Immobilization of laccase for biotechnology applications

Abstract

Laccase played an important role in the decolorization of wide spectrum dyes as a low-cost and environmentally friendly technology. Laccase was immobilized in alginate beads and immobilization conditions were identified. 25 mg/ml laccase enzyme encapsulation efficiencies of using the prepared bead was calculated as approximately 94%. At the end of the 10 days of storage, the free laccase and immobilized laccase retained about 8.08% and 80.83%, respectively. The decolorization of the dye (Direct Blue 2) was around 86% for immobilized enzyme at 45°C. In the study, compared to the free enzyme, high activity, stable, reusable immobilized enzyme preparation was prepared.

Keywords::

• Alginate
• Direct Blue 2
• dye decolorization
• enzyme immobilization
• laccase

Introductıon

Laccase is oxidoreductase belonging to the multinuclear copper-containing oxidases; it catalyzes the monoelectronic oxidation of suitable substrate molecules such as phenols, substituted polyphenols, aromaticamines, benzenethiols and a series of other oxidizable compounds (Wang et al. 2008). Laccase has low substrate specificity, so it has been used in biosensor, biofuel cell, biotransformation and wastewater treatment (Wang et al. 2010). Laccase played an important role in the decolorization of wide spectrum dyes as a low-cost and environmentally friendly technology. If free laccase is used in process applications, it is often easily inactivated in wastetreatment for the wide variety of treating conditions and is also difficult to be separated from the residual reaction system for reuse (Wang et al. 2008). For this reason, laccase was immobilized using different materials including alginate/gelatin blent with PEG (Wang et al. 2008), magnetic mesoporous silica nanoparticles (Wang et al. 2010), porous glass beads (Champagne and Ramsay 2010), microfibers (Dai et al. 2010), silica nanoparticles (Galliker et al. 2010), non-porous poly(GMA/EGDMA) beads (Arıca et al. 2009), epoxy-activated carriers (Kunamneni et al. 2008), magnetic nano-composite (Xıao et al. 2006), modified chitosan (Yang et al. 2006), magnetic chitosan microspheres (Jiang et al. 2005), poly (4-vinyl pyridine) grafted magnetic beads (Bayramoğlu et al. 2010), green coconut fiber (Cristovao et al. 2011), κ-carrageenan-based semi-interpenetrating polymer (Makas et al. 2010).

Dyes are extensively used in several industries including textile, paper, printing, cosmetics, and pharmaceuticals (Kunamneni et al. 2008). Nowadays, the share of dyes from industrial chemicals that pollute the water ecosystems is enormous. The majority of this amount is used in the textile industry. Textile wastewater pollution parameter of the first visible coloration. Many of the dyes have very complex chemical structures, resistant to purification of toxic substances. For dye removal from wastewater, some methods are widely used such as coagulation, floculation, membrane filtration, and adsorption (Gupta and Suhas 2009). Azo dyes Azo (-N = N-) group are known by the presence of coatings. Azo dyes 60–70% world market has a market share of pollutant impact and water only, but also in creating a complex chemical structure (Figure 1) that the removal is difficult.

Figure 1. Direct Blue 2 chemical structure.

In this paper, laccase was immobilized in alginate beads and the conditions for immobilization and characterization of the free and immobilized enzyme were studied. The reusability and stability (pH stability, thermal stability and storage stability) of immobilized laccase were also studied and were compared with free enzyme. Immobilized and free laccase then tested in the decoloration of textile dye, Direct Blue 2 using synthetic textile wastewater.

Materials and methods

Laccase used in this study was provided by Novozymes Company for the grant. ABTS(2,2’-azino-di-3-ethyl-benzo-Thiazole-sulphonate) was purchased from Sigma Chem. Co. Company. The other chemicals and solvents were of analytical grade and no further purification was required.

Preparation of alginate beads

A solution of alginate (1.0, 1.5, 2.0, 2.5, and 3.0%) was prepared which contained a certain amount of laccase (5–50 mg/ml) and 2 ml of this solution was then added dropwise into 0.5 M CaCl₂ solution in the ice bath (Baysal 2007). During the process, the suspension medium was stirred with a mechanical stirrer at 150 rpm. Following incubation for 4 h to complete gelation, beads were separated by using nuche filtration. After that, beads were washed three times with distilled water. Laccase activity and protein amounts were determined in order to investigate the encapsulation efficiency for each sample. Beads size was calculated by measuring the increase in volume. The process was repeated ten times and the particule sizes were determined 1.34 nm for alginate beads, 1.50 nm for alginate-laccase beads. Protein determinations were performed spectrophotometrically by the method of Bradford (Bradford 1976). 0.02–0.5 mg/ml albumin and protein amounts were calculated for each sample plotted with the help of the Standard curve.

