- Email: [email protected]
Immobilization of laccase onto chitosan beads to enhance its capability to degrade synthetic dyes
International Biodeterioration & Biodegradation 110 (2016) 69e78
Contents lists available at ScienceDirect
International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Immobilization of laccase onto chitosan beads to enhance its capability to degrade synthetic dyes Fei Zheng a, 1, Bao-Kai Cui a, 1, Xue-Jun Wu a, Ge Meng a, Hong-Xia Liu b, Jing Si a, * a b
Institute of Microbiology, Beijing Forestry University, Beijing 100083, China College of Forestry, Beijing Forestry University, Beijing 100083, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 November 2015 Received in revised form 4 March 2016 Accepted 7 March 2016 Available online xxx
Loss in activity and denaturation remain key challenges to the potential use of laccase in industrial applications. One of the most important aims of enzyme technology is to enhance the stability and reusability of enzymes through immobilization processes. Here, a purified laccase (Tplac) from the white rot fungus Trametes pubescens was entrapped onto chitosan beads with the crosslinker glutaraldehyde, in order to improve the stability and recovery rate of Tplac, and was applied in decolorization of various synthetic dyes. The optimal conditions for Tplac immobilized onto chitosan beads were 0.8% (v/v) glutaraldehyde concentration, 3 h crosslinking time, 2 mL enzyme solution (approximately 43.672 U/mL), and 4 h immobilization time. The pH adaptability and resistance to thermal denaturation of immobilized Tplac were considerably enhanced compared with free Tplac, and both the operational stability and durability during multiple reuses were superior to those of free Tplac; after six cycles of continuous use, the activity of immobilized enzyme remained above 60%. Also, immobilized Tplac was able to degrade various synthetic dyes, especially metal-complex dye Acid Black 172. Results of this study demonstrated that, alongside the better stability and reusability of immobilized Tplac, the immobilized enzyme could be used in many applications. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Laccase Immobilization Stability Reusability Dye degradation
1. Introduction Fungal laccases have been the subject of increased reports in recent decades, particularly laccase from the white rot fungus Trametes pubescens. The wide substrate specificity and high catalytic efficiency of fungal laccases make them particularly valuable for an extensive variety of industrial applications, including biopulping in the paper industry, improving the properties of fibers, food processing, biosynthesis, energy exploitation, environmental protection, and biodetection (Arora et al., 2002; Si et al., 2013a; Kuhar et al., 2015). Among these applications, it is promising to use laccase as a biocatalyst for degradation of synthetic dyes (Wangpradit and Chitprasert, 2014; Yavuz et al., 2014; Adnan et al., 2015). However, previous researches revealed that the use of free
* Corresponding author. P.O. Box 61, Beijing Forestry University, No. 35 Tsinghua East Road, Haidian District, Beijing 100083, China. E-mail address: [email protected] (J. Si). 1 Fei Zheng and Bao-Kai Cui contributed equally to this work and shared the first author. http://dx.doi.org/10.1016/j.ibiod.2016.03.004 0964-8305/© 2016 Elsevier Ltd. All rights reserved.
laccase in these processes presented some problems, such as loss in enzyme activity, sensitivity to environmental conditions, and low storage stability, which seriously restricted the efficiency and ndez et al., 2013). reusability of laccase in practice (Fern andez-Ferna Emerging immobilization technology is an effective means to resolve these problems. With this technology, laccase can be immobilized on a support by physical or chemical methods to form insoluble enzyme derivatives, which exhibit improved thermal and storage stability combined with good performance for reusability; such immobilized laccase have potentials in many applications (Spinelli et al., 2013; Rahmani et al., 2015). Various methods of laccase immobilization have been studied extensively, including adsorption, covalent binding, entrapment, ndez et al., 2013; Rahmani et al., and crosslinking (Fern andez-Ferna 2015; Sun et al., 2015). Entrapment is considered to be a good choice, since it is a mild process that causes little damage to the ^ssi et al., native structure of the enzyme (Basak et al., 2014; Daa 2014). During this process, a suitable support is required to trap the enzyme molecules or enzymatic preparations within a matrix. Gelatin, sodium alginate ([C6H7NaO6]n), starch ([C6H10O5]n), chitosan ([C6H11NO4]n), polyvinyl alcohol ([C2H4O]n), polyacrylamide
70
F. Zheng et al. / International Biodeterioration & Biodegradation 110 (2016) 69e78
([C3H5NO]n), photosensitive resin, nylon membrane, cellulose acetate ([C6H7O2(OH)3-m(OOCCH3)m], m ¼ 0e3), ethyl cellulose n ([C6H7O2(OC2H5)3]n), etc., are all frequently used supports (Dura et al., 2002). Chitosan, extracted from chitin by >50% deacetylation, is the only natural basic polysaccharide, and has attracted extensive attention from pharmaceutical researchers because of its good biocompatibility and low toxicity (Singh et al., 2008; Nidheesh et al., 2015). By using chitosan to incorporate enzymes or cells, higher enzyme activity can be retained (Jiang et al., 2005). Although entrapment is relatively simple and inexpensive, the support swells and cracks easily, leading to leakage of the cells and enzymes, thereby decreasing the enzyme activity and immobilization efficiency (Hou et al., 2014). Through crosslinking, enzyme molecules are firmly attached to the appropriate support by a crosslinker containing two or more functional groups (Da^ assi et al., 2014). The crosslinker can increase the strength of the support by forming a spatial grid structure. The most widely used crosslinker is glutaraldehyde; it contains two aldehyde groups, which can react with amino groups on an enzyme to form a Schiff base (RC¼N), so that the enzyme is fixed to the support with the crosslinker acting as a bridge (Migneault et al., 2004; Palvannan et al., 2014). Advantages of crosslinking are that the connection between the enzyme and substrate is firm, giving good stability and reusability. However, there are also some drawbacks: the reaction conditions are relatively difficult to control in crosslinked support preparation, and the crosslinking reaction often cause structural denaturation of enzyme molecules resulting ^ssi et al., 2014). in deleterious effects to active sites (Daa In this study, to enhance stability and durability, T. pubescens laccase (Tplac), which had been purified by ammonium sulfate precipitation, anionic exchange, and sepharose chromatography, was entrapped onto chitosan beads using the crosslinker glutaraldehyde. Beyond this, the major objective of the study was to obtain the maximum degradation capability of Tplac for synthetic dyes by optimizing the immobilization process factors, i.e., concentration of crosslinker, crosslinking time, volume of enzyme, and immobilization time, and to elaborate how the properties of the immobilized enzyme were influenced by the factors such as pH, temperature, storage time, and continuous use. It is shown that the results will be valuable in facilitating the use of chitosan-immobilized laccase in practical applications in the field of environmental protection. The whole scheme of the immobilization procedure is illustrated in Fig. 1.
2. Materials and methods 2.1. Dyes and chemicals Chitosan and 2,20 -azino-bis(3-ethylbenzothiazoline-6sulphonic acid) (ABTS) were purchased from SigmaeAldrich (St Louis, MO, USA). All other reagents and chemicals were of the highest available purity. Deionized water was used for preparation of all aqueous solutions. Table 1 lists the dyes used, their color index (C.I.) numbers, C.I. names, chemical classes, structures, and adsorption wavelengths. Dye solutions were prepared by dissolving an appropriate amount of dye in deionized water and filtering through a 0.22-mm membrane to remove bacteria. The desired pH was adjusted with 1 M NaOH and 1 M HCl. 2.2. Fungal strain and purification of laccase T. pubescens Cui 7571 used in the present study is a white rot fungal strain isolated from Chebaling Nature Reserve, Guangdong Province, China. It was selected due to the laccase production during the decolorization of synthetic dyes (Si et al., 2013b). The liquid cultivation of this fungal strain and the purified procedure of its laccase (Tplac) were presented by Si et al. (2013a). Laccase activity was estimated by measuring the increase in absorbance at 420 nm with ABTS as substrate, as described by Kalyani et al. (2008). One unit of laccase activity was defined as the amount of enzyme that oxidized 1 mmol of ABTS per minute at 25 C (ε420nm ¼ 36,000 L mol1 cm1). 2.3. Immobilization of Tplac An aliquot of chitosan powder (0.25 g) was dissolved in 10 mL of 1.0% (v/v) acetic acid, and left to stand for 1 h after being stirred uniformly, so that it was thoroughly dissolved in a viscous state. The resulting solution was added dropwise into a coagulant batch of 2.0 M NaOH using a 0.4 mm-inner diameter syringe to produce spherical beads. During this process, the dropping speed was controlled at 10e12 drops per minute, and the mixture was stirred constantly when adding the chitosan to prevent the viscous solution from coagulating before forming beads. Thereafter, the beads (2.20 ± 0.21 mm) were harvested by filtration, washed several times with deionized water until neutrality, and dried at room temperature to constant weight.
Fig. 1. Experimental design of laccase immobilization in this study.
F. Zheng et al. / International Biodeterioration & Biodegradation 110 (2016) 69e78
71
Table 1 Decolorization of various synthetic dyes by immobilized laccase Tplac from white rot fungus Trametes pubescens. Dye
Color index number
Color index Chemical class name
Reactive Brilliant Blue X-BR
61205
Reactive Blue 4
Remazol Brilliant Blue R
61200
Congo Red
Structure
Maximum adsorption wavelength (nm)
% Decolorization Immobilized laccase
Free laccase
Anthraquinone
603
52.26 ± 6.84
47.15 ± 6.23
Reactive Blue 19
Anthraquinone
592
48.23 ± 7.17
46.28 ± 7.84
22120
Direct Red 28
Azo
497
54.24 ± 7.45
50.53 ± 7.61
Acid Black 172
15711
Acid Black 172
Azo (metal-complex)
597
68.84 ± 6.68
56.34 ± 7.12
Methylene Blue
52015
Basic Blue 9 Cyanine
664
25.39 ± 7.52
17.24 ± 7.63
Neutral Red
50040
Basic Red 5 Heterocycle
553
44.58 ± 6.46
36.54 ± 6.78
Indigo Blue
73000
Vat Blue 1
Indigo
610
45.12 ± 6.61
38.12 ± 6.51
Naphthol Green B
10020
Acid Green 1
Nitroso (metalcomplex)
714
37.18 ± 7.46
30.51 ± 7.41
Direct Fast Blue FBL 74190
Direct Blue 199
Phthalocyanine (metal-complex)
608
56.28 ± 6.81
48.81 ± 6.91
(continued on next page)
72
F. Zheng et al. / International Biodeterioration & Biodegradation 110 (2016) 69e78
Table 1 (continued ) Dye
Crystal Violet
Color index number
Color index Chemical class name
42555
Basic Violet Triphenylmethane 3
Structure
Glutaraldehyde was used as the crosslinker for laccase immobilization. Chitosan beads (5.0 g) obtained above were mixed with 5.0 mL of glutaraldehyde solution and crosslinked for 3 h after oscillation for 5 min at room temperature. Afterwards, the crosslinked chitosan beads were washed several times with deionized water to remove excess glutaraldehyde. Tplac was immobilized onto glutaraldehyde-crosslinked chitosan beads at 1 g:1 mL at room temperature with shaking speed at 150 rpm. After a 6-h immobilization, the beads were washed with deionized water, dried at room temperature to constant weight, and stored in dry conditions until use. Control experiments were performed with incubation of free enzyme under the same conditions to quantify enzyme denaturation during the immobilization process.
