Catalytic potential of laccase immobilized on transition metal oxides nanomaterials: Degradation of alizarin red S dye

Accepted Manuscript Title: Catalytic potential of laccase immobilized on transition metal oxides nanomaterials: Degradation of alizarin red S dye Authors: Manviri Rani, Uma Shanker, Amit K. Chaurasia PII: DOI: Reference:

S2213-3437(17)30216-6 http://dx.doi.org/doi:10.1016/j.jece.2017.05.026 JECE 1629

To appear in: Received date: Revised date: Accepted date:

20-1-2017 20-4-2017 18-5-2017

Please cite this article as: Manviri Rani, Uma Shanker, Amit K.Chaurasia, Catalytic potential of laccase immobilized on transition metal oxides nanomaterials: Degradation of alizarin red S dye, Journal of Environmental Chemical Engineeringhttp://dx.doi.org/10.1016/j.jece.2017.05.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Highlights 

A comprehensive study on catalytic application of immobilized laccase on hazardous dye



Successful immobilization of laccase on green synthesized ZnO and MnO2 nanomaterial



Activity and stability: lac-ZnO>lac-MnO2>free lac



Lac-ZnO as potential catalyst for ARS due to enhanced enzyme loading



Quick, green approach and mineralization of ARS dye in water

Catalytic potential of laccase immobilized on transition metal oxides nanomaterials: Degradation of alizarin red S dye Manviri Rani, Uma Shanker, Amit K. Chaurasia

*

Department of Chemistry Dr B R Ambedkar National Institute of Technology Jalandhar-144011, Punjab, India

* Corresponding Author Dr Uma Shanker (Assistant Professor) Office Number-CE-306 Department of Chemistry Dr B R Ambedkar National Institute of Technology Jalandhar, Jalandhar, GT Road bypass, Jalandhar-144011, Punjab, India Email: [email protected], [email protected] Contact number: +91- 7837-588-168 (Mobile) +91-0181-269-301-2258 (Office) Fax: +91-0181-269-0932

Abstract Recently, nanoparticle-based immobilization of biocatalytic systems is getting interested in bioremediation efficiency. Therefore, green synthesized ZnO (<50 nm) and MnO2 (<10 nm) nanoparticles were chelated with Cu2+ to immobilize laccase (Lac) through metal affinity adsorption. Nanospheres (~300 nm) of lac-ZnO and nanoclusters of lac-MnO2(<50 nm) were confirmed by SEM. Lac was strongly immobilized on ZnO followed by MnO2 and their activities were doubled than free lac prepared by solid state fermentation. Further, catalytic potential of lac-ZnO and lac-MnO2 was examined for in vitro degradation of alizarin red S dye in simulatedwater. Degradation of dye was highest with lac-ZnO (95%) followed by lac-MnO2 (85%) and free lac (49%) at initial pH (~7.0), dye (20 mg/L) and catalyst (50 mg). Control experiment indicated that lac-ZnO was the better catalyst than ZnO and free lac. MnO2 seemed to enhance stability and activity of lac- MnO2 over free lac. Moreover, a combination of nanomaterial and enzyme is required for achieving biocompatibility and inert condition without denaturing of the enzyme. Overall, ZnO and MnO2 are potential for large-scale lac immobilization with improved properties and reuse. Lac-ZnO and lac-MnO2 may be used as important adsorbents in waste water treatment with a bright future.

Keywords: laccase immobilization, ZnO/MnO2 nanoparticles, dye removal.

1. Introduction Enzymes with different catalytic activity and selectivity offer enormous potential which further affected by their low stabilities and requires enzyme immobilization. Recently, nano-structured materials, such as carbon nanomaterials, nano-sized polymer beads, and metal nanoparticles, have been utilized as immobilization matrices for enzymes [1, 2]. They made convenient of handling of the enzyme with ease of separation, reuse, low product cost and high thermal and pH stability [3, 4]. For example, the potential of horseradish peroxidase (HRP) was enhanced after immobilization with graphene oxide (GO) [5]or single-wall carbon nanotubes [6]. Nanomaterials with unique properties are known to be potential catalysts in various commercial applications. Moreover, their combinations with enzyme provide a biocompatible and inert environment, i.e. they do not interfere with the native structure of the enzyme, and thereby, could compromise its biological activity [7]. Laccases (benzenediol: oxygenoxidoreductase; E.C. 1.10.3.2) are Cucontaining oxidase enzymes (extracellular proteins, but intracellular also). The most common laccase producers are nearly all wood-rotting fungi, such as Trametes (Versicolor, hirsute, ochracea,

