Azo dyes are widely used in textile industries. A significant portion of these recalcitrant dyes are being discharged to the natural waters. Due to their low biodegradability they pose serious pollution problems if untreated. In this work, decolourization studies of Acid Red 1 (AR1) by laccase enzyme immobilized onto zein–polyvinyl pyrrolidone (PVP) composite nanofiber is done. The nanofibers were characterized by scanning electron microscopy (SEM) and Fourier-transform infrared (FTIR) analysis. pH and temperature profiles of immobilized enzyme were found to be broader than its free counterpart. The Km value was found to be 0.243 mM for free laccase and 0.958 mM for immobilized laccase. Similarly, Vmax for the free enzyme was 3.572 U/mg compared to 2.48 U/mg of immobilized laccase. The relative activity of immobilized laccase was 64.91% after storage for 30 days at room temperature while it was 28.64% for free laccase. The temperature and pH for AR 1 decolorization were optimized and was found to be 60 °C and 5, respectively. Also, decolorization percentage was found to be 91.67% for immobilized laccase and 72.03% of free laccase in the presence of natural mediators like vanillin. From phytotoxicity studies it was found that the germination rate, shoot and root length was increased compared to untreated dye. Therefore, zein–PVP nanofiber immobilized laccase could be an ideal candidate for the textile dye decolorization.

  • Textile effluents cause serious pollution problem in the environment.

  • Effectiveness of laccase enzyme in the decolorization of Acid Red-1 is studied.

  • Laccase was used either in the free form as well as immobilized in the zein–polyvinyl pyrrolidone nanofiber membrane.

  • The immobilized enzyme showed greater capacity for decolorization.

The usage of dyes has increased enormously in various aspects of life. The textile industry is one major sector that uses dyes which can range from harmless to non-degradable. Untreated, non-degradable dyes when released into the water bodies contribute to pollution and affect human health. Azo dyes are popular and extensively used in textile industries due to easy and affordable methods of synthesis and increased stability (Singh & Singh 2017). However, the carcinogenic, mutagenic, and lethal effects of synthetic azo dyes have been well documented. Synthetic dyes result in the production of highly colored effluent which are resistant to degradation. Colored effluents discharged from these industries causes water pollution, environmental hazards, rise in chemical oxygen demand (COD) and biological oxygen demand (BOD) and, therefore, addressing the issue of treating and removing dyes from the textile effluent remains a challenging task (Sarkar et al. 2017).

Various physico-chemical strategies used for decolorizing textile effluents are coagulation, flocculation, activated carbon adsorption and reverse osmosis (Hooshmandfar et al. 2016). However, these processes consume excessive amounts of chemicals and also produce large amounts of sludge and do not remove all dyes, thus preventing recycling of treated effluent. The excessive use of chemicals demands increased cost and results in secondary pollution (Kothari & Shah 2020). The physical methods can only transfer the dyestuff from one form to another leaving the pollution problem unsolved. This necessitates the requirement of new and efficient technologies for the elimination of dyes from effluents. The genetic diversity and metabolic versatility of microorganisms makes them an attractive option of treatment to remediate the pollution caused by dyes. In recent times, biological methods of removal have been researched and investigated as it offers environmental friendly strategies for decolorizing and detoxifying these recalcitrant compounds (Vasantharaj et al. 2017). Moreover, degradation products are found to be less toxic by employing bio based approaches.

Of late, enzyme based strategies to degrade dyes is well worked on. The important features that make enzymes more suitable in comparison to conventional catalysts are: they are biodegradable and able to function at different substrate concentrations, viable over wide range of pH, temperature and salt concentrations, low level of sludge formation and are easy to control. Also, enzymes possess a broad range of specificity of substrate, and easiness in immobilization. By nature, the efficacy is high, too. The immobilization of enzymes on a suitable support matrix enables the industry to accelerate many reactions and prolonged work. Laccase is polyphenol oxidase which catalyzes the oxidation of ortho and para phenols, polyphenols, aminophenols, lignin, as well as aryl diamines (Jones & Solomon 2015). They have been noticed by the researchers due to their capability to decolorize and detoxify the textile industry effluent. Studies show that acid red dyes are degraded to compounds such as aniline, benzene and pyrocatechol. The various factors make laccase a promising and versatile enzyme with plenty of biotechnological applications. By the use of mediators in combination with laccase, the range of substrates can be extended further.