2.4. Influences of conditions on immobilization The influences of glutaraldehyde concentration (0.1e1.0%, v/v), crosslinking time (1e10 h), enzyme volume (1.0e8.0 mL), and immobilization time (2e14 h) on the relative activity of immobilized Tplac were investigated. Experiments were all performed in triplicate. Laccase activity at an optimum condition was defined as 100%.
Free and immobilized Tplac were kept in storage for 30 days, and the laccase activities were determined on days 3, 5, 7, 10, 20, and 30. Experiments were all performed in triplicate, and laccase activity at the optimum pH and temperature on day 0 was defined as 100%.
Immobilized laccase
Free laccase
595
20.81 ± 6.95
14.45 ± 7.16
Immobilized laccase was reused for 14 cycles and the residual activity was measured with respect to the initial one. After each cycle, the immobilized enzyme was filtered, washed with 0.1 M citrate-phosphate buffer (pH 5.0), and reintroduced into fresh reaction medium. Experiments were all performed in triplicate, and laccase activity at the optimum pH and temperature in the first cycle was defined as 100%. 2.8. Dye decolorization capacity of immobilized Tplac The decolorization capacity of various structurally distinct dyes by immobilized Tplac was monitored by the decrease in absorbance at the maximum absorption wavelength of each dye (Table 1). The 10-mL reaction mixtures for dye decolorization contained 1.0 U/mL pure enzyme solution and 50.0 mg/L dye (Reactive Brilliant Blue XBR, Remazol Brilliant Blue R, Congo Red, Acid Black 172, Methylene Blue, Neutral Red, Indigo Blue, Naphthol Green B, Direct Fast Blue FBL, and Crystal Violet) in 0.1 M citrate-phosphate buffer (pH 5.0). The mixtures were incubated in a dark chamber at 50 C with shaking speed at 150 rpm. The negative control contained all components except the enzyme solution. Experiments were all performed in triplicate. Decolorization efficiency is expressed in terms of percentage and calculated as
%Decolorization ¼
2.6. Storage stability of immobilized Tplac
% Decolorization
2.7. Reusability of immobilized Tplac
2.5. pH and thermal stability of immobilized Tplac The influence of pH on Tplac activity was determined in citratephosphate buffer within the pH range 1.0e13.0 at 25 C using 1.0 mM ABTS as substrate. The pH stability of free or immobilized laccase was assessed by pre-incubating the enzyme in buffer at 25 C for 72 h; then the residual laccase activity was determined. The optimum temperature for Tplac was examined in citratephosphate buffer from 10 to 90 C at the optimum pH using 1.0 mM ABTS as the substrate. The thermal stability of free or immobilized laccase was evaluated by pre-incubating the enzyme in buffer (optimum pH) at different temperatures from 10 to 90 C for 2 h; then the residual laccase activity was determined. Aliquots of samples of 5.0 mL were taken at regular intervals and centrifuged at 12,000 rpm for 20 min; the supernatant was then used for laccase activity determination. Experiments were all performed in triplicate. Laccase activity at the optimum pH or temperature was defined as 100%.
Maximum adsorption wavelength (nm)
Ai Ao 100% Ai
where Ai is the initial absorbance of the synthetic dye, and Ao is the final absorbance of the dye after decolorization. 2.9. Identification of degradation metabolites After decolorization of Acid Black 172, the metabolites formed were extracted three times with an equal volume of ethyl acetate, with vigorous shaking. The combined organic phase was filtered over Na2SO4 on filter paper and concentrated in a rotary vacuum evaporator. GCeMS analysis was carried out using a QP 2010 mass spectrophotometer (Shimadzu model No. U-2800). The ionization voltage was 70 eV and the temperature of the injection port was 280 C. GC was conducted in temperature programming mode with a Resteck column (0.25 30 nm, XTI-5). The initial column temperature was 80 C for 2 min, which was then increased to 280 C linearly at 10 C per min and held for 7 min. The GCeMS interface was maintained at 290 C and helium was used as the carrier gas at a flow rate of 1.0 mL/min with a 30 min run time. 2.10. Statistical analysis Data were subjected to statistical analysis by one-way analysis of variance and the TukeyeKramer comparison test using SPSS18.0
F. Zheng et al. / International Biodeterioration & Biodegradation 110 (2016) 69e78
software. P values < 0.05 (denoted by *) and <0.01 (**) were considered significant and highly significant respectively. 3. Results and discussion 3.1. Influences of conditions on immobilization 3.1.1. Glutaraldehyde concentration The effectiveness of immobilization of laccase is mainly dependent on the crosslinker. Within a certain concentration range, the crosslinker can enhance the extent of crosslinking, thus stimulating laccase immobilization. However, a higher concentration of crosslinker may lead to a decline in immobilized laccase activity, due to enzyme denaturation, obstruction of active sites of the enzyme by crosslinker structure, and/or an insufficient amount of laccase immobilization (Zhang et al., 2009a; Sathishkumar et al., 2014). The data of relative laccase activity versus crosslinker concentration are presented in Fig. 2A. The relative activity of immobilized T. pubescens laccase Tplac increased with increasing concentration of glutaraldehyde from 0.1 to 0.8% (v/v), and the maximum relative laccase activity was obtained with the use of 0.8% glutaraldehyde. Afterwards, the laccase activity decreased sharply as the glutaraldehyde concentration increased from 0.8 to
73
1.0% (v/v). Better laccase activity observed at relatively moderate crosslinker concentrations can be explained by the attractive forces between the glutaraldehyde and the enzyme molecules. At a certain concentration, the glutaraldehyde can adequately react with the enzyme to form a Schiff base, so that more enzyme molecules are attached to the support with the glutaraldehyde acting as a bridge (Songulashvili et al., 2012; Barbosa et al., 2014). As the glutaraldehyde concentration increases further, crosslinking reactions often cause conformational changes of enzyme molecules, resulting in damage to the active site, thus inhibiting the immo^ssi et al., 2014). According to the data in bilization efficiency (Daa Fig. 2A, from hereon, immobilization was carried out using a crosslinker glutaraldehyde concentration of 0.8% (v/v). 3.1.2. Crosslinking time Crosslinking time also significantly influences the immobilization process. As depicted in Fig. 2B, the maximum relative activity of immobilized T. pubescens laccase Tplac was obtained after crosslinking for 3 h, indicating that this reaction time is suitable for adsorption of enzyme molecules onto chitosan beads in grid number and size (Sathishkumar et al., 2014). The spatial grid structure of the support increases with the extended period, leading to restricted conditions for more enzyme molecules to
Fig. 2. Influences of (A) glutaraldehyde concentration, (B) crosslinking time, (C) enzyme volume, and (D) immobilization time on the activity of immobilized laccase Tplac from white rot fungus Trametes pubescens. Each value is the mean value ± standard error mean of triplicate.
74
F. Zheng et al. / International Biodeterioration & Biodegradation 110 (2016) 69e78
access (Kaspar et al., 2013). Hence, an appropriate crosslinking time is crucial for maintaining high laccase activity after immobilization.
3.1.3. Enzyme volume Results on the influence of enzyme volume on the relative activity of immobilized T. pubescens laccase Tplac are given in Fig. 2C. Better immobilization efficiency of laccase Tplac was obtained using 2.0 mL of enzyme solution, corresponding to 43.672 U/mL of laccase activity, which could be attributed to the equilibrium reached by the dissociative amino and aldehyde groups on Tplac and the support respectively. A lower amount of enzyme could not couple as effectively with the support. On the contrary, an excess of enzyme could lead to overloading of the support, thereby resulting ^ssi et al., 2014; Sathishkumar in a decrease in laccase activity (Daa et al., 2014).
3.1.4. Immobilization time Immobilization time is another key variable during the enzyme immobilization process. Too short an immobilization time means that most of the laccase molecules do not have enough time to penetrate the grid structure of the support, while too long an immobilization time leads to laccase molecules on the support being too crowded to make optimal contact with their substrate, resulting in a decline in enzyme activity (Halder et al., 2014). Additionally, if too much laccase is bound to the support, rising temperature as a result of chemical reaction catalyzed by closepacked laccase molecules is another reason for reduction of laccase activity (Xu et al., 2013). As can be seen from Fig. 2D, an apparent peak in the relative activity of T. pubescens laccase Tplac was detected after immobilization for 4 h, demonstrating that the appropriate enzyme molecules have access to the interior structure of the support for laccase immobilization (Sathishkumar et al., 2014). As the immobilization time increased, the quantity of enzyme adsorbed onto the support increased, and was influenced by the external environment more easily; therefore, the activity of the immobilized laccase decreased significantly (Halder et al., 2014). When the effect of the volume of enzyme attached was greater than the influence of the environment on enzyme activity, the immobilized laccase activity gradually increased again, and thus a small, second peak was found after 12 h of immobilization (Fig. 2D), after which the activity decreased again.
3.2. pH dependence of activity and pH stability of immobilized Tplac In Fig. 3A it is shown that immobilized laccase Tplac exhibited activity over a broad pH range from 3.5 to 12.0, with the optimum pH at 5.0, 0.5 pH units higher than that of the free enzyme (optimum pH 4.5). These observations suggested that immobilization treatment of the laccase improves its adaptive capacity to an alkaline environment, and broadens the pH range for catalysis (Catapane et al., 2013). As pH increased, declining trends were observed in the relative activities of both free and immobilized Tplac, but less for the latter than for the former with respect to the degree and speed of decline, implying that immobilized Tplac is more resistant to alkali conditions. This might be due to that the inner surface of the chitosan support contains relatively abundant negatively charged amino groups, and thus it could act as a buffer, causing the pH in proximity to the enzyme molecules to be higher than in the surrounding liquid, providing a relatively favorable microenvironment for catalysis. Overall, the immobilized laccase suffers less interference from the external environment than free laccase, and possesses higher tolerance to acidic and alkali conditions, and thus wider applicability in catalysis (Yan et al., 2014). The microenvironment has a remarkable influence on the catalytic activity of enzymes, especially in extreme conditions that may generally cause enzyme inactivation. In this study, the pH stabilities of free and immobilized laccase Tplac from T. pubescens were also investigated. Enzyme was incubated at various pH values for an extended period and the activity was then determined. As displayed in Fig. 3B, free Tplac was very stable over a broad pH range, from 4.5 to 9.0, maintaining more than 75% of its original activity after incubation at 25 C for 72 h. While the relative activity of immobilized T. pubescens Tplac was >80% in the same conditions. A sharp decline in relative laccase activity was observed when the pH was <4.0 or >11.0. Demarche et al. (2012) explicated that the main reason why the pH stability of laccase changes after immobilization is proton production by the support. The electronwithdrawing effect of C¼O groups on the support surface could stimulate eCH2 groups to more easily release Hþ, resulting in a pH in the support microenvironment lower than that in the overall system, thus altering the pH stability range of the immobilized laccase. In contrast to free Tplac, immobilized Tplac is stable at higher pHs, which improved its values in practical applications.
