Enhanced Laccase Activity and Stability as Crosslinked Enzyme Aggregates on Magnetic Copper Ferrite Nanoparticles for Biotechnological Processes

Highly stable and reusable magnetic crosslinked enzyme aggregates (m‐CLEAS) of laccase are synthesized with simultaneous improved enzymatic activity. Magnetic copper ferrite nanoparticles (CFNPs) were synthesized by solvothermal procedure with an average size of ~8 nm. The nanometric m‐CLEAS were formed by co‐aggregation of enzyme with CFNPs and crosslinked using glutaraldehyde. Different mass ratios of CFNPs:Laccase were assayed (1 : 2, 1 : 3, and 1 : 6), where 1 : 6 resulted in the highest activity recovery (97 %). The m‐CLEAS showed an average size of ~239 nm, ~24 % enzyme immobilization efficiency, and loading as high as 1.75 g of protein per g of support. As expected, m‐CLEAS oxidized the substrate with a higher transformation rate (kcat) and catalytic efficiency (kcat/Km) than the free enzyme. m‐CLEAS showed superior storage and thermostability compared to free enzyme and non‐magnetic CLEAS. In particular, the m‐CLEAS showed ~150 % and ~100 % residual activity after 30 days of storage at 4 °C and room temperature, respectively. Furthermore, m‐CLEAS showed good recyclability, retaining ~78 % and ~54 % laccase activity after 5 and 10 cycles of reuse, respectively. This work highlights the facile and cost‐effective synthesis of nanometric m‐CLEAS with exceptional storage stability and simultaneously improved laccase activity, making them suitable for a range of green industrial processes.