However, the use of indigenous microbial enzymes suffers some disadvantages under certain process conditions. To overcome these restrictions, immobilization of enzyme has been suggested which improves the performances of biocatalysts. Immobilized enzyme performance, to a great extent, depends on the structure of support. Studies reveal that nano structured supports were found to preserve catalytic activity and ensure efficiency of immobilized enzyme to a great level (Lev et al. 2014; Maryskova et al. 2019). Likewise, nanofibrous support provides greater porosity and good interconnectivity there by acting as one of the ideal systems for immobilization.

In the present study, zein–polyvinyl pyrrolidone nanofibrous mat was produced by electrospinning and characterized. This was used as a scaffold to immobilize laccase enzymes by crosslinking with glutaraldehyde. The immobilized enzyme in nanofiber was used for the decolorization studies of Acid red-1 (AR-1). Many earlier reports tried to study the degradation of AR-1 by indigenous bacteria like Stenotrophomonas sp as well as fenton-like methods but this work proposed to use enzyme-based nanofabrication for decolorization.

Chemicals and reagents

Trametes versicolor laccase (LAC) (EC 1.10.3.2), zein (Z), polyvinyl pyrrolidone (PVP), dimethylformamide, glutaraldehyde, guaiacol, amidonaphthol red G (MW: 509.42) (Acid red-1) were purchased from Sigma Aldrich (USA). Ethyl alcohol, citric acid, sodium citrate, glycine (C2H5NO2), HCl, acetic acid (CH3COOH), sodium acetate (C2H3NaO2), monosodium phosphate (NaH2PO4), disodium phosphate (Na2HPO4), sodium carbonate (Na2CO3), sodium bicarbonate (Na2HPO4). All the chemicals used in the experimental study were of analytical grade.

Preparation of Zein–PVP (Z-PVP) composite nano fibrous membrane by electrospinning

PVP (3 g) was dissolved in dimethyl formamide. To this mixture, 3 g zein was added and mixed thoroughly for about 50 min to obtain a homogeneous mixture. This Z–PVP mixture was taken in plastic syringe which was then pumped through a tiny nozzle having a 1.2 mm inner diameter. A copper pin from the high-voltage generator was attached to the needle. The solution was subjected to a 20 kV voltage with a 15 cm tip-to-target gap and a flow rate of 0.5 mL/h. The fibers collected from the surface of a rotating mandrel at 600 rpm were covered with aluminium foil. The nanofibrous membrane obtained by electro spinning was vacuum dried for 12 h at 60 °C.

Z–PVP composite nano fibers characterization

The surface morphology of nanofibers was determined by scanning electron microscopy (SEM) which is one of the commonly used methods to characterize the nanofibers. The fiber diameter, uniformity of the surface and smoothness of surface are often examined by SEM analysis. Fourier transform infrared (FTIR) spectroscopy was used to examine the chemical assembly of the nanofiber (Perkin Elmer Spectrum IR Version 10.6.0). Samples were mixed with KBr and the prepared pellet was used for FTIR spectra. Spectra in the 400 to 4,000 cm−1 range were measured.

Laccase immobilization on electrospun Z-PVP nanofibrous membranes

Immobilization was carried out using 5 mg weighted stand pieces of Z–PVP nanofiber membrane. The pieces were mixed with glutaraldehyde (GA) solution (250 mL, 2%v/v) in a conical flask and were placed on a rocker (100 rpm) at 4 °C overnight. The nanofibers activated by GA were taken in new tubes and washed thoroughly with sodium acetate buffer (10 mM, pH-4.5) to remove excess GA. It was then combined with laccase solution (2 mg/mL) at 4 °C overnight in a rocker (50 rpm). The laccase immobilized nanofibrous membranes (Z-PVP-LAC) was washed with buffer solution to remove the proteins. The Bradford (1976) method was used to determine the protein content in the solution, using bovine serum albumin (BSA) as the reference. The amount of immobilized enzyme, the efficiency of immobilization, and the enzyme loading were all measured. Immobilization yield and loading efficiency are calculated using the formula:

Laccase assay

Laccase activity was determined using Guaiacol (2 mM) as substrate. The oxidation of guaiacol by laccase leads to the development of reddish brown color which was observed at 450 nm. The reaction mixture consisted of 1 mL substrate, 3 mL acetate buffer, and 1 mL enzyme. The blank solution contained 1 mL of distilled water instead of enzyme. Both were incubated at 30 °C for 15 min before being spectrophotometrically analyzed at 450 nm. The amount of enzyme needed to oxidise 1 mM of guaiacol per minute is measured in International Units (IU), with 1 IU equaling the amount of enzyme required to oxidize 1 mM of guaiacol per minute.

The formula for calculating laccase operation in U/mL is:
where E.A = enzyme activity (U/mL), A = absorbance at 450 nm, V = total volume of reaction mixture (mL), v = enzyme volume (mL), t = incubation time (min) and e = extinction coefficient (M−1 cm−1).

Effect of pH and temperature on enzyme activity

The optimum pH for both free laccase and immobilized laccase (Z–PVP–LAC) was investigated by calculating the relative activity using respective enzymes, 2 mM guaiacol and various buffers of pH in the range 3–8 and incubating the mixture for 15 min. The impact of temperature on enzyme activity was measured by calculating the relative enzymatic activity at pH 4.5 and within the temperature range of 20–80 °C for 15 min. The relative activity is calculated as the ratio of the activity of sample to that of control and is expressed as the percentage (Sathishkumar et al. 2014).

Storage stability of free enzyme and Z–PVP–LAC

Storage stability of free enzyme and Z–PVP–LAC was determined by incubating the samples in 50 mM sodium acetate buffer (pH 4.5) at room temperature for 30 days. The residual enzyme activity was measured by guaiacol assay at 6-day intervals.

Dye decolorization studies using free and immobilized enzyme

The decolorization of Acid red-1 (AR-1) by both free and Z–PVP–LAC was studied using a reaction mixture containing 50 mg/L of the dye. The decolorization efficiency of laccase enzyme was calculated. AR-1 solution was made by dissolving the dye in acetate buffer (100 mM, pH 5.0). After that, the dye solution was applied to the immobilized enzyme, and the reaction mixture was incubated for 3 h at 35 °C and 50 rpm. The absorbance was measured by withdrawing samples at 30 min interval and the percentage of decolorization was determined using the below equation:
where Ai and At are the initial and final absorbance, respectively. The same steps were followed to find the decolorization potential of free enzyme. The experiments were done in triplicates.

Effect of temperature, pH and mediator concentration on decolorization of AR-1

The effect of temperature on decolorization was calculated by incubating the reaction mixtures consisting of free laccase and Z–PVP–LAC with dye (concentration: 50 mg/L) at different temperatures (10–80 °C). Effect of pH on decolorization of dye was carried out with pH values ranging from 3 to 10. The different aliquots of reaction mixtures were inserted with the buffers and incubated for 3 h and the decolorization percentage was determined. The effect of mediators on dye decolorization was studied by adding 0.25 mL of vanillin of different concentrations (0.1 mM to 0.6 mM) to the reaction mixtures containing free and immobilized enzyme. The change in decolorization percentage with respect to time was determined by incubating the reaction mixture in the presence and absence of vanillin at optimum conditions.

Determination of kinetic parameters of free enzyme and Z-PVP-LAC

Km (Michaelis–Menten constant) and Vmax (maximum reaction rate) of free and immobilized enzymes were studied using different concentrations of substrates ranging from 0.1 to 1 mM under optimum conditions. Km and Vmax values were found from the Lineweaver–Burk plot.

Reusability analysis of Z-PVP-LAC

The reusability of Z–PVP–LAC was assessed using AR-1 as the substrate at ambient temperature. After each run, the Z–PVP–LAC membranes were washed using the buffer solution until no traces of dye was detected in the washings.