Fig. 3. pH stability of immobilized and free laccase Tplac from white rot fungus Trametes pubescens. (A) activity assay at the given pH at 25 C; (B) activity assay after incubation at the given pH for 72 h at 25 C. Each value is the mean value ± standard error mean of triplicate.
F. Zheng et al. / International Biodeterioration & Biodegradation 110 (2016) 69e78
75
3.3. Thermal stability of immobilized Tplac The adaptability of laccase from various sources to temperature differs (Santos et al., 2007; Wu and Nian, 2014). Results in Fig. 4A showed that immobilized Tplac performed its maximal activity at 60 C; the activity increased with temperature up to this point. It was suggested that the immobilization process notably enhances Tplac's thermal tolerance. This may be assigned to that the characteristic embedding structure of the support effectively protects the laccase, and affords a stable microenvironment for it (Zhang et al., 2009b). Furthermore, after immobilization, interactions among enzyme molecules and between the enzyme and substrate and the enzyme and the support enhance the rigidity of the molecular structure of the enzyme, thereby reinforcing its capability to resist thermal denaturation. In contrast, free enzymes are more easily denatured (Yang et al., 2006; Nair et al., 2013). After pre-incubation at 60e70 C for 2 h, the relative activity of immobilized Tplac remained >50%, while the free laccase activity decreased by 50% from the level of 72.31%, as presented in Fig. 4B, which revealed that after an incubation period at elevated temperature, immobilized laccase is more stable than free laccase (Addorisio et al., 2013). At tested temperatures above 60 C, and particularly above 75 C, the activities of both free and immobilized Tplac decreased rapidly. Nevertheless, both the rate and degree of decline of the activity of the immobilized laccase were much lower than those of free laccase. Through attachment to an appropriate support, the structural rigidity and thermal resistance of the enzyme molecules can be strengthen obviously (Nair et al., 2013). Hence, at the same temperature, relatively poor durability of free ^ssi et al., laccase is detected compared to immobilized enzyme (Daa 2014).
3.4. Storage stability of immobilized Tplac Storage stability is one of the most important parameters by which to evaluate performance because immobilized enzymes are susceptible to decreased activity after prolonged storage ndez-Fern (Ferna andez et al., 2013). As depicted in Fig. 5, downward trends were presented for the activities of both free and immobilized Tplac on storage for up to one month. Apparently, the decrease in activity of immobilized T. pubescens laccase was smaller in terms of both amplitude and rate. After 10 days, the remaining
Fig. 5. Storage stability of immobilized and free laccase Tplac from white rot fungus Trametes pubescens. Each value is the mean value ± standard error mean of triplicate.
relative activity of immobilized Tplac was above 50%; after 30 days, it was above 40%, whereas the activity of free laccase lost approximately 85% of its initial activity over the same period. These results indicated that, in terms of storage stability, immobilized Tplac is significantly superior to free laccase, which provides the possibility of long-term preservation for further use (Nair et al., 2013).
3.5. Reusability of immobilized Tplac The feasibility of regeneration of immobilized laccase for repeated use is one of the most important indicators for reducing the overall cost of enzymatic applications, especially in industry ndez-Ferna ndez et al., 2013). According to the data on the (Ferna reusability of immobilized laccase Tplac (Fig. 6), after three cycles of continuous use, the relative activity of immobilized Tplac was >90%, and it remained 60% after six cycles. The relative activity of immobilized T. pubescens laccase decreased with increased use, dropping to around 20% of its original activity after 14 cycles at room temperature. This decrease in enzyme activity could be ascribed to inactivation and loss of enzyme molecules during each
Fig. 4. Thermal stability of immobilized and free laccase Tplac from white rot fungus Trametes pubescens. (A) activity assay at the given temperature at the optimum pH; (B) activity assay after incubation at the given temperature for 2 h at the optimum pH. Each value is the mean value ± standard error mean of triplicate.
76
F. Zheng et al. / International Biodeterioration & Biodegradation 110 (2016) 69e78
dyes were decolorized to various extents in the absence of redox mediators, and the removal efficiency of the dyes depended on their different chemical structures. Surprisingly, the immobilized Tplac exerted higher catalytic efficiency toward all the tested dyes. Among them, better decolorization of metal-complex dye Acid Black 172 (decolorization efficiency 68.84%) was observed after reaction for 48 h, which was 1.22-fold higher than that by free laccase. These data implied that dye affinity is different for different enzymatic complexes, underlining the influence of immobilization on the laccase's behavior (Rodríguez-Couto, 2014). 3.7. Identification of degradation metabolites
Fig. 6. Reusability of immobilized laccase Tplac from white rot fungus Trametes pubescens. Each value is the mean value ± standard error mean of triplicate.
cycle (Sadighi and Faramarzi, 2013). 3.6. Dye decolorization capacity of immobilized Tplac The decolorization capacity of immobilized laccase Tplac was assessed using various dyes (Table 1). In general, the catalytic efficiency of an enzyme may be altered after immobilization treatment. To confirm it, dye degradation by immobilized laccase was compared with that by free laccase. It is shown in Table 1 that the
GCeMS analysis is a versatile technique to identify and quantify chemical compounds in complex forms (Schauer et al., 2005). As displayed in Table 2, the degradation metabolites of metal-complex dye Acid Black 172 were identified by organic solvent and GCeMS analysis. Based on the above data, possible partial pathways of biodegradation of Acid Black 172 by immobilized T. pubescens laccase Tplac were proposed (Fig. 7), in which 2-Nitronaphthalene (III), 1-Naphthalene diazonium (VII), 6-Nitro-2-naphthol (VIII), and 2-Naphthol (IX) are the main degradation products of Acid Black 172. Remarkably, 1-Amino-6-nitro-2-naphthol-4-sulfonate (I), 1-Diazonium-2-naphthol (IV), 6-Nitro-2-naphthol-4-sulfonate (V), and 1-Amino-2-naphthol (VI) could not be detected after 72 h suggesting that these intermediates might be eventually transferred into stable compounds by desulfonation, deamination, and dehydroxylation reactions. However, the intermediate II was not found. The possible reason should be that most of it was rapidly degraded to small molecules (Tan et al., 2013). It is clear that the first step of biodegradation in the present study is initiated by
Table 2 Degradation metabolites of metal-complex dye Acid Black 175 obtained from GCeMS analysis. No.
Retention time (min)
Major fragment m/z
Calculated mass
Compound
I
18.245
284
284
1-Amino-6-nitro-2-naphthol-4-sulfonate
III
17.268
173
173
2-Nitronaphthalene
IV
17.648
172
172
1-Diazonium-2-naphthol
V
16.441
269
269
6-Nitro-2-naphthol-4-sulfonate
VI
14.584
159
159
1-Amino-2-naphthol
VII
20.452
156
156
1-Naphthalene diazonium
VIII
22.474
189
189
6-Nitro-2-naphthol
IX
19.334
144
144
2-Naphthol
Chemical structure
F. Zheng et al. / International Biodeterioration & Biodegradation 110 (2016) 69e78
77
Na O O
+
Na
ON
S
ONO2
Cr3+
N
O
N N
O2N OO
Acid Black 172
O-
S O O
Na
Asymmetric cleavage by chitosan-immobilized laccase Decolorization
Na O
O S
ON
O
Cr3+
N NO2
NH2
O2N O-
HO NH2
I
O
Oxidative cleavage
Na
Desulfonation
HO NH
N
NH2 OH
NO2
OH
Deamination
IV Dehydroxylation
II
S O O
1-Amino-6-nitro-2-naphthol-4-sulfonate
O-
O
1-Diazonium-2-naphthol
S O
O
VI
V
1-Amino-2-naphthol
Na
6-Nitro-2-naphthol-4-sulfonate NO2
Deamination
Dehydroxylation
III Desulfonation
2-Nitronaphthalene N
OH
NH
IX 2-Naphthol VII
HO
1-Naphthalene diazonium
NO2
VIII
6-Nitro-2-naphthol Fig. 7. Proposed pathways of biodegradation of metal-complex dye Acid Black 172 by immobilized laccase Tplac from white rot fungus Trametes pubescens.
asymmetric reduction of azo groups (N¼N), which requires electrons and protons for the reduction process (Nam and Renganathan, 2000; Du et al., 2013). Laccase, a class of multicopper oxidase containing four histidine-rich copper binding domains at the reaction center, is capable of using molecular oxygen as a co-substrate to oxidize an array of compounds by a radicalcatalyzed reaction mechanism (Thurston, 1994). Therefore, the enzyme can donate electrons to azo dye that is able to accept electrons via a nonspecific interaction mechanism, resulting in production of colorless solution (Jadhav et al., 2008; Tahmasbi et al., 2016). Obviously, the proposed partial pathways were only dependent on GCeMS analysis results and relevant literature. It still needs to be investigated systematically.
using the crosslinker glutaraldehyde. Optimization experiments indicated that the stability was dependent on the concentration of crosslinker, crosslinking time, volume of enzyme, and immobilization time. Moreover, the immobilized laccase was less sensitive to changes in pH and temperature, and to storage time, compared with free laccase, thereby exhibiting better stability and reusability. Catalytic performance of immobilized Tplac was evaluated by degradation of various synthetic dyes, showing that the removal efficiency of metal-complex dye Acid Black 172 by immobilized Tplac (68.84%) was 1.22 times higher than that by free laccase. These results suggested that immobilization is feasible to improve the conformational stability of laccase for many industrial applications.