Introduction
The ongoing industrial transition to a more sustainable, circular economy, which is an increasing trend, is currently stimulating the demands of enzymes.Currently valued at $ 5.5 billion, the global enzyme market is expected to grow to $ 7.0 billion by 2023. [1]Consequently, the need for enhanced enzyme activity, stability, recoverability, and reusability with resilience to various operating variables is essential.Laccase is a multi-copper oxidase enzyme that is involved in several important biological processes, like the breakdown of complex polymeric biomolecules, such as lignin.It catalyzes the one-electron oxidation of aromatic compounds with the four-electron reduction of oxygen to water, meeting the criteria of green chemistry.Moreover, it has a wide range of substrates, including aromatic hydroxyl and amine groups, polyphenols, and methoxy-substituted monophenols.These outstanding features make it very important in green industrial processes, including textile/pulp bleaching, bioremediation, biomass delignification, and organic synthesis. [2,3]mobilizing laccase on insoluble support has become a popular method to improve enzyme stability, reusability, and bioactivity.A variety of carriers including membrane, [4] nanoparticles, [5,6] microspheres, [7] zeolites, [8] and nanofibers [9] have been developed so far.Amongst them, magnetic nanoparticles (MNPs) have attracted much interest due to the added advantage of facile separation and recovery from reaction media that lowers operating costs compared to traditional methods like filtration and centrifugation. [10,11]However, bare MNPs have an inert surface and have a propensity to aggregate in aqueous suspensions due to their substantial surface energy and attractive forces, making it difficult to immobilize enzymes directly on the surface.][14] Nevertheless, many immobilization methods lead to a significant loss in catalytic activity due to the enzyme's conformational changes.Moreover, the choice of nanosupport plays an important role as it may influence the final enzymatic activity.
It is well known that copper is an inducer influences the laccase activity.In fact, various copper-based nanomaterials are reported to significantly improve the enzymatic activity. [15,16]In recent work, copper ferrite (CuFe 2 O 4 ) magnetic nanoparticles (CFNPs) with a functional layer of 3-aminopropyltriethoxysilane (APTES) are used to immobilize laccase by covalent conjugation using glutaraldehyde as a cross-linker. [13,17]The laccase showed superior kinetic potential and stability.Moreover, CFNPs are well known for their photocatalytic properties, and Fenton-like reactions, consequently, are also applied in the degradation of organic pollutants present in wastewater. [18]Besides, its lowcost production is useful for translational applications.
On the other hand, cross-linked enzyme aggregates (CLEAS), first described in the early 2000s, are formed by a simple and low-cost process involving the enzyme precipitation into a water-miscible solvent, a high concentration of salts or nonionic polymers, and then the physical aggregates are crosslinked.The process results in high enzyme loading with preserved pre-organized superstructure and, thus, enzymatic activity.However, CLEAS technology faces challenges like mass transfer limitations and mechanical stability that could be addressed by recoverable magnetic CLEAS (m-CLEAS).The preparation of m-CLEAS involves the precipitation of the enzyme in the presence of MNPs and a cross-linking with a bifunctional linker.The m-CLEAs have lower particle sizes than standard CLEAS, and show high activity and recoverability, offering a unique amalgam of biotransformation and downstream processing. [19]arious reports explain the immobilization of laccase on magnetite nanoparticles (MNPs) via covalent conjugation, coentrapment in mesoporous structures, or magnetic hierarchical structures to obtain both activity and magnetic properties. [8,12,20]lthough these methods provide improved stability under variable conditions, however, decrease in activity has been found in most of the cases.Moreover, the reproducibility of such immobilization techniques is another challenge.On the other hand, reports on m-CLEAS or even CLEAS of laccase are scarce.CLEAS of laccase from Coriolopsis polyzona was synthesized for the transformation of endocrine disruptor compounds. [21]Sheldon and group synthesized CLEAS of laccase from three different sources: Trametes versicolor, Trametes villosa, and Agaricus bisporus with improved stability. [22]Laccase from Coriolopsis polyzona was immobilized as CLEAS using chitosan as a crosslinker. [23][26][27] Recently, a comparative analysis of CLEAS and m-CLEAS of different enzymes, including laccase on maghemite nanoparticles, was performed, highlighting the better stability of m-CLEAS. [28]n this work, m-CLEAS of laccase of Coriolopsis gallica are synthesized using CFNPs as magnetic core and analyzed for catalytic activity, stability at variable conditions, and recyclability for its potential application in industrial biotechnological processes.The efficiency of m-CLEAS is compared with free enzyme and standard non-magnetic CLEAS.The obtained nanometric m-CLEAS showed a superior enzyme loading with more than 100 % storage stability after 30 days at 4 °C and room temperature, probably the highest reported so far.Furthermore, the applicability of m-CLEAS in degrading endocrine disruptors, dangerous contaminants from industrial wastewater is also analyzed.To the best of our knowledge, this is the first study to highlight the potential of m-CLEAS of laccase on copper ferrite nanoparticles for industrial bioprocessing.

Synthesis and characterization of CFNPs
The core CFNPs were synthesized by one-step solvothermal synthesis due to the known advantages like easy scale-up production, use of less harmful reagents, high yield, and good size control.The X-ray diffraction (XRD) depicted a crystalline structure with the diffraction peaks at 30.2°(220), 35.5°(311), 43.4°(400), 57.2°(511), 62.8°(440) and 74.2°(533) corresponding to the cubic phase of CuFe 2 O 4 (JCPDS PDF 025-0283) with slight impurities of tetragonal phase (JCPDS PDF 034-0425) (Figure 1a).The TEM analysis showed the crystalline structure with spherical morphology forming an ordered cluster-like structure, attributed to the magnetic attractions of the particle (Figure 1 b).The size analysis of over 100 nanoparticles gave the average diameter as ~8 nm (Figure 1c), which is in the similar size range (~12 nm) reported earlier. [29]Figure 1d shows the magnetic separation of nanoparticles in aqueous suspension.

