Instantaneous synthesis of organic-inorganic laccase-cobalt phosphate hybrid nanoflowers: Structural and biocatalytic characterization

Instantaneous synthesis of organic-inorganic laccase-cobalt phosphate hybrid nanoflowers: Structural and biocatalytic characterization | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Instantaneous synthesis of organic-inorganic laccase-cobalt phosphate hybrid nanoflowers: Structural and biocatalytic characterization Khashayar Vojdanitalab, Hossein Jafari-Nodoushan, Somayeh Mojtabavi, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1351173/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Organic-inorganic hybrid nanoflowers (HNFs) have been synthesized by soft biomineralization procedures and are mainly used in biocatalysis and biosensing. Previously-reported methods for the synthesis of HNFs have so far required a 3-day incubation, bath sonication, or shear stress tension, which possibly introduces damage to the organic component. In this study, a novel method for instant fabrication of laccase@Co 3 (PO 4 ) 2 •HNFs was developed without using harsh conditions. The prepared HNFs were assembled instantly by the “concentrated method,” which resulted in the fast growth of the flower-shaped nanostructures by a higher collision rate of the primary nucleation sites. The obtained results indicated that catalytic efficiency and enzymatic activity of laccase@Co 3 (PO 4 ) 2 •HNFs were 113% and 110%, respectively, compared to the free enzyme. Also, the stability of the immobilized enzyme was enhanced by 400% in basic pH values. The activity of laccase@Co 3 (PO 4 ) 2 •HNFs declined to 50% of the initial value after 10 reusability cycles, indicating successful immobilization of the enzyme. Structural studies revealed a 32% increase in the α-helix content after hybridization with cobalt phosphate, which improved the activity and stability of the immobilized laccase. Furthermore, the fabricated HNFs exhibited a considerable ability to remove moxifloxacin as an emerging pollutant. The antibiotic (10 mg/L) was removed by 24% and 75% after 24 h through adsorption and biodegradation mechanisms, respectively. This study introduces a new method for synthesizing HNFs, which could be used for the instant fabrication of efficient biocatalysts, biosensors, and adsorbents to be potentially employed in industrial, biomedical, and environmental applications. Bioremoval Enzyme immobilization Hybrid nanoflowers Laccase Moxifloxacin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Over the past two decades, various heterogeneous biocatalysts, immobilized enzymatic active substances, have been developed for environmental and industrial applications 1 . The preparation of new platforms based on organic-inorganic hybrid materials for the immobilization of enzymes is of great interest. Organic-inorganic hybrid nanoflowers (HNFs), the hierarchical three-dimensional nanostructures that were initially discovered in 2012 2 . HNFs, composed of enzyme(s) and inorganic component(s), have a large surface area, facile synthesis procedure, and hierarchical porous structures 3 . After incorporating enzymes into HNFs, their activity, stability, and reusability have notably increased, owing to low mass transfer limitations arising from their high surface-to-volume ratio and cooperative effects between enzymes and metal ions 4 . For instance, the incorporation of lipase into Zn 3 (PO 4 ) 2 •HNFs resulted in a 147% enhancement of the enzyme activity. Also, the storage stability of the lipase-containing HNFs was three times higher than free lipase 5 . Carbonic anhydrase (CA) was successfully immobilized into Ca 8 H 2 (PO 4 ) 6 •HNFs and exhibited remarkable stability against elevated temperatures (30–80°C). CA@Ca 8 H 2 (PO 4 ) 6 •HNFs retained 100% of their initial CO 2 hydration activity after 5 reusability cycles 6 . Accidental addition of copper sulfate to phosphate buffer saline (PBS) in the presence of bovine serum albumin (BSA) resulted in the synthesis of the flower-shaped BSA@Cu 3 (PO 4 ) 2 •HNFs 2 . Three approaches have been reported for the preparation of HNFs, including one-step precipitation 2 , ultrafast sonication 7 , and shear stress-mediated synthesis 8 . In the one-step precipitation method, a facile and straightforward strategy, fabrication of HNFs requires a long incubation time (1 ~ 3 days). As long synthesis procedure may potentially reduce the stability of the organic constituent, serious attempts have been established to shorten the assembling time. A rapidly ultrafast sonochemical synthesis of laccase@Cu 3 (PO 4 ) 2 •HNFs was reported using sonication of the reaction mixture within 5 min at room temperature 7 . Introducing shear stress to the organic component and inorganic precursors to synthesize copper-based HNFs was recently reported 8 . Considering the fact that HNFs are at the early stages of development, the formation mechanism of HNFs has not been accurately established. However, it is believed that the formation of primary nucleation sites of metal phosphate(s) and organic molecule(s) (e.g., protein, DNA, etc.) and time-dependent anisotropic growth of these primary nucleation sites 9 are responsible for the formation of the flower-like structures. It seems that by introducing kinetic energy to the precursors, provided by ultrasonication and vortexing, HNFs assembling time was reduced. As efficient biocatalysts, HNFs have been used for the removal of emerging contaminants such as industrial dyes. However, the ability of HNFs for the removal of antibiotics have not been reported previously 4 . Moxifloxacin, as a fourth-generation fluoroquinolone (FQ), is responsible for more than 34.6% of the total FQs consumption in China. It is mostly used for the treatment of pneumonia and skin infections. Recently, due to its usage in severe acute respiratory syndrome coronavirus 2, moxifloxacin consumption has temporarily increased. Therefore, consumption, release, and accumulation of moxifloxacin in the environment may be threatening 10 . Moxifloxacin is also the most toxic FQs against the growth of Pseudokirchneriella subcaptitata 11 . Likewise, it exhibited significant negative effects on the growth and reproduction of Ceriodaphnia dubia and Daphnia manga 12 . Therefore, increased consumption and release of moxifloxacin in the environment may be threatening to the ecosystem and human health. The efficiency of current approaches for the elimination of antibiotics from wastewaters is limited 13 . In this regard, various techniques for the removal of antibiotics have been so far established such as biocatalysis 14 , photocatalysis 15 , electrocatalysis 16 , etc. Laccases, an oxidoreductase enzyme, has been broadly used for environmental and industrial applications 17 , 18 . Free and immobilized laccases have been incorporated for bioremoval of a wide range of pollutants such as bisphenol A, crystal violet, acid orange-7, levofloxacin, etc. 19 – 22 In this study, a novel method for the fast synthesis of HNFs was proposed without incorporating kinetic energy. Laccase@Co 3 (PO 4 ) 2 •HNFs were instantly synthesized through rapid anisotropic growth of laccase-cobalt phosphate nanocrystals, and a new mechanism was proposed for the synthesis of HNFs. Biocatalytic properties of laccase@Co 3 (PO 4 ) 2 •HNFs were investigated, and the structure of the hybridized enzyme was also characterized. The synthesized HNFs were then employed to remove moxifloxacin from aqueous media. The degradation products were identified, and their toxicity against four bacteria involved in the degradation of organic compounds was assessed. 2. Experimental 2.1. The enzyme and chemicals Laccase from Trametes versicolor (0.5 U/mg) and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were purchased from Sigma-Aldrich (St. Louis, Mo, USA). Cobalt (II) chloride hexahydrate (CoCl 2 .6H 2 O) was obtained from Merck (Darmstadt, Germany). All other applied reagents and chemicals were of analytical grade without further purification. Moxifloxacin was kindly gifted from Soha Pharmaceutical Company (Tehran, Iran). 2.2. Instant synthesis of cobalt-based hybrid nanoflowers with laccase Regardless of the conventional methods, an instant strategy was applied for the construction of HNFs. The synthesis procedure was accomplished by the addition of 0.1 mL of CoCl 2 solution into 0.9 mL of phosphate buffer (pH 7.4) containing 0.1 U mL − 1 of laccase. Instantly a large number of purple precipitates appeared in the solution. Proper concentration of CoCl 2 and molarity of phosphate buffer were subsequently optimized against highest activity recovery (n = 3, p -value < 0.05). The precipitates were centrifuged at 6000 g and washed three times with phosphate buffer. The purple products were marked as laccase@Co 3 (PO 4 ) 2 •HNFs. 2.3. Characterization of laccase@Co 3 (PO 4 ) 2 •HNFs Scanning electron microscopy (SEM) images (Tescan, MIRA II, Czech Republic) were applied to indicate the flower-shaped morphology of the fabricated HNFs. Energy dispersive X-Ray spectroscopy (EDX, Tescan, MIRA II, Czech Republic) and elemental mapping were incorporated to determine the composition and spatial distribution of elements in the constructed HNFs, respectively. Fourier transform infrared (FTIR) spectroscopy (Shimadzu, Equinox 55, Japan) was utilized for analyzing functional groups of organic and inorganic constituents of HNFs. The samples were dispersed in pressed KBr disks, and the spectra were recorded at 4000–400 cm − 1 . Brunauer-Emmett-Teller (BET) analysis was performed (Microtrac, BELSORP MINI X, Japan) after degassing the samples under N 2 . X-ray diffraction (XRD) analysis was carried out to identify the crystalline phase of the inorganic component using an X-ray diffractometer (Philips, PW1730, Philips, Eindhoven, Netherlands). 2.4. Laccase activity assay Laccase activity was spectroscopically measured following by oxidation of ABTS as the substrate 23 . Laccase@Co 3 (PO 4 ) 2 •HNFs and the free enzyme were incubated with ABTS (50 µM, 1 mL) at 40°C in a shaker incubator. After 15 min incubation, the optical density of the supernatants was recorded at 420 nm. One unit of activity was defined as the amount of laccase capable of oxidizing 1 µmol ABTS to the colored product in 1 min. Effect of pH and temperature on the activity of both laccase@Co 3 (PO 4 ) 2 •HNFs and the free form of laccase was determined in the temperature range of 25–55°C (interval 10°C) and pH range of 2.5–9.5 (interval 1) (n = 3, p -value < 0.05). 2.5. Determination of immobilization yield and efficiency After preparing laccase@Co 3 (PO 4 ) 2 •HNFs, the concentration of residual protein in the supernatant was estimated by the bicinchoninic acid (BCA) method (n = 3, p -value < 0.05) 24 . Immobilization yield (IY) was calculated using Eq. 1, where Y 0 and Y 1 represent the amount of immobilized enzyme in HNFs before and after the synthesis procedure, respectively (n = 3, p -value < 0.05). IY (%)= [(Y 0 -Y 1 )/Y 0 ]×100 Eq. 1 The immobilization efficiency (IE) was obtained by Eq. 2, where E 0 and E t demonstrate the activity of free laccase and laccase@Co 3 (PO 4 ) 2 •HNFs, respectively. IE (%)= [(E 0 -E t )/E 0 ]×100 Eq. 2 2.6. Evaluation of the kinetic parameters of the constructed HNFs In order to assess the influence of immobilization on laccase, kinetic parameters of laccase@Co 3 (PO 4 ) 2 •HNFs and the free laccase were evaluated. The activity of both the free and immobilized biocatalysts was determined in the presence of ABTS concentrations (20–100 µM). Lineaweaver-Burke plot was incorporated for evaluation of kinetic parameters such as Michaelis constant ( K m ) and maximum velocity ( V max ). Turnover number ( K cat ) and catalytic efficiency were calculated based on Eqs. 3 and 4 25–27 ; where [E] is the concentration of enzyme. K cat = V max /[E] Eq. 3 Catalytic efficiency = K cat / K m Eq. 4 2.7. Reusability and stability studies In order to evaluate the reusability of the constructed biocatalyst, repeated measurement of the activity of laccase@Co 3 (PO 4 ) 2 •HNFs was examined 2 . The fabricated HNFs were incubated in 1 mL phosphate buffer (10 mM, pH 7.4) containing ABTS (0.5 mM) at 40°C for 15 min. After centrifugation of the mixture, OD 420 of the supernatant was recorded, and the precipitates were washed three times with phosphate buffer (10 mM, pH 7.4). The procedure repeated to the point that the activity of HNFs dropped to below 50% of its initial value (n = 3, p -value < 0.05). The stability of the free laccase and HNFs was assessed as a function of recovered activity after 3 h incubation at temperatures ranging 25–55°C (interval 10°C) and a wide pH range of 2.5–9.5 (interval 1). Storage stability of the free laccase and HNFs were obtained by measurement of laccase activity during storage at 4°C (n = 3, p -value < 0.05). 