Phytotoxicity

The phytotoxicity test was carried out using Vigna radiata seeds (10 numbers) placed on sterilized filter paper kept in sterilized Petri plates at room temperature. The seeds were treated with the untreated dye and the laccase treated dye to study the phytotoxic nature of the dye and its metabolites. The seeds treated with distilled water were used as the control. The percentage germination and length of plumule (part of a seed embryo that develops into the shoot) and radical was noted after 7 days.

The efficiency of immobilized enzymes, on a suitable matrix, for various activities have been advocated by several reports. The key to the effectiveness of the enzyme depends on the substrate selected for immobilization. The advent of nanotechnology and the benefits offered is also combined along with immobilization in the present investigation to study the efficacy of laccase. The nano fibrous supports show many advantages when compared with other nano structured supports because of its greater porosity and interconnectivity. However, some nano structured materials possess some disadvantages, too. Lower enzyme activity resulting from immobilization of enzyme in mesoporous silica is an example. But electrospun nanofibers are able to overcome these limitations and act as a promising candidate for enzyme immobilization. At present, nanofibers are used as substrates for enzyme immobilization. Previous research has shown that enzymes immobilized on electrospun materials have higher activity than their unimmobilized counterparts (Li et al. 2019a, 2019b). Also, recent studies have utilized polyvinyl pyrrolidone as a spinning agent to produce magnetite PVP composite to form magnetic nanofiber composite for catalyst immobilization purposes (Tomasz & Andrea 2019). In this study, we demonstrate for the first time, to the best of our knowledge, a novel approach of using Z–PVP electrospun fibers for covalent immobilization of laccase.

Considering the economical and environmental perspectives, electrospinning of natural biopolymers such as zein is gaining much attention. Zein is a corn protein and obtained as a by-product of the alcohol industry. One of the approaches to improve the properties of zein nanofiber is to blend with another material that should be strong enough. In this study, PVP was incorporated with zein because of some of its distinctive properties, such as being non-toxic, biocompatible, water-soluble and hydrophilic. PVP nanofiber-based materials find uses in biomedical research like drug delivery and drug release systems.

Characterization of nanofibrous membranes

The SEM micrographs of electro-spun fibrous membranes from zein and PVP composite (Z–PVP) used for this study are provided in Figure 1. From the figure, it was observed that the morphology of Z–PVP appeared as smooth fibers and was free of beads. The average diameter of nanofiber was found to be 2 μm. Average diameter of nanofibers was found to be varied from less than 1 to 2 μm. Some studies showed that as zein concentration increased, the diameter of nanofibers increased dramatically. Song et al. (2010) found that the average diameter of composite fibers of zein-chitosan-PVP increased significantly from 2.9 to 4.8 μm with increase in zein concentration from 150 to 200 g/L (Song et al. 2010). Polymer solution with higher viscosity results in the formation of fibers with large diameters and smooth surface during electrospinning (Elashmawi et al. 2014).

Figure 1

(a) Scanning electron microscopic (SEM) image of Z–PVP composite; (b) electrospun fiber after immobilization.

Figure 1

(a) Scanning electron microscopic (SEM) image of Z–PVP composite; (b) electrospun fiber after immobilization.

Close modal

The chemical analysis of zein, PVP, laccase and the Z–PVP nanofibers were performed using FTIR in order to substantiate the presence of functional groups in nanofibers. The FTIR spectra were measured in the range 1,000–4,000 cm−1 and the results are provided in Figure 2. It has been mentioned that the FTIR is advantageous as it requires less time and less sample and can provide a spectrum of protein at a wide range of environments.

Figure 2

(a)FTIR spectrum of zein; (b) PVP; (c) Z–PVP nanofibrous membrane; (d) laccase; (e) Z–PVP nanofibrous membrane after immobilization with laccase

Figure 2

(a)FTIR spectrum of zein; (b) PVP; (c) Z–PVP nanofibrous membrane; (d) laccase; (e) Z–PVP nanofibrous membrane after immobilization with laccase