villosa, gallica), Cerrena maxima, Coriolopsis polyzona, Lentinus tigrinus and

Pleurotus eryngium [8, 9]. There are essentially three possible roles of fungal laccase: pigment formation, lignin degradation and detoxification [10]. Since the nineteenth century, they are studied for catalyzes the four one-electron oxidation of electron-rich compounds (phenolic) with a simultaneous four-electron reduction of molecular dioxygen to water [11,12]and their applications in several industrial sectors [13,14]. Immobilized laccase with improved catalytic ability and stability via adsorption, entrapment, encapsulation, covalent binding and self-immobilization has been used in a wide range of

commercial applications [15]. Adsorption is a relatively simple and inexpensive method for laccase immobilization and may, therefore, have a higher commercial potential than other methodologies [16,17]. Earlier, different types of supports, such as activated carbon [18], porous glass [19], kaolinite [20], polymer beads and membranes [21, 22], magnetic chitosan [23], and polystyrene microspheres [24] were used. Nowadays, nanomaterials with unique properties are getting the attention for enzyme immobilization. Pang et al. [25] found that MWNTs and graphene are good material for high enzyme loading capacity and activity than C60 because of aggregation of the nanoparticles that reduce the available adsorption space and substrate accessibility. Immobilized laccase on bacterial nanocellulose (BNC) show the antimicrobial effects in Gram-positive (92%) and Gram-negative (26%) bacteria [26]. Due to TiO2's chemical stability, ease of functionalization, and architecture, it is highly used among transition metal oxides (TMO) for immobilization of laccase [27]. TiO2 functionalized PES (polyethersulfone) membrane showed better enzyme immobilization efficiency than the non-functionalized membrane. Improved performance of immobilized laccase on amine-functioned magnetic Fe3O4 nanoparticles modified with polyethyleneimine has been studied [28]. Laccase from Trametes versicolor immobilized onto chitosan coated magnetite nanoparticles by using reversed phase suspension adsorption retained about 71% of its initial activity at the end of its 30th repeated use [29]. However, the studies of laccase immobilized on TMO nanoparticles, especially MnO2 and ZnO have not been reported yet. TMOs are an important class of materials due to their several attractive properties such as magnetic, electronic and optical [30, 31]. Das et al. [32] carried out the degradation of chlorpyrifos using laccase immobilized magnetic iron nanoparticles. Within 3 hours of treatment at pH 3.0 and temperature 60 0C, >90% degradation was achieved. Biodegradation of more than 90% of 2,4-dinitrophenol was achieved within 12 hours using

laccase immobilized on nano-porous silica beads [33]. Immobilization of laccase onto titania nanoparticles resulted in >80% degradation of three anionic dyes, namely, direct red 31, acid blue 92 and direct green 6 within 60 min [34]. Direct immobilization of crude laccase on titania nanoparticles shows a significant potential for cost-effective practical applications such as waste water treatment [35]. Recently, authors reported the catalytic application of MnO2 and ZnO in degradation of various aromatic amines and dyes from wastewater [36, 37]. The problem of waste water containing hazardous dyes is a major issue of environmental concern. Industries such as textiles, cosmetics, paper, printing, etc., involve the use of dyes in form of their aqueous solution and discharge them untreated into the water reservoirs. This resulted in dark blue coloration of the water and affected the health of the local residents and farmers residing nearby. In addition to this, dyeing industries pose serious risks to the labors working there. High risks of tumors are identified in dye workers in Japan. Also, in the USA, deaths of the dye workers due to various cancers, lung and cerebrovascular diseases are 40 times higher than in the general population [38]. Moreover, use of reactive dyes worldwide has increased from 60,000 tons in 1988 to 178,000 tons in 2004 [39]. The clinical and immunological investigation revealed that about 15% of 400 workers exposed to reactive dyes experienced nasal and respiratory problems [40]. In order to regulate negative impact of dyes on living species, their removal using effective removal techniques are imperative. This motivated us to carry out a comprehensive study on immobilization of laccase by TMO (ZnO and MnO2) nanoparticles and verify its potential in degradation of ARS dye in simulated water. ARS (1, 2-dihydroxy-9, 10-anthra-quinonesulfonic acid sodium salt) is one of the most widely employed anthraquinone dye. It is water-soluble, carcinogenic, and resistant to