4. Conclusions
Acknowledgments
The immobilized laccase Tplac from white rot fungus T. pubescens was fabricated by entrapment onto chitosan beads
We express our gratitude to Prof. Yu-Cheng Dai (Beijing Forestry University, China) and Dr. Hai-Jiao Li (Chinese Center for Disease
78
F. Zheng et al. / International Biodeterioration & Biodegradation 110 (2016) 69e78
Control and Prevention, China) for improving the manuscript. This study was supported by the Fundamental Research Funds for the Central Universities (No. BLX2014-36) and the National Natural Science Foundation of China (No. 31422001). References Addorisio, V., Sannino, F., Mateo, C., Guisan, J.M., 2013. Oxidation of phenyl compounds using strongly stable immobilized-stabilized laccase from Trametes versicolor. Process Biochem. 48, 1174e1180. Adnan, L.A., Sathishkumar, P., Yusoff, A.R., Hadibarata, T., 2015. Metabolites characterisation of laccase mediated Reactive Black 5 biodegradation by fast growing ascomycete fungus Trichoderma atroviride F03. Int. Biodeterior. Biodegrad. 104, 274e282. Arora, D.S., Chander, M., Gill, P.K., 2002. Involvement of lignin peroxidase, manganese peroxidase and laccase in degradation and selective ligninolysis of wheat straw. Int. Biodeterior. Biodegrad. 50, 115e120. Torres, R., Rodrigues, R.C., FernandezBarbosa, O., Ortiz, C., Berenguer-Murcia, A., Lafuente, R., 2014. Glutaraldehyde in bio-catalysts design: a useful crosslinker and a versatile tool in enzyme immobilization. RSC Adv. 4, 1583e1600. Basak, B., Bhunia, B., Dey, A., 2014. Studies on the potential use of sugarcane bagasse as carrier matrix for immobilization of Candida tropicalis PHB5 for phenol biodegradation. Int. Biodeterior. Biodegrad. 93, 107e117. Catapane, M., Nicolucci, C., Menale, C., Mita, L., Rossi, S., Mita, D.G., Diano, N., 2013. Enzymatic removal of estrogenic activity of nonylphenol and octylphenol aqueous solutions by immobilized laccase from Trametes versicolor. J. Hazard. Mater. 248249, 337e346. ^ssi, D., Rodríguez-Couto, S., Nasri, M., Mechichi, T., 2014. Biodegradation of Daa textile dyes by immobilized laccase from Coriolopsis gallica into Ca-alginate beads. Int. Biodeterior. Biodegrad. 90, 71e78. Demarche, P., Junghanns, C., Mazy, N., Agathos, S.N., 2012. Design-of-experiment strategy for the formulation of laccase biocatalysts and their application to degrade bisphenol A. New Biotechnol. 30, 96e103. Du, L.N., Zhao, M., Li, G., Xu, F.C., Chen, W.H., Zhao, Y.H., 2013. Biodegradation of malachite green by Micrococcus sp. strain BD15: biodegradation pathway and enzyme analysis. Int. Biodeterior. Biodegrad. 78, 108e116. Dur an, N., Rosa, M.A., D'Annibale, A., Gianfreda, L., 2002. Applications of laccases and tyrosinases (phenoloxidases) immobilized on different supports: a review. Enzyme Microb. Technol. 31, 907e931. Moldes, D., 2013. Recent developments ndez-Fern Ferna andez, M., Sanrom an, M.A., and applications of immobilized laccase. Biotechnol. Adv. 31, 1808e1825. Halder, S.K., Maity, C., Jana, A., Ghosh, K., Das, A., Paul, T., das Mohapatra, P.K., Pati, B.R., Mondal, K.C., 2014. Chitinases biosynthesis by immobilized Aeromonas hydrophila SBK1 by prawn shells valorization and application of enzyme cocktail for fungal protoplast preparation. J. Biosci. Bioeng. 117, 170e177. Hou, J., Dong, G., Ye, Y., Chen, V., 2014. Laccase immobilization on titania nanoparticles and titania-functionalized membranes. J. Membr. Sci. 452, 229e240. Jadhav, S.U., Kalme, S.D., Govindwar, S.P., 2008. Biodegradation of methyl red by Galactomyces geotrichum MTCC 1360. Int. Biodeterior. Biodegrad. 62, 135e142. Jiang, D.S., Long, S.Y., Huang, J., Xiao, H.Y., Zhou, J.Y., 2005. Immobilization of Pycnoporus sanguineus laccase on magnetic chitosan microspheres. Biochem. Eng. J. 25, 15e23. Kalyani, D.C., Patil, P.S., Jadhav, J.P., Govindwar, S.P., 2008. Biodegradation of reactive textile dye Red BL1 by an isolated bacterium Pseudomonas sp. SUK1. Bioresour. Technol. 99, 4635e4641. e rov pa nek, F., 2013. Effect of Kaspar, O., Toka a, V., Nyanhongo, G.S., Gübitz, G., St cross-linking method on the activity of spray-dried chitosan microparticles with immobilized laccase. Food Bioprod. Process. 91, 525e533. Kuhar, F., Castiglia, V., Levin, L., 2015. Enhancement of laccase production and malachite green decolorization by co-culturing Ganoderma lucidum and Trametes versicolor in solid-state fermentation. Int. Biodeterior. Biodegrad. 104, 238e243. Migneault, I., Dartiguenave, C., Bertrand, M.J., Waldron, K.C., 2004. Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking. BioTechniques 37, 790e802. Nair, R.R., Demarche, P., Agathos, S.N., 2013. Formulation and characterization of an immobilized laccase biocatalyst and its application to eliminate organic micropollutants in wastewater. New Biotechnol. 30, 814e823. Nam, S., Renganathan, V., 2000. Non-enzymatic reduction of azo dyes by NADH. Chemosphere 40, 351e357. Nidheesh, T., Kumar, P.G., Suresh, P.V., 2015. Enzymatic degradation of chitosan and production of D-glucosamine by solid substrate fermentation of exo-b-D-glucosaminidase (exochitosanase) by Penicillium decumbens CFRNT15. Int. Biodeterior. Biodegrad. 97, 97e106. Palvannan, T., Saravanakumar, T., Unnithan, A.R., Chung, N.J., Kim, D.H., Park, S.M.,
2014. Efficient transformation of phenyl urea herbicide chloroxuron by laccase immobilized on zein polyurethane nanofiber. J. Mol. Catal. B Enzym. 99, 156e162. Rahmani, K., Faramarzi, M.A., Mahvi, A.H., Gholami, M., Esrafili, A., Forootanfar, H., Farzadkia, M., 2015. Elimination and detoxification of sulfathiazole and sulfamethoxazole assisted by laccase immobilized on porous silica beads. Int. Biodeterior. Biodegrad. 97, 107e114. Rodríguez-Couto, S., 2014. Decolouration of industrial metal-complex dyes in successive batches by active cultures of Trametes pubescens. Biotechnol. Rep. 4, 156e160. Sadighi, A., Faramarzi, M.A., 2013. Congo red decolorization by immobilized laccase through chitosan nanoparticles on the glass beads. J. Taiwan Inst. Chem. Eng. 44, 156e162. Santos, A.M.P., Oliveira, M.G., Maugeri, F., 2007. Modelling thermal stability and activity of free and immobilized enzymes as a novel tool for enzyme reactor design. Bioresour. Technol. 98, 3142e3148. Sathishkumar, P., Kamala-Kannan, S., Cho, M., Kim, J.S., Hadibarata, T., Salim, M.R., Oh, B.T., 2014. Laccase immobilization on cellulose nanofiber: the catalytic efficiency and recyclic application for simulated dye effluent treatment. J. Mol. Catal. B Enzym. 100, 111e120. Schauer, N., Steinhauser, D., Strelkov, S., 2005. GC-MS libraries for the rapid identification of metabolites in complex biological samples. FEBS Lett. 579, 1332e1337. Si, J., Peng, F., Cui, B.K., 2013a. Purification, biochemical characterization and dye decolorization capacity of an alkali-resistant and metal-tolerant laccase from Trametes pubescens. Bioresour. Technol. 128, 49e57. Si, J., Cui, B.K., Dai, Y.C., 2013b. Decolorization of chemically different dyes by whiterot fungi in submerged cultures. Ann. Microbiol. 63, 1099e1108. Singh, T., Vesentini, D., Singh, A.P., Daniel, G., 2008. Effect of chitosan on physiological, morphological, and ultrastructural characteristics of wood-degrading fungi. Int. Biodeterior. Biodegrad. 62, 116e124. z-Tobo n, G.A., Jaspers, C., Penninckx, M.J., 2012. ImmobiSongulashvili, G., Jimene lized laccase of Cerrena unicolor for elimination of endocrine disruptor micropollutants. Fungal Biol. 116, 883e889. Spinelli, D., Fatarella, E., di Michele, A., Pogni, R., 2013. Immobilization of fungal (Trametes versicolor) laccase onto Amberlite IR-120 H beads: optimization and characterization. Process Chem. 48, 218e223. Sun, H., Yang, H., Huang, W., Zhang, S., 2015. Immobilization of laccase in a spongelike hydrogel for enhanced durability in enzymatic degradation of dye pollutants. J. Colloid Interface Sci. 450, 353e360. Tahmasbi, H., Khoshayand, M.R., Bozorgi-Koushalshahi, M., Heidary, M., GhaziKhansari, M., Faramarzi, M.A., 2016. Biocatalytic conversion and detoxification of imipramine by the laccase-mediated system. Int. Biodeterior. Biodegrad. 108, 1e8. Tan, L., Ning, S., Zhang, X., Shi, S., 2013. Aerobic decolorization and degradation of azo dyes by growing cells of a newly isolated yeast Candida tropicalis TL-F1. Bioresour. Technol. 138, 307e313. Thurston, C.F., 1994. The structure and function of fungal laccase. Microbiology 140, 19e26. v.: inWangpradit, R., Chitprasert, P., 2014. Chitosan-coated Lentinus polychrous Le tegrated biosorption and biodegradation systems for decolorization of anionic reactive dyes. Int. Biodeterior. Biodegrad. 93, 168e176. Wu, Y.R., Nian, D.L., 2014. Production optimization and molecular structure characterization of a newly isolated novel laccase from Fusarium solani MAS2, an anthracene-degrading fungus. Int. Biodeterior. Biodegrad. 86, 382e389. Xu, P., Zeng, G., Huang, D., Hu, S., Feng, C., Lai, C., Zhao, M., Huang, C., Li, N., Wei, Z., Xie, G., 2013. Synthesis of iron oxide nanoparticles and their application in Phanerochaete chrysosporium immobilization for Pb(II) removal. Colloids Surfaces A Physicochem. Eng. Aspects 419, 147e155. Yan, J., Chen, D., Yang, E., Niu, J., Chen, Y., Chagan, I., 2014. Purification and characterization of a thermotolerant laccase isoform in Trametes trogii strain and its potential in dye decolorization. Int. Biodeterior. Biodegrad. 93, 186e194. Yang, W.Y., Min, D.Y., Wen, S.X., Jin, L., Rong, L., Tetsuo, M., Bo, C., 2006. Immobilization and characterization of laccase from Chinese Rhus vernicifera on modified chitosan. Process Biochem. 41, 1378e1382. Yavuz, M., Kaya, G., Aytekin, Ç., 2014. Using Ceriporiopsis subvermispora CZ-3 laccase for indigo carmine decolourization and denim bleaching. Int. Biodeterior. Biodegrad. 88, 199e205. Zhang, J., Xu, Z., Chen, H., Zong, Y., 2009a. Removal of 2,4-dichlorophenol by chitosan-immobilized laccase from Coriolus versicolor. Biochem. Eng. J. 45, 54e59. Zhang, Y.W., Zhang, Y., Wang, H., Yan, B.N., Shen, G.L., Yu, R.Q., 2009b. An enzyme immobilization platform for biosensor designs of direct electrochemistry using flower-like ZnO crystals and nano-sized gold particles. J. Electroanal. Chem. 627, 9e14.