Close modal

The FTIR spectra of zein nanoparticles (Figure 2(a)) exhibit a peak at 3,425 cm−1 and a typical peak at 1,639 cm−1. The former corresponds to the carboxyl group while the latter corresponds to an amide peak that corresponds to carbonyl C = O stretching vibration and both C–N stretching and C–N–H in plane bending. The presence of amide I peak obtained in the range 1,633–1,641 cm−1 is correlated to the presence of α helix. This finding is consistent with those of Miao et al., who found identical peaks for zein procured from Sigma Aldrich (Miao et al. 2017). The FTIR spectrum of pure PVP (Figure 2(b)) indicated a characteristic peak at around 1,647 cm−1, which can be attributed to the pyrrolidone group's C = O stretching vibration. Also, the band at 2,960 cm−1 can be endorsed to the asymmetric CH2 of pyrrole ring. The peak at 1,647 cm−1 clearly corroborates with the C = O groups of pure PVP and these vibrations are dependent on the molecular weight of PVP. The bands at 1,430 cm−1can be related to the C–H deformation modes of the CH2 group. Furthermore, absorption bands at 1,295 cm−1 linked to C–N bending vibrations in the pyrrolidone structure were discovered. These results are supported with the studies carried out by Safo et al. who reported similar peaks with pure PVP (Safo et al. 2019)

The FTIR spectrum of Z–PVP (Figure 2(c)) showed clear peaks at 3,453 cm−1 which is noted in both zein and PVP. Small peaks were noticed at 2,958 and 2,872 that very well corresponds to peaks of PVP. The peak at 1,646 cm−1 relates to C = C stretching vibrations. Smaller peaks in the region of 1,455, 1,291, 1,160, and 1,115 cm−1 which were absent in zein but present in PVP states that there is a clear association between zein and PVP. The spectrum of Z–PVP–LAC (Figure 2(e)) shows the disappearance of certain peaks observed in that of Z–PVP cross linked with glutaraldehyde. Moreover, the presence of a new peak at 1,531 cm−1 is ascribed to the N–H band which authorizes the attachment of enzymes on nanofibrous membranes (Liu et al. 2014). A slight shift was noted in the absorption band from 1,455 cm−1 upon laccase immobilization on activated membrane. Peaks at 3,455 cm−1 and 563 cm−1 and two small peaks at 2,051 cm−1 and 1,029 cm−1 correspond to the peaks observed in the FTIR spectra of laccase (Figure 2(d)), indicating its immobilization on the nanofibers.

Laccase immobilization on to Z–PVP nano fibrous membrane

The amount of laccase which got immobilized onto Z–PVP nanofiber was measured and it was found to be 0.273 mg (5.75 U) enzyme to 5 mg of Z–PVP nanofiber. The immobilization yield was found to be 72.5% and enzyme loading efficiency was calculated as 87.52%. A similar approach was used to calculate the loading efficiency and immobilization yield (Zhang et al. 2009).

Effect of pH and temperature on enzyme activity

Studies prove that enzymes activity is modified by changes in pH. That pH at which the enzyme is found most active is known as optimum pH. The effect of pH on free enzyme and Z–PVP–LAC was evaluated (Figure 3(a)). Free laccase showed 100% relative activity at pH 4 and Z–PVP–LAC showed 100% relative activity at pH 5, after which there was a decline in activity. At pH 10 free laccase had an activity of 5.12% and Z–PVP–LAC had 7.14% activity. In a previous study of the laccase immobilization on cellulose nanofiber for simulated dye effluent treatment, a similar pH (4–5.5) was pointed out as optimal (Sathishkumar et al. 2014).

Figure 3

(a) Effect of pH on enzyme activity; (b) effect of temperature on enzyme activity.

Figure 3

(a) Effect of pH on enzyme activity; (b) effect of temperature on enzyme activity.

Close modal

The slight change in optimum pH may be due to the fact that the microenvironment of Z–PVP–LAC and bulk solution possess uneven distribution of H+ and OH– ions because to electrostatic interactions with the nanofiber matrix (Abdullah et al. 2007). Additionally, pH dependence of immobilized laccase is wider than free laccase, which shows that immobilization method preserves enzyme activity in a varied pH range. Therefore, the electrospun Z–PVP nanofiber surface has enough charge density to alter the enzyme microenvironment.