Synthesis and characterization of CLEAS and m-CLEAS
The CLEAS were synthesized by first precipitating the enzyme and then crosslinking with glutaraldehyde (GA).It is well reported that the enzymes could be precipitated using watermiscible organic solvent or by salt saturation of the solution. [19]hen salt is used, the binding interaction of kosmotropic ions with water becomes higher than water itself, increasing the surface tension of the solution and, in turn, competing with the protein surface for water hydration.The protein molecules selfassociate and precipitate since there is less water left to hydrate the protein surface.On the other hand, organic solvents lower the solution's dielectric constant, becoming a less effective solvent for the protein.Thus, the increase in the proteinÀ protein interaction results in protein precipitation.The efficiency of different precipitating agents was tested in the absence of CFNPs, where ammonium sulfate gave the maximum activity recovery of ~70 %, while acetone resulted in ~58 %.The other organic solvents gave less than 50 % activity recovery (Figure 2a).However, in the full process for the formation of the m-CLEAS in the presence of CFNPs and crosslinking with glutaraldehyde, the preparation precipitated with saturated ammonium sulfate showed very low activity recovery (~4 %) upon magnetic separation.On the contrary, non-magnetic CLEAS was obtained in a high yield with ~60 % activity recovery.This could be due to the aggregation or incompatibility of CFNPs in high salt concentrations, leading to poor attraction between laccase and nanosupport.Therefore, acetone with the second highest activity recovery, was chosen as a precipitating agent for synthesis.Acetone is widely used in various industrial applications, including pharmaceutics, cosmetics, paints and coatings, plastics, rubber, electronics, textiles, etc.Its uses are diverse due to its solvency, volatility, and ability to dissolve a wide range of substances.On the other hand, ethanol as a precipitating agent gave enzyme aggregates with ~45 % activity recovery and could also be used as an alternative.
After precipitation, the preparation was cross-linked with glutaraldehyde.Glutaraldehyde (GA) is a universal homodimeric linker and binds with the amine groups at neutral pH.It is more effective than other aldehydes at producing thermally and chemically durable crosslinks. [19]Nevertheless, an overcrosslinking can lead to an irreversible enzyme deactivation.Thus, to ensure catalytic stability, the effective concentration of GA is therefore crucial.It has been found that 5 mM GA concentration is enough to saturate the superficial amino groups of physical aggregates and retain the tertiary structure of the protein. [22,25]The concentrations higher than 10 mM showed a loss of enzymatic activity due to the reduction of molecule flexibility caused by extensive cross-linking. [22]In this work, CLEAS and m-CLEAS were synthesized by acetone precipitation and cross-linking with 6 mM GA for a reaction time of 16 h at 4 °C (Figure 2b).Different mass ratios of CFNPs:Laccase were tested (1 : 2, 1 : 3, and 1 : 6), and the activity recovery was evaluated.The activity recovery of m-CLEAS produced with 1 : 2, 1 : 3, and 1 : 6 ratios was ~52 %, ~67 %, and ~97 % with ~50 %, ~40 %, and ~29 % immobilization efficiency, respectively (Figure 3a).The immobilization efficiency was estimated by protein concentration determination by Bradford assay.The non-magnetic CLEAS showed ~64 % immobilization efficiency and ~55 % activity recovery.Interestingly, the percentage activity recovery of 1 : 3 and 1 : 6 m-CLEAS was higher than the immobilization efficiency, suggesting enhanced relative activity of the enzyme in lower concentrations.This could be due to the Cu 2 + ions supply from the CFNPs support to the laccase molecules, increasing the enzyme activity.The enzyme loading was estimated as 1, 1.2 and 1.75 g of enzyme per g of magnetic nanoparticles from 1 : 2, 1 : 3 and 1 : 6 mass ratios, respectively.Wang et al. observed an opposite trend where increasing the mass ratio of MNPs to laccase showed higher activity. [27]On the other hand, Muthuvelu et al. analyzed the effect of Cu 2 + ion concentration on the activity of laccase, and 15 mM CuSO4 was found to increase the relative activity by 16 %, while at higher concentrations, the activity decreased.[13] Thus, it is anticipated that 1 : 6 mass ratio of CFNPs:Laccase provides an appropriate copper ions concentration for the activity enhancement.Furthermore, comparing the findings reported in the earlier literature, the achieved loading of enzyme per g of support was superior in this work (Table 1).Even though all the tested ratios of CFNPs:Laccase gave good results in terms of enzyme loading and activity recovery, 1 : 6 ratio with the best activity was chosen for further analysis.