Contents lists available at ScienceDirect
International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Immobilization of laccase onto chitosan beads to enhance its capability to degrade synthetic dyes Fei Zheng a, 1, Bao-Kai Cui a, 1, Xue-Jun Wu a, Ge Meng a, Hong-Xia Liu b, Jing Si a, * a b
Institute of Microbiology, Beijing Forestry University, Beijing 100083, China College of Forestry, Beijing Forestry University, Beijing 100083, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 November 2015 Received in revised form 4 March 2016 Accepted 7 March 2016 Available online xxx
Loss in activity and denaturation remain key challenges to the potential use of laccase in industrial applications. One of the most important aims of enzyme technology is to enhance the stability and reusability of enzymes through immobilization processes. Here, a purified laccase (Tplac) from the white rot fungus Trametes pubescens was entrapped onto chitosan beads with the crosslinker glutaraldehyde, in order to improve the stability and recovery rate of Tplac, and was applied in decolorization of various synthetic dyes. The optimal conditions for Tplac immobilized onto chitosan beads were 0.8% (v/v) glutaraldehyde concentration, 3 h crosslinking time, 2 mL enzyme solution (approximately 43.672 U/mL), and 4 h immobilization time. The pH adaptability and resistance to thermal denaturation of immobilized Tplac were considerably enhanced compared with free Tplac, and both the operational stability and durability during multiple reuses were superior to those of free Tplac; after six cycles of continuous use, the activity of immobilized enzyme remained above 60%. Also, immobilized Tplac was able to degrade various synthetic dyes, especially metal-complex dye Acid Black 172. Results of this study demonstrated that, alongside the better stability and reusability of immobilized Tplac, the immobilized enzyme could be used in many applications. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Laccase Immobilization Stability Reusability Dye degradation
1. Introduction Fungal laccases have been the subject of increased reports in recent decades, particularly laccase from the white rot fungus Trametes pubescens. The wide substrate specificity and high catalytic efficiency of fungal laccases make them particularly valuable for an extensive variety of industrial applications, including biopulping in the paper industry, improving the properties of fibers, food processing, biosynthesis, energy exploitation, environmental protection, and biodetection (Arora et al., 2002; Si et al., 2013a; Kuhar et al., 2015). Among these applications, it is promising to use laccase as a biocatalyst for degradation of synthetic dyes (Wangpradit and Chitprasert, 2014; Yavuz et al., 2014; Adnan et al., 2015). However, previous researches revealed that the use of free
* Corresponding author. P.O. Box 61, Beijing Forestry University, No. 35 Tsinghua East Road, Haidian District, Beijing 100083, China. E-mail address: [email protected] (J. Si). 1 Fei Zheng and Bao-Kai Cui contributed equally to this work and shared the first author. http://dx.doi.org/10.1016/j.ibiod.2016.03.004 0964-8305/© 2016 Elsevier Ltd. All rights reserved.
laccase in these processes presented some problems, such as loss in enzyme activity, sensitivity to environmental conditions, and low storage stability, which seriously restricted the efficiency and ndez et al., 2013). reusability of laccase in practice (Fern andez-Ferna Emerging immobilization technology is an effective means to resolve these problems. With this technology, laccase can be immobilized on a support by physical or chemical methods to form insoluble enzyme derivatives, which exhibit improved thermal and storage stability combined with good performance for reusability; such immobilized laccase have potentials in many applications (Spinelli et al., 2013; Rahmani et al., 2015). Various methods of laccase immobilization have been studied extensively, including adsorption, covalent binding, entrapment, ndez et al., 2013; Rahmani et al., and crosslinking (Fern andez-Ferna 2015; Sun et al., 2015). Entrapment is considered to be a good choice, since it is a mild process that causes little damage to the ^ssi et al., native structure of the enzyme (Basak et al., 2014; Daa 2014). During this process, a suitable support is required to trap the enzyme molecules or enzymatic preparations within a matrix. Gelatin, sodium alginate ([C6H7NaO6]n), starch ([C6H10O5]n), chitosan ([C6H11NO4]n), polyvinyl alcohol ([C2H4O]n), polyacrylamide
70
F. Zheng et al. / International Biodeterioration & Biodegradation 110 (2016) 69e78
([C3H5NO]n), photosensitive resin, nylon membrane, cellulose acetate ([C6H7O2(OH)3-m(OOCCH3)m], m ¼ 0e3), ethyl cellulose n ([C6H7O2(OC2H5)3]n), etc., are all frequently used supports (Dura et al., 2002). Chitosan, extracted from chitin by >50% deacetylation, is the only natural basic polysaccharide, and has attracted extensive attention from pharmaceutical researchers because of its good biocompatibility and low toxicity (Singh et al., 2008; Nidheesh et al., 2015). By using chitosan to incorporate enzymes or cells, higher enzyme activity can be retained (Jiang et al., 2005). Although entrapment is relatively simple and inexpensive, the support swells and cracks easily, leading to leakage of the cells and enzymes, thereby decreasing the enzyme activity and immobilization efficiency (Hou et al., 2014). Through crosslinking, enzyme molecules are firmly attached to the appropriate support by a crosslinker containing two or more functional groups (Da^ assi et al., 2014). The crosslinker can increase the strength of the support by forming a spatial grid structure. The most widely used crosslinker is glutaraldehyde; it contains two aldehyde groups, which can react with amino groups on an enzyme to form a Schiff base (RC¼N), so that the enzyme is fixed to the support with the crosslinker acting as a bridge (Migneault et al., 2004; Palvannan et al., 2014). Advantages of crosslinking are that the connection between the enzyme and substrate is firm, giving good stability and reusability. However, there are also some drawbacks: the reaction conditions are relatively difficult to control in crosslinked support preparation, and the crosslinking reaction often cause structural denaturation of enzyme molecules resulting ^ssi et al., 2014). in deleterious effects to active sites (Daa In this study, to enhance stability and durability, T. pubescens laccase (Tplac), which had been purified by ammonium sulfate precipitation, anionic exchange, and sepharose chromatography, was entrapped onto chitosan beads using the crosslinker glutaraldehyde. Beyond this, the major objective of the study was to obtain the maximum degradation capability of Tplac for synthetic dyes by optimizing the immobilization process factors, i.e., concentration of crosslinker, crosslinking time, volume of enzyme, and immobilization time, and to elaborate how the properties of the immobilized enzyme were influenced by the factors such as pH, temperature, storage time, and continuous use. It is shown that the results will be valuable in facilitating the use of chitosan-immobilized laccase in practical applications in the field of environmental protection. The whole scheme of the immobilization procedure is illustrated in Fig. 1.
2. Materials and methods 2.1. Dyes and chemicals Chitosan and 2,20 -azino-bis(3-ethylbenzothiazoline-6sulphonic acid) (ABTS) were purchased from SigmaeAldrich (St Louis, MO, USA). All other reagents and chemicals were of the highest available purity. Deionized water was used for preparation of all aqueous solutions. Table 1 lists the dyes used, their color index (C.I.) numbers, C.I. names, chemical classes, structures, and adsorption wavelengths. Dye solutions were prepared by dissolving an appropriate amount of dye in deionized water and filtering through a 0.22-mm membrane to remove bacteria. The desired pH was adjusted with 1 M NaOH and 1 M HCl. 2.2. Fungal strain and purification of laccase T. pubescens Cui 7571 used in the present study is a white rot fungal strain isolated from Chebaling Nature Reserve, Guangdong Province, China. It was selected due to the laccase production during the decolorization of synthetic dyes (Si et al., 2013b). The liquid cultivation of this fungal strain and the purified procedure of its laccase (Tplac) were presented by Si et al. (2013a). Laccase activity was estimated by measuring the increase in absorbance at 420 nm with ABTS as substrate, as described by Kalyani et al. (2008). One unit of laccase activity was defined as the amount of enzyme that oxidized 1 mmol of ABTS per minute at 25 C (ε420nm ¼ 36,000 L mol1 cm1). 2.3. Immobilization of Tplac An aliquot of chitosan powder (0.25 g) was dissolved in 10 mL of 1.0% (v/v) acetic acid, and left to stand for 1 h after being stirred uniformly, so that it was thoroughly dissolved in a viscous state. The resulting solution was added dropwise into a coagulant batch of 2.0 M NaOH using a 0.4 mm-inner diameter syringe to produce spherical beads. During this process, the dropping speed was controlled at 10e12 drops per minute, and the mixture was stirred constantly when adding the chitosan to prevent the viscous solution from coagulating before forming beads. Thereafter, the beads (2.20 ± 0.21 mm) were harvested by filtration, washed several times with deionized water until neutrality, and dried at room temperature to constant weight.
Fig. 1. Experimental design of laccase immobilization in this study.
F. Zheng et al. / International Biodeterioration & Biodegradation 110 (2016) 69e78
71
Table 1 Decolorization of various synthetic dyes by immobilized laccase Tplac from white rot fungus Trametes pubescens. Dye
Color index number
Color index Chemical class name
Reactive Brilliant Blue X-BR
61205
Reactive Blue 4
Remazol Brilliant Blue R
61200
Congo Red
Structure
Maximum adsorption wavelength (nm)
% Decolorization Immobilized laccase
Free laccase
Anthraquinone
603
52.26 ± 6.84
47.15 ± 6.23
Reactive Blue 19
Anthraquinone
592
48.23 ± 7.17
46.28 ± 7.84
22120
Direct Red 28
Azo
497
54.24 ± 7.45
50.53 ± 7.61
Acid Black 172
15711
Acid Black 172
Azo (metal-complex)
597
68.84 ± 6.68
56.34 ± 7.12
Methylene Blue
52015
Basic Blue 9 Cyanine
664
25.39 ± 7.52
17.24 ± 7.63
Neutral Red
50040
Basic Red 5 Heterocycle
553
44.58 ± 6.46
36.54 ± 6.78
Indigo Blue
73000
Vat Blue 1
Indigo
610
45.12 ± 6.61
38.12 ± 6.51
Naphthol Green B
10020
Acid Green 1
Nitroso (metalcomplex)
714
37.18 ± 7.46
30.51 ± 7.41
Direct Fast Blue FBL 74190
Direct Blue 199
Phthalocyanine (metal-complex)
608
56.28 ± 6.81
48.81 ± 6.91
(continued on next page)
72
F. Zheng et al. / International Biodeterioration & Biodegradation 110 (2016) 69e78
Table 1 (continued ) Dye
Crystal Violet
Color index number
Color index Chemical class name
42555
Basic Violet Triphenylmethane 3
Structure
Glutaraldehyde was used as the crosslinker for laccase immobilization. Chitosan beads (5.0 g) obtained above were mixed with 5.0 mL of glutaraldehyde solution and crosslinked for 3 h after oscillation for 5 min at room temperature. Afterwards, the crosslinked chitosan beads were washed several times with deionized water to remove excess glutaraldehyde. Tplac was immobilized onto glutaraldehyde-crosslinked chitosan beads at 1 g:1 mL at room temperature with shaking speed at 150 rpm. After a 6-h immobilization, the beads were washed with deionized water, dried at room temperature to constant weight, and stored in dry conditions until use. Control experiments were performed with incubation of free enzyme under the same conditions to quantify enzyme denaturation during the immobilization process.
