Laccase-conjugated amino-functionalized nanosilica for efficient degradation of Reactive Violet 1 dye
Immobilization of enzyme with nanostructures enhances its ideal characteristics, which may allow the enzyme to become more stable and resistant. The present investigation deals with the formulation of laccase nanosilica conjugates to overcome the problems associated with its stability and reusability. Synthesized nanosilica and laccase nanoparticles were spherical shaped, with the mean size of 220 and 615 nm, respectively. Laccase nanoparticles had an optimum temperature of 55 °C and pH 4.0 for the oxidation of ABTS. Laccase nanoparticle retained 79% of residual activity till 20th cycle. It also showed 91% of its initial activity at lower temperatures even after 60 days. Laccase nanoparticles were applied for Reactive Violet 1 degradation wherein 96.76% of decolourization was obtained at pH 5.0 and 30 °C within 12 h. Toxicity studies on microbes and plants suggested that the degraded metabolites were less toxic than control dye. Thus, the method applied for immobilization increased storage stability and reusability of laccase, and therefore, it can be utilized for efficient degradation of azo dyes.
KeywordsBiocatalyst Ganoderma cupreum AG-1 Laccase nanoparticle Nano-immobilization Reactive Violet 1
Enzymes catalyse more specific reactions inside living systems under mild conditions; therefore, they are efficient alternate for chemical catalysts . The economy of biocatalytic process can be improved by enzyme reusability and stability by means of immobilization [2, 3]. The challenges of using immobilized enzymes are identifying new matrix materials with appropriate structural characteristics, such as morphology and surface functionality .
Recently, carbon nanotubes, nanosized polymer beads, and metal nanoparticles are utilized as immobilization matrices of enzymes . The use of nanomaterials not only offers advantages such as large surface area, increased mechanical strength, and effective enzyme loading, but also exhibits high catalytic efficiency [6, 7]. Enzyme nano-immobilization can be performed by either physical or chemical modification. Although physical nano-immobilization leads to weaker interaction with enzymes, conformation of the enzyme is unaffected. Chemical nano-immobilization method changes the enzyme conformation, but it provides the strong covalent bond formation. 3-Aminopropyltriethoxisaline (APTES) is the aldehyde anchoring chemical, which is subsequently attached and formed covalent bond. This modification provides strong cross-linking with protein amino surface with nanoparticles. The catalytic efficiency of an enzyme decreases due to the steric effects, limited freedom of active site, and diffusion barriers when enzyme conjugated with nanostructures [8, 9]. In addition, the binding affinity of peroxidase was decreased by immobilizing it on graphene (graphane) oxide by electrostatic interactions . However, the effect of nanomaterials, substrate acted on enzyme activity, and interface nature between enzyme and nanomaterials has not been fully elucidated .
Laccases (E.C. 184.108.40.206) catalyse the removal of hydrogen atom from hydroxyl group of o- and p- substituted mono-phenolic and poly-phenolic substrates and from aromatic amines by one electron abstraction to form free radicals as well as capable of further reaction such as depolymerisation, re-polymerization, demethylation, or quinone formation [11, 12]. Immobilized laccase has a wide range of commercial applications such as formulation of biosensors and biofuel cells, toxic pollutant degradation, and transformation of industrially and medicinally important compounds [4, 13, 14, 15]. Laccases have been successfully immobilized with nanomaterials such as nanoparticles, nano-composite, carbon nanotubes, and nanogels [16, 17, 18].
Laccase nano-immobilization is a promising strategy for recovering laccase on amino-functionalized nanosilica. The present study is aimed to fabricate and characterize laccase-conjugated amino-functionalized nanosilica. Furthermore, prepared nano-biocatalyst was applied for the degradation of Reactive Violet 1 (RV 1) dye. Based on several analytical procedures such as UV–visible spectrophotometry, HPTLC, and GC–MS/MS analysis, we demonstrated the produced nano-biocatalyst was efficient for decolourizing and degrading RV 1 dye. Toxicity assessment of RV 1 dye and degraded metabolites were also evaluated on microbes and on plants.
Materials and methods
2, 2-Azino-bis (3-ethylbenzthiozoline-6-sulphonic acid) (ABTS), tetraethyl orthosilicate (TEOS), APTES, and Sephadex G-75 were purchased from Sigma-Aldrich (St. Louis, USA). Fungal media, agar–agar, and other chemicals were purchased from Hi-media Labs. (Mumbai, India). RV 1 dye was procured from Meghmani Enterprise Pvt. Ltd. (Ahmedabad, India). All other reagents used were of analytical grade with highest purity.