Kinetic constants
The Michaelis-Menten constant, K m is a fundamental constant in enzyme catalysis that illustrates the affinity of the enzyme for the substrate.Using Lineweaver-Burk plots, the K m and maximal enzyme-catalyzed reaction rate (V max ) of free laccase, CLEAS, and m-CLEAS were estimated (Figure 3b).Table 2 shows the kinetic parameters obtained for different preparations.As  expected, the enzyme immobilization increased the affinity constant (K m ) with a greater extent in CLEAS.However, the V max of m-CLEAS was ~2.6-fold higher than free laccase, while CLEAS showed a similar maximum transformation rate.The catalytic efficiency (k cat /K m ) of m-CLEAS was ~1.4-fold higher than free enzyme, and CLEAS showed a ~3.6-fold decrease in catalytic efficiency compared to laccase.Our results indicate that CFNPs seem to be a better candidate as solid support for laccase immobilization, as the copper ions from the crystal lattice could strongly induce laccase activity.A non-magnetic bioconjugated laccase on CuO nanocages exhibited up to 18-times higher catalytic rate than the free enzyme. [16]In addition, Muthuvelu et al. also found an increase in catalytic efficiency of laccase covalently conjugated to CFNPs compared to MNPs as solid support due to the presence of copper ions in the crystal lattice of CFNPs. [13]Patel et al. reported the covalent conjugation of laccase (R. vernicifera) on amine-functionalized copper containing magnetic nanoparticles (Cu/Fe 2 O 4 NPs) with 140 % activity recovery and observed ~1.4-fold higher V max of immobilized enzyme. [17]In the case of m-CLEAS, Kumar et al. reported ~4.5fold higher catalysis of m-CLAES of laccase (T.versicolor) on MNPs resulting in 8-fold higher catalytic efficiency. [25]][28] However, these values should be taken cautiously because they are highly dependent on the conditions in which the enzymatic reactions were evaluated.

Physicochemical characterization of m-CLEAS
The TEM analysis of m-CLEAS (1 : 6) showed the presence of nanometric clusters of nanoparticles with a layer of organic coating corresponding to protein (Figure 4a).The analysis of at least 100 aggregates gave an average size of ~239 nm (Figure 4b).As the magnetic nanoparticles show a propensity to aggregate, the < 10 nm size of CFNPs obtained by solvothermal synthesis was useful for synthesizing m-CLEAS clusters in the nanometric range.The m-CLEAS are generally obtained in the μm range. [19]The nanometric size is advantageous due to the higher surface area for catalytic reactions, which could also contribute to the higher catalytic efficiency obtained for m-CLEAS in this work.The zeta potential analysis showed an increase in the negative surface charge of laccase after immobilization as CLEAS and m-CLEAS (Figure 4c).

Thermal stability
The thermal stability of biocatalysts was analyzed after 1 h incubation at pH 4.5 and variable temperatures of 25, 30, 40, and 70 °C.CLEAS and free laccase showed a similar trend in catalytic activity with 100 % at 40 °C and showed a significant decrease at higher temperatures (Figure 5a).Interestingly, the m-CLEAS was stable for a wider window of temperatures retaining ~100 % activity at 30 and 40 °C, which decreased to only to ~88 % at 50 °C and to ~30 % at 70 °C, showing higher thermostability than free laccase.This higher thermal stability of m-CLEAS is promising for bioremediation and industrial bioprocessing.The increase in thermostability can be attributed to the higher rigidity obtained after enzyme immobilization by cross-linking. [19,25,27] activity profile The active sites of enzymes are composed of ionizable groups that must be in the proper ionic form for the conformation of the active site.This conformation should be maintained to allow the substrate binding and its catalytic transformation.Thus, it is unsurprising that pH influences the rate of enzymecatalyzed reactions.The pH profile of free enzyme, CLEAS, m-CLEAS was determined by 1-hour incubation at different pHs.
The stability of enzymes in pH is linked to the origin of the enzyme.Fungal laccases such C. gallica laccase used in this work exhibit maximum catalytic properties at a pH range between 3.5 and 5, while plant laccases have an optimal pH of around 7. [14,32] The pH effect assay demonstrates the good activity of biocatalysts in acidic conditions where a maximum relative activity was observed at pH 4 (Figure 5b).At pH 3, the free laccase and m-CLEAS showed ~56 % of the maximal activity, and CLEAS showed ~80 %.At pH 5, CLEAS showed ~87 %, m-CLEAS ~73 %, and free enzyme showed ~62 % of the maximal activity.After pH 5 the activity of all the preparations decreased significantly.The trend of pH activity profile of all preparations was like free enzyme, but still, slightly higher activity was found after immobilization at pH 5.