2.4. Influences of conditions on immobilization The influences of glutaraldehyde concentration (0.1e1.0%, v/v), crosslinking time (1e10 h), enzyme volume (1.0e8.0 mL), and immobilization time (2e14 h) on the relative activity of immobilized Tplac were investigated. Experiments were all performed in triplicate. Laccase activity at an optimum condition was defined as 100%.
Free and immobilized Tplac were kept in storage for 30 days, and the laccase activities were determined on days 3, 5, 7, 10, 20, and 30. Experiments were all performed in triplicate, and laccase activity at the optimum pH and temperature on day 0 was defined as 100%.
Immobilized laccase
Free laccase
595
20.81 ± 6.95
14.45 ± 7.16
Immobilized laccase was reused for 14 cycles and the residual activity was measured with respect to the initial one. After each cycle, the immobilized enzyme was filtered, washed with 0.1 M citrate-phosphate buffer (pH 5.0), and reintroduced into fresh reaction medium. Experiments were all performed in triplicate, and laccase activity at the optimum pH and temperature in the first cycle was defined as 100%. 2.8. Dye decolorization capacity of immobilized Tplac The decolorization capacity of various structurally distinct dyes by immobilized Tplac was monitored by the decrease in absorbance at the maximum absorption wavelength of each dye (Table 1). The 10-mL reaction mixtures for dye decolorization contained 1.0 U/mL pure enzyme solution and 50.0 mg/L dye (Reactive Brilliant Blue XBR, Remazol Brilliant Blue R, Congo Red, Acid Black 172, Methylene Blue, Neutral Red, Indigo Blue, Naphthol Green B, Direct Fast Blue FBL, and Crystal Violet) in 0.1 M citrate-phosphate buffer (pH 5.0). The mixtures were incubated in a dark chamber at 50 C with shaking speed at 150 rpm. The negative control contained all components except the enzyme solution. Experiments were all performed in triplicate. Decolorization efficiency is expressed in terms of percentage and calculated as
%Decolorization ¼
2.6. Storage stability of immobilized Tplac
% Decolorization
2.7. Reusability of immobilized Tplac
2.5. pH and thermal stability of immobilized Tplac The influence of pH on Tplac activity was determined in citratephosphate buffer within the pH range 1.0e13.0 at 25 C using 1.0 mM ABTS as substrate. The pH stability of free or immobilized laccase was assessed by pre-incubating the enzyme in buffer at 25 C for 72 h; then the residual laccase activity was determined. The optimum temperature for Tplac was examined in citratephosphate buffer from 10 to 90 C at the optimum pH using 1.0 mM ABTS as the substrate. The thermal stability of free or immobilized laccase was evaluated by pre-incubating the enzyme in buffer (optimum pH) at different temperatures from 10 to 90 C for 2 h; then the residual laccase activity was determined. Aliquots of samples of 5.0 mL were taken at regular intervals and centrifuged at 12,000 rpm for 20 min; the supernatant was then used for laccase activity determination. Experiments were all performed in triplicate. Laccase activity at the optimum pH or temperature was defined as 100%.
Maximum adsorption wavelength (nm)
Ai Ao 100% Ai
where Ai is the initial absorbance of the synthetic dye, and Ao is the final absorbance of the dye after decolorization. 2.9. Identification of degradation metabolites After decolorization of Acid Black 172, the metabolites formed were extracted three times with an equal volume of ethyl acetate, with vigorous shaking. The combined organic phase was filtered over Na2SO4 on filter paper and concentrated in a rotary vacuum evaporator. GCeMS analysis was carried out using a QP 2010 mass spectrophotometer (Shimadzu model No. U-2800). The ionization voltage was 70 eV and the temperature of the injection port was 280 C. GC was conducted in temperature programming mode with a Resteck column (0.25 30 nm, XTI-5). The initial column temperature was 80 C for 2 min, which was then increased to 280 C linearly at 10 C per min and held for 7 min. The GCeMS interface was maintained at 290 C and helium was used as the carrier gas at a flow rate of 1.0 mL/min with a 30 min run time. 2.10. Statistical analysis Data were subjected to statistical analysis by one-way analysis of variance and the TukeyeKramer comparison test using SPSS18.0
F. Zheng et al. / International Biodeterioration & Biodegradation 110 (2016) 69e78
software. P values < 0.05 (denoted by *) and <0.01 (**) were considered significant and highly significant respectively. 3. Results and discussion 3.1. Influences of conditions on immobilization 3.1.1. Glutaraldehyde concentration The effectiveness of immobilization of laccase is mainly dependent on the crosslinker. Within a certain concentration range, the crosslinker can enhance the extent of crosslinking, thus stimulating laccase immobilization. However, a higher concentration of crosslinker may lead to a decline in immobilized laccase activity, due to enzyme denaturation, obstruction of active sites of the enzyme by crosslinker structure, and/or an insufficient amount of laccase immobilization (Zhang et al., 2009a; Sathishkumar et al., 2014). The data of relative laccase activity versus crosslinker concentration are presented in Fig. 2A. The relative activity of immobilized T. pubescens laccase Tplac increased with increasing concentration of glutaraldehyde from 0.1 to 0.8% (v/v), and the maximum relative laccase activity was obtained with the use of 0.8% glutaraldehyde. Afterwards, the laccase activity decreased sharply as the glutaraldehyde concentration increased from 0.8 to
73
1.0% (v/v). Better laccase activity observed at relatively moderate crosslinker concentrations can be explained by the attractive forces between the glutaraldehyde and the enzyme molecules. At a certain concentration, the glutaraldehyde can adequately react with the enzyme to form a Schiff base, so that more enzyme molecules are attached to the support with the glutaraldehyde acting as a bridge (Songulashvili et al., 2012; Barbosa et al., 2014). As the glutaraldehyde concentration increases further, crosslinking reactions often cause conformational changes of enzyme molecules, resulting in damage to the active site, thus inhibiting the immo^ssi et al., 2014). According to the data in bilization efficiency (Daa Fig. 2A, from hereon, immobilization was carried out using a crosslinker glutaraldehyde concentration of 0.8% (v/v). 3.1.2. Crosslinking time Crosslinking time also significantly influences the immobilization process. As depicted in Fig. 2B, the maximum relative activity of immobilized T. pubescens laccase Tplac was obtained after crosslinking for 3 h, indicating that this reaction time is suitable for adsorption of enzyme molecules onto chitosan beads in grid number and size (Sathishkumar et al., 2014). The spatial grid structure of the support increases with the extended period, leading to restricted conditions for more enzyme molecules to
Fig. 2. Influences of (A) glutaraldehyde concentration, (B) crosslinking time, (C) enzyme volume, and (D) immobilization time on the activity of immobilized laccase Tplac from white rot fungus Trametes pubescens. Each value is the mean value ± standard error mean of triplicate.
74
F. Zheng et al. / International Biodeterioration & Biodegradation 110 (2016) 69e78
access (Kaspar et al., 2013). Hence, an appropriate crosslinking time is crucial for maintaining high laccase activity after immobilization.
3.1.3. Enzyme volume Results on the influence of enzyme volume on the relative activity of immobilized T. pubescens laccase Tplac are given in Fig. 2C. Better immobilization efficiency of laccase Tplac was obtained using 2.0 mL of enzyme solution, corresponding to 43.672 U/mL of laccase activity, which could be attributed to the equilibrium reached by the dissociative amino and aldehyde groups on Tplac and the support respectively. A lower amount of enzyme could not couple as effectively with the support. On the contrary, an excess of enzyme could lead to overloading of the support, thereby resulting ^ssi et al., 2014; Sathishkumar in a decrease in laccase activity (Daa et al., 2014).
3.1.4. Immobilization time Immobilization time is another key variable during the enzyme immobilization process. Too short an immobilization time means that most of the laccase molecules do not have enough time to penetrate the grid structure of the support, while too long an immobilization time leads to laccase molecules on the support being too crowded to make optimal contact with their substrate, resulting in a decline in enzyme activity (Halder et al., 2014). Additionally, if too much laccase is bound to the support, rising temperature as a result of chemical reaction catalyzed by closepacked laccase molecules is another reason for reduction of laccase activity (Xu et al., 2013). As can be seen from Fig. 2D, an apparent peak in the relative activity of T. pubescens laccase Tplac was detected after immobilization for 4 h, demonstrating that the appropriate enzyme molecules have access to the interior structure of the support for laccase immobilization (Sathishkumar et al., 2014). As the immobilization time increased, the quantity of enzyme adsorbed onto the support increased, and was influenced by the external environment more easily; therefore, the activity of the immobilized laccase decreased significantly (Halder et al., 2014). When the effect of the volume of enzyme attached was greater than the influence of the environment on enzyme activity, the immobilized laccase activity gradually increased again, and thus a small, second peak was found after 12 h of immobilization (Fig. 2D), after which the activity decreased again.
3.2. pH dependence of activity and pH stability of immobilized Tplac In Fig. 3A it is shown that immobilized laccase Tplac exhibited activity over a broad pH range from 3.5 to 12.0, with the optimum pH at 5.0, 0.5 pH units higher than that of the free enzyme (optimum pH 4.5). These observations suggested that immobilization treatment of the laccase improves its adaptive capacity to an alkaline environment, and broadens the pH range for catalysis (Catapane et al., 2013). As pH increased, declining trends were observed in the relative activities of both free and immobilized Tplac, but less for the latter than for the former with respect to the degree and speed of decline, implying that immobilized Tplac is more resistant to alkali conditions. This might be due to that the inner surface of the chitosan support contains relatively abundant negatively charged amino groups, and thus it could act as a buffer, causing the pH in proximity to the enzyme molecules to be higher than in the surrounding liquid, providing a relatively favorable microenvironment for catalysis. Overall, the immobilized laccase suffers less interference from the external environment than free laccase, and possesses higher tolerance to acidic and alkali conditions, and thus wider applicability in catalysis (Yan et al., 2014). The microenvironment has a remarkable influence on the catalytic activity of enzymes, especially in extreme conditions that may generally cause enzyme inactivation. In this study, the pH stabilities of free and immobilized laccase Tplac from T. pubescens were also investigated. Enzyme was incubated at various pH values for an extended period and the activity was then determined. As displayed in Fig. 3B, free Tplac was very stable over a broad pH range, from 4.5 to 9.0, maintaining more than 75% of its original activity after incubation at 25 C for 72 h. While the relative activity of immobilized T. pubescens Tplac was >80% in the same conditions. A sharp decline in relative laccase activity was observed when the pH was <4.0 or >11.0. Demarche et al. (2012) explicated that the main reason why the pH stability of laccase changes after immobilization is proton production by the support. The electronwithdrawing effect of C¼O groups on the support surface could stimulate eCH2 groups to more easily release Hþ, resulting in a pH in the support microenvironment lower than that in the overall system, thus altering the pH stability range of the immobilized laccase. In contrast to free Tplac, immobilized Tplac is stable at higher pHs, which improved its values in practical applications.