2.8. The enzyme structural studies In order to evaluate the conformational changes of laccase after immobilization, far-UV circular dichroism (CD) and fluorescence spectroscopies were utilized. CD spectra of laccase@Co 3 (PO 4 ) 2 •HNFs and the free enzyme were recorded by a Jasco 725 spectrophotometer in a 2 mm path-length cell, and the absorbance was recorded at 190–240 nm. The fluorescence property of tryptophan is dependent on the 3rd structure of a protein. In this regard, the tryptophan fluorescence intensity of the free laccase and laccase@Co 3 (PO 4 ) 2 •HNFs were recorded by a Hitachi 850 spectrofluorometer after excitation at 285 nm 23 . Finally, thermogravimetry analysis (TGA) was using performed using a thermogravimetric analyzer (Perkin Elmer STA 6000, USA) for evaluation of thermal decomposition of both pure cobalt phosphate and laccase@Co 3 (PO 4 ) 2 •HNFs. The experiment was conducted under an N 2 atmosphere in the temperature range of 25–600°C by a heating rate of 10°C/min. 2.9. Bioremoval experiments 2.9.1. Bioremoval of moxifloxacin Moxifloxacin was incubated with laccase@Co 3 (PO 4 ) 2 •HNFs and 1-hydroxybenzotriazole (HBT) as the laccase mediator in phosphate buffer (10 mM, pH 4.5) at 40°C under stirring condition (100 rpm) 28 . After determining the period of incubation, the concentration of moxifloxacin was quantified by high-performance liquid chromatography (HPLC) coupled with UV detector, and the removal percentage was calculated based on Eq. 5 (n = 3, p -value < 0.05); as C 0 and C t represent the concentration of moxifloxacin before and after removal experiments, respectively. Removal (%)= (C 0 -C t /C 0 )×100 Eq. 5 2.9.2. Sample preparation and quantification of moxifloxacin After the removal procedure, the pH of the reaction mixture was set to 7.95 (isoelectric point of moxifloxacin) by phosphate buffer (100 mM, 7.95). Then, the mixture was extracted with chloroform under vigorous vortex three times after the addition of ciprofloxacin as the internal standard. The extracted chloroform evaporated to dryness by a rotary evaporator instrument, and the remaining powder was dissolved in 1 mL of the HPLC mobile phase. A Knauer HPLC-UV system (Berlin, Germany) was incorporated for moxifloxacin quantification using a PDA 2800 detector, a Smartline 1000 pump, and ChromGate software (version 3.3.1). The mobile phase consisted of methanol (45%) and 10 mM phosphate buffer (pH 3.0) (55%). Samples were injected by Smartline autosampler 3950 to a Eurospher 100 C18 reversed-phase column (250 × 4.6 mm). 2.9.3. Identification of biotransformation products Biotransformation products of moxifloxacin were identified by liquid chromatography coupled by mass spectroscopy (LC-MS). The apparatus was an Agilent 1200 series LC system coupled with an Agilent 6520 quadrupole time of flight tandem MS instrument featured by an electrospray ion source (Agilent, Waldbronn, Germany). A 2.1–100 mm Nucleosil 100 − 3 C18 HD column (Macherey-Nagel, Düren, Germany) was applied with a flow rate of 0.35 mL min − 1 . Deionized water + 0.1% formic acid (40%) and acetonitrile (60%) were used as mobile phase. 2.9.4. Toxicity of the bioremoval procedure Based on previous studies, elimination of toxicity is not guaranteed by degradation of antibiotics in catalytic procedures 29 . Accordingly, the toxicity of moxifloxacin degradation products was examined against some bacteria, including Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC 25,922, Staphylococcus epidermidis ATCC 49,619, and Staphylococcus aureus ATCC 6538, which are responsible for the bioremoval of organic pollutants in the environment. In this regard, the fresh culture of each bacterium was seeded in a nutrient broth and incubated at 37°C overnight. Then, each bacterium was treated with a mixture of degradation products, and the optical density (OD) of each bacterium was evaluated at 600 nm after determining the incubation time periods at 37°C. Growth inhibition of moxifloxacin degradation products, moxifloxacin as the positive control, and sterile water as the negative control were measured by plotting OD 600 against incubation time (n = 3, p -value < 0.05) 22 . 2.10. Analysis of the experiments The experiments were conducted in triplicate, and results were reported as mean ± standard deviation. Statistical significance among the mean value of experimental results was calculated by two-way ANOVA (± standard deviation) and p-value < 0.05 regarded as significant. 3. Results And Discussion 3.1. Instant synthesis of laccase@Co 3 (PO 4 ) 2 •HNFs HNFs have been routinely constructed with the addition of metal ions to enzyme-containing PBS followed by a 3-day incubation at room temperature, vigorous vortexing, or bath sonication, which introduces significant stress to the organic constituent 2 , 7 , 9 , 30 . In the present study, the concentration of CoCl 2 and molarity of phosphate buffer (pH 7.4) were optimized to instantly fabricate HNFs and avoid incorporating stress-inducing conditions in the synthesis procedure. After the addition of CoCl 2 to laccase-containing phosphate buffer, the purple precipitates were immediately formed. The precipitate was washed with deionized water three times and designated as laccase@Co 3 (PO 4 ) 2 •HNFs. As shown in Fig. 1 a, the recovered activity increased gradually at the higher molarity of phosphate buffer. However, the highest activity recovery was obtained with HNFs prepared with 0.16 M phosphate buffer and 30 mM CoCl 2 . Effects of bath sonication (60 kHz, 13.8 W) or incubation at 25°C (24–72 h) on the activity recovery were also evaluated. The results (Fig. 1 b) demonstrated that further incubation reduced the recovered activity. Also, sonication of the reaction mixture (up to 20 min, 5 min intervals) did not increase the recovered activity. The mentioned synthesis method is referred to as the “concentrated method” in the present study for comparison with the up to now reported and traditional procedures. The present explanation of the formation mechanism of HNFs is based on the nucleation of primary metal phosphate-protein nanocrystals and subsequent anisotropic growth of the hybrid nanoaggregates. Due to the high surface energy of the primary protein-inorganic nanocrystals, they tend to attach and form the nano-sheet structures, which finally form the ultimate structure of HNFs 3 . The growth of nucleation sites of HNFs could be facilitated by increasing the collision rate of the primary crystals 31 . It seems that introduction of certain amounts of kinetic energy, provided by long-term incubation (24–72 h at 25°C), bath sonication, microwave heating, and shear stress, to the synthesis mixture (organic component, metal, and phosphate ions) could induce the formation of HNFs. The number of primary nucleation sites increased by incorporating a high concentration of Cobalt (II) and phosphate ions in the reaction mixture. Hence, based on Eq. 6, higher numbers of nanocrystals possibly collide together more frequently, and HNFs were prepared very fast 31 . In Eq. 6, z is collision frequency, D is the particle diameter, ῡ represents the mean velocity of dispersed particles, N is the total number of particles, and V is the total volume of the reaction mixture. z= (√2πD 2 ῡN)×V − 1 Eq. 6 Hybrid nanoflowers have been synthesized in a short period of time through vigorous vortexing 8 and also bath sonication 7 of the precursors, which increased the mean velocity of the dispersed particles (ῡ). Based on Eq. 6, after the increase in the mean velocity of dispersed organic-inorganic particles by vortexing or bath sonication, the collision frequency increases, which results in fast anisotropic growth of the HNFs. 3.2. Characterization of laccase@Co 3 (PO 4 ) 2 •HNFs The flower-shaped morphology of the fabricated HNFs (Fig. 2 a) was confirmed by SEM image. The size of laccase@Co 3 (PO 4 ) 2 •HNFs ranged 0.5–3 µm and was assembled by interconnected nano-sheets resulting high surface-to-volume ratio (Fig. 2 b). Cobalt phosphate nanoflowers (NFs) were also prepared through the same synthetic route without the incorporation of laccase in the reaction mixture. As shown in Fig. S1 and S2, the cobalt phosphate•NFs also exhibited flower-like morphology. Some of the previous studies reported that the flower-shaped morphology of HNFs is due to the presence of inorganic constituent(s) 32 , 33 . Also, it was reported that the 3D structure of the incorporated organic component could alter the morphology of HNFs 34 . However, inorganic copper phosphate was prepared by the same synthesis procedure, which exhibited the same flower-shaped morphology as protein@Cu 3 (PO 4 ) 2 •HNFs 35 – 38 . All in all, it could be concluded that some metal phosphates have flower-like morphology 39 that could be modified after hybridization with proteins. EDX analysis of laccase@Co 3 (PO 4 ) 2 •HNFs and cobalt phosphate nanoflowers are exhibited in Fig. 2 d and Fig. S4, respectively. The weight percentage of elements in both constructed HNFs and cobalt phosphate is summarized in Table S1. The presence of nitrogen atoms in the prepared HNFs confirms the immobilization of laccase into the support. Elemental map analysis (Fig. 2 c) also provides information on the homogenous distribution of Co, P, N, and O atoms in the constructed HNFs. In order to identify the inorganic part of laccase@Co 3 (PO 4 ) 2 •HNF, the XRD analysis was performed. The obtained peaks of laccase@Co 3 (PO 4 ) 2 •HNF were in good agreement with the standard cobalt phosphate pattern (JCPDS 00-027-1120) (Fig. 3 ). Mean pore diameter, total pore volume, and specific surface area of the fabricated HNFs and Co 3 (PO 4 ) 2 •NFs are summarized in Table S2, and corresponding BET plots are shown in Fig. S5. The high specific surface area (17.42 m 2 /g) is in agreement with the nano-sized width of petals in the SEM image of laccase@Co 3 (PO 4 ) 2 . As shown, the specific surface area of Co 3 (PO 4 ) 2 •NFs and the synthesized HNFs was almost identical. The presence of type IV isotherm in the BET plots is due to the mesoporous structure of the synthesized materials. As a rule of thumb, the higher surface area of the immobilized enzyme increases the accessibility of substrate to the enzyme and enhances the biocatalytic efficiency 40 . The same results were reported for laccase@Cu 3 (PO 4 ) 2 •HNFs (10.2 m 2 /g) and GOx@Cu 3 (PO 4 ) 2 •HNFs (17.9 m 2 /g) 41,42 . FTIR spectra of laccase, Co 3 (PO 4 ) 2 , and laccase@Co 3 (PO 4 ) 2 •HNFs are represented in Fig. 4 a. As shown, characteristic vibrational frequencies in the region 725–1300 cm − 1 are due to the presence of phosphate groups 43 . Peaks at 2980–3600 cm − 1 attributed to the presence of CH 2 and CH 3 groups in laccase@Co 3 (PO 4 ) 2 •HNFs 43 . The broad band in 3200–3500 cm − 1 corresponds to O-H starching band 44 . A peak at 1640 cm − 1 is attributed to the amide (II) band of laccase; however, the peak is overlapped with entrapped water peak of laccase@Co 3 (PO 4 ) 2 •HNFs at 1640 cm − 1 45,46 . 