Storage stability
The production cost of enzymes has an evident impact on the economics of industrial enzymatic bioprocesses.Therefore, it is imperative to determine how long industrial enzymes may be stored without losing their catalytic activity.A crucial advantage of CLEAS over free enzymes is their storage stability, as free enzymes can rapidly lose their activity.The free laccase, CLEAS, and m-CLEAS were incubated at both 4 °C and room temperature (22 °C) for 30 days, and their residual activity was analyzed once a week.At room temperature (RT) a decrease in activity of ~50 % for free laccase, ~17 % for CLEAS, and only ~3 % for m-CLEAS was observed during approximately the first 10 days of incubation.In the fourth week, the downward trend in the activity from free laccase and CLEAS was seen reaching only ~12 % of the initial activity.Exceptionally, m-CLEAS showed an increase of ~11 % activity.At 4 °C after the first 10 days, the activity of free laccase and CLEAS decreased by ~10 and 31 %, respectively, while m-CLEAS increased by ~8 %.In the fourth week, free laccase showed ~26 %, and CLEAS showed ~52 % residual activity.The m-CLEAS showed an improvement with ~137 % of initial activity.Overall, the decrease in the activity of free laccase and CLEAS occurred in a lower percentage at 4 °C.Importantly, the activity of m-CLEAS improved to a greater extent at 4 °C.The storage stability of m-CLEAS of this work was superior to the m-CLEAS of laccase on magnetite and maghemite nanoparticles (Table 3).
Laccases are multicopper oxidases containing four catalytic copper atoms involved in the oxidation substrates.The catalytically active sites of laccases contain four copper ions that are located in three sites: one in T1 site, one in the T2 site and two copper in the T3 site.The T1 copper ions are tightly bound and T2 ions are relatively unstable.The loss of T2 ions can occur during the purification process or during storage for a few days.It has been reported that the removal of T2 ion inactivates the enzyme, and its reconstitution could be achieved after incubation with Cu 2 + ions.Thus, it is anticipated that the increase in activity after 4 weeks of storage could be due to the copper ions supply to the T2 active site from the copper ions released from nanoparticles. [13,33]

Recyclability
The capacity to recycle biocatalysts in industrial bioprocessing can result in considerable cost savings, improved productivity, and more environmentally friendly and sustainable production methods.The reusability of m-CLEAS was investigated by magnetic separation of the biocatalyst from the reaction mixture and reusing it for another round of reaction.A total of ten cycles of reaction were monitored for oxidation of ABTS at pH 4.5 and room temperature.After the third cycle, the recovered activity slightly decreased to ~85 %, while it retained 78 % activity after 5th cycle of reuse (Figure 5e).Overall, a gradual decrease in activity was observed in consecutive cycles and was found to be more significant after 5th cycle, finally retaining up to ~54 % residual activity after 10 cycles.The decreased activity after reuse could be attributed to enzyme deactivation due to the accumulation of reaction products or physical damage due to the mechanical stress caused by shaking and repeated washings, or partial denaturation of the enzyme due to repeated use.Earlier, the laccase functionalized on CFNPs was recycled and reused up to 6 effective cycles, which is comparable to this work. [13]On the other hand, m-CLEAS of laccase on MNPs were effectively used for up to 4 cycles. [26,27]Contrary, Kumar et al. showed higher stabilization, retaining ~86 % of the initial activity in m-CLEAS of laccase immobilized on MNPs after 10 cycles. [25]n the other hand, reports suggest that the immobilization of laccase on non-magnetic support shows lower activity recovery after 5 cycles compared to m-CLEAS as they are recovered by centrifugation. [8,33,34]Centrifugal force can cause aggregation of the various forms of immobilized laccase, resulting in decreased activity and accentuating mass transfer limitations.Therefore, magnetic nanoparticles provide an added advantage for the facile reusability of biocatalysts.