The effect of temperature on activity was studied by incubating the mixture in the temperature range 20–80 °C (Figure 3(b)). The maximum activity for the free laccase was found at 50 °C whereas Z–PVP–LAC showed maximum activity at 60 °C. A decrease in activity was found for free as well as immobilized enzyme thereafter. The results given in this study is in line with the findings on the optimum temperature for the immobilization of laccase on polyacrylamide and polyacrylamide – κ – carrageenan based polymer networks by Gökgöz and Altinok in which it is reported that optimum temperature for free laccase as 45 °C and that for immobilized laccase as 60 °C (Gökgöz & Altinok 2012). Another study also stated that laccase immobilized on modified cellulose nanofibers showed maximum activity upto 70 °C (Bansal et al. 2018). Further, an optimum temperature of 50 °C was observed in the laccase immobilized in methacrylate acrylate microspheres (Mazlan & Hanifah 2017). Immobilized laccase can withstand high temperatures when compared with free enzyme which could be because of the variation in physical and chemical properties of immobilized enzyme (Ndlovu et al. 2020). The conformational flexibility might be reduced by covalent bond forming amino groups of immobilized laccase. This increases activation energy for the molecule and thus reorganizes conformation of substrate.

Storage stability

In view of practical applications, evaluation of storage stability and reusability are of significance. Immobilized biocatalysts are expected to show great catalytic activity even after several days of storage (Talebi et al. 2016). Figure 4 represents the activity of free laccase and Z–PVP–LAC at room temperature when stored for a period of 30 days. The relative activity percentage of the free enzyme was 100%, 84.31%, 58.72%, 39.21% and 28.64% respectively, for each 6-day interval and for Z–PVP–LAC it was found to be 100%, 89.25%, 82.57%, 73.43% and 64.91%, respectively. The obtained result proves that the enzyme stability during storage was enhanced by the immobilization method compared with the free enzyme. These results suggest that immobilization extensively prevented enzyme deactivation and improved its storage stability. The enzyme leakage might be reduced due to the stable binding of enzyme to the nanofiber which increased the stability of immobilized enzyme. Similar findings have been pointed out by Rouhani et al. who showed that free laccase showed only 45% activity after 30 days, while graphene oxide-CuFe2O4-laccase had retained 83% of its initial activity (Rouhani et al. 2018). Storage stability determined in this work is in accordance with that of laccase immobilized in copper alginate beads in our previous report. The enzyme entrapped in copper alginate showed greater storage stability than free laccase enzyme. After the first week, the activity of the free enzyme was 72.14% and 94.38% for immobilized enzyme. The conclusion from these results is immobilization of enzymes serves as the best method for improving the storage stability.

Figure 4

Storage stability analysis of free and immobilized enzyme.

Figure 4

Storage stability analysis of free and immobilized enzyme.

Close modal

Decolorization studies of AR 1

In order to study the decolorization potential of immobilized laccase enzyme, the dye sample was treated with Z–PVP–LAC as a preliminary study. The result showed that 38% decolorization was achieved after 30 min incubation even in the absence of optimum parameters for enzyme activity. Therefore, conditions were optimized to achieve better decolorization by changing the temperature and pH.

Effect of temperature and pH on decolorization

The reaction mixture was incubated at various temperatures to study the percentage decolorization. The result (Figure 5(a)) shows a higher decolorization of 80.45% at 60 °C by Z–PVP–LAC and 57.25% decolorization by free laccase at 50 °C. The decolorization percentage dropped to 2.1% for free laccase and 19.23% for Z–PVP–LAC at 80 °C. The amount of decolorization of dyes dropped below 20% by elevating the temperature above 70 °C. This could be due to the inactivation of enzymes at high temperatures. Chaurasia et al. have reported the optimum activity of laccase isolated from Phellinus linteus MTCC-1175 to be 45 °C (Chaurasia et al. 2013). Also, a study by Baldrian (2006) demonstrated that laccase activity was maximum at the temperature range of 50–70 °C. On the other hand, Lentinulaedodes laccases showed optimal temperature at 70 °C (Sun et al. 2011).

Figure 5

(a) Effect of temperature on decolorization of AR-1; (b) effect of pH on decolorization of AR-1.

Figure 5

(a) Effect of temperature on decolorization of AR-1; (b) effect of pH on decolorization of AR-1.