degradation because of complex aromatic structure, high thermal and optical stability [41]. ZnO and MnO2 nanoparticles used were synthesized by green methods. For maximum degradation of ARS, various process parameters such as the concentration of mixed dye solution, the dose amount of catalysts used, pH of the dye solution and contact time were optimized. Adsorption isotherm, kinetics, and thermodynamic studies were investigated in order to understand the adsorbing behavior, degradation reaction and feasibility of reaction catalyzed by immobilized enzyme. The results further enhance the importance of MO and several other nanomaterials in immobilization of enzyme as well as their catalytic efficiency in degradation of various harmful pollutants. 2. Material and Methods 2.1 Reagents All the chemicals used were of analytical grade and Millipore water was used throughout the studies. Agar, malt extract, citric acid, dextrose, gallic acid and potassium hydroxide were purchased from S. D. Fine Chem. Ltd. Mumbai, India. The white rot fungus Trametes versicolor MTCC-138 was purchased from microbial type culture collection (MTCC) Chandigarh, India and maintained on Glucose, Yeast Extract and Agar (GYEA) at 4 °C and sub-cultured every 30 days. ABTS (2,2-Azino-Bis(3-Ethylbenzene-6-sulfonic acid) diammonium salt) used as a substrate for laccase activity was purchased from AMRESCO Genetix Biotech Asia Pvt. Ltd. New Delhi, India. Zinc nitrate, potassium permanganate, manganese chloride, sodium hydroxide and alizarin red S were purchased from Merck, Ltd. Sapindus mukorossi was purchased from the local market Jalandhar, India. Triplicates of each sample (laccase production and ARS photodegradation) were prepared for calculation of the standard deviation and to check the reproducibility of the data.

2.2 Method for laccase production Laccase was produced with the method reported by Bakkiyaraj et al. [42] with some modification. It is based on solid state fermentation (SSF) using lignocellulosic material (orange peelings), basal medium as a nutrient substrate, fungus Trametes versicolor MTCC-138 and gallic acid as an inducer. The actively grown Petri plates were used to prepare spore suspension by the addition of sterile water. From this, 10 mL of spore suspension was transferred to 100 mL of GYEA medium and incubated at 25 °C and three days old culture of Trametes versicolor was used as inoculum (Fig. S1). Basal media (growth medium) composition is made of Glucose (Ddextrose; 20g/L), Peptone (bacteriological; 10g/L), Malt extracts (5g/L), and CuSO4 (0.005g/L). The basal media was inoculated with 3 ml of inoculum and incubated at 25oC on a rotary shaker at120 rpm for 11 days [43]. Orange peelings (Citrus sinensis) were chopped (2-4mm size) followed by soaking in 81.17 mM of KOH (30 mL) for 1h (to reduce the organic acid content) and washed several times with millipore water and dried at moderate temperature. A mixture of 15 g pretreated orange peelings and 50 mL of basal medium was autoclaved in 250 mL flask at 121 °C for 15 minutes and solidified (agar acts as a solidifying agent). Then three day old actively grown inoculum were transferred and culture was incubating at the constant temperature of 25°C. Inducer gallic acid (0.5 mM) was sprayed over the grown spores of Trametes versicolor fungus on third day of the fermentation. The same experiment was employed on 5 flasks namely, 3rd day, 6th day, 9th day, 11th day, 12th day, 13th day and 15th day, respectively and then analyzed the result to obtained maximum laccase production. To each culture broth (flask), 100 mL of citrate-phosphate buffer (pH 5) was added and shaken (1 hour) at 150 rpm on the shaker incubator at 4°C followed by filtration of culture suspension. Later, liquid part of the culture was centrifuged at 8000 rpm for 20 minutes at 4°C and supernatant as