Laccase production and purification
Solid-state fermentation strategy was used for laccase production using Ganoderma cupreum AG-1. An Erlenmeyer flask containing wheat straw (5 gm) was moistened with Asther’s medium to give a final substrate to moisture ratio of 1:4 . Actively grown fungal mycelia (5 agar plugs; 8 mm diameter) was inoculated on moistened wheat straw and incubated it for 16 days at 30 °C. Extrudates were extracted by squeezing fermented wheat straw using muslin cloth and centrifuged at 8000 rpm and 4 °C for 15 min. The obtained supernatant was precipitated using ammonium sulfate saturation procedure. Furthermore, precipitated protein was dialyzed overnight in Na-acetate buffer and purified using gel filtration chromatography (Sephadex G-75) at pH 5.0 (Na-acetate buffer, 50 mM).
Laccase assay was performed by incubating purified laccase (100 µl) with ABTS (1 mM, 100 µl) in 800 µl of Na-acetate buffer (50 mM, pH 5.0). The rate of substrate oxidation was monitored for purified and laccase nanoparticle at 420 nm (ε = 36,000 cm/M) in a UV–visible spectrophotometer following the method described by Rudakiya and Gupte . One unit of enzyme activity (U) is defined as the amount of enzyme oxidizes 1 μM of ABTS per min.
Laccase nanoparticle preparation
The nanosilica was chemically synthesized according to the method described by Stöber et al. . Preparation of nanosilica was carried out by mixing TOES (4 ml) and NH4OH (3.3 ml, 24% w/v) in ethanol (47 ml) at 20 °C for 24 h with moderate stirring. The resulting suspension was transferred to filtration assembly equipped with cellulose filtration membrane (cut off; 50 kDa) and washed thoroughly with water. With this suspension, APTES (300 µl) was added and reaction was maintained under vigorous stirring for 20 h. Prepared suspension was washed three times with Sorenson’s phosphate buffer (pH 7.0) and collected in the pellet form by centrifuging it for 3 min at 10,000 rpm. After washing step, pellet of nanosilica was dried under reduced pressure and produced nanoparticles are called amino-functionalized nanosilica.
Characterization of laccase nanoparticles
Structural and functional characterization
Structural elucidation of nanosilica and laccase nanoparticles was acquired using microscopic, spectroscopic, and light scattering techniques. The size and shape of nanosilica and laccase nanoparticles were analysed using transmission electron microscopy (Tecnai 20, Philips, Holland). The interaction of laccase with amino-functionalized nanosilica was studied by Fourier Transform Infrared Spectroscopy (Spectrum GX, Perkin Elmer, USA). The average size of nanosilica and laccase nanoparticles was observed using dynamic light scattering analyzer (Zetasizer S-90, UK).
Temperature and pH optima
Temperature optimum was examined by incubating the purified laccase and laccase nanoparticles in Na-acetate buffer (50 mM, pH 5.0) at different temperatures (30–70 °C). The pH optima of purified laccase and laccase nanoparticles were determined by monitoring the oxidation of ABTS in the pH range from 2.0 to 8.0 at 30 °C. Na-acetate (50 mM, pH 3–5) and Na-phosphate (50 mM, pH 6–8) buffers were used to maintain the pH. Residual activities of purified laccase and laccase nanoparticles were measured after 1 h of incubation.
Temperature and pH stability
Thermal stability of purified laccase and laccase nanoparticles was determined at different temperatures (30–70 °C) in Na-acetate buffer (50 mM, pH 5.0) for 24 h. The pH stability was studied by incubating laccase nanoparticles and purified laccase in Na-acetate buffer at different pH values (3.0, 4.0, and 5.0) for 24 h at 30 °C. Residual activities were measured periodically at an interval of 4 h under standard assay conditions.
Storage stability and reusability
Purified laccase and laccase nanoparticles were stored at 4 and −20 °C and residual activities were measured periodically at an interval of 10 days for 60 days under standard assay conditions. Reusability of laccase nanoparticles (100 mg) was determined by incubating it with ABTS in Na-acetate buffer at 30 °C for 3 min. Operational stability were carried out for 20 cycles wherein samples were withdrawn after an interval of 3 min and absorbance was measured at 420 nm. Laccase nanoparticles were collected by centrifuging (10,000 rpm, 10 min) and washing twice with Na-acetate buffer. Subsequently, washed laccase nanoparticles were re-suspended in a fresh substrate solution and assayed subsequently.
Kinetic studies of laccase nanoparticle
The kinetic parameter of purified laccase and laccase nanoparticles was determined at 30 °C using ABTS (0.05–100 mM) in Na-acetate buffer (50 mM, pH 5.0). Kinetic parameter Km and Vmax were obtained using Line weaver-Burk plots. All assays were performed in triplicates.
RV 1 dye decolourization experiments
Physic-chemical parameters, i.e., temperature and pH were monitored to study the effect on decolourization of RV 1. Effect of pH on RV 1 decolourization was determined by measuring % decolourization in 2.0–7.0 pH range. The effect of temperature on decolourization of RV 1 was determined by measuring % decolourization at different temperature in the range of 20–60 °C.