Fig. 3. pH stability of immobilized and free laccase Tplac from white rot fungus Trametes pubescens. (A) activity assay at the given pH at 25 C; (B) activity assay after incubation at the given pH for 72 h at 25 C. Each value is the mean value ± standard error mean of triplicate.
F. Zheng et al. / International Biodeterioration & Biodegradation 110 (2016) 69e78
75
3.3. Thermal stability of immobilized Tplac The adaptability of laccase from various sources to temperature differs (Santos et al., 2007; Wu and Nian, 2014). Results in Fig. 4A showed that immobilized Tplac performed its maximal activity at 60 C; the activity increased with temperature up to this point. It was suggested that the immobilization process notably enhances Tplac's thermal tolerance. This may be assigned to that the characteristic embedding structure of the support effectively protects the laccase, and affords a stable microenvironment for it (Zhang et al., 2009b). Furthermore, after immobilization, interactions among enzyme molecules and between the enzyme and substrate and the enzyme and the support enhance the rigidity of the molecular structure of the enzyme, thereby reinforcing its capability to resist thermal denaturation. In contrast, free enzymes are more easily denatured (Yang et al., 2006; Nair et al., 2013). After pre-incubation at 60e70 C for 2 h, the relative activity of immobilized Tplac remained >50%, while the free laccase activity decreased by 50% from the level of 72.31%, as presented in Fig. 4B, which revealed that after an incubation period at elevated temperature, immobilized laccase is more stable than free laccase (Addorisio et al., 2013). At tested temperatures above 60 C, and particularly above 75 C, the activities of both free and immobilized Tplac decreased rapidly. Nevertheless, both the rate and degree of decline of the activity of the immobilized laccase were much lower than those of free laccase. Through attachment to an appropriate support, the structural rigidity and thermal resistance of the enzyme molecules can be strengthen obviously (Nair et al., 2013). Hence, at the same temperature, relatively poor durability of free ^ssi et al., laccase is detected compared to immobilized enzyme (Daa 2014).
3.4. Storage stability of immobilized Tplac Storage stability is one of the most important parameters by which to evaluate performance because immobilized enzymes are susceptible to decreased activity after prolonged storage ndez-Fern (Ferna andez et al., 2013). As depicted in Fig. 5, downward trends were presented for the activities of both free and immobilized Tplac on storage for up to one month. Apparently, the decrease in activity of immobilized T. pubescens laccase was smaller in terms of both amplitude and rate. After 10 days, the remaining
Fig. 5. Storage stability of immobilized and free laccase Tplac from white rot fungus Trametes pubescens. Each value is the mean value ± standard error mean of triplicate.
relative activity of immobilized Tplac was above 50%; after 30 days, it was above 40%, whereas the activity of free laccase lost approximately 85% of its initial activity over the same period. These results indicated that, in terms of storage stability, immobilized Tplac is significantly superior to free laccase, which provides the possibility of long-term preservation for further use (Nair et al., 2013).
3.5. Reusability of immobilized Tplac The feasibility of regeneration of immobilized laccase for repeated use is one of the most important indicators for reducing the overall cost of enzymatic applications, especially in industry ndez-Ferna ndez et al., 2013). According to the data on the (Ferna reusability of immobilized laccase Tplac (Fig. 6), after three cycles of continuous use, the relative activity of immobilized Tplac was >90%, and it remained 60% after six cycles. The relative activity of immobilized T. pubescens laccase decreased with increased use, dropping to around 20% of its original activity after 14 cycles at room temperature. This decrease in enzyme activity could be ascribed to inactivation and loss of enzyme molecules during each
Fig. 4. Thermal stability of immobilized and free laccase Tplac from white rot fungus Trametes pubescens. (A) activity assay at the given temperature at the optimum pH; (B) activity assay after incubation at the given temperature for 2 h at the optimum pH. Each value is the mean value ± standard error mean of triplicate.
76
F. Zheng et al. / International Biodeterioration & Biodegradation 110 (2016) 69e78
dyes were decolorized to various extents in the absence of redox mediators, and the removal efficiency of the dyes depended on their different chemical structures. Surprisingly, the immobilized Tplac exerted higher catalytic efficiency toward all the tested dyes. Among them, better decolorization of metal-complex dye Acid Black 172 (decolorization efficiency 68.84%) was observed after reaction for 48 h, which was 1.22-fold higher than that by free laccase. These data implied that dye affinity is different for different enzymatic complexes, underlining the influence of immobilization on the laccase's behavior (Rodríguez-Couto, 2014). 3.7. Identification of degradation metabolites
Fig. 6. Reusability of immobilized laccase Tplac from white rot fungus Trametes pubescens. Each value is the mean value ± standard error mean of triplicate.
cycle (Sadighi and Faramarzi, 2013). 3.6. Dye decolorization capacity of immobilized Tplac The decolorization capacity of immobilized laccase Tplac was assessed using various dyes (Table 1). In general, the catalytic efficiency of an enzyme may be altered after immobilization treatment. To confirm it, dye degradation by immobilized laccase was compared with that by free laccase. It is shown in Table 1 that the
GCeMS analysis is a versatile technique to identify and quantify chemical compounds in complex forms (Schauer et al., 2005). As displayed in Table 2, the degradation metabolites of metal-complex dye Acid Black 172 were identified by organic solvent and GCeMS analysis. Based on the above data, possible partial pathways of biodegradation of Acid Black 172 by immobilized T. pubescens laccase Tplac were proposed (Fig. 7), in which 2-Nitronaphthalene (III), 1-Naphthalene diazonium (VII), 6-Nitro-2-naphthol (VIII), and 2-Naphthol (IX) are the main degradation products of Acid Black 172. Remarkably, 1-Amino-6-nitro-2-naphthol-4-sulfonate (I), 1-Diazonium-2-naphthol (IV), 6-Nitro-2-naphthol-4-sulfonate (V), and 1-Amino-2-naphthol (VI) could not be detected after 72 h suggesting that these intermediates might be eventually transferred into stable compounds by desulfonation, deamination, and dehydroxylation reactions. However, the intermediate II was not found. The possible reason should be that most of it was rapidly degraded to small molecules (Tan et al., 2013). It is clear that the first step of biodegradation in the present study is initiated by
Table 2 Degradation metabolites of metal-complex dye Acid Black 175 obtained from GCeMS analysis. No.
Retention time (min)
Major fragment m/z
Calculated mass
Compound
I
18.245
284
284
1-Amino-6-nitro-2-naphthol-4-sulfonate
III
17.268
173
173
2-Nitronaphthalene
IV
17.648
172
172
1-Diazonium-2-naphthol
V
16.441
269
269
6-Nitro-2-naphthol-4-sulfonate
VI
14.584
159
159
1-Amino-2-naphthol
VII
20.452
156
156
1-Naphthalene diazonium
VIII
22.474
189
189
6-Nitro-2-naphthol
IX
19.334
144
144
2-Naphthol
Chemical structure
F. Zheng et al. / International Biodeterioration & Biodegradation 110 (2016) 69e78
77
Na O O
+
Na
ON
S
ONO2
Cr3+
N
O
N N
O2N OO
Acid Black 172
O-
S O O
Na
Asymmetric cleavage by chitosan-immobilized laccase Decolorization
Na O
O S
ON
O
Cr3+
N NO2
NH2
O2N O-
HO NH2
I
O
Oxidative cleavage
Na
Desulfonation
HO NH
N
NH2 OH
NO2
OH
Deamination
IV Dehydroxylation
II
S O O
1-Amino-6-nitro-2-naphthol-4-sulfonate
O-
O
1-Diazonium-2-naphthol
S O
O
VI
V
1-Amino-2-naphthol
Na
6-Nitro-2-naphthol-4-sulfonate NO2
Deamination
Dehydroxylation
III Desulfonation
2-Nitronaphthalene N
OH
NH
IX 2-Naphthol VII
HO
1-Naphthalene diazonium
NO2
VIII
6-Nitro-2-naphthol Fig. 7. Proposed pathways of biodegradation of metal-complex dye Acid Black 172 by immobilized laccase Tplac from white rot fungus Trametes pubescens.
asymmetric reduction of azo groups (N¼N), which requires electrons and protons for the reduction process (Nam and Renganathan, 2000; Du et al., 2013). Laccase, a class of multicopper oxidase containing four histidine-rich copper binding domains at the reaction center, is capable of using molecular oxygen as a co-substrate to oxidize an array of compounds by a radicalcatalyzed reaction mechanism (Thurston, 1994). Therefore, the enzyme can donate electrons to azo dye that is able to accept electrons via a nonspecific interaction mechanism, resulting in production of colorless solution (Jadhav et al., 2008; Tahmasbi et al., 2016). Obviously, the proposed partial pathways were only dependent on GCeMS analysis results and relevant literature. It still needs to be investigated systematically.
using the crosslinker glutaraldehyde. Optimization experiments indicated that the stability was dependent on the concentration of crosslinker, crosslinking time, volume of enzyme, and immobilization time. Moreover, the immobilized laccase was less sensitive to changes in pH and temperature, and to storage time, compared with free laccase, thereby exhibiting better stability and reusability. Catalytic performance of immobilized Tplac was evaluated by degradation of various synthetic dyes, showing that the removal efficiency of metal-complex dye Acid Black 172 by immobilized Tplac (68.84%) was 1.22 times higher than that by free laccase. These results suggested that immobilization is feasible to improve the conformational stability of laccase for many industrial applications.
