INTRODUCTION:
Laccases (EC 1.10.3.2) are oxidoreductases that have massive prospectives in bioremediation processes mainly due to their high relative non-specific oxidation capacity, the lack of a requirement for cofactors, and the use of readily available oxygen as an electron acceptor1. Being selective, energy saving, and biodegradable, laccase-based biocatalysts fit well with the development of highly efficient, sustainable, and eco-friendly industries2. The promise of laccase for the decolorization of different structural dyes and textile effluents has been established over the last few years3-4. The commercialization of enzymatic processes for wastewater treatment is hampered by a lack of availability, high price and/or limited stability under operational conditions of the enzyme in question.
These drawbacks can be overcome by novel techniques of enzyme immobilization which leads to higher activity and stability, trouble-free recovery and recyclability3-4.
Insolubilization is the key to enable cost-effective application in a wide variety of industries including waste water treatment. Magnetic supports have recently been used for immobilization of enzymes5. Magnetic beads are small, globular, iron oxide containing particles, available in nanometer to hundreds of micrometer diameter range. Magnetic bio-separation technology is a promising strategy for recovering the immobilized enzymes on magnetic nanoparticles using an external magnetic field for recycled usage and enhancement of mechanical strength. This manuscript addresses the recent advancement in exploiting MNPs as a host for immobilization of Pleurotus ostreatus laccase and makes a detailed comparison between free enzyme and enzyme immobilized on glutaraldehyde modified MNPs. The prospective applications of surface functionalized MNPs in the decolorization of dyes are also discussed.
MATERIALS AND METHODS:
Chemicals:
Trametes versicolor laccase, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), bovine serum albumin (BSA) and remazol brilliant blue R (RBBR) were purchased from Sigma-Aldrich (Saint-Louis, MO, USA) . All other chemicals were of analytical grade.
Synthesis and characterization of magnetite nanoparticles:
Magnetite nanoparticles (MNPs) were synthesized by adding ammonium hydroxide (NH4OH) into a mixed solution of 0.1 M ferric chloride (FeCl3.6H2O) and 0.2 M ferrous sulphate (FeSO4.7H2O) in 25 ml deionized water until a precipitate was obtained at room temperature. In order to remove the residual ions, the precipitate was centrifuged and washed several times with deionized water until a pH value of 7 was obtained; the powder was dried at 100 oC for 2 h. The collected products were characterized using scanning electron microscope (SEM) and energy dispersive X-ray spectrometer (EDX) element analysis. The shape and surface morphology of the MNPs were characterized by Leo Gemini 1530 SEM analysis at an accelerating voltage of 10keV. Element analysis of MNP was carried out using EDX which allowed identifying the particular elements present in a sample and their relative proportions.
The magnetite nanoparticles (5 mg) were mixed with 1 mL of free laccase in buffer solution (5 mg/mL, 0.1 M acetate buffer and pH 5.6) and shaken for 30 min at 20 oC. Glutaraldyhyde was added into the suspension up to the final concentration of 5 mM and stirred for 24 h at 20 oC. After cross-linking, magnetic nanobiocatalyst were separated using magnet, washed three times with buffer and stored in 0.1 M acetate buffer (pH 4.0) at 4 oC. The washings were collected and no residual activity and proteins were found according to the activity assay by Lowry's method which convinced us that the initial 5 mg of free laccase was completely insolubilized as 5 mg magnetic laccase.
Enzyme activity and protein concentration assay:
Laccase activity was measured by monitoring the oxidation of 2, 2’-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) to its cation radical (ABTS+) at 420 nm (εmax= 3.6 x 104 M-1 cm-1)6. The assay mixture contained 1 mM ABTS and pH 4.0 (0.1 M acetate buffer). The temperature was set at 20 oC. One unit (U) of activity is defined as the amount of enzyme forming 1 mol of ABTS+ per min. Protein concentration was measured using Lowry's method.
Properties of free and immobilized biocatalyst:
The effect of optimum pH and temperature on the activity of the free and immobilized laccase was determined by measuring the ABTS oxidation in the pH range of 3.0-8.0 at 20 °C. The buffers used were glycine-HCl (pH 3.0), sodium acetate (pH 4.0, 5.0), Sodium phosphate (6.0, 7.0) and Tris-HCl (pH 8.0, 9.0). To determine the optimum temperature of free and nanobiocatlayst, their activities were measured in the temperature range (20–60 oC) at pH 4.0. Relative activities (calculated as the ratio of the enzyme activity measured at a specific pH and temperature to the maximal activity of the enzyme) were plotted against specific pH and temperature. Thermal stability of free laccase, and magnetic laccase at 60◦C was compared to that of free laccase at different time intervals at pH 4.0. Samples were taken at specified time intervals and cooled instantly in an ice bath and assayed for activity.