Analysis of degradation metabolites
The degraded metabolites were extracted in ethyl acetate, concentrated in a rotary vacuum evaporator, and re-dissolved in a small volume of ethyl acetate. HPTLC analysis was performed to compare the Rf values of RV 1 dye and degraded metabolites on pre-coated silica gel 60 F254 plate. The chromatogram was observed at 254 nm using Camag TLC scanner 3. Identification of RV 1 dye and decolourized metabolites was conducted using the GC–MS/MS analysis with 30 m fused silica column (HP-5 30 m × 0.53 mm; Agilent Technologies, USA). The temperature of injection port was at 275 °C and performed at 70 eV.
Toxicity analysis of degraded metabolites
Microbial toxicity assessment of RV 1 (500 mg/l) and degraded metabolites was conducted using micro-organisms like Bacillus subtilis, Streptococcus aureus, Salmonella typhi, Escherichia coli, Rhizobacter radiobacter, and Azotobacter sp. The antibacterial action of RV 1 dye and degraded metabolites was studied using well diffusion method wherein inhibition zone surrounding the well represented the toxicity index. The phytotoxicity assessment of dye (500 and 1000 mg/l) as well as degraded metabolites was carried out on Pennisetum glacum and Vigna radiate . Ten seeds of respective plants were germinated in small pots; daily supplemented with relevant contents (distilled water, dye, or degraded metabolites) to seeds and maintaining light (12 h) and temperature (30 °C) in a controlled environment. Toxic effects were measured in terms of % germination, plumule length, and radical length of plant after 7 days.
Results and discussion
Effect of laccase concentration cross-linking with nanosilica
Characterization of laccase nanoparticle
Temperature and pH optima
The oxidation reaction for purified laccase and laccase nanoparticles had maximum activity at pH 3.0 and 4.0, respectively (Fig. 4b). Almost 15% of relative activity was retained at pH 8.0, while purified laccase was inactive. The results showed one unit shift in optimum pH towards higher value after cross-linking of laccase on nanosilica. This may be attributed due to nanosilica that has been changed micro-environment of laccase, the ionic interaction between enzyme, and charged surfaces of nanosilica. Laccase nanoparticle showed more than 90% residual activity in pH range of 3–5, while purified laccase obtained a comparable decrease in residual activity within the same pH range. Similarly, 0.5 and 1.5 unit shift of optimum pH after covalent immobilization of laccase on Fe3O4-CS-CCn and Fe3O4-CS-EDAC support, respectively . Wang et al.  also reported a shift in optimum pH for the oxidation of catechol upon immobilization of laccase on magnetic mesoporous nanosilica.
Temperature and pH stability
Storage stability of laccase nanoparticle
Reusability of laccase nanoparticle
Laccase is an expensive biocatalyst; the reuse of catalyst makes the enzymatic process economically viable to cut down production cost. Results depicted that more than 87.6% of laccase activity was retained till 16th cycle. Thereafter, a gradual decrease in laccase activity was observed after each cycle and almost 79% of residual activity was retained till 20th cycle (Fig. 7c). Laccase cross-linked with nanosilica was reasonably stable when repeatedly used for catalysis of ABTS oxidation. This property of laccase nanoparticle is beneficial for the application in a batch or in a continuous mode. Bayramoglu et al.  reported 81% residual activity of laccase immobilized on CHX-g-p(IA)-Cu(II) membrane, after 10th cycle of syringaldazine oxidation. Liu et al.  reported 50% residual activity of laccase immobilized on CMMC support after 10th cycle of ABTS oxidation.
Kinetic studies of laccase nanoparticle
The kinetic parameters such as Km and Vmax vary considerably depending upon the types of enzymes, which support materials and process conditions. Lower Vmax may have resulted from mass transfer limitations and reduction in enzyme–substrate affinities after immobilization. Reduction in Vmax after laccase immobilization has also been reported by various researchers. The apparent Km of immobilized laccase nanoparticles was found to be 0.5 mM which was 21.05% higher than purified laccase (0.19 mM). Vmax of laccase nanoparticle was found to be 3.58 × 102 mM/min, which is only 29.01% of Vmax value of the purified laccase (12.34 × 102 mM/min). The results depicted that the catalytic efficiency of laccase nanoparticle was lower than the purified laccase. The lower catalytic efficiency of laccase nanoparticle may be due to the rigid conformation of enzyme in nanoparticle form, which will not allow any changes in the conformation of active site to accommodate the substrates. When enzyme cross-linked with nanoparticle, neighbouring enzyme molecules sterically hinder the active centres of enzyme, so all enzyme molecules may not be available for the substrates, resulting the lower catalytic efficiency of enzyme. A lower catalytic efficiency of laccase crystal had been observed by Roy et al.  wherein Vmax of CLEC laccase was only 12% than the Vmax of native laccase. Huang et al.  also reported lower catalytic efficiency of CuTAPc-Fe3O4 nanoparticle laccase as compared to free laccase, who reported 88.88% higher Km and 43.33% lower Vmax value of CuTAPc-Fe3O4 nanoparticle laccase as compared to free laccase.
