Simple fabrication of polymer-based Trametes versicolor laccase for decolorization of malachite green
Highlights
► A highly efficient composite biocatalyst D201_Lac was fabricated through a simple adsorption process for dye decoloration. ► D201_Lac possesses outstanding stability and enhanced resistance against pH and temperature fluctuation. ► Negligible activity loss of the resultant D201-Lac was observed for batch continuous 320-h operation.
Introduction
Laccase (benzenediol:oxygen oxidoreducatase, EC 1.10.3.2), which is widespread in fungi, is a group of polyphenol oxidases containing copper atoms. Laccase from fungi is proved effective in oxidation of various phenolic compounds with the comitant reduction of oxygen to water (Karam and Nicell, 1997). The substrate range of fungal laccase can be extended by inclusion of a mediator, such as 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS), 1-hydroxybenzotriazole (Hobt), etc. (Srebotnik and Hammel, 2000, Cabana et al., 2009). The potential applications of fungal laccase in biodegradation of pollutants (Rodriguez Couto and Toca Herrera, 2006) have been well recognized. It is worth noting that laccase is often used to decolorize dye effluents and has excellent performance (Camarero et al., 2005, Chhabra et al., 2009, Khlifi et al., 2010). However, the use of laccase in large-scale industrial applications is still restricted by its relatively poor stability against environmental factors such as pH and temperature. Also, laccase is well soluble in water. Its recovery and reuse is still a challenge because laccase is currently of high cost. Previous studies (Peralta-Zamora et al., 2003, Cabana et al., 2009, Arica et al., 2009, Rekuc et al., 2010) proved that laccase immobilization is favorable to enhance its resistance against severe environmental factors (such as extreme pH or temperature) and to achieve a simple separation from the reaction systems, which is particularly significant for its recovery and reuse and for development of continuous bioprocessess for practical use.
In review of the methods for enzyme immobilization onto carriers, adsorption is obviously a basic and simple one. Various enzymes have been immobilized onto carriers by adsorption (Lei et al., 2002, Lei et al., 2006, Bayramoglu et al., 2011). However, leaching is a troublesome problem in the application of the resultant composite catalysts with the above-mentioned materials as carriers, especially when used in a wide pH range or at high ionic strengths. For example, Qiu et al. (2009) have ever immobilized laccase onto nanoporous gold by means of physical adsorption. As the obtained enzyme composite was introduced into a phosphate–citric acid buffer solution (50 mM, pH 4.4) for 1 h at 4 °C, a considerable amount laccase was leached out from the composite. Wang and Caruso (2004) reported that substantial amounts (up to 87%) of catalase adsorbed onto mesoporous silicas (2 nm in diameter) would be desorbed when merely immersed in 50 mM phosphate buffered saline for 48 h. To improve the stability of the enzyme composites based on adsorption technique, several strategies, either strengthening the interactions between carriers and enzymes or designing special structures to restrict enzymes within nanopores/nanochannels, have been proposed in recent years, including modifying carriers with amine or carboxylate groups, cross-linking enzymes inside nanochannels/nanopores, partially closing pore openings by silylation of the pre-loaded enzymes, and deposition of layers to cover the pore openings (Lee et al., 2009, Hartmann and Jung, 2010). As expected, they are efficient in stabilizing the enzyme within the frameworks of carriers. However, the excessive reactions used in these strategies are very likely to inactivate or even denature the enzymes. Besides, how to control the reaction extent is a challenging task. For example, the method to cross link the enzyme molecules via reagents after they entered the nanochannels/nanopores of carriers, which was considered as “the most promising novel approach” by Hartmann and Jung (2010), encountered a bottleneck in controlling the size of the cross-linked enzyme aggregates. Moreover, how to get facile substrate diffusion to the active sites of the cross-linked enzymes is a problem.
Ease in fabrication is an important aspect in the potential industrialization of a material. To take advantage of the facility of the adsorption method in fabrication of composite catalysts, selection of efficient carriers turns to be the key issue. The leaching problem of the aforementioned composites might come from the drawbacks of the carriers: their structures are rigid and the interactions between their frameworks with enzymes are weak van der Waals forces, therefore, they cannot effectively hold the enzymes. Macroporous ion exchange resins are well-known to have the following unique structural and chemical properties: (a) a broad pore size distribution from <0.2 to hundreds of nanometer which allows enzyme molecules of various size to enter in, (b) abundant electric surface groups which offer strong electrostatic attraction to enzymes, and (c) cross-linking nature which is believed extraordinarily appropriate to enhance and stabilize long-chain polymeric substances therein (Chen et al., 2010). Therefore, we expect that macroporous ion exchange resins might be a better type of carriers.
In the present study, our efforts were focused on the fabrication of a resin–enzyme composite catalyst through a facile adsorption process. D201, a strongly basic anion-exchange resin containing covalently bonded quaternary amine groups in a macroporous polystyrene skeleton with chloride as counter-ion, was selected as the carrier. Trametes versicolor laccase (69 kD) was employed as the active catalytic component. D201 was selected as the host carrier because T. versicolor laccase is electronegative in neutral aqueous system and D201 contains plentiful electropositive groups. Besides, D201 is widely used in wastewater treatment and commercially available, which makes the present study more valuable for potential practical application. The resistance of the resultant enzyme composites toward changes in solution pH and temperature were evaluated. Decoloration of malachite green (MG), a widely used and high aquatic toxic triarylmethane dye (Srivastava et al., 2004), by the resultant biocatalyst was performed to evaluate its stability and efficiency for potential application.
Section snippets
Materials
T. versicolor laccase was provided by Sigma–Aldrich. D201was provided by Zhengguang Electrical Resin Co. Ltd. (Hangzhou, China). Prior to use, D201 was subjected to extraction with ethanol in a Soxhlet apparatus and then vacuum desiccated at 45 °C for 24 h. The spherical beads ranging from 0.6 to 0.7 mm in diameter were sieved for further use.
Other chemicals, including 2,6-dimethoxyphenol (DMP), 1-hydroxybenzotriazole (Hobt), and MG, are of analytical grade and were purchased from Sigma–Aldrich.
Characteristics of D201_Lac
The encapsulation of laccase into D201 beads had little effect on their shape and diameter, while changed the color from white to yellowish-brown. TEM images of D201_Lac and D201 in Fig. S1 (in Supplementary materials) indicated that both single laccase molecules (6.5 × 5.5 × 4.5 nm) (Qiu et al., 2009) and laccase aggregates (of 2–3 molecules) were present in D201_Lac. Similar conclusions could also be obtained from the variation in pore size distribution before and after laccase encapsulation, as
Conclusions
A hybrid biocatalyst, D201_Lac, was fabricated in this work. It is noteworthy that the fabrication was achieved through a simple mixing process. The resultant composite has outstanding performance: enhanced stability, resistance and highly reusable. The resultant biocatalyst possessed new features, such as rigidity and bulky, thanks to the host carrier, D201 resin. The ease in production and good performance in cyclic runs are important merits for a material to be used in environmental
Acknowledgements
We acknowledge the financial support from NSFC (21177059/51078179), Fundamental Research Funds for the Central University of China, and New Century Excellent Talents in University of China (NCET-10).
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