3.3. Structural studies CD spectra were applied to elucidate the effect of immobilization on the secondary structure of laccase (Fig. S6). As summarized in Table 1 , the conformation of the free native laccase was mainly composed of β-sheets (46.4%), β-turns (12.3%), and random coils (41.4%). Nevertheless, the amount of α-helix in the secondary structure of laccase increased remarkably (32%) after the immobilization of laccase in cobalt-based HNFs. The content of β-turns decreased slightly; however, β-sheets content (10.3%) of the laccase was significantly reduced. Laccase from T. versicolor is mainly composed of β-sheets, β-turns, and other structures, as reported previously 47 , 48 . Higher α-helix contents result in a more rigid protein structure, arising from the formation of a higher number of hydrogen bonds. Thus, by decreasing β-sheets and increasing α-helix contents, the laccase secondary structure became more rigid 49 . At first glance, it is not far-fetched that enzyme activity diminishes completely after such alteration in the protein structure. However, the immobilization of enzymes may significantly change the protein structure without loss of activity. For instance, by the preparation of cross-linked enzyme aggregates, the 3D structure of the enzyme is remarkably changed. Cross-linked laccase aggregated have been used for immobilization of the enzyme; meanwhile, the biocatalytic activity of the enzyme was preserved 50 – 52 . In order to verify the effect of any change in laccase tertiary structure, fluorescence spectroscopy was employed. The surrounding microenvironment highly influences the fluorescence properties of tryptophan; consequently, any change in the tryptophan microenvironment represents protein folding. As shown in Fig. S7, the fluorescence intensity of laccase@Co 3 (PO 4 ) 2 •HNFs was lower than the free enzyme, which indicates the considerable change in the 3rd protein structure 53 . The results indicate that immobilization of laccase into cobalt phosphate HNFs increased the content of α-helix, which ultimately changes the 3rd structure of the protein. As shown in Fig. S8, the weight loss of laccase@Co 3 (PO 4 ) 2 •HNFs was higher than the pure inorganic nanoflowers, which indicates that 5% of the weight of HNFs is composed of laccase. The total weight loss of laccase@Co 3 (PO 4 ) 2 •HNFs upon decomposition was 32.02%. The initial weight loss of samples (6% at 100–160°C) was due to the evaporation of crystal water entrapped in cobalt phosphate. At the second stage, the weight loss is attributed to the decomposition of immobilized laccase, which was 22%. Table 1 Secondary structures content of the free laccase and laccase@Co 3 (PO 4 ) 2 •HNFs. Type of catalyst α-Helix (%) β-Sheet (%) β-Turns (%) Random coils (%) The free laccase 5 44.4 11.3 39.4 Laccase@Co 3 (PO 4 ) 2 •HNFs 32 10.3 6.5 51.2 * p < 0.05 vs. the free laccase 3.4. Enzyme activity of laccase@Co 3 (PO 4 ) 2 •HNFs against temperatures and pH values In order to determine the optimum conditions for the activity of laccase@Co 3 (PO 4 ) 2 •HNFs, the enzyme activity was measured in a broad range of pH and temperature. As shown in Fig. 5 a and 5 b, the activity of the laccase@Co 3 (PO 4 ) 2 •HNFs in basic pH values was remarkably higher than the free enzyme. However, maximum activity of both laccase@Co 3 (PO 4 ) 2 •HNFs and the free enzyme was obtained at a pH range of 4.5–5.5 and 45°C. This phenomenon could be due to the higher surface area of laccase@Co 3 (PO 4 ) 2 •HNFs that enhance the enzyme activity 54 . It is believed that high temperatures and extreme acidic or basic pH reduce the activity and stability of the enzymes by induction of conformational changes 55 . However, it was shown that hybridization of nitrile hydratase (NHase) with cobalt phosphate complexes increased the stability of NHase against temperature 56 . Similarly, the activity of laccase was increased by 165% after incorporation into laccase@Cu 3 (PO 4 ) 2 •HNFs 55 . Stable coordination of laccase with cobalt phosphate probably results in higher operational stability of the enzyme. 3.5. Estimation of immobilization yield, efficacy, and kinetic parameters Due to the improved enzymatic activity of the synthesized HNFs in basic pH values, kinetic properties of the immobilized and free laccase were evaluated in pH 4.5 and 7.4. Immobilization yield, efficiency, and kinetic properties of instantly-prepared laccase@Co 3 (PO 4 ) 2 •HNFs and the free enzyme are summarized in Table 2 . Immobilization efficiency, V max and K m of the biocatalysts were different in basic and acidic conditions. The V max value of the laccase@Co 3 (PO 4 ) 2 •HNFs at pH 7.4 was 40% higher than pH 4.5; however, the maximum velocity of the free enzyme declined 21.2% at pH 7.4. Likewise, the efficiency of the immobilization procedure was increased by 25% in pH 7.4. The results indicated that K cat of laccase@Co 3 (PO 4 ) 2 •HNFs was higher than the free enzyme at pH 7.4; while, the free enzyme exhibited superior K cat over laccase@Co 3 (PO 4 ) 2 •HNFs at pH 4.5. These results are in alignment with the effect of pH on the activity of both free and immobilized laccase. The Michaelis constant ( K m ) indicates the affinity of the enzyme toward the substrate, and a low K m value indicates the high affinity of the enzyme toward the substrate in both pH values. The improved accessibility of laccase@Co 3 (PO 4 ) 2 •HNFs to the substrate (compared to the free enzyme) could be attributed to the high surface area of the flower-shaped structures that reduces the mass transfer limitations 54 . Normally, K m , V max , and the optimum conditions for reaching the highest catalytic efficiency enzymes may alter after immobilization 57 . Maximum activity of fungal laccases has been reported to be obtainable at a pH range of 3 − 5 58 . One of their main limitations is an insufficient activity in neutral and alkaline conditions despite the high redox potential of fungal laccases (490–790 mV). In this regard, pH-tolerant laccases are desirable for industrial and environmental purposes 59 . Hybridization of laccase with cobalt phosphate nanocrystals enhanced the tolerance of laccase from T. versicolor toward alkaline conditions, which could improve their desirability for industrial and environmental applications 60 . Table 2 Kinetic parameters of the free laccase and laccase@Co 3 (PO 4 ) 2 •HNFs. Parameter The free laccase Laccase@Co 3 (PO 4 ) 2 •HNFs pH pH 4.5 7.4 4.5 7.4 V max (µmol/min) 8.4 ± 0.4 6.2 ± 0.2 4.7 ± 0.2 6.6 ± 0.3 K m (µM) 147.9 ± 8.5 118.5 ± 5.3 85.4 ± 4.3 110.7 ± 4.6 K cat × 10 − 2 (S − 1 ) 6.5 ± 0.2 4.8 ± 0.2 3.6 ± 0.1 5.1 ± 0.1 Catalytic efficiency × 10 − 4 ( K cat / K m ) 4.4 ± 0.3 4.0 ± 0.2 4.2 ± 0.3 4.6 ± 0.4 Immobilization yield (%) - - 65.3 ± 7.6 65.3 ± 7.6 Immobilization efficacy (%) - - 86 ± 5.3 110 ± 4.9 3.6. Reusability and stability of laccase@Co 3 (PO 4 ) 2 •HNFs Reusability is an important feature of biocatalysts that reduces their utilization cost, especially on the industrial scale 61 . However, in the present study, measurement of the reusability for laccase@Co 3 (PO 4 ) 2 •HNFs provided evidence on the successful immobilization of laccase into the matrix of cobalt phosphate. In order to evaluate the reusability of laccase@Co 3 (PO 4 ) 2 •HNFs, the enzyme activity was consecutively measured by ABTS as the substrate. As shown in Fig. 4 b, after 4 consecutive reuse cycles, the activity of the laccase@Co 3 (PO 4 ) 2 •HNFs did not significantly change. However, the residual activity declined to 50% of its initial value after 10 cycle reuses. The results indicate that laccase@Co 3 (PO 4 ) 2 •HNFs exhibited remarkable reusability, and the enzyme was successfully immobilized in the matrix of cobalt phosphate. Laccase-based HNFs were exhibited promising reusability; for instance, laccase and lysin-copper HNFs retained 60% of initial activity after 6 cycles of reusability 62 . In another study, laccase-copper HNFs retained 50% of the initial activity after 10 reusability runs; however, the glutaraldehyde-treated HNFs retained almost 100% of their initial activity at the same conditions 63 . As shown in Fig. 5 c and 5 d, the stability of laccase@Co 3 (PO 4 ) 2 •HNFs against elevated temperatures and basic pH values was higher than the free laccase. Laccase@Co 3 (PO 4 ) 2 •HNFs retained 50% of their activity after incubation at pH 9.5 for 3 h, where the free laccase almost lost all of its activity. It should be noted that the stability of laccase@Co 3 (PO 4 ) 2 •HNFs was lower than the free enzyme in acidic pH values due to the solubility of cobalt phosphate in acidic pH 64 . No data is available for comparison. As mentioned in 3.3, the α-helix content of laccase was increased after hybridization with cobalt phosphate. Copper ions of laccase, which are involved in the catalytic activity of the enzyme, are present in β-sheets, which are mainly composed of conserved polar amino acids 65 . The stability and activity of laccase have been reported to be increased when the content of α-helix increased and β-sheets decreased 22 , 49 , 66 . A more rigid protein structure aligned with increasing in α-helix content could explain the higher stability of laccase HNFs than the free enzyme 22 . However, the stability of enzymes immobilized on cobalt-based supports was enhanced 67 . The thermal stability of laccase-based HNFs was previously reported 54 , 55 . Immobilization of laccase into laccase@Co 3 (PO 4 ) 2 •HNFs increased the storage stability of the enzyme. As shown in Fig. 4 c, after 40 days of storage at 25°C, the prepared HNFs retained 20% of the initial activity, while the free enzyme lost 58% of the initial activity. 3.7. Bioremoval of moxifloxacin As shown in Fig. 4 d, laccase@Co 3 (PO 4 ) 2 •HNFs were able to degrade moxifloxacin significantly. However, due to the propensity of FQ antibiotics to adsorb onto inorganic surfaces, the possible adsorption of moxifloxacin on the synthesized HNFs was evaluated 13,68−70 . In order to specifically determine the adsorption of moxifloxacin on the constructed HNFs, the immobilized laccase was deactivated by sequential freeze and thawing cycles. Adsorption of moxifloxacin on the surface of HNFs reached a plateau after 18 h of incubation. The maximum adsorptive capacity of inactivated laccase@Co 3 (PO 4 ) 2 •HNFs for moxifloxacin was found to be 0.8 mg/g. The laccase@Co 3 (PO 4 ) 2 •HNFs completely removed moxifloxacin from the reaction mixture. Due to the high surface of HNFs, the adsorption of organic and inorganic components is not farfetched. There are some reports on the ability of HNFs for adsorption of cadmium and doxorubicin 36 , 71 . 3.8. Identification of degradation products After the bioremoval procedure, the mixture of moxifloxacin biotransformed products was subjected to LC-MS analysis. The main by-products, their retention times, and corresponding mass to charge ration (m/z) are presented in Fig. S9. Based on the LC-MS results, five plausible pathways for the degradation of moxifloxacin are proposed (Fig. 6 ). Interestingly, the biocatalyst replaced the fluorine atoms from moxifloxacin, which is responsible for the low biodegradability of FQs, with hydroxyl groups (compound III; m/z + 1 = 262, R t = 6 min) 72 . As shown, ring-opening of piperazine moiety and further demethylation reactions were responsible for appearing compound VI (m/z = 307, trace) and compound II (m/z + 1 = 280, R t = 3.9 min), respectively (pathway I). In pathway II, moxifloxacin underwent defluorination and cleavage of the piperazine moiety (compound VII; m/z = 388, trace) and further transformed to compound V (m/z = 358, R t = 9 min) after dihydroxylation and demethylation 73 . In the 3rd pathway, by oxidation and opening of piperazine ring, compound VIII (m/z = 418, trace) was formed, which finally converted to compound IV (m/z = 294, R t = 8.7 min) after subsequent oxidation steps. In the next pathway, compound VIII (m/z = 418, trace) was formed, and by complete cleavage of piperazine group, compound I was transformed to compound IX (m/z = 307, trace) that was transformed to compound VIII (m/z = 294, R t = 8.7 min) after demethylation and cracking of cyclopropane ring (pathway IV). In the final proposed mechanism, moxifloxacin fluor atom was substituted with OH (compound X, m/z = 400, trace) and further, by loss of piperazine group and demethylation, converted to compound III (m/z + 1 = 262, R t = 6 min) (pathway V). 