Transformation of endocrine disruptor compounds
As a proof of concept, the application of developed m-CLEAS is tested for the degradation of endocrine disruptor compounds (EDC).These molecules can negatively affect the normal operation of the endocrine system, which might have an impact on both human and animal development, reproduction, and other hormonal functions.These disruptors are often synthetic compounds with estrogenic activity that may be found in various products.For example, Bisphenol A (BPA) is used for the synthesis of different plastics, Triclosan (TCS) is used as an antimicrobial additive in plastics and cleaning products, and Nonylphenol (NP) is used in the production of industrial chemicals like antioxidants, lubricating oil and mainly surfactants.While 17β-estradiol (E2) is a natural hormone, however, its exposure at high levels may cause disruptions in the endocrine system.Its extensive use as synthetic estrogens in pharmaceutical companies can cause water contamination, significantly affecting aquatic life. [35,36]Laccase has been earlier used for the transformation of EDCs to polymeric derivatives, reducing the endocrinic effect. [35]Thus, m-CLEAS of laccase are This work also analyzed for their application in EDC degradation and compared with the free enzyme.
Free laccase and m-CLEAS were incubated with BPA, NP, E2 and TCS in acetate buffer (pH 4.5), having 25 % acetonitrile for 20 min at room temperature.The reaction medium turned turbid during the reaction, which was centrifuged and the percentage transformation was analyzed in the supernatant by HPLC.The m-CLEAS could transform EDCs at a significantly higher rate than free laccase, while the transformation of TCS was non-significant until 20 min incubation time (Figure 6).The estimated transformation rates were similar for BPA, E2 and NP with values of 0.23 U/mg, 0.3 U/mg, and 0.32 U/mg, respectively, where U represents μM/min.The extent of transformation for BPA, E2 and NP was ranging from ~45-60 % with m-CLEAS and ~30-45 % with free laccase, supporting the potential of m-CLEAS for degradation of EDC contaminants.The transformation efficiency of m-CLEAS was ~1.5 fold higher than free laccase, which is a similar trend found in enzyme kinetics analysis.However, the rate of reaction was slower in TCS.Within 20 minutes, m-CLEAS could transform ~13 % (0.07 U/mg) while laccase transformed ~7 % (0.04 U/mg).A similar trend was also seen in the earlier study where EDCs were transformed using laccase.The catalytic efficiency (k cat /K m ) of laccase for BPA, E2, NP was analogous, ranging from 20-42 s À 1 mM À 1 , while it was 1.5 s À 1 mM À 1 for TCS, which is one order of magnitude lower than other EDCs. [35]Overall, the developed m-CLEAS showed higher efficiency in transforming EDCs than free enzyme.

Conclusions
The enzymatic processes are unbeatable in terms of green chemistry.Enzymes perform catalytic transformations in aqueous media, mild reaction conditions of temperature and pressure, and low reagents concentrations, essential requirements for sustainable industrial bioprocesses.However, the extended use of natural biocatalysts is limited by the lability of proteins.Here, we have designed and produced highly stable and reusable magnetic crosslinked enzyme aggregates (m-CLEAS) of laccase by co-aggregation of the enzyme with magnetic copper ferrite nanoparticles (CFNPs) and crosslinking using glutaraldehyde with nanometric dimensions.The m-CLEAS showed improved catalytic performance and thermal and storage stabilities than the free enzyme and standard CLEAS.In addition, the magnetic properties of the biocatalyst permit its facile recovery from the reaction medium just using a magnetic field.The improved properties could be attributed to the copper ions supply from copper-containing magnetic support (CFNPs), which seems to act as an activity inducer for laccase regenerating the enzymatic activity lost due to the loss of copper ions in the active site.These results suggest that m-CLEAS have the potential for various sustainable bioprocesses, which was evidenced by the higher efficiency in degrading potential endocrine disruptors compared to free laccase.Moreover, the facile and cost-effective synthesis could have a significant impact on enzymatic catalysis in industrial applications.