Analysis of enzyme activity

Activity of the free and immobilized laccase was assayed using 1 mM ABTS in distilled water as substrate. The reaction mixture consisting 3 ml of 0.1 M asetate buffer, ph 4.5 containing enzyme was initiated by adding 0.3 ml of ABTS (Erden et al. 2009). Free enzyme by the addition of 10 sec intervals at 420 nm Perkin Elmayer UV/VIS absorbance measurements were taken by spectrophotometer. 10 beads, 2.7 ml acetate buffer and 0.3 ml of ABTS were incubated with shaking at 100 rpm for 5 min for immobilization laccase activity. To separate the beads at the end of time, the upper phase at 420 nm absorbance was measured. The molar extinction coefficient of ABTS is 36000 l/(mol.cm). One activity unit of laccase was defined as the amount of enzyme required to catalyze 1 mmol/ml of substrate per minute.

Determination of pH and thermal profile

Enzyme activity as a function of pH was determined at 25°C in 0.1 M different buffer solutions. The range of pH 3–5 in acetate buffer, pH 6–8 in phosphate buffer, pH 9 in tris/HCl buffer was used as a buffer solution. The temperature activity profiles of free and immobilized enzymes were determined in the range 8–60°C at pH 3.5.

Stability tests

Thermal stability was determined by incubating the free and immobilized laccase in the acetate buffers, pH 3.5 at different temperature for 30 minutes and then laccase activity was assayed at 25°C in standard conditions with ABTS.

pH stability of free and immobilized laccase was studied by incubating at 25°C in buffers of varying pH from 3.0 to 10.0 for 4 h, and then the residual activity was determined in standard conditions with ABTS.

For testing the storage stability of free and immobilized laccase was stored in a refrigerator at 4°C for several days. Free laccases were stored in distilled water (1 mg/ml) at 4°C. İmmobilized beads were stored under slightly wet conditions. Then the residual activity of the enzyme was measured in standard conditions with ABTS.

Reusability

The reusability of the immobilized laccase was studied by cycles of ABTS oxidation due to its importance for industry to reduce the processing cost. 10 beads in 0.1 M acetate buffer pH 3.5 were added 0.3 ml ABTS (1 mM) and activity was measured under standard conditions. At the end of each oxidation cycle, the immobilized laccase was washed three times with distilled water and the procedure repeated with a fresh aliquot of substrate and activity measurements were repeated several times.

Dye decolorization

Direct Blue 2, dye material from the wavelength of maximum absorbance wavelength scans was identified. 0.01–0.1mg/ml dye amounts were calculated for each sample plotted with help of the Standard curve. Synthetic textile waste water containing dye was used. Synthetic wastewater was prepared as described in the outline below (Muda et al. 2010). The beads with immobilized laccase (29.95U) and free laccase (0.94 ml, 31.86 U/mg protein) were added into 50 ml textile waste water containing dye. The reaction mixture was incubated at 25°C for free enzyme and at 45°C for immobilized enzyme and laccase-free alginate beads. Specific periods of time by measuring absorbance at 575 nm dye samples taken from the removal efficiencies were calculated.

Synthetic Textile Waste Water Processing (Muda et al. 2010):

NH₄Cl 0.16 g/L, KH₂PO₄ 0.23 g/L, K₂HPO₄ 0.58 g/L, CaCl₂.2H₂O 0.07 g/L, MgSO₄.7H₂O 0.09 g/L, EDTA 0.02 g/L, Glucose 0.5 g/L, ethanol 0.125 g/L, sodium acetate 0.5 g/L, element 1ml/L(element: H₃BO₃ 0.15 g/L, FeCl₃.4H₂O 1.5 g/L, ZnCl₂ 0.12 g/L, MnCl₂.4H₂O 0.12 g/L, CuCl₂.2H2O 0.03 g/L, NaMoO₄.2H₂O 0.06 g/L, CoCl₂.6H₂O 0.15 g/L ve KI 0.03 g/L) and dye 50 mg/L.

Results and discussıon

Optimization of immobilization conditions

Prepared by using 1% and 1.5% alginate beads were forms of corruption. Viscous solution in the syringe on the instillation of 2.5% alginate used was difficult. There was a problem dropping alginate used above 2.5% so 2.5% was chosen as optimum amount of alginate.