3.9. Toxicity of the biodegradation products Diminishing toxicity of pollutants is not always guaranteed by degradation procedures; even there are reports on an increase in toxicity of pollutants after degradation 74 , 75 . In order to examine the efficiency of the synthesized HNFs, the toxicity of moxifloxacin and its metabolites against four bacterial strains was evaluated. As shown in Table S3, the toxicity of the degradation products was effectively decreased after treatment with laccase@Co 3 (PO 4 ) 2 •HNFs. Treatment of moxifloxacin with the constructed HNFs reduced the GI% for P . aeruginosa by 67%. GI% reduction for S . aureus, S. epidermidis , and E . coli was 24.6%, 48.7%, and 12%, respectively. Some of the FQs degradation products exhibited antimicrobial activity. Due to the enhancement of DNA gyrase activity by fluorine, the defluorination of moxifloxacin significantly reduced its biological activity. Also, most of the identified degradation products possess lower hydrophobicity, which may limit their diffusion through the cell membrane 73 , 76 , 77 . 4. Conclusion This study reported a facile method for the instant synthesis of HNFs. Contrary to previously-reported synthesis procedures, high concentrations of phosphate and cobalt ions were incorporated for the synthesis of HNFs. The formation of primary nucleation sites (protein-inorganic nanocrystals) and their anisotropic growth are the main steps involved in the synthesis of HNFs by the “concentrated method.” Rapid fabrication of HNFs was achieved by increasing the number of primary nanocrystals, which improved their collision rate and accelerated the growth of HNFs. Catalytic efficiency, kinetic parameters, and stability of laccase were enhanced after the immobilization of enzymes into HNFs. Based on CD and fluorimetry spectra, higher α-helix content of laccase@Co 3 (PO 4 ) 2 •HNFs than the free enzyme is responsible for the enhanced stability of the constructed HNFs. The ability of laccase@Co 3 (PO 4 ) 2 •HNFs to remove moxifloxacin was also assessed. The antibiotic was removed through adsorption and biodegradation mechanisms. The toxicity of degradation products against two G + and two G bacteria was remarkably lower than parent fluoroquinolone. Declarations Data availability All data generated or analysed during this study are included in this published article (and its Supplementary Information files). Author contributions Khashayar Vojdanitalab: Conceptualization, Methodology, Investigation, and Writing - Original Draft. Hossein Jafari: Data curation, Writing- Original draft preparation, Visualization, and Software. 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Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 04 May, 2022 Reviews received at journal 02 Mar, 2022 Reviewers agreed at journal 19 Feb, 2022 Reviewers invited by journal 19 Feb, 2022 Editor assigned by journal 19 Feb, 2022 Editor invited by journal 19 Feb, 2022 Submission checks completed at journal 19 Feb, 2022 First submitted to journal 11 Feb, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1351173","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":85078296,"identity":"f2745555-df13-4be6-ac97-7cfcd936047d","order_by":0,"name":"Khashayar Vojdanitalab","email":"","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Khashayar","middleName":"","lastName":"Vojdanitalab","suffix":""},{"id":85078297,"identity":"3e6567c6-4e84-4fcd-99dc-c4af202166ac","order_by":1,"name":"Hossein Jafari-Nodoushan","email":"","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Hossein","middleName":"","lastName":"Jafari-Nodoushan","suffix":""},{"id":85078298,"identity":"2c740541-44e0-4b7f-a5e1-8c859ed1beb9","order_by":2,"name":"Somayeh Mojtabavi","email":"","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Somayeh","middleName":"","lastName":"Mojtabavi","suffix":""},{"id":85078299,"identity":"6c126c00-f6f1-4d43-a74d-7ca88bf3652b","order_by":3,"name":"Mahtab Shokri","email":"","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mahtab","middleName":"","lastName":"Shokri","suffix":""},{"id":85078300,"identity":"d1fb7476-ad87-4746-bf15-b99e2ea27e09","order_by":4,"name":"Hoda Jahandar","email":"","orcid":"","institution":"Islamic Azad University","correspondingAuthor":false,"prefix":"","firstName":"Hoda","middleName":"","lastName":"Jahandar","suffix":""},{"id":85078301,"identity":"6fd36494-8e30-48be-9577-0e7ca241340f","order_by":5,"name":"Mohammad Ali Faramarzi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYBAC9gYogx8uxExAC88BKEOyjWQtBseIdRgP+9mHn27U3LM3vt9juvEHg508AzvvA/xaeNKNpXOOFTObHeMxu83DkGzYwMxugFeLPUMag3QOWwIbWAvQIwkMzGwEHMb/jPl3zr8EHuM2HrObPxjqidAikcYmnduWIGHAxmN2g4fhMDFanrFZ5/YlGEgcSyu7zWNw3LCNsMPSmG/nfEuw528+vO3mj4pqeX5+ooMbDIBhRcCOUTAKRsEoGAXEAAAgKjR9JhMtSwAAAABJRU5ErkJggg==","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Mohammad","middleName":"Ali","lastName":"Faramarzi","suffix":""}],"badges":[],"createdAt":"2022-02-11 17:59:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1351173/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1351173/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":18496185,"identity":"edcf58d0-9f39-4c1f-8e54-6b75da9015aa","added_by":"auto","created_at":"2022-02-22 21:09:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":39314,"visible":true,"origin":"","legend":"\u003cp\u003eOptimization of the synthesis procedure.\u003cstrong\u003e \u003c/strong\u003eThe synthesis of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e•HNFs by optimizing the concentration of phosphate and cobalt (II) ions (a), the influence of incubation (at 25 °C) as traditional procedures for the construction of HNFs on the recovered activity of the heterogeneous biocatalyst (b).\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-1351173/v1/6715fc8f5e498166b9486ea3.png"},{"id":18496187,"identity":"3312edeb-b894-48c4-88e9-62db42f357c2","added_by":"auto","created_at":"2022-02-22 21:09:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":159422,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscopy (SEM) imaging. SEM image (a, b), elemental map (c), and Energy dispersive X-Ray spectroscopy (EDX) analysis (d) of the constructed laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e•HNFs.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-1351173/v1/226ad317b844111e8876e834.png"},{"id":18496085,"identity":"95fa6f06-d018-4c27-bded-5dc52b8c6ede","added_by":"auto","created_at":"2022-02-22 21:06:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":45000,"visible":true,"origin":"","legend":"\u003cp\u003eX-ray diffraction (XRD) analysis. X-ray spectrum of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e•HNFs (a), XRD pattern of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e•HNFs and the standard Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e (JCPDS–00–027–1120) (b).\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-1351173/v1/11c1690f445a49c780e3d6ca.png"},{"id":18496186,"identity":"aa73ee44-c775-4613-bbdd-a148e85e5a28","added_by":"auto","created_at":"2022-02-22 21:09:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":33062,"visible":true,"origin":"","legend":"\u003cp\u003eFT-IR analysis. The spectra of Fourier transform infrared (FTIR) spectroscopy of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e•HNFs (solid line), Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2 \u003c/sub\u003e(long dash line), and the free enzyme (dash-dot) (a). Reusability of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e•HNFs (b). The activity of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e•HNFs declined to under 50% of initial activity after 11 reusability cycles. Storage stability of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e•HNFs and the free laccase after 40 days of storage at 4 °C (c). Bioremoval of moxifloxacin by laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e•HNFs and inactivated laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e•HNFs. The experiments were conducted at 40 °C in phosphate buffer (10 mM, pH 4.5) (d).\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-1351173/v1/830b12fba2e3b54c3c891b57.png"},{"id":18496441,"identity":"69095688-07fa-4dce-82b1-df1b4bfc8ed6","added_by":"auto","created_at":"2022-02-22 21:12:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":47843,"visible":true,"origin":"","legend":"\u003cp\u003eStability and activity studies. Effect of pH and temperature on the activity (a) and stability of the free enzyme (c). Influence of pH and temperature on the activity (b) and stability of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e•HNFs (d).\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-1351173/v1/aa993aac0e6b4aeadafbc423.png"},{"id":18496081,"identity":"900520cf-0519-4947-99ca-281d76c1ea7b","added_by":"auto","created_at":"2022-02-22 21:06:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":122111,"visible":true,"origin":"","legend":"\u003cp\u003eMechanism of moxifloxacin degradation. Proposed moxifloxacin biotransformation pathways by laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e•HNFs.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-1351173/v1/c71811f177e6df6173bb5bf5.png"},{"id":18496442,"identity":"0cbb358d-0b44-4b81-8f28-f5f447d4539b","added_by":"auto","created_at":"2022-02-22 21:12:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":940848,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1351173/v1/b1d4c546-cc76-4517-93a7-cc74ca666660.pdf"},{"id":18496082,"identity":"53e66a2d-1185-4952-bc6c-6a7e8a7c6661","added_by":"auto","created_at":"2022-02-22 21:06:31","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1631258,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-1351173/v1/012a3a971dfe86c5ac0e5bb8.