Protein and Reagents
The laccase of Coriolopsis gallica (EC 1.10.3.2) was obtained and purified by the standardized protocol reported previously. [16,37]To prevent Cu atom losses in the active site, the laccase was stored in the buffer containing 10 μM CuSO

Material characterization
To determine the crystalline structure of the synthesized CFNPs, Xray diffraction analysis was performed on PANalytical AERIS with Xray radiation Cu Kα (λ = 0.15406 nm).The morphology of the synthesized particles was determined by transmission electron microscopy (Hitachi, H-7500) while the analysis was performed using copper grids (100-mesh) coated with formvar/carbon support film (Ted-Pella, USA).UV-Vis analysis of the samples was conducted on spectrophotometer (Perkin Elmer Lambda 25 UV/vis).Hydrodynamic radii and the zeta potential of the synthesized particles was determined by Zetasizer Nano ZS (Malvern Panalytical).

Synthesis of copper ferrite nanoparticles (CFNPs)
The copper ferrite nanoparticles (CFNPs) were synthesized via one step solvothermal method according to the previously reported with minor changes. [29]Briefly, FeCl 3 • 6H 2 O (0.54 g), CuCl 2 • 2H 2 O (0.17 g) and sodium acetate (0.75 g) were added to ethylene glycol (25 mL) and deionized water (10 mL) and kept under stirring for 2 h.Then the homogenized solution was transferred into a Teflon sealed hydrothermal chamber and placed in the oven at 180 °C for 24 h.After the incubation, the precipitate was magnetically separated from the solution and washed three times with deionized water and two times with 70 % ethanol.Finally, the powdered nanoparticles were obtained by drying at 100 °C.

Synthesis of cross-linked enzyme aggregates (CLEAS)
The synthesis of CLEAS was optimized by using different precipitating agents like ammonium sulphate, acetone, acetonitrile and ethanol.A solution containing precipitating agent (1.2 mL) and glutaraldehyde (4 μL, 25 % w/v H 2 O) was placed under constant stirring on an ice bath.Then a laccase solution (400 μL, 1 mg/mL) was added dropwise to the above mixture and the mixture was kept under stirring for 2 h at room temperature, and overnight at 4 °C.Thereafter, the prepared CLEAS were collected by centrifugation (13,000 rpm at 4 °C for 30 min) and washed thrice with deionized water.The final synthesis was performed using acetone and to the obtained CLEAS were stored in 70 % acetone (400 μL) and kept at À 20 °C until use.The final synthesis was performed in triplicates.

Synthesis of magnetic CLEAS (m-CLEAS)
The m-CLEAS were synthesized like the non-magnetic CLEAS.The synthesis was optimized by varying the w/w ratio of CFNPs:Laccase (1 : 2, 1 : 3, and 1 : 6) to obtain the best catalytic activity.The protein precipitation was done using acetone (70 % in total reaction volume).The CFNPs were resuspended in water using ultrasonicator.The mixture of laccase and CFNPs in deionized water was prepared and again sonicated in water bath sonicator for 15 min, and kept in ice cold conditions.The ice-cold acetone was then added dropwise with constant agitation.After 30 min, glutaraldehyde (6 mM in final reaction volume) was added for crosslinking the protein aggregates.The reaction mixture was left under stirring for 2 h at room temperature, and overnight at 4 °C.Finally, the prepared m-CLEAS were washed thrice with deionized water by magnetic separation.The m-CLEAS were stored at 4 °C after resuspending it with deionized water.The synthesis was performed in triplicates.

Determination of enzyme activity
The catalytic activities of free laccase, CLEAS and m-CLEAS were determined spectrophotometrically by the oxidation of ABTS (ɛ436 = 29,300 M À 1 cm À 1 ).The catalytic reaction was carried out in 1 mL reaction mixtures containing ABTS (500 μM) in acetate buffer (0.05 M, pH 4.5) at room temperature.The reaction was initiated by addition of respective sample (20 μL, 0.1 mg/mL) and the extent of transformation was monitored at 436 nm using an Agilent 8453 UVvis spectrophotometer.The amount of enzyme required to oxidize 1 μmol of substrate per minute was used to define one unit of enzyme activity.The kinetic constants of maximum reaction rate (V max ) and Michaelis-Menten (K m ) were obtained by varying the concentration of ABTS (0.002 to 8 mM) and fitting the data on a Michaelis-Menten equation.
The percentage enzymatic activity recovery in CLEAS and m-CLEAS was determined by using Equation (1): [38] % Activity recovery ¼