Different laccase concentrations were used in enzyme immobilization. As is shown in Figure 2, maximum encapsulation efficiency was determined by using 25 mg/ml laccase. Encapsulation efficiency was 94% for 25 mg/ml laccase but it was 69% for 50 mg/ml laccase.

Figure 2. Effect of laccase concentration on encapsulation efficiency.

Protein determinations have been carried out using the Bradford method with albumin standard curve, y = 2.067 × R² = 0.9913.

Characterization of free and ımmobilized enzyme

Entrapment is physical enclosure of the enzyme in the microspaces formed in the matrix structure. So, the three dimensional structure of the enzymes may not be affected upon immobilization and optimum pH of the immobilized enzyme can be observed to be similar to that of the free enzyme. As shown in Figure 3, the optimum pH value is 3.5 for free and immobilized enzyme (Figure 3). Literature data are analyzed, the maximum activity of immobilized laccase in the pH range 3–5 has been reported (Jiang et al. 2005, Kunamneni et al. 2008, Dai et al. 2010, Champagne and Ramsay 2010). Free enzyme showed maximum activity at 25°C while optimum temperature value for the immobilized enzyme has been identified as 45°C (Figure 4).

Figure 3. Effect of pH on enzyme activity.

Figure 4. Effect of temperature on enzyme activity.

Stability tests

pH stability data for free and immobilized enzyme are shown in Figure 5. Compared to the free enzyme, immobilized enzyme stability in acidic pH values is greater and is about 30%. Stability of the immobilized enzyme in the range pH 9–10 of about 20% is higher than the free enzyme. PH stability was found to be increased after the immobilization of laccase. The thermal stability is shown in Figure 6. Deterioration of bead structures is higher at 60°C. The residual activity for free enzyme is 45% at 60°C but it is 85% for immobilized enzyme. As is shown in Figure 6, the free enzyme immobilization is compared to the increased thermal stability. Similarly, thermal and pH stability of laccase after immobilization compared to free enzyme studies indicate an increase as found in the literature data (Corman et al. 2010).

Figure 5. pH stability.

Figure 6. Thermal stability.

Storage stability is one of the most important parameters to be considered in immobilized enzyme product. At the end of the 10 days of storage, the free laccase and immobilized laccase retained about 8.08% and 80.83%, respectively.

Reusability

Reusability of immobilized enzymes is an important aspect for industrial applications, because immobilized enzymes decrease the cost of production due to their repeated, continuous or batch uses. After the 4th use, the residual activities were found to be 80% (Figure 7). Upon repeated uses (10th), either blocking of some pores of beads by substrate or product may take place.

Figure 7. Reusability of immobilized laccase.

Dye decolorization

Dye decolorization by free and immobilized laccase and alginate beads were investigated and the results are given in Figure 8. As is shown in Figure 8, a high decolorization percentage of Direct Red 23 was obtained for immobilized laccase at 45°C(optimum temperature of immobilized enzyme). The decolorization of the dye was around 16% for alginate beads, 50% for free enzyme at 25°C, 59% for immobilized enzyme at 25°C and 86% for immobilized enzyme at 45°C.

Figure 8. Dye decolorization.

The concentrations of the dye samples can be calculated using standart curve, y = × R² = 1.0.

Conclusion

Dyes are widely used in various industries such as dyestuffs, textiles and leather. These dyes may cause suspected carciogenic and genotoxic effects. Overall at present there are more than 100.000 commercial dyes with a rough estimated production of 7.10⁵–1.10⁶ tons per year. Laccase played an important role in the decolorization of wide spectrum dyes. If free laccase is used in process applications, it is often easily inactivated in waste treatment for the wide variety of treating conditions and is also difficult to be separated from the residual reaction system for reuse. For this reason, laccase was immobilized and for enzyme immobilization non-toxic, biodegradable, and affordable, alginate copolymer was selected. Based on above results, the immobilized laccase was more stable during operation and storage compared to the free enzyme. Compared to the free enzyme, immobilized enzyme stability in acidic pH values was greater and was about 30%. The immobilization allowed enzyme reuse and has been shown to improve its stability, which could be a potential advantage in waste water treatment.

Acknowledgement

The authors thank Novozymes (İstanbul-TÜRKİYE) for laccase.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

This research was financially supported by İzmir Özel Tevfik Fikret Fen Lisesi, İzmir-TÜRKİYE.