Close modal

The effect of pH on decolorization is given in Figure 5(b). With respect to increase in pH, the percentage decolorization increased and reached a maximum point for both free laccase and Z–PVP–LAC. For free laccase, the percentage decolorization was 14.27% at pH 3 and maximum decolorization was found at pH 4 (59.72%). But for Z–PVP–LAC it was 17.23% at pH 3 and maximum decolorization at pH 5 (84.73%). There were also reports which stated that deviation from optimum pH adversely affects the decolorization percentage. This may be due to the fact that the diffusion of dye molecules across the cell membrane may be affected by changes in pH, which is considered as the rate-limiting step for the decolorization.

Kinetic constants of free and immobilized enzyme

In order to study the affinity of laccase towards its substrate, and the changes in the catalytic efficiency upon immobilization by cross linking, the Michaelis–Menten constant (Km) and maximum rate of reaction (Vmax) were calculated. A lower Km value indicates a higher substrate affinity. The Km value for laccase was measured by recording the oxidation of guaiacol in pH 4.5 at 40 °C. Km values were found to be 0.243 mM for free enzyme and 0.958 mM for Z–PVP–LAC, respectively. Higher value of Km was shown by Z–PVP–LAC than free laccase. This may be due to conformational changes in enzyme or resistance to diffusion of substrate molecules to the active site of immobilized enzyme (Zhang & Rochefort 2011). Factors such as increased resistance to the transfer of substrate or interaction between the substrate and nanofibrous membrane or changes in the conformation of enzyme may be the reason for higher Km value for immobilized enzyme (El-Aassar et al. 2013). Additional studies have also stated an increase in Km because of immobilization. It clearly indicates that immobilized enzyme has an apparent low affinity towards its substrate with respect to its free form. Increase in Km upon immobilization is reported by Gahlout et al. in kinetic study of laccase from Ganodermacupreum AG-1 in hydrogels (Gahlout et al. 2014). The maximum reaction velocity, Vmax for free enzyme was higher (3.572 U/mg) compared to that of Z–PVP–LAC (2.48 U/mg). This may be because of the presence of steric hindrances in the system. Immobilization process may block laccase active sites and thereby decrease enzyme substrate affinity and maximize reaction rate (Antecka et al. 2018). Low Vmax for laccase immobilized on both surface modified and unmodified activated carbon fibers were reported by (Zhang et al. 2018).

Effect of mediator concentration and incubation time

The mediator is a low molecular weight compound acting as a carrier for electrons. The presence of redox mediators could expand the activity profile of laccase towards many recalcitrant compounds. The influence of mediator concentration was studied using different concentrations of vanillin (0.1–0.6 mM) in the reaction mixture. The result (Figure 6(a) and 6(b)) demonstrates that the percentage degradation increased with increase in vanillin concentration. The highest decolorization percentage was observed at 0.6 mM. Earlier studies have shown that the presence of mediators results in decreased the potential energy of laccase oxidation reactions and significant increase in oxidation efficiency. Studies on the decolorization of Azure B, Xylidine, and Gentian Violet by laccase from T. trogii in the presence of synthetic mediators and natural laccase mediators shows that higher mediator concentrations resulted in high decolorization, indicating that the reactive species evolved by the mediator's oxidation are involved in the reaction. With respect to time, the decolorization percentage was found increasing for free and immobilized laccase in the presence of mediators compared to the same without mediator. The immobilized laccase of Trametes pubescens was used in two experiments, with findings of 69 and 77.5% decolorization in 48 and 96 h, respectively (Zheng et al. 2016; Ma et al. 2018).

Figure 6

(a) Effect of mediator concentration on decolorization of AR-1; (b) effect of incubation time on decolorization of AR-1.

Figure 6

(a) Effect of mediator concentration on decolorization of AR-1; (b) effect of incubation time on decolorization of AR-1.