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Instantaneous synthesis of organic-inorganic laccase-cobalt phosphate hybrid nanoflowers: Structural and biocatalytic characterization","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eOver the past two decades, various heterogeneous biocatalysts, immobilized enzymatic active substances, have been developed for environmental and industrial applications\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The preparation of new platforms based on organic-inorganic hybrid materials for the immobilization of enzymes is of great interest. Organic-inorganic hybrid nanoflowers (HNFs), the hierarchical three-dimensional nanostructures that were initially discovered in 2012\u003csup\u003e2\u003c/sup\u003e. HNFs, composed of enzyme(s) and inorganic component(s), have a large surface area, facile synthesis procedure, and hierarchical porous structures\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. After incorporating enzymes into HNFs, their activity, stability, and reusability have notably increased, owing to low mass transfer limitations arising from their high surface-to-volume ratio and cooperative effects between enzymes and metal ions\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. For instance, the incorporation of lipase into Zn\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs resulted in a 147% enhancement of the enzyme activity. Also, the storage stability of the lipase-containing HNFs was three times higher than free lipase\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Carbonic anhydrase (CA) was successfully immobilized into Ca\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e6\u003c/sub\u003e\u0026bull;HNFs and exhibited remarkable stability against elevated temperatures (30\u0026ndash;80\u0026deg;C). CA@Ca\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e6\u003c/sub\u003e\u0026bull;HNFs retained 100% of their initial CO\u003csub\u003e2\u003c/sub\u003e hydration activity after 5 reusability cycles\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Accidental addition of copper sulfate to phosphate buffer saline (PBS) in the presence of bovine serum albumin (BSA) resulted in the synthesis of the flower-shaped BSA@Cu\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Three approaches have been reported for the preparation of HNFs, including one-step precipitation\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, ultrafast sonication\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, and shear stress-mediated synthesis\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. In the one-step precipitation method, a facile and straightforward strategy, fabrication of HNFs requires a long incubation time (1\u0026thinsp;~\u0026thinsp;3 days). As long synthesis procedure may potentially reduce the stability of the organic constituent, serious attempts have been established to shorten the assembling time. A rapidly ultrafast sonochemical synthesis of laccase@Cu\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs was reported using sonication of the reaction mixture within 5 min at room temperature\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Introducing shear stress to the organic component and inorganic precursors to synthesize copper-based HNFs was recently reported\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eConsidering the fact that HNFs are at the early stages of development, the formation mechanism of HNFs has not been accurately established. However, it is believed that the formation of primary nucleation sites of metal phosphate(s) and organic molecule(s) (e.g., protein, DNA, etc.) and time-dependent anisotropic growth of these primary nucleation sites\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e are responsible for the formation of the flower-like structures. It seems that by introducing kinetic energy to the precursors, provided by ultrasonication and vortexing, HNFs assembling time was reduced. As efficient biocatalysts, HNFs have been used for the removal of emerging contaminants such as industrial dyes. However, the ability of HNFs for the removal of antibiotics have not been reported previously\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMoxifloxacin, as a fourth-generation fluoroquinolone (FQ), is responsible for more than 34.6% of the total FQs consumption in China. It is mostly used for the treatment of pneumonia and skin infections. Recently, due to its usage in severe acute respiratory syndrome coronavirus 2, moxifloxacin consumption has temporarily increased. Therefore, consumption, release, and accumulation of moxifloxacin in the environment may be threatening\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Moxifloxacin is also the most toxic FQs against the growth of \u003cem\u003ePseudokirchneriella subcaptitata\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Likewise, it exhibited significant negative effects on the growth and reproduction of \u003cem\u003eCeriodaphnia dubia\u003c/em\u003e and \u003cem\u003eDaphnia manga\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Therefore, increased consumption and release of moxifloxacin in the environment may be threatening to the ecosystem and human health. The efficiency of current approaches for the elimination of antibiotics from wastewaters is limited\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In this regard, various techniques for the removal of antibiotics have been so far established such as biocatalysis\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, photocatalysis\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, electrocatalysis\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, etc. Laccases, an oxidoreductase enzyme, has been broadly used for environmental and industrial applications\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Free and immobilized laccases have been incorporated for bioremoval of a wide range of pollutants such as bisphenol A, crystal violet, acid orange-7, levofloxacin, etc.\u003csup\u003e\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn this study, a novel method for the fast synthesis of HNFs was proposed without incorporating kinetic energy. Laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs were instantly synthesized through rapid anisotropic growth of laccase-cobalt phosphate nanocrystals, and a new mechanism was proposed for the synthesis of HNFs. Biocatalytic properties of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs were investigated, and the structure of the hybridized enzyme was also characterized. The synthesized HNFs were then employed to remove moxifloxacin from aqueous media. The degradation products were identified, and their toxicity against four bacteria involved in the degradation of organic compounds was assessed.\u003c/p\u003e"},{"header":"2. Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. The enzyme and chemicals\u003c/h2\u003e \u003cp\u003eLaccase from \u003cem\u003eTrametes versicolor\u003c/em\u003e (0.5 U/mg) and 2,2\u0026rsquo;-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were purchased from Sigma-Aldrich (St. Louis, Mo, USA). Cobalt (II) chloride hexahydrate (CoCl\u003csub\u003e2\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003eO) was obtained from Merck (Darmstadt, Germany). All other applied reagents and chemicals were of analytical grade without further purification. Moxifloxacin was kindly gifted from Soha Pharmaceutical Company (Tehran, Iran).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Instant synthesis of cobalt-based hybrid nanoflowers with laccase\u003c/h2\u003e \u003cp\u003eRegardless of the conventional methods, an instant strategy was applied for the construction of HNFs. The synthesis procedure was accomplished by the addition of 0.1 mL of CoCl\u003csub\u003e2\u003c/sub\u003e solution into 0.9 mL of phosphate buffer (pH 7.4) containing 0.1 U mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of laccase. Instantly a large number of purple precipitates appeared in the solution. Proper concentration of CoCl\u003csub\u003e2\u003c/sub\u003e and molarity of phosphate buffer were subsequently optimized against highest activity recovery (n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The precipitates were centrifuged at 6000 \u003cem\u003eg\u003c/em\u003e and washed three times with phosphate buffer. The purple products were marked as laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Characterization of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs\u003c/h2\u003e \u003cp\u003eScanning electron microscopy (SEM) images (Tescan, MIRA II, Czech Republic) were applied to indicate the flower-shaped morphology of the fabricated HNFs. Energy dispersive X-Ray spectroscopy (EDX, Tescan, MIRA II, Czech Republic) and elemental mapping were incorporated to determine the composition and spatial distribution of elements in the constructed HNFs, respectively. Fourier transform infrared (FTIR) spectroscopy (Shimadzu, Equinox 55, Japan) was utilized for analyzing functional groups of organic and inorganic constituents of HNFs. The samples were dispersed in pressed KBr disks, and the spectra were recorded at 4000\u0026ndash;400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Brunauer-Emmett-Teller (BET) analysis was performed (Microtrac, BELSORP MINI X, Japan) after degassing the samples under N\u003csub\u003e2\u003c/sub\u003e. X-ray diffraction (XRD) analysis was carried out to identify the crystalline phase of the inorganic component using an X-ray diffractometer (Philips, PW1730, Philips, Eindhoven, Netherlands).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Laccase activity assay\u003c/h2\u003e \u003cp\u003eLaccase activity was spectroscopically measured following by oxidation of ABTS as the substrate\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs and the free enzyme were incubated with ABTS (50 \u0026micro;M, 1 mL) at 40\u0026deg;C in a shaker incubator. After 15 min incubation, the optical density of the supernatants was recorded at 420 nm. One unit of activity was defined as the amount of laccase capable of oxidizing 1 \u0026micro;mol ABTS to the colored product in 1 min. Effect of pH and temperature on the activity of both laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs and the free form of laccase was determined in the temperature range of 25\u0026ndash;55\u0026deg;C (interval 10\u0026deg;C) and pH range of 2.5\u0026ndash;9.5 (interval 1) (n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Determination of immobilization yield and efficiency\u003c/h2\u003e \u003cp\u003eAfter preparing laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs, the concentration of residual protein in the supernatant was estimated by the bicinchoninic acid (BCA) method (n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003csup\u003e24\u003c/sup\u003e. Immobilization yield (IY) was calculated using Eq.\u0026nbsp;1, where Y\u003csub\u003e0\u003c/sub\u003e and Y\u003csub\u003e1\u003c/sub\u003e represent the amount of immobilized enzyme in HNFs before and after the synthesis procedure, respectively (n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eIY (%)= [(Y\u003csub\u003e0\u003c/sub\u003e-Y\u003csub\u003e1\u003c/sub\u003e)/Y\u003csub\u003e0\u003c/sub\u003e]\u0026times;100 Eq.\u0026nbsp;1\u003c/p\u003e \u003cp\u003eThe immobilization efficiency (IE) was obtained by Eq.\u0026nbsp;2, where E\u003csub\u003e0\u003c/sub\u003e and E\u003csub\u003et\u003c/sub\u003e demonstrate the activity of free laccase and laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs, respectively.\u003c/p\u003e \u003cp\u003eIE (%)= [(E\u003csub\u003e0\u003c/sub\u003e-E\u003csub\u003et\u003c/sub\u003e)/E\u003csub\u003e0\u003c/sub\u003e]\u0026times;100 Eq.\u0026nbsp;2\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Evaluation of the kinetic parameters of the constructed HNFs\u003c/h2\u003e \u003cp\u003eIn order to assess the influence of immobilization on laccase, kinetic parameters of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs and the free laccase were evaluated. The activity of both the free and immobilized biocatalysts was determined in the presence of ABTS concentrations (20\u0026ndash;100 \u0026micro;M). Lineaweaver-Burke plot was incorporated for evaluation of kinetic parameters such as Michaelis constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e) and maximum velocity (\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e). Turnover number (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e) and catalytic efficiency were calculated based on Eqs.\u0026nbsp;3 and 4\u003csup\u003e25\u0026ndash;27\u003c/sup\u003e; where [E] is the concentration of enzyme.\u003c/p\u003e \u003cp\u003e \u003cem\u003eK\u003c/em\u003e \u003csub\u003ecat\u003c/sub\u003e= \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e/[E] Eq.\u0026nbsp;3\u003c/p\u003e \u003cp\u003eCatalytic efficiency\u0026thinsp;=\u0026thinsp;\u003cem\u003eK\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e/\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e Eq.