such (containing laccase) was investigated for further activity test. Laccase production was found maximum at 12th day of fermentation with Laccase enzyme activity 32781 U/l with 2,2’-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and Michaelis–Menten parameter was Km and Vm was 0.381 mM and 2.24 U/mg respectively. In view of sufficient enzyme activity and limitation on research facility, the laccase as such was used for further studies. Mostly laccase are extracellular with a considerable level of stability, which generally reduce the use of advanced purification procedures [44, 45] 2.3 Assay for laccase activity Laccase activity was optimized using colorimetric method [42, 46]. In brief, 200 µL of ABTS (4.2 mM) was added to 200 µL enzyme suspensions and makeup to 700 µL by citrate buffer. The enzymatic reactions were carried out at constant temperature (25°C) in an incubator. With high laccase activity, a blue-green color appeared almost immediately which was monitored using the spectrophotometer (λmax = 420 nm). The rate of color formation is proportional to the enzyme activity where one unit of laccase activity was defined as the amount of enzyme required to oxidize 1 µmol of ABTS per minute. Enzyme activity calculation:-

The Michaelis–Menten kinetics parameter Km and Vm of the laccase were obtained by non-linear curve fitting of the plot of reaction rate verses ABTS concentration at 1, 3, 5, 7, 10, 15, 20, 25 mM (i.e., by Lineweaver -Burk plots). The reciprocal of the Michaelis-Menten equation is called Lineweaver-Burk plot. ([ ])

Where V is the reaction rate, and [S] is the substrate concentration. Vm is the maximum rate achieved by the bio-catalytic system. The Michaelis constant Km is the substrate concentration at which reaction rate is half of Vm and often is used as the indication of the enzymes affinity to the substrate.

2.4 ZnO and MnO2 nanoparticles More details about the green synthesis and characterization were reported by our research group [36, 37]. In brief, 100 mL of 0.1M aqueous NaOH solution (0.2 M KMnO4 in case of MnO2) was slowly added to equivalent volume of respective metal salt solution (0.05M of Zn(NO3)2; 0.3 M of MnCl2) containing sapindus mukorossi under continuous magnetic stirring at a molar ratio of 2:3 at room temperature. The precipitate of ZnO (white) was filtered, washed and heated at 5000 C for 4 hours. MnO2 (black) obtained was dried at 80 0C for about 12 hours. High crystalline long uniform channels of ZnO (<50 nm; nanotubes) were obtained with the unsymmetrical distribution throughout the lattice. The average particle size in case of MnO2 nanoparticles was extremely small (<10 nm). (Fig. 2a, 2c and Fig S2). Characterized metal oxides nanoparticles were used for immobilizing laccase via adsorption technique. 2.5 Immobilization of laccase on nanoparticles Immobilization of laccase enzyme on metal oxides nanoparticles was carried out by physical route i.e. adsorption (a non-covalent and reversible process). Nanoparticles of ZnO and MnO2 (50 mg) were added to 200 µL of purified laccase freshly prepared in 0.1 M citrate-phosphate buffer (pH 5.0) and the mixture is gently shaken for 1 h at 160 rpm at room temperature, as adsorption equilibrium is achieved after 30 min of incubation. For a control, 200 µL of free

enzyme was used. The samples were subsequently centrifuged for 5 min, and the supernatants were removed. Three washes with 1 mL of buffer (0.1 M, pH 7.0) were performed to remove unbound enzyme. After immobilization solution was filtered out, and dried at ambient temperature. The activity of immobilized laccase enzyme was determined with the same procedure used in free laccase. The immobilized forms were characterized using FT-IR (Fig. S3), and FE-SEM (Fig. 2) techniques and applied for photodegradation of toxic ARS dye. 2.6 Equation used for evaluating degradation parameters of ARS dye on free and immobilized laccase Adsorption equilibrium isotherm study Adsorption data was represented through Langmuir adsorption isotherms which assume the formation of a monolayer of solute molecules on the surface of the adsorbent [47, 48]. A typical graph of Ce/Xe v/s Ce of the solute was a straight line.

or (

)

Where Ce is the equilibrium concentration of the dye solution; Xe the amount of dye adsorbed per gram weight of adsorbent; Xm the amount of dye adsorbed at saturation; kL the Langmuir adsorption constant. Kinetics studies Under optimized conditions removal of ARS dye by immobilized laccase follows pseudosecond-order kinetics i.e., plot between t/qe and t was the best fit.