Close modal

Reusability

Reusability is an added feature of immobilized enzymes. As shown in Figure 7, the decolorization rate of AR-1 by Z–PVP–LAC was 69.71% for the first cycle and reduced to 43.24% after five batch operation cycles. The decrease in the percentage decolorization after each cycle may be due to the loss of enzyme during the process and inactivation of enzymes used. Comparable results were pointed out by Zdarta et al. in the degradation studies of naproxen and diclofenac using laccase immobilized in electrospun nanofibers (Zdarta et al. 2019). The results given by Li et al. (2019a, 2019b) also exhibited that immobilization of laccase onto modified PU/RC (polyurethane-RC/2-hydroxy methacrylate) nanofiber improves the reusability in the removal of bisphenol A. In another report, leaching of immobilized carbonic anhydrase from polylactic acid nanofibrous membranes modified by graphene oxide was found to be less due to steady covalent bonds formed between the enzyme and the support (Sahoo et al. 2015). Recyclability studies of laccase immobilized in dendritic nanofibrous membranes for the removal of bisphenol A by Koloti et al. pointed out a removal efficiency of up to 79% even after four filtration cycles (Koloti et al. 2017).

Figure 7

Reusability analysis of Z–PVP–LAC.

Figure 7

Reusability analysis of Z–PVP–LAC.

Close modal

Phytotoxicity

The phytotoxicity test was carried out using Vigna radiata seeds. The seeds were exposed to treated and untreated AR-1 samples. The parameters used to assess phytotoxicity are germination percentage, and shoot and root length of germinated seeds (Table 1). The seeds kept as control showed 100% germination, higher shoot and root lengths but seeds treated with untreated dye showed 70% germination and decreased root and shoot length compared with treated dye. The percentage germination of seeds exposed to treated dye was 40%. The results suggest that AR1 treated with laccase reduced the toxicity of dye. The results show that the seeds exposed to laccase treated dye could achieve 77.41% of the shoot length and 82.329% shoot length as that of control seeds, whereas the seeds exposed to untreated dye could achieve only 56.31% shoot length and 60.64% root length as that of control seeds which is supported through the findings from Bilal et al. (2016) and Iqbal et al. (2017).

Table 1

Phytotoxicity of untreated and biodegraded AR-1 along with control

ParametersControlUntreated dyeTreated dye
Germination (%) 100 40 70 
Shoot length (cm) 6.73 ± 0.526 3.79 ± 0.329 5.21 ± 0.217 
Root length (cm) 4.98 ± 0.27 2.02 ± 0.474 4.1 ± 0.579 
ParametersControlUntreated dyeTreated dye
Germination (%) 100 40 70 
Shoot length (cm) 6.73 ± 0.526 3.79 ± 0.329 5.21 ± 0.217 
Root length (cm) 4.98 ± 0.27 2.02 ± 0.474 4.1 ± 0.579 

These results indicate the possibility of using immobilized laccase as an effective option for decolorization of AR-1. AR-1 being most commonly used in textile effluent, definitely requires a treatment method before being released into water bodies to prevent further pollution associated health risks arising out of these water sources for the flora and fauna and mankind at large.

In the present study, laccase enzyme is immobilized onto zein–PVP nanofibrous membrane and used for the decolorization of the azo dye AR-1. The SEM and FTIR characterization studies clearly depict the successful immobilization of enzyme onto nanofibrous membrane. The effect of pH, temperature, incubation time and mediator concentration was studied on both free and immobilized enzyme. The optimum pH and temperature for free laccase were 4 and 50 °C, respectively, and for Z–PVP–LAC it was found as 5 and 60 °C, respectively. Analysis of storage stability shows that immobilized enzyme can be stored without much loss of actual activity even for a period of 30 days. The dye decolorization studies indicate that at optimum temperature and pH, the decolorization percentage was higher for Z–PVP–LAC compared with that of free laccase. Vanillin is the natural mediator used in this study and the presence of mediator boosted the decolorization rate. Z–PVP–LAC could be used repeatedly for five batch operation cycles with the decolorization percentage of 43.24% for the fifth cycle. The phytotoxicity analysis showed that 70% germination occurred for seeds exposed to enzyme treated dye and only 40% for those exposed to untreated dye. The root and shoot length was also found to be higher for enzyme treated dye than untreated dye. Thus, immobilization of laccase onto nanofibrous membranes offers a good choice for textile effluent decolorization.

All relevant data are included in the paper or its Supplementary Information.

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