\u0026nbsp;4\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Reusability and stability studies\u003c/h2\u003e \u003cp\u003eIn order to evaluate the reusability of the constructed biocatalyst, repeated measurement of the activity of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs was examined\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The fabricated HNFs were incubated in 1 mL phosphate buffer (10 mM, pH 7.4) containing ABTS (0.5 mM) at 40\u0026deg;C for 15 min. After centrifugation of the mixture, OD\u003csub\u003e420\u003c/sub\u003e of the supernatant was recorded, and the precipitates were washed three times with phosphate buffer (10 mM, pH 7.4). The procedure repeated to the point that the activity of HNFs dropped to below 50% of its initial value (n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The stability of the free laccase and HNFs was assessed as a function of recovered activity after 3 h incubation at temperatures ranging 25\u0026ndash;55\u0026deg;C (interval 10\u0026deg;C) and a wide pH range of 2.5\u0026ndash;9.5 (interval 1). Storage stability of the free laccase and HNFs were obtained by measurement of laccase activity during storage at 4\u0026deg;C (n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. The enzyme structural studies\u003c/h2\u003e \u003cp\u003eIn order to evaluate the conformational changes of laccase after immobilization, far-UV circular dichroism (CD) and fluorescence spectroscopies were utilized. CD spectra of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs and the free enzyme were recorded by a Jasco 725 spectrophotometer in a 2 mm path-length cell, and the absorbance was recorded at 190\u0026ndash;240 nm. The fluorescence property of tryptophan is dependent on the 3rd structure of a protein. In this regard, the tryptophan fluorescence intensity of the free laccase and laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs were recorded by a Hitachi 850 spectrofluorometer after excitation at 285 nm\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Finally, thermogravimetry analysis (TGA) was using performed using a thermogravimetric analyzer (Perkin Elmer STA 6000, USA) for evaluation of thermal decomposition of both pure cobalt phosphate and laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs. The experiment was conducted under an N\u003csub\u003e2\u003c/sub\u003e atmosphere in the temperature range of 25\u0026ndash;600\u0026deg;C by a heating rate of 10\u0026deg;C/min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Bioremoval experiments\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.9.1. Bioremoval of moxifloxacin\u003c/h2\u003e \u003cp\u003eMoxifloxacin was incubated with laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs and 1-hydroxybenzotriazole (HBT) as the laccase mediator in phosphate buffer (10 mM, pH 4.5) at 40\u0026deg;C under stirring condition (100 rpm)\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. After determining the period of incubation, the concentration of moxifloxacin was quantified by high-performance liquid chromatography (HPLC) coupled with UV detector, and the removal percentage was calculated based on Eq.\u0026nbsp;5 (n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05); as C\u003csub\u003e0\u003c/sub\u003e and C\u003csub\u003et\u003c/sub\u003e represent the concentration of moxifloxacin before and after removal experiments, respectively.\u003c/p\u003e \u003cp\u003eRemoval (%)= (C\u003csub\u003e0\u003c/sub\u003e-C\u003csub\u003et\u003c/sub\u003e/C\u003csub\u003e0\u003c/sub\u003e)\u0026times;100 Eq.\u0026nbsp;5\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.9.2. Sample preparation and quantification of moxifloxacin\u003c/h2\u003e \u003cp\u003eAfter the removal procedure, the pH of the reaction mixture was set to 7.95 (isoelectric point of moxifloxacin) by phosphate buffer (100 mM, 7.95). Then, the mixture was extracted with chloroform under vigorous vortex three times after the addition of ciprofloxacin as the internal standard. The extracted chloroform evaporated to dryness by a rotary evaporator instrument, and the remaining powder was dissolved in 1 mL of the HPLC mobile phase. A Knauer HPLC-UV system (Berlin, Germany) was incorporated for moxifloxacin quantification using a PDA 2800 detector, a Smartline 1000 pump, and ChromGate software (version 3.3.1). The mobile phase consisted of methanol (45%) and 10 mM phosphate buffer (pH 3.0) (55%). Samples were injected by Smartline autosampler 3950 to a Eurospher 100 C18 reversed-phase column (250 \u0026times; 4.6 mm).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.9.3. Identification of biotransformation products\u003c/h2\u003e \u003cp\u003eBiotransformation products of moxifloxacin were identified by liquid chromatography coupled by mass spectroscopy (LC-MS). The apparatus was an Agilent 1200 series LC system coupled with an Agilent 6520 quadrupole time of flight tandem MS instrument featured by an electrospray ion source (Agilent, Waldbronn, Germany). A 2.1\u0026ndash;100 mm Nucleosil 100\u0026thinsp;\u0026minus;\u0026thinsp;3 C18 HD column (Macherey-Nagel, D\u0026uuml;ren, Germany) was applied with a flow rate of 0.35 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Deionized water\u0026thinsp;+\u0026thinsp;0.1% formic acid (40%) and acetonitrile (60%) were used as mobile phase.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.9.4. Toxicity of the bioremoval procedure\u003c/h2\u003e \u003cp\u003eBased on previous studies, elimination of toxicity is not guaranteed by degradation of antibiotics in catalytic procedures\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Accordingly, the toxicity of moxifloxacin degradation products was examined against some bacteria, including \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e ATCC 9027, \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25,922, \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e ATCC 49,619, and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 6538, which are responsible for the bioremoval of organic pollutants in the environment. In this regard, the fresh culture of each bacterium was seeded in a nutrient broth and incubated at 37\u0026deg;C overnight. Then, each bacterium was treated with a mixture of degradation products, and the optical density (OD) of each bacterium was evaluated at 600 nm after determining the incubation time periods at 37\u0026deg;C. Growth inhibition of moxifloxacin degradation products, moxifloxacin as the positive control, and sterile water as the negative control were measured by plotting OD\u003csub\u003e600\u003c/sub\u003e against incubation time (n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003csup\u003e22\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Analysis of the experiments\u003c/h2\u003e \u003cp\u003eThe experiments were conducted in triplicate, and results were reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical significance among the mean value of experimental results was calculated by two-way ANOVA (\u0026plusmn;\u0026thinsp;standard deviation) and p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 regarded as significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results And Discussion","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Instant synthesis of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs\u003c/h2\u003e \u003cp\u003eHNFs have been routinely constructed with the addition of metal ions to enzyme-containing PBS followed by a 3-day incubation at room temperature, vigorous vortexing, or bath sonication, which introduces significant stress to the organic constituent\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In the present study, the concentration of CoCl\u003csub\u003e2\u003c/sub\u003e and molarity of phosphate buffer (pH 7.4) were optimized to instantly fabricate HNFs and avoid incorporating stress-inducing conditions in the synthesis procedure. After the addition of CoCl\u003csub\u003e2\u003c/sub\u003e to laccase-containing phosphate buffer, the purple precipitates were immediately formed. The precipitate was washed with deionized water three times and designated as laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, the recovered activity increased gradually at the higher molarity of phosphate buffer. However, the highest activity recovery was obtained with HNFs prepared with 0.16 M phosphate buffer and 30 mM CoCl\u003csub\u003e2\u003c/sub\u003e. Effects of bath sonication (60 kHz, 13.8 W) or incubation at 25\u0026deg;C (24\u0026ndash;72 h) on the activity recovery were also evaluated. The results (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) demonstrated that further incubation reduced the recovered activity. Also, sonication of the reaction mixture (up to 20 min, 5 min intervals) did not increase the recovered activity. The mentioned synthesis method is referred to as the \u0026ldquo;concentrated method\u0026rdquo; in the present study for comparison with the up to now reported and traditional procedures. The present explanation of the formation mechanism of HNFs is based on the nucleation of primary metal phosphate-protein nanocrystals and subsequent anisotropic growth of the hybrid nanoaggregates. Due to the high surface energy of the primary protein-inorganic nanocrystals, they tend to attach and form the nano-sheet structures, which finally form the ultimate structure of HNFs\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The growth of nucleation sites of HNFs could be facilitated by increasing the collision rate of the primary crystals\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. It seems that introduction of certain amounts of kinetic energy, provided by long-term incubation (24\u0026ndash;72 h at 25\u0026deg;C), bath sonication, microwave heating, and shear stress, to the synthesis mixture (organic component, metal, and phosphate ions) could induce the formation of HNFs. The number of primary nucleation sites increased by incorporating a high concentration of Cobalt (II) and phosphate ions in the reaction mixture. Hence, based on Eq.\u0026nbsp;6, higher numbers of nanocrystals possibly collide together more frequently, and HNFs were prepared very fast\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In Eq.\u0026nbsp;6, z is collision frequency, D is the particle diameter, ῡ represents the mean velocity of dispersed particles, N is the total number of particles, and V is the total volume of the reaction mixture.\u003c/p\u003e \u003cp\u003ez= (\u0026radic;2πD\u003csup\u003e2\u003c/sup\u003eῡN)\u0026times;V\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Eq.\u0026nbsp;6\u003c/p\u003e \u003cp\u003eHybrid nanoflowers have been synthesized in a short period of time through vigorous vortexing\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e and also bath sonication\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e of the precursors, which increased the mean velocity of the dispersed particles (ῡ). Based on Eq.