Where qe and qt are the removal capacity at equilibrium and at time t respectively (mg/g), C o is initial concentration, (mg/L), Ct is concentration at time t, (mg/L), V is volume of adsorbent (g) and W is amount of adsorbent (g). Degradation rate h (mg g-1 min-1) is expressed as h = K2qe2 Second order constant K2 (g mg-1 min-1). Thermodynamic studies The thermodynamic parameters (H and S) for the removal of ARS dye were calculated by determining

adsorption equilibrium constant (K; L mg−1) by the non-linear form of Liu

isotherm model [49].

Where Kg (or K) is the Liu equilibrium constant (L mg-1); nL is dimensionless exponent of the Liu equation, and Qmax is the maximum adsorption capacity of the adsorbent (mg g−1) assuming a monolayer of adsorbate uptake by the adsorbent. From the linear plot of lnK and 1/T, enthalpy and entropy of adsorption were obtained.

H (KJ mol-1) – enthalpy; S (KJ mole-1 K-1) – entropy; T - Temperature (k) and R (8.314 J mole-1 K-1) - Universal gas constant.

Parameters derived from adsorption isotherm studies, kinetic studies and thermodynamic studies were presented in Table S1. 3. Result and Discussion 3.1 Production and activity assay for free and immobilized laccase Using Trametes versicolor, orange peeling, basal medium and gallic acid by solid state fermentation at 25 oC,

laccase production is found maximum on 12th day culture broth (Fig. 1).

It is indicated by maximum laccase activity (16713 U/l) with ABTS substrate at room temperature (25oC) and pH 5 with (extinction coefficient=36000 M

-1

cm-1) after 12th-day of

fermentation. Many laccase are found to have an optimum pH in the acidic range of pH 3.0 to 6.0 [27]. Using ABTS, Michaelis-Menton parameters of free and immobilized laccase were calculated from Lineweaver-Burk plots and followed the order: laccase immobilized on ZnO (Km :2.75 mM; Vm :146 U/mg protein; activity: 32781 U/l ) > laccase immobilized on MnO2 (Km :1.23 mM; Vm :65 U/mg protein; 24812 U/ l) >free laccase (Km :0.381 mM; Vm :2.24 U/mg protein). The kinetic constants of the free and immobilized form of laccase by adsorption indicated activity of laccase was increased upon immobilization. We expected that high surface area of the nanoparticles enhanced enzyme loading, stability, and activity by increasing the available adsorption space and substrate accessibility. The Km value for the immobilized laccase on bacterial nanocellulose membranes or TiO2 functionalized PES (polyethersulfone) or MWCNT was found to be higher (more than two times) than that of the free enzyme[27,28, 50]. 3.2 Characterization of immobilized laccase on Nanoparticles 3.2.1 FT-IR Spectra (Fig S3) In laccase immobilized ZnO nanoparticles, a variable band at 2223 cm-1 found for –CN- is due to amino acids. The variable –C-C- stretching is also found as 2119 cm-1 due to alkynes group and

430 cm-1 peak is due to Zn-O stretching. A strong band at 1015 cm-1was observed for nitrile, alkyl halide, carbonyl and carboxylic acid. For laccase immobilized MnO2 nanoparticles a strong broad band at 497 and 412 cm-1 were observed for Mn-O stretching frequencies. A broad O-H stretch band was found at 3256 cm-1 due presence of alcohol or carboxylic acid groups. A variable band at 2121 cm-1 is found for variable –C=C- is due to acids. A band at 1599 cm-1 might be due to –NH- deformation or -C=C- of the amino acid group. A band at 1406 cm-1 for variable –CH and –C=C- was found due to the carboxylic acid. Therefore, FT-IR spectra revealed various functional groups such as hydroxyl, carboxylic, nitrile, alkynes, Alkyl Halide and carbonyl group present on the surface of nano-ZnO and MnO2, thus confirming immobilization. 3.2.2 FE-SEM Analysis (Fig. 2) In order to investigate morphology, orientation and elemental composition of particles (before and after immobilization) FE-SEM analysis was performed. Results reveal that there are significant changes in the morphology of the ZnO and MnO2 particles. It was observed that after immobilization, the morphology of ZnO completely changed to nanospheres (200 to 300 nm) that might be due to adsorption followed by chelation of Cu2+ of laccase to ZnO and MnO2 i.e., the formation of coordinate bonds between ZnO or MnO2 and Cu2+ may be due to metal affinity adsorption [28, 51]. Nano clusters of immobilized MnO2were formed with average size below 50 nm. Change in morphology and increased particle size clearly revealed the successfull immobilization of laccase on both ZnO and MnO2 nanomaterials with stronger absorption and high enzyme loading. The EDS (Energy Dispersive X-Ray Spectroscopy) analysis of laccase immobilized nanoparticles indicated the presence of additional peaks corresponding to ions such as C, P, K