Protein immobilization efficiency
The protein immobilization was estimated by Bradford assay using bovine serum albumin (BSA) as a standard.The protein concentration was directly determined for free enzyme and CLEAS.However, for m-CLEAS the protein concentration was estimated as the unbound protein in the supernatant ([P] s ) and then subtracted from the total amount of protein initially used in the reaction for the synthesis ([P] 0 ). [8,26]The samples (100 μL) were mixed with BioRad reagent (900 μL) in a plastic cuvette and after 10 min the absorption was measured at 595 nm using UV-Vis spectrophotometer.The protein concentration was determined by the interpolation from a calibration curve prepared with bovine serum albumin (BSA).The immobilization efficiency was then estimated by Equation ( 2): where [P] f is the amount of protein in aggregates ([P] 0 -[P] s ), and [P] 0 is the initial protein amount in the immobilizing reaction.

Stability studies (i) Storage stability
The free laccase, CLEAS and m-CLEAS were stored in sodium acetate buffer (0.1 M, pH 4.5) at two different temperatures: 4 °C and at room temperature.The catalytic activity of the samples was tested at 7-days interval for at least 30 days.To estimate the residual activity, the activity of each sample at day 0 was considered as 100 %.

(ii) Effect of pH
To determine the influence of pH, the samples and the free enzyme were incubated for 1 hour in buffer solutions of different pH values, at room temperature.Sodium acetate buffer (0.1 M) was used for pH values 3-5, modifying the pH with acetic acid, while phosphate buffer (0.1 M) was used for pH 6 and 7.After that, the samples were tested to determine the relative laccase activity, using 100 % as the maximal activity.

(iii) Effect of temperature
The effect of temperature on samples and free enzyme was determined by incubating all samples in a buffer solution of sodium acetate (0.1 M pH 4.5) for 60 minutes, without substrate, at 25, 30, 40, 50 and 70 °C.The residual activity was calculated by considering 100 % activity of the respective samples at room temperature at time 0.

Recyclability
The residual activity of m-CLEAS samples was evaluated for up to 10 cycles of recycling.The 5 mL reaction mixture contained m-CLEAS (1 mg) and ABTS (500 μM).The reaction was incubated under mechanical stirring for 30 min and the residual activity was estimated considering the 100 % as the initial activity.After each cycle, the m-CLEAS were separated from reaction using a magnet and washed with acetate buffer three times prior to use in next cycle with a fresh substrate solution.

Figure 1 .
Figure 1.Characterization of magnetic CFNPs: (a) X-ray diffraction analysis; (b) TEM micrographs in panoramic and high-resolution view; (c) Size distribution analysis of at least 100 nanoparticles; (d) the photo of the magnetic separation of nanoparticles from aqueous medium.

Figure 2 .
Figure 2. Synthesis of CLEAS and m-CLEAS.(a) Effect of different precipitating agents on the recovered activity of CLEAS.The standard bars derived from analysis in duplicates; (b) schematic diagram of synthesis of m-CLEAS.

Figure 3 .
Figure 3. (a) Effect of different mass ratios of CFNPs: Laccase on activity recovery and immobilization efficiency; (b) Lineweaver-Burk plots of free laccase, CLEAS and m-CLEAS.The standard bars are derived from analysis in triplicates.

Figure 5 .
Figure 5. Stability analysis of free and immobilized laccase.(a) Thermal stability; (b) pH activity profile; (c) storage stability at 4 °C; storage stability at room temperature; (e) recyclability.Standard deviation obtained from analysis in triplicates.

Table 1 .
Comparison of enzyme loading and activity recovery of laccase immobilized in different magnetic supports.

Table 2 .
Kinetic parameters of free and immobilized laccase for the oxidation of ABTS at pH 4.5, 22 °C.

Table 3 .
Comparison of storage stability of different m-CLEAS of laccase.