\u0026nbsp;6, after the increase in the mean velocity of dispersed organic-inorganic particles by vortexing or bath sonication, the collision frequency increases, which results in fast anisotropic growth of the HNFs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Characterization of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs\u003c/h2\u003e \u003cp\u003eThe flower-shaped morphology of the fabricated HNFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) was confirmed by SEM image. The size of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs ranged 0.5\u0026ndash;3 \u0026micro;m and was assembled by interconnected nano-sheets resulting high surface-to-volume ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Cobalt phosphate nanoflowers (NFs) were also prepared through the same synthetic route without the incorporation of laccase in the reaction mixture. As shown in Fig. S1 and S2, the cobalt phosphate\u0026bull;NFs also exhibited flower-like morphology. Some of the previous studies reported that the flower-shaped morphology of HNFs is due to the presence of inorganic constituent(s)\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Also, it was reported that the 3D structure of the incorporated organic component could alter the morphology of HNFs\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. However, inorganic copper phosphate was prepared by the same synthesis procedure, which exhibited the same flower-shaped morphology as protein@Cu\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs\u003csup\u003e\u003cspan additionalcitationids=\"CR36 CR37\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. All in all, it could be concluded that some metal phosphates have flower-like morphology\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e that could be modified after hybridization with proteins. EDX analysis of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs and cobalt phosphate nanoflowers are exhibited in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed and Fig. S4, respectively. The weight percentage of elements in both constructed HNFs and cobalt phosphate is summarized in Table S1. The presence of nitrogen atoms in the prepared HNFs confirms the immobilization of laccase into the support. Elemental map analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) also provides information on the homogenous distribution of Co, P, N, and O atoms in the constructed HNFs. In order to identify the inorganic part of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNF, the XRD analysis was performed. The obtained peaks of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNF were in good agreement with the standard cobalt phosphate pattern (JCPDS 00-027-1120) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Mean pore diameter, total pore volume, and specific surface area of the fabricated HNFs and Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;NFs are summarized in Table S2, and corresponding BET plots are shown in Fig. S5. The high specific surface area (17.42 m\u003csup\u003e2\u003c/sup\u003e/g) is in agreement with the nano-sized width of petals in the SEM image of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e. As shown, the specific surface area of Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;NFs and the synthesized HNFs was almost identical. The presence of type IV isotherm in the BET plots is due to the mesoporous structure of the synthesized materials. As a rule of thumb, the higher surface area of the immobilized enzyme increases the accessibility of substrate to the enzyme and enhances the biocatalytic efficiency\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. The same results were reported for laccase@Cu\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs (10.2 m\u003csup\u003e2\u003c/sup\u003e/g) and GOx@Cu\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs (17.9 m\u003csup\u003e2\u003c/sup\u003e/g)\u003csup\u003e41,42\u003c/sup\u003e. FTIR spectra of laccase, Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e, and laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs are represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea. As shown, characteristic vibrational frequencies in the region 725\u0026ndash;1300 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are due to the presence of phosphate groups \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Peaks at 2980\u0026ndash;3600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e attributed to the presence of CH\u003csub\u003e2\u003c/sub\u003e and CH\u003csub\u003e3\u003c/sub\u003e groups in laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. The broad band in 3200\u0026ndash;3500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponds to O-H starching band\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. A peak at 1640 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is attributed to the amide (II) band of laccase; however, the peak is overlapped with entrapped water peak of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs at 1640 cm\u003csup\u003e\u0026minus;\u0026thinsp;1 45,46\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Structural studies\u003c/h2\u003e \u003cp\u003eCD spectra were applied to elucidate the effect of immobilization on the secondary structure of laccase (Fig. S6). As summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the conformation of the free native laccase was mainly composed of β-sheets (46.4%), β-turns (12.3%), and random coils (41.4%). Nevertheless, the amount of α-helix in the secondary structure of laccase increased remarkably (32%) after the immobilization of laccase in cobalt-based HNFs. The content of β-turns decreased slightly; however, β-sheets content (10.3%) of the laccase was significantly reduced. Laccase from \u003cem\u003eT. versicolor\u003c/em\u003e is mainly composed of β-sheets, β-turns, and other structures, as reported previously\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Higher α-helix contents result in a more rigid protein structure, arising from the formation of a higher number of hydrogen bonds. Thus, by decreasing β-sheets and increasing α-helix contents, the laccase secondary structure became more rigid\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. At first glance, it is not far-fetched that enzyme activity diminishes completely after such alteration in the protein structure. However, the immobilization of enzymes may significantly change the protein structure without loss of activity. For instance, by the preparation of cross-linked enzyme aggregates, the 3D structure of the enzyme is remarkably changed. Cross-linked laccase aggregated have been used for immobilization of the enzyme; meanwhile, the biocatalytic activity of the enzyme was preserved\u003csup\u003e\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. In order to verify the effect of any change in laccase tertiary structure, fluorescence spectroscopy was employed. The surrounding microenvironment highly influences the fluorescence properties of tryptophan; consequently, any change in the tryptophan microenvironment represents protein folding. As shown in Fig. S7, the fluorescence intensity of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs was lower than the free enzyme, which indicates the considerable change in the 3rd protein structure\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. The results indicate that immobilization of laccase into cobalt phosphate HNFs increased the content of α-helix, which ultimately changes the 3rd structure of the protein. As shown in Fig. S8, the weight loss of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs was higher than the pure inorganic nanoflowers, which indicates that 5% of the weight of HNFs is composed of laccase. The total weight loss of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs upon decomposition was 32.02%. The initial weight loss of samples (6% at 100\u0026ndash;160\u0026deg;C) was due to the evaporation of crystal water entrapped in cobalt phosphate. At the second stage, the weight loss is attributed to the decomposition of immobilized laccase, which was 22%.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSecondary structures content of the free laccase and laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eType of catalyst\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eα-Helix (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eβ-Sheet (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eβ-Turns (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRandom coils (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThe free laccase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e44.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e39.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLaccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e51.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003cb\u003e*\u003c/b\u003e \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. the free laccase\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Enzyme activity of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs against temperatures and pH values\u003c/h2\u003e \u003cp\u003eIn order to determine the optimum conditions for the activity of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs, the enzyme activity was measured in a broad range of pH and temperature. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, the activity of the laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs in basic pH values was remarkably higher than the free enzyme. However, maximum activity of both laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs and the free enzyme was obtained at a pH range of 4.5\u0026ndash;5.5 and 45\u0026deg;C. This phenomenon could be due to the higher surface area of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs that enhance the enzyme activity\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. It is believed that high temperatures and extreme acidic or basic pH reduce the activity and stability of the enzymes by induction of conformational changes\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. However, it was shown that hybridization of nitrile hydratase (NHase) with cobalt phosphate complexes increased the stability of NHase against temperature\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Similarly, the activity of laccase was increased by 165% after incorporation into laccase@Cu\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Stable coordination of laccase with cobalt phosphate probably results in higher operational stability of the enzyme.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Estimation of immobilization yield, efficacy, and kinetic parameters\u003c/h2\u003e \u003cp\u003eDue to the improved enzymatic activity of the synthesized HNFs in basic pH values, kinetic properties of the immobilized and free laccase were evaluated in pH 4.5 and 7.4. Immobilization yield, efficiency, and kinetic properties of instantly-prepared laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs and the free enzyme are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Immobilization efficiency, \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e and \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e of the biocatalysts were different in basic and acidic conditions. The \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e value of the laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs at pH 7.4 was 40% higher than pH 4.5; however, the maximum velocity of the free enzyme declined 21.2% at pH 7.4. Likewise, the efficiency of the immobilization procedure was increased by 25% in pH 7.4. The results indicated that \u003cem\u003eK\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs was higher than the free enzyme at pH 7.4; while, the free enzyme exhibited superior \u003cem\u003eK\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e over laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs at pH 4.5. These results are in alignment with the effect of pH on the activity of both free and immobilized laccase. The Michaelis constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e) indicates the affinity of the enzyme toward the substrate, and a low \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e value indicates the high affinity of the enzyme toward the substrate in both pH values. The improved accessibility of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs to the substrate (compared to the free enzyme) could be attributed to the high surface area of the flower-shaped structures that reduces the mass transfer limitations\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Normally, \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e, \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e, and the optimum conditions for reaching the highest catalytic efficiency enzymes may alter after immobilization\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Maximum activity of fungal laccases has been reported to be obtainable at a pH range of 3\u0026thinsp;\u0026minus;\u0026thinsp;5\u003csup\u003e58\u003c/sup\u003e. One of their main limitations is an insufficient activity in neutral and alkaline conditions despite the high redox potential of fungal laccases (490\u0026ndash;790 mV). In this regard, pH-tolerant laccases are desirable for industrial and environmental purposes\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Hybridization of laccase with cobalt phosphate nanocrystals enhanced the tolerance of laccase from \u003cem\u003eT. versicolor\u003c/em\u003e toward alkaline conditions, which could improve their desirability for industrial and environmental applications\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eKinetic parameters of the free laccase and laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eThe free laccase\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eLaccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u003cb\u003epH\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cb\u003epH\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4.5\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e7.4\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e4.5\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e7.4\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e (\u0026micro;mol/min)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e4.