and Na. This confirms the successful immobilization of laccase on to the nanoparticles. In immobilized laccase-ZnO; Zn, O, C, P and K showed 28.46, 38.64, 21.58, 9.19 and 2.13 weight (%) and 8.71, 48.32, 35.95, 5.94, 1.09 atomic (%), respectively. Whereas, in immobilized laccase-MnO2; Mn, O, P, K, C, and Na exhibited 53.33, 24.56, 8.39, 5.69, 6.65 and 1.39 weight (%) and 27.45, 43.43, 7.66, 4.11, 15.66 and 1.70 atomic (%), respectively.

3.3 Analysis for photodegradation studies for ARS dye using immobilized laccase The photodegradation of ARS dye using immobilized laccase on ZnO and MnO2 nanoparticles as well as free laccase (control) was carried out in simulated water. The average temperature (C) and light intensity (W/m2) were 31.1±1.7 (range: 27.0-34.0) and 452±183 W/m2 (range: 153-811).To get maximum degradation (%), concentration of ARS dye(5–25 ppm), contact time(20-60 min), catalyst dosage(10-50 mg) and initial pH (2.0–9.0), were optimized in view of the high catalytic efficiency of both forms of laccase (free and immobilized) observed under sunlight exposure. The simulated experiment was designed as follows: free and immobilized laccase (10-50 mg) were added to 10 mL of ARS solution (5-25 mg/L). To carry out the control experiments ZnO and MnO2 (10-50 mg) were individually added to ARS solution (5-25 mg/L). All the samples were kept under bright sunlight. At different time intervals (20, 40 and 60 minutes), 2 ml of the aliquots was centrifuged at 10,000 rpm for 10 min and absorbance is measured and converted into concentration using calibration graph. % degradation of ARS dye was calculated as follows:-

3.3.1 Effect of Initial pH

pH of the solution is the most important parameter for the photocatalytic efficiency. In each case, dye removal increased with the raising pH up to 7.0, after that it got decreased (Fig. 3a). At high pH values, the activity of the enzyme decreases when the hydroxide anion binds to the T2/T3 copper center of laccase, which interrupts the transfer of the electron from T1 to the T2/T3 center. The rate of oxidation and the reaction products can differ based on the reaction pH and the substrate used to assay the enzyme [44]. Maximum decrease at neutral pH may be due to the ion screening effects of H+ ions under acidic conditions and OH- ions in the alkaline medium which may have the adverse impact on the adsorption and hence less dye decoloration was observed [52]. The degradation of ARS dye at neutral pH is laccase immobilized on ZnO (90%)>MnO2(82%)> free laccase (45%). 3.3.2 Effect of catalyst dose The rate of removal of harmful dye as a function of an initial amount of photocatalyst has been examined. The results (Fig. 3b) showed that removal of dye continuously increased with increasing dosage of the catalysts, reached the higher value (at 50 mg dose amount) and then becomes constant. The probable reason could be that as the dose increases, the number of active sites on catalyst penetrated by photons becomes increased. No further change in degradation occurred because of blocked active sites in the limited size of the vessel [52]. The degradation of ARS dye at 50 mg catalyst dose was: laccase immobilized on ZnO (94%)>MnO 2(79%)> free laccase (45%) 3.3.3 Effects of concentration of ARS dye To estimate the initial concentration of dye which can be effectively degraded using synthesized nanoparticles, experiments were carried out for various concentrations of the dye solution (5-25 mg/L). Maximum removal of dye was obtained at 5 mg/L (Fig. 3c), which is probably due to the

