figure 4c and Figure 4d the highest decolorization percent in the case of both synthetic dyes was obtained at optimum temperature of Pv L activity (40°C). At elevated temperature of 70°C, relative decolorization of both dyes was decreased below 20%. However, in the case of immobilized Pv L above 75% of initial decolorization remained at temperature of 70°C (Figure 4d). In the study of Sadighi et al.  it was found that the laccase immobilized on chitosan nanoparticles retained decolorization activity of Congo red at 95°C, and eventually lost 66% of decolorization activity at 100°C.
Effect of HBT on laccase-mediated decolorization
Reusability of immobilized laccase
Kinetic parameters of free and immobilized laccases
Kinetic parameters of free and immobilized laccases toward two studied dyes of AB25 and AO7 were summarized in Table 1. Since anthraquinoid dyes especially those with amine groups on the ring are more suitable substrates than azo dyes for laccase , K m value of both free and immobilized laccases for AB25 is lower than AO7 and also the degradation of AB25 is faster than AO7, i.e., the Vmax for AB25 is higher than AO7. On the other hand, the higher K m value of immobilized Pv L toward both applied dyes means lower affinity of immobilized laccase for applied dyes compared to free laccase. The obtained results are in agreement with the results achieved by Rekuc et al.  indicating 3.4 fold increase in K m value of immobilized laccase compared to free laccase. Such observation might be due to structural change in the adsorption process of laccase onto solid support and low accessibility of the substrate to the active site of the immobilized laccase [23, 33].
The purified laccase of P. variabile, recently studied for its capacity in the removal of aromatic compounds (e.g., chlorophenol pollutants, hazardous synthetic dyes, and pharmaceutical agents), was immobilized on porous silica beads and applied for decolorization of two synthetic dyes of Acid Blue 25 (an anthraquinone dye) and Acid Orange 7 (an azo dye). pH and thermal stability of immobilized enzyme was improved compare to free laccase. Both applied dyes were efficiently decolorized using immobilized laccase. Application of the laccase-HBT system improved the decolorization of both synthetic dyes using free and immobilized laccases.
This work was financially supported by the Biotechnology Research Center, Tehran University of Medical Sciences, Tehran, Iran and Institute for Environmental Research, Tehran University of Medical Sciences.
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