4. Conclusions
Acknowledgments
The immobilized laccase Tplac from white rot fungus T. pubescens was fabricated by entrapment onto chitosan beads
We express our gratitude to Prof. Yu-Cheng Dai (Beijing Forestry University, China) and Dr. Hai-Jiao Li (Chinese Center for Disease
78
F. Zheng et al. / International Biodeterioration & Biodegradation 110 (2016) 69e78
Control and Prevention, China) for improving the manuscript. This study was supported by the Fundamental Research Funds for the Central Universities (No. BLX2014-36) and the National Natural Science Foundation of China (No. 31422001). References Addorisio, V., Sannino, F., Mateo, C., Guisan, J.M., 2013. Oxidation of phenyl compounds using strongly stable immobilized-stabilized laccase from Trametes versicolor. Process Biochem. 48, 1174e1180. Adnan, L.A., Sathishkumar, P., Yusoff, A.R., Hadibarata, T., 2015. Metabolites characterisation of laccase mediated Reactive Black 5 biodegradation by fast growing ascomycete fungus Trichoderma atroviride F03. Int. Biodeterior. Biodegrad. 104, 274e282. Arora, D.S., Chander, M., Gill, P.K., 2002. Involvement of lignin peroxidase, manganese peroxidase and laccase in degradation and selective ligninolysis of wheat straw. Int. Biodeterior. Biodegrad. 50, 115e120. Torres, R., Rodrigues, R.C., FernandezBarbosa, O., Ortiz, C., Berenguer-Murcia, A., Lafuente, R., 2014. Glutaraldehyde in bio-catalysts design: a useful crosslinker and a versatile tool in enzyme immobilization. RSC Adv. 4, 1583e1600. Basak, B., Bhunia, B., Dey, A., 2014. Studies on the potential use of sugarcane bagasse as carrier matrix for immobilization of Candida tropicalis PHB5 for phenol biodegradation. Int. Biodeterior. Biodegrad. 93, 107e117. Catapane, M., Nicolucci, C., Menale, C., Mita, L., Rossi, S., Mita, D.G., Diano, N., 2013. Enzymatic removal of estrogenic activity of nonylphenol and octylphenol aqueous solutions by immobilized laccase from Trametes versicolor. J. Hazard. Mater. 248249, 337e346. ^ssi, D., Rodríguez-Couto, S., Nasri, M., Mechichi, T., 2014. Biodegradation of Daa textile dyes by immobilized laccase from Coriolopsis gallica into Ca-alginate beads. Int. Biodeterior. Biodegrad. 90, 71e78. Demarche, P., Junghanns, C., Mazy, N., Agathos, S.N., 2012. Design-of-experiment strategy for the formulation of laccase biocatalysts and their application to degrade bisphenol A. New Biotechnol. 30, 96e103. Du, L.N., Zhao, M., Li, G., Xu, F.C., Chen, W.H., Zhao, Y.H., 2013. Biodegradation of malachite green by Micrococcus sp. strain BD15: biodegradation pathway and enzyme analysis. Int. Biodeterior. Biodegrad. 78, 108e116. Dur an, N., Rosa, M.A., D'Annibale, A., Gianfreda, L., 2002. Applications of laccases and tyrosinases (phenoloxidases) immobilized on different supports: a review. Enzyme Microb. Technol. 31, 907e931. Moldes, D., 2013. Recent developments ndez-Fern Ferna andez, M., Sanrom an, M.A., and applications of immobilized laccase. Biotechnol. Adv. 31, 1808e1825. Halder, S.K., Maity, C., Jana, A., Ghosh, K., Das, A., Paul, T., das Mohapatra, P.K., Pati, B.R., Mondal, K.C., 2014. Chitinases biosynthesis by immobilized Aeromonas hydrophila SBK1 by prawn shells valorization and application of enzyme cocktail for fungal protoplast preparation. J. Biosci. Bioeng. 117, 170e177. Hou, J., Dong, G., Ye, Y., Chen, V., 2014. Laccase immobilization on titania nanoparticles and titania-functionalized membranes. J. Membr. Sci. 452, 229e240. Jadhav, S.U., Kalme, S.D., Govindwar, S.P., 2008. Biodegradation of methyl red by Galactomyces geotrichum MTCC 1360. Int. Biodeterior. Biodegrad. 62, 135e142. Jiang, D.S., Long, S.Y., Huang, J., Xiao, H.Y., Zhou, J.Y., 2005. Immobilization of Pycnoporus sanguineus laccase on magnetic chitosan microspheres. Biochem. Eng. J. 25, 15e23. Kalyani, D.C., Patil, P.S., Jadhav, J.P., Govindwar, S.P., 2008. Biodegradation of reactive textile dye Red BL1 by an isolated bacterium Pseudomonas sp. SUK1. Bioresour. Technol. 99, 4635e4641. e rov pa nek, F., 2013. Effect of Kaspar, O., Toka a, V., Nyanhongo, G.S., Gübitz, G., St cross-linking method on the activity of spray-dried chitosan microparticles with immobilized laccase. Food Bioprod. Process. 91, 525e533. Kuhar, F., Castiglia, V., Levin, L., 2015. Enhancement of laccase production and malachite green decolorization by co-culturing Ganoderma lucidum and Trametes versicolor in solid-state fermentation. Int. Biodeterior. Biodegrad. 104, 238e243. Migneault, I., Dartiguenave, C., Bertrand, M.J., Waldron, K.C., 2004. Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking. BioTechniques 37, 790e802. Nair, R.R., Demarche, P., Agathos, S.N., 2013. Formulation and characterization of an immobilized laccase biocatalyst and its application to eliminate organic micropollutants in wastewater. New Biotechnol. 30, 814e823. Nam, S., Renganathan, V., 2000. Non-enzymatic reduction of azo dyes by NADH. Chemosphere 40, 351e357. Nidheesh, T., Kumar, P.G., Suresh, P.V., 2015. Enzymatic degradation of chitosan and production of D-glucosamine by solid substrate fermentation of exo-b-D-glucosaminidase (exochitosanase) by Penicillium decumbens CFRNT15. Int. Biodeterior. Biodegrad. 97, 97e106. Palvannan, T., Saravanakumar, T., Unnithan, A.R., Chung, N.J., Kim, D.H., Park, S.M.,
2014. Efficient transformation of phenyl urea herbicide chloroxuron by laccase immobilized on zein polyurethane nanofiber. J. Mol. Catal. B Enzym. 99, 156e162. Rahmani, K., Faramarzi, M.A., Mahvi, A.H., Gholami, M., Esrafili, A., Forootanfar, H., Farzadkia, M., 2015. Elimination and detoxification of sulfathiazole and sulfamethoxazole assisted by laccase immobilized on porous silica beads. Int. Biodeterior. Biodegrad. 97, 107e114. Rodríguez-Couto, S., 2014. Decolouration of industrial metal-complex dyes in successive batches by active cultures of Trametes pubescens. Biotechnol. Rep. 4, 156e160. Sadighi, A., Faramarzi, M.A., 2013. Congo red decolorization by immobilized laccase through chitosan nanoparticles on the glass beads. J. Taiwan Inst. Chem. Eng. 44, 156e162. Santos, A.M.P., Oliveira, M.G., Maugeri, F., 2007. Modelling thermal stability and activity of free and immobilized enzymes as a novel tool for enzyme reactor design. Bioresour. Technol. 98, 3142e3148. Sathishkumar, P., Kamala-Kannan, S., Cho, M., Kim, J.S., Hadibarata, T., Salim, M.R., Oh, B.T., 2014. Laccase immobilization on cellulose nanofiber: the catalytic efficiency and recyclic application for simulated dye effluent treatment. J. Mol. Catal. B Enzym. 100, 111e120. Schauer, N., Steinhauser, D., Strelkov, S., 2005. GC-MS libraries for the rapid identification of metabolites in complex biological samples. FEBS Lett. 579, 1332e1337. Si, J., Peng, F., Cui, B.K., 2013a. Purification, biochemical characterization and dye decolorization capacity of an alkali-resistant and metal-tolerant laccase from Trametes pubescens. Bioresour. Technol. 128, 49e57. Si, J., Cui, B.K., Dai, Y.C., 2013b. Decolorization of chemically different dyes by whiterot fungi in submerged cultures. Ann. Microbiol. 63, 1099e1108. Singh, T., Vesentini, D., Singh, A.P., Daniel, G., 2008. Effect of chitosan on physiological, morphological, and ultrastructural characteristics of wood-degrading fungi. Int. Biodeterior. Biodegrad. 62, 116e124. z-Tobo n, G.A., Jaspers, C., Penninckx, M.J., 2012. ImmobiSongulashvili, G., Jimene lized laccase of Cerrena unicolor for elimination of endocrine disruptor micropollutants. Fungal Biol. 116, 883e889. Spinelli, D., Fatarella, E., di Michele, A., Pogni, R., 2013. Immobilization of fungal (Trametes versicolor) laccase onto Amberlite IR-120 H beads: optimization and characterization. Process Chem. 48, 218e223. Sun, H., Yang, H., Huang, W., Zhang, S., 2015. Immobilization of laccase in a spongelike hydrogel for enhanced durability in enzymatic degradation of dye pollutants. J. Colloid Interface Sci. 450, 353e360. Tahmasbi, H., Khoshayand, M.R., Bozorgi-Koushalshahi, M., Heidary, M., GhaziKhansari, M., Faramarzi, M.A., 2016. Biocatalytic conversion and detoxification of imipramine by the laccase-mediated system. Int. Biodeterior. Biodegrad. 108, 1e8. Tan, L., Ning, S., Zhang, X., Shi, S., 2013. Aerobic decolorization and degradation of azo dyes by growing cells of a newly isolated yeast Candida tropicalis TL-F1. Bioresour. Technol. 138, 307e313. Thurston, C.F., 1994. The structure and function of fungal laccase. Microbiology 140, 19e26. v.: inWangpradit, R., Chitprasert, P., 2014. Chitosan-coated Lentinus polychrous Le tegrated biosorption and biodegradation systems for decolorization of anionic reactive dyes. Int. Biodeterior. Biodegrad. 93, 168e176. Wu, Y.R., Nian, D.L., 2014. Production optimization and molecular structure characterization of a newly isolated novel laccase from Fusarium solani MAS2, an anthracene-degrading fungus. Int. Biodeterior. Biodegrad. 86, 382e389. Xu, P., Zeng, G., Huang, D., Hu, S., Feng, C., Lai, C., Zhao, M., Huang, C., Li, N., Wei, Z., Xie, G., 2013. Synthesis of iron oxide nanoparticles and their application in Phanerochaete chrysosporium immobilization for Pb(II) removal. Colloids Surfaces A Physicochem. Eng. Aspects 419, 147e155. Yan, J., Chen, D., Yang, E., Niu, J., Chen, Y., Chagan, I., 2014. Purification and characterization of a thermotolerant laccase isoform in Trametes trogii strain and its potential in dye decolorization. Int. Biodeterior. Biodegrad. 93, 186e194. Yang, W.Y., Min, D.Y., Wen, S.X., Jin, L., Rong, L., Tetsuo, M., Bo, C., 2006. Immobilization and characterization of laccase from Chinese Rhus vernicifera on modified chitosan. Process Biochem. 41, 1378e1382. Yavuz, M., Kaya, G., Aytekin, Ç., 2014. Using Ceriporiopsis subvermispora CZ-3 laccase for indigo carmine decolourization and denim bleaching. Int. Biodeterior. Biodegrad. 88, 199e205. Zhang, J., Xu, Z., Chen, H., Zong, Y., 2009a. Removal of 2,4-dichlorophenol by chitosan-immobilized laccase from Coriolus versicolor. Biochem. Eng. J. 45, 54e59. Zhang, Y.W., Zhang, Y., Wang, H., Yan, B.N., Shen, G.L., Yu, R.Q., 2009b. An enzyme immobilization platform for biosensor designs of direct electrochemistry using flower-like ZnO crystals and nano-sized gold particles. J. Electroanal. Chem. 627, 9e14.