fact that at low dye concentration more number of active sites are available for adsorption on the catalyst. As the concentration increases, the active sites for adsorption present on nanoparticles get blocked, thus, the rate of dye removal decreased [53].The degradation of ARS dye was: laccase immobilized on ZnO (95%)>MnO2(84%)> free laccase (49%) 3.3.4 Effect of contact time The effect of contact time was studied using 20 mg/L initial dye concentration with optimum dose 50 mg of immobilized laccase at neutral pH. Results revealed that within 60 minutes, 83% and 71% ARS degradation was achieved using immobilized ZnO and MnO2 nanoparticles, respectively, and then attained equilibrium. Initially, degradation is faster due to the availability of the active site and then attained equilibrium at the later stage (Fig. 3d). Within 80 minutes, free laccase could be able to maximum degradation (48%) of ARS which further no changed even if increasing the exposure time. Overall, the concentration of ARS was found to decrease continuously with increase in the time interval. Under optimized conditions, the photocatalytic removal capacity of various nanostructures was observed to be: immobilized ZnO (95%) >immobilized MnO2(85%)> free laccase (49%). Degradation of ARS with immobilized laccase on ZnO is even higher than control ZnO (88%) but almost comparable with MnO2(86%)[36, 37]. The presence of MnO2 is enhancing the stability and activity of laccase against ARS dye. Moreover, the combinations of MnO2 with enzyme provide a biocompatible and inert environment and also avoided direct exposure of metals or their oxides [7]. Langmuir isotherm of ARS clearly shows that adsorption is fast in all the cases and the isotherms are regular, positive and concave to the concentration axis (Fig. 4a). Negative values of ΔG suggest that the dye adsorption is a spontaneous process and decrease in value of ΔS favored the strong adsorption of dye on laccase immobilized

nanoparticles (Fig. 4c). Pseudo-second-order degradation kinetics of ARS on immobilized laccase suggesting that in the removal of dye from aqueous solution both the nanoparticles as well as the laccase are playing an equally important role. (Fig. 4b). 3.3.5 Reaction mechanism MOs (ZnO and MnO2) being semiconducting in nature are able to generate electron-hole pair (in conduction and valence band, respectively) upon photo-illumination. The interaction of these electron-hole pairs with water produces active species OH• which breaks the large harmful organic dyes (Fig. 5). First, adsorption of the dye takes place on the surface of adsorbent (immobilized laccase), followed by dye degradation process. Overall, the generation of electronhole pairs cum OH• is responsible for the photo degradation of different organic dyes as well as other organic pollutants [52]. 3.3.6. Photocatalytic degradation products of dye using immobilized laccase Possible degradation pathways involving oxidation or hydrolysis of ARS dye by immobilized laccase has been presented by Figures 6. Under neutral conditions (pH~7.0) solid-support oxidative process resulted in the formation of colorless products which got deposited over the surface of immobilized laccase. Under visible radiation, ARS (1; mol.wt. = 342) is oxidized by O2 radical at C-12 position into phthalic acid (2a; m/z=166) and hydroxyl intermediates (2b) which finally gets mineralized into CO2 and SO42- ions[54]. The major oxidative agent OH• (highly active species) change complex ARS dye into

smaller by-products

such as

benzoquinone (3b), but-2-enal (4b; m/z=71), sulfur trioxide (5; m/z=83), and 2,3-dimethyl cyclohexa-2,5-diene-1,4-dione (6; m/z=140)[52].

Attack of OH• at C-5 position converted ARS

into 6-formyl-3,4-dihydroxy-5,8-dioxo-5,8-dihydrophthalene-2-sulphonic acid (4a; m/z=295) via formation of intermediate (4; 3,4,5-trihydroxy-9,10-dioxo-dihydroanthracene-2-sulfonic acid).

Formation of CO2 and SO42- ions suggest complete mineralization of the dye[55]. The representative mass spectra of possible degradation products of ARS are given in Figures 7. 4. Conclusions Laccase was successfully immobilized on both ZnO and MnO2 nanomaterials with stronger absorption (high enzyme loading) on ZnO indicated by the change in morphology (nanospheres) and increased particle size. Due to enhanced activity with enzyme stability than free laccase, laccase-ZnO was found to a potential catalyst in quantitative removal of ARS dye from water. Laccase- MnO2 has better activity than free lac but no significant difference was observed in degradation of ARS with laccase- MnO2 and control MnO2 . These points clearly highlight the importance of nanomaterial to increase stability and activity of free enzyme in addition to biocompatibility and inert environment without denature of enzyme. Formation of small nontoxic by-products (but-2-enal, sulfur trioxide, and benzoquinone) showed the efficiency of immobilized enzyme. Adsorption of ARS on to immobilized laccase was regular, monolayer, spontaneous and naturally favored process. Further spread of immobilized enzyme-MO nanoparticles utilization in waste water treatment needs to be explored more effectively. Supporting data Other details of characterization were provided in supporting information Acknowledgements Authors are thankful to DST-FIST New Delhi for providing support in the procurement of UV and IR instruments. References: 1. C.R. Martin, P. Kohli, The emerging field of nanotube biotechnology, Nat. Rev. Drug. Discov. 2 (2003) 29-37