Decolourization of RV 1 dye using laccase nanoparticle
Laccase nanoparticle showed more than 75% decolourization of RV 1 dye within a pH range of 3.0–6.0 (Fig. 8b). The optimum pH for dye decolourization was 4.0 wherein 96.85% of decolourization of RV 1 dye was obtained. However, decolourization of RV 1 dye was 31% at neutral (pH 7.0). Usluoglu and Arabaci  reported pH 4.0 as an optimum pH for maximum decolourization of acid and metal complex dyes by phenol oxidase immobilized on to alginate beads. Mogharabi et al.  reported pH 8.0 as optimum pH for the decolourization of dye solution of various textile dyes by laccase immobilized on alginate gelatin gel.
The optimum temperature for maximum decolourization (96.78%) of RV 1 by laccase nanoparticle was 30 °C (Fig. 8c). Decolourization of RV 1 by laccase nanoparticle was also comparable to the incubation temperature range from 25 to 50 °C. However, further increase in temperature results in the decreased decolourization of RV 1. The decrease in decolourization might be observed due to the unfolding or degradation of laccase at high temperature. Similar results have been observed by Mogharabi et al.  for the decolourization of crystal violet dye by laccase in alginate–gelatin mixed gel.
Analysis of degraded metabolites
Toxicity analysis of degraded metabolites
In general, treated/untreated effluent of dyes directly releases to the environment, so it shows the toxicity towards plants, animals, and microbes. Among them, microbes and plants have been used for decades as a biosensor for the toxicity of environmental pollutants [28, 41]. Evaluating the microbial toxicity of RV 1 dye on agricultural important micro-organisms like R. radiobacter and Azotobacter sp. showed inhibition zone 10.5 ± 0.8 and 12.4 ± 0.5 mm, respectively. Degraded metabolites did not show inhibition zone against these bacteria. B. subtilis (15.9 ± 0.9 mm), S. aureus (15.2 ± 1.0 mm), S. typhi (15.1 ± 1.2 mm), and E. coli (14.3 ± 0.8 mm) were also significantly inhibited by RV1 dye. In contrast, degraded metabolites exhibited less zone of inhibition in case of B. subtilis (7.8 ± 1.0 mm), E. coli (8.1 ± 0.9 mm), and S. typhi (10.2 ± 0.8 mm), while S. aureus (12.9 ± 1.4 mm) depicted almost similar inhibition zone. Growth of B. cereus and Azotobacter sp. was unaffected when both bacteria were tested against degraded metabolites of Synozol red HF-6BN using Aspergillus niger and Nigrospora sp. .
Phytotoxicity study of untreated and laccase nanoparticle treated RV 1 dye on V. radiate and P. glacum
RV1 dye (500/gl)
RV1 dye (1000/gl)
RV1 dye (500/gl)
RV1 dye (1000/gl)
20.8 ± 1.3
13.5 ± 1.4
19.5 ± 1.7
11.5 ± 1.4
16.4 ± 1.5
18.9 ± 2.1
12.4 ± 1.6
16.5 ± 1.4
9.1 ± 1.1
14.4 ± 1.7
6.3 ± 1.6
4.4 ± 0.9
5.6 ± 0.8
3.9 ± 0.9
4.1 ± 0.7
8.1 ± 1.4
4.2 ± 1.1
6.6 ± 1.1
3.2 ± 0.8
5.1 ± 1.7
Overall, amino-functionalized nanosilica was synthesized chemically and anchored covalently using glutaraldehyde to laccase enzyme. Due to the immobilization with nanosilica, laccase nanoparticles obtained higher efficiency at higher pH and temperature compared to purified laccase. Laccase nanoparticles displayed higher stability with repeated use, long-term storage, and thermal stability than purified laccase. Furthermore, prepared nano-conjugates efficiently cleaved RV 1 dye and transformed into less-toxic products, which represented an additional advantage for waste water treatment. Though laccase nanoparticles obtained lower catalytic efficiency than purified laccase, laccase nanoparticles are promising biocatalysts that can be used for the elimination of synthetic dyes.
The authors are very obliged to the Department of Biotechnology (DBT Sanction No. BT/PR9134/BCE/08/543/2007) and Ministry of Science and Technology, New Delhi, for their financial support. The authors also wish to acknowledge SICART, V.V. Nagar, Gujarat, for providing the necessary instrumentation facilities.
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