Dye Decolorization by magnetic nanobiocatalyst:
Remazol brilliant blue R (RBBR) was used at a concentration of 50 ppm, for the decolorization experiments. Decolorization was expressed as percent decrease of absorbance at the respective absorbance maxima: 595 nm for RBBR. The batch decolorization reaction was carried out in 10 ml reaction mixtures containing 50 ppm dye prepared in 0.1M phosphate buffer (pH 7.0), final Magnetic laccase activity (0.2 U) and incubated in the dark at 20 ◦C for 300 min. After decolorization of the dye solutions, magnetic nanobiocatalysts were separated using a magnet. Controls were performed by using heat-inactivated enzymes after incubation at 100 oC for 10 min were used to measure decolorization of dye at different time intervals. The average decolorization rate (μg min−1) was calculated as follows:
Average decolorization rate = 
where C is the initial concentration of dye (mg L-1) and % D is the dye decolorization (%) after a time t (h). The decolorization was monitored by scanning the UV-vis spectrum between 400 and 800 nm using spectrophotometer.
The effect of laccase concentration was tested at different levels 0.1–0.3 U mL-1 with fixed RBBR dye concentration of 50 ppm. In order to investigate the tolerance of the laccase against the dyes, the decolorization was carried out with varying initial concentration solutions (50-200 ppm) of RBBR in the reaction mixture with different incubation time.
Operational stability of magnetic laccase for RBBR decolorization:
A batch-wise approach was adopted to investigate the operational stability of magnetic laccase. M- laccase (50 mg) was evaluated for activity after repeated performance of the same biocatalyst in the oxidation of 50 ppm of RBBR. After enzymatic reaction for 90 min at 28 oC, magnetic nanobiocatalysts were easily recovered using an external magnetic field and then, the recovered biocatalyst was washed three times with freshly prepared 0.1 M acetate buffer (pH 4.0) and subsequently used in the next reaction.
RESULTS AND DISCUSSION:
Magnetic laccase preparation:
From the SEM analysis, agglomerations of magnetic nanosized particles were clearly visible (Fig.1a), and EDX analysis indicated that these agglomerations were predominantly ferrous irons (Fig.1b). The obtained nanoparticles are in the range of sizes 48-74 nm and few particles are agglomerated. It may be noted that the size of the MNPs obtained from SEM is in good agreement with the size. The optimization of magnetic nanobiocatalyst preparation was performed separately for each step consisting of functionalization of MNPs according to the charge properties of the targeted enzyme followed by precipitation of free enzyme on surface modified MNPs and subsequent covalent cross-linking of laccase. Increase in the cross-linking time led to increase in activity recovery of 84 %, but this relationship reached a plateau at approximately 16 h. The apparent activity of the magnetic laccase was around 3.9±0.17 U/mg and the activity recovery close to 84% was achieved during preparation.
Figure 1. (a) Representative SEM image of MNPs (b) EDX profile of MNPs.
Effect of pH and temperature on the activity of free and insolubilized laccase:
The two important factors directly influencing the ABTS oxidation by laccase are the pH and temperature. The ABTS oxidation by free laccase is strongly dependent on temperature. The laccase activity of free enzyme and magnetic biocatalyst increased gradually with temperature and a maximum activity was obtained at 30 oC. The pH is one of the major parameters capable of shifting enzymatic activities in aqueous solution. The optimum pH of the free enzyme was at pH 4.0 which shifted to pH 6.0 for magnetic laccase, due to the structural conformational change which reflects the relative activity of the enzyme (Fig. 2a). Moreover, the enzyme can be utilized even in non-usual situations, like acidic conditions and high temperatures, which are usually encountered in bioremediation processes.
Thermal stability of the magnetic nanobiocatalyst:
To evaluate the thermal stability of the free laccase, magnetic laccase was incubated at 60 ◦C. The magnetic laccase prepared was thermally more stable than the free enzyme. The half life of laccase at 60oC was prolonged 4 times from 30 min (free laccase) to 120 min (Magnetic laccase) (Fig. 2b). Magnetic laccase was thermostable up to 60oC and above this temperature; the enzyme activity started decreasing as the incubation time increased. The enrichment in thermal stability may be due to (1) the ordered arrangement of the molecules by inter and intramolecular cross-links within and between the aggregates, giving rigidity to the three-dimensional arrangement of the molecules and (2) due to supplementary ionic and hydrophobic contacts between the enzyme molecule4. The above results showed that thermal stability of laccase was higher than the free enzyme and it makes magnetic laccase promising for industrial and bioremediation applications.
Figure 2. pH activity profiles for free and insoluble laccase and thermal stability of the free and insoluble laccase at 50 oC. The experiments were done in triplicate and the error bar represents the percentage error in each set of determinations.