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Figures

2.2

Laccase Production

2.0

Absorbance (a.u.)

1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

Incubation time (days)

Fig. 1. Incubation time analysis for maximum laccase production

Fig. 2. FE-SEM images of (a) ZnO (b) laccase immobilized on ZnO (c) MnO2 and (d) laccase immobilized on MnO2

(b)

(a) 100

100

Laccase Immobilized on ZnO Laccase Immobilized on ZnO Free laccase

Free laccase

80

% Degradation

% Degradation

80

Laccase Immobilized on ZnO Laccase Immobilized on MnO2

60

40

20

60

40

20

0

0

2

3

4

5

6

7

8

9

10

20

pH

30

40

50

Catalyst dose (mg)

(d)

(c) 120

100 Laccase Immobilized on ZnO Laccase Immobilized on MnO2

100

Laccase Immobilized on ZnO Laccase Immobilized on ZnO Free laccase

90

Free laccase

% Degradation

% Degradation

80 80

60

40

70

60

50 20

40

0

30 5

10

15

Conc (mg/L)

20

25

20

40

60

80

100

120

Contact time (min)

Fig. 3. (a) Effect of pH (b) catalyst dose (c) dye concentration (d) contact time on photodegradation of ARS dye

(a)

(b)

0.5 18

Laccase Immobilized on ZnO Laccase Immobilized on MnO2 0.4

16

Laccase Immobilized on ZnO Laccase Immobilized on MnO2

14

Free laccase

Free laccase 12

0.3

1/q t

8 6 4

0.1

2 0

0.0

-2

0.5

1.0

1.5

2.0

2.5

-4

3.0

-20

0

20

1/Ce

40

60

80

100

120

140

Time (min)

(c)

2.0 Laccase immobilized on ZnO Laccase immbilized on MnO2

1.8

Free laccase 1.6 1.4

ln K

1/qe

10

0.2

1.2 1.0 0.8 0.6 0.4 0.00310

0.00315

0.00320

0.00325

0.00330

0.00335

0.00340

1/T

Fig. 4. (a) Langmuir isotherms (b) Pseudo-second-order kinetic model (c) Thermodynamic model for free laccase and laccase immobilized on metal oxides nanoparticle

* O

O ONa S O

O

O

+ h

OH

OH O

O

OH

Alizarin Red S (ARS) dye

Laccase immobilized MO + h h+ + H2O

OH

ARS Dye (excited state)

Laccase immobilized MO(e- CB + h+VB) H+ + OH OH-

* O

O ONa S O

O

+

O

OH

+ OH

OH

ARS Dye (excited state)

*

HO ONa S O OH

OH O

ONa S O

ARS Dye (colourless form)

Degradation products (CO2, H2O)

Fig. 5. Photocatalytic degradation of ARS in presence of laccase immobilized MO nanoparticles.

CO2 OH O

O

O

HO

O

H

H

HO3S

CH3

O m/z=295(4a)

O m/z=108 (3b)

OH O

H+

m/z=71 (4b)

OH

O

HO

H+

H3C

OH HO3S O Intermediate (4) Intermediate (3a)

H3C O m/z=140 (6)

Oxidation OH

O

OH O OH OH +

O m/z=166(2a)

HO

H+

Na-O3S

O S OO m/z=83 (5)

O Alizarin Red S Mol. wt.=342(1)

OH O HO +

Na-O3S O

OH HO

OH

NaO3S

OH

Possible mineralized products CO2, SO42-

Hydroxyl intermediate(2b)

O2

Fig. 6. Proposed degradation pathway of ARS using immobilized laccase

Fig. 7. Representative mass spectra of various degraded products of ARS dye

31