Decolorization of RBBR by magnetic nanobiocatalyst:
Incubation of RBBR in the presence of laccase resulted in detectable reduction in absorbance at 595 nm of the reaction mixture within 15 min of initiation in the absence of redox mediators. The time course of the dyes transformation by the enzymatic reaction was monitored by UV-vis spectroscopy. The UV-vis spectrum showed dramatic changes in the enzymatically treated dyes. Absorbance at λ max continued to decrease with time of incubation, associated with oxidation of the dye. In order to discover the possible mechanism of dye decolorization, various analytical techniques were used to identify the metabolites generated by enzyme-mediated processes (Kumar et al., 2012). The disappearance of the sharp peak (at λmax) is observed after decolorization of the dyes and further evidence of the removal of the dye can be observed with an increase in absorbance towards UV region. Peaks initially observed at 595 nm (0h) decreased without any shift in λmax up to complete decolorization of RBBR. These results suggested that the decrease in the absorbance was due to degradation of dye by the magnetic nanobiocatalyst laccase. At 50 ppm of RBBR concentration, 84 % decolorization was achieved after 60 min and complete decolorization occurred within 120 min (Fig.1). Faster decolorization due to the presence of electron donating methyl and methoxy groups on the dyes that have enhanced laccase activity7.
Decolorization process was affected by various physico-chemical parameters like initial dye concentration, enzyme and incubation time. Fig 3a shows the time decolorization reduced with increase in initial RBBR concentration. At initial MG concentrations between 50 and 100 ppm, complete decolorization occurred within 120 min. As the dye concentration increased from 100 to 200 ppm the decolorization decreased from 97 to 84 % after 2 h (Fig. 3a). The decrease in decolorization efficiency might be due to the degraded products and inhibition of enzyme with excess dyes8. These results indicate that decolorization time decreased with increase in dye concentration.
The effect of enzyme dosage was carried out at a fixed initial dye concentration (50 ppm) tested at different laccase activity levels 0.1–0.3 U/mL with different time intervals at 37oC, pH 6.0. Fig. 3b shows significant influence on enzyme concentration on decolorization of dyes. The increase in an enzyme concentration from 0.1–0.3 U/mL resulted in a gradual increase in the decolorization rates of both dyes. 0.3 U/mL enzyme activity achieved a 100% decolorization of MG for a contact time of 75 min, whereas 0.2 U/mL displays complete decolorization after 120 min (Fig. 3b). Kammoun et al., 2009 also showed rapid decolorization of dyes at higher enzyme concentration. The rate of decolorization decreased when enzyme activity was lower than 0.1U/mL and 84 % was decolorized over 300 min under the standard incubation conditions.
Figure 3. Biocatalytic degradation of RBBR by magnetic nanobiocatalyst laccase. (a) initial dye concentration; (b) enzyme concentration
Operational stability of magnetic laccase for RBBR decolorization:
The reusability of the magnetic nanobiocatalysts was examined for repeated applications in a batch reactor. Preserving the activity of the magnetic laccase on repeated use is shown in Fig.4. In order to independently evaluate the capacity of immobilized laccase to degrade dyes, we used the average decolorization rate (ADR) parameter. In the case of immobilized laccase, the ADR values calculated for RBBR are: 0.070 mg/min. The magnetic laccase showed continuous decrease in decolorization rate of 50 ppm RBBR after 10 cycles, which is derived from continuous leaching of laccase. On the other hand, MNP clearly stabilized the laccase activity for many iterative cycles of enzyme reaction and separation using a magnet. For example, 86 % of initial laccase activity was preserved after 10 cycles of ABTS oxidation and separation. The results show that about further immobilization of laccase in MNPs prevented the leaching of laccase and also stabilized activity of the iterative recycling of laccase. However, it must be pointed out that, unlike immobilized laccase, the free enzyme is difficult to recover for reutilization purposes.
Figure 4. Effect of the operational stabiity on the RBBR decolorization by magnetic nanobiocatalyst laccase with initial activity, 0.6 U/mg; pH 7.0 and 20oC
CONCLUSIONS:
In conclusion, we have exemplified a more facile and economically viable procedure for fabricating robust magnetic nanobiocatalyst technology with highly catalytically active laccase. This simple and efficient technology not only has the potential to solve the mechanical stability problems associated with laccase, but also greatly improve the thermal and operational stability. The present study demonstrates the ability of magnetic laccase to decolorize and transform the anthraquinone dye, giving it an advantage for treatment of dye containing wastewaters. UV-Vis spectroscopy analysis reveals the high potential of magnetic nanobiocatlyst for dye decolorization. From an industrial point of view, it would be simpler to add biocatalyst into the wastewater and easily recover it by magnet. An attractive feature of this enzyme is the fact that, it does not require redox mediators to function efficiently in dye decolorization. These encouraging results enable a step forward towards the industrial application of laccase at a more cost effective and easier manner, compared to other methods.
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Received on 19.08.2013 Accepted on 01.09.2013
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Research J. Engineering and Tech. 4(4): Oct.-Dec., 2013 page 272-278