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e6.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e (\u0026micro;M)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e147.9\u0026thinsp;\u0026plusmn;\u0026thinsp;8.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e118.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e85.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e110.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e (S\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e5.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCatalytic efficiency \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e/ \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e4.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e4.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eImmobilization yield (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e65.3\u0026thinsp;\u0026plusmn;\u0026thinsp;7.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e65.3\u0026thinsp;\u0026plusmn;\u0026thinsp;7.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eImmobilization efficacy (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e86\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e110\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Reusability and stability of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs\u003c/h2\u003e \u003cp\u003eReusability is an important feature of biocatalysts that reduces their utilization cost, especially on the industrial scale\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. However, in the present study, measurement of the reusability for laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs provided evidence on the successful immobilization of laccase into the matrix of cobalt phosphate. In order to evaluate the reusability of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs, the enzyme activity was consecutively measured by ABTS as the substrate. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, after 4 consecutive reuse cycles, the activity of the laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs did not significantly change. However, the residual activity declined to 50% of its initial value after 10 cycle reuses. The results indicate that laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs exhibited remarkable reusability, and the enzyme was successfully immobilized in the matrix of cobalt phosphate. Laccase-based HNFs were exhibited promising reusability; for instance, laccase and lysin-copper HNFs retained 60% of initial activity after 6 cycles of reusability\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. In another study, laccase-copper HNFs retained 50% of the initial activity after 10 reusability runs; however, the glutaraldehyde-treated HNFs retained almost 100% of their initial activity at the same conditions\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, the stability of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs against elevated temperatures and basic pH values was higher than the free laccase. Laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs retained 50% of their activity after incubation at pH 9.5 for 3 h, where the free laccase almost lost all of its activity. It should be noted that the stability of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs was lower than the free enzyme in acidic pH values due to the solubility of cobalt phosphate in acidic pH\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. No data is available for comparison. As mentioned in 3.3, the α-helix content of laccase was increased after hybridization with cobalt phosphate. Copper ions of laccase, which are involved in the catalytic activity of the enzyme, are present in β-sheets, which are mainly composed of conserved polar amino acids\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. The stability and activity of laccase have been reported to be increased when the content of α-helix increased and β-sheets decreased\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. A more rigid protein structure aligned with increasing in α-helix content could explain the higher stability of laccase HNFs than the free enzyme\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. However, the stability of enzymes immobilized on cobalt-based supports was enhanced\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. The thermal stability of laccase-based HNFs was previously reported\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Immobilization of laccase into laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs increased the storage stability of the enzyme. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, after 40 days of storage at 25\u0026deg;C, the prepared HNFs retained 20% of the initial activity, while the free enzyme lost 58% of the initial activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Bioremoval of moxifloxacin\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs were able to degrade moxifloxacin significantly. However, due to the propensity of FQ antibiotics to adsorb onto inorganic surfaces, the possible adsorption of moxifloxacin on the synthesized HNFs was evaluated\u003csup\u003e13,68\u0026minus;70\u003c/sup\u003e. In order to specifically determine the adsorption of moxifloxacin on the constructed HNFs, the immobilized laccase was deactivated by sequential freeze and thawing cycles. Adsorption of moxifloxacin on the surface of HNFs reached a plateau after 18 h of incubation. The maximum adsorptive capacity of inactivated laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs for moxifloxacin was found to be 0.8 mg/g. The laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs completely removed moxifloxacin from the reaction mixture. Due to the high surface of HNFs, the adsorption of organic and inorganic components is not farfetched. There are some reports on the ability of HNFs for adsorption of cadmium and doxorubicin\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Identification of degradation products\u003c/h2\u003e \u003cp\u003eAfter the bioremoval procedure, the mixture of moxifloxacin biotransformed products was subjected to LC-MS analysis. The main by-products, their retention times, and corresponding mass to charge ration (m/z) are presented in Fig. S9. Based on the LC-MS results, five plausible pathways for the degradation of moxifloxacin are proposed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Interestingly, the biocatalyst replaced the fluorine atoms from moxifloxacin, which is responsible for the low biodegradability of FQs, with hydroxyl groups (compound III; m/z\u0026thinsp;+\u0026thinsp;1\u0026thinsp;=\u0026thinsp;262, R\u003csub\u003et\u003c/sub\u003e = 6 min)\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. As shown, ring-opening of piperazine moiety and further demethylation reactions were responsible for appearing compound VI (m/z\u0026thinsp;=\u0026thinsp;307, trace) and compound II (m/z\u0026thinsp;+\u0026thinsp;1\u0026thinsp;=\u0026thinsp;280, R\u003csub\u003et\u003c/sub\u003e = 3.9 min), respectively (pathway I). In pathway II, moxifloxacin underwent defluorination and cleavage of the piperazine moiety (compound VII; m/z\u0026thinsp;=\u0026thinsp;388, trace) and further transformed to compound V (m/z\u0026thinsp;=\u0026thinsp;358, R\u003csub\u003et\u003c/sub\u003e = 9 min) after dihydroxylation and demethylation\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. In the 3rd pathway, by oxidation and opening of piperazine ring, compound VIII (m/z\u0026thinsp;=\u0026thinsp;418, trace) was formed, which finally converted to compound IV (m/z\u0026thinsp;=\u0026thinsp;294, R\u003csub\u003et\u003c/sub\u003e = 8.7 min) after subsequent oxidation steps. In the next pathway, compound VIII (m/z\u0026thinsp;=\u0026thinsp;418, trace) was formed, and by complete cleavage of piperazine group, compound I was transformed to compound IX (m/z\u0026thinsp;=\u0026thinsp;307, trace) that was transformed to compound VIII (m/z\u0026thinsp;=\u0026thinsp;294, R\u003csub\u003et\u003c/sub\u003e = 8.7 min) after demethylation and cracking of cyclopropane ring (pathway IV). In the final proposed mechanism, moxifloxacin fluor atom was substituted with OH (compound X, m/z\u0026thinsp;=\u0026thinsp;400, trace) and further, by loss of piperazine group and demethylation, converted to compound III (m/z\u0026thinsp;+\u0026thinsp;1\u0026thinsp;=\u0026thinsp;262, R\u003csub\u003et\u003c/sub\u003e = 6 min) (pathway V).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.9. Toxicity of the biodegradation products\u003c/h2\u003e \u003cp\u003eDiminishing toxicity of pollutants is not always guaranteed by degradation procedures; even there are reports on an increase in toxicity of pollutants after degradation\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e,\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. In order to examine the efficiency of the synthesized HNFs, the toxicity of moxifloxacin and its metabolites against four bacterial strains was evaluated. As shown in Table S3, the toxicity of the degradation products was effectively decreased after treatment with laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs. Treatment of moxifloxacin with the constructed HNFs reduced the GI% for \u003cem\u003eP\u003c/em\u003e. \u003cem\u003eaeruginosa\u003c/em\u003e by 67%. GI% reduction for \u003cem\u003eS\u003c/em\u003e. \u003cem\u003eaureus, S. epidermidis\u003c/em\u003e, and \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e was 24.6%, 48.7%, and 12%, respectively. Some of the FQs degradation products exhibited antimicrobial activity. Due to the enhancement of DNA gyrase activity by fluorine, the defluorination of moxifloxacin significantly reduced its biological activity. Also, most of the identified degradation products possess lower hydrophobicity, which may limit their diffusion through the cell membrane\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e,\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e,\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study reported a facile method for the instant synthesis of HNFs. Contrary to previously-reported synthesis procedures, high concentrations of phosphate and cobalt ions were incorporated for the synthesis of HNFs. The formation of primary nucleation sites (protein-inorganic nanocrystals) and their anisotropic growth are the main steps involved in the synthesis of HNFs by the \u0026ldquo;concentrated method.\u0026rdquo; Rapid fabrication of HNFs was achieved by increasing the number of primary nanocrystals, which improved their collision rate and accelerated the growth of HNFs. Catalytic efficiency, kinetic parameters, and stability of laccase were enhanced after the immobilization of enzymes into HNFs. Based on CD and fluorimetry spectra, higher α-helix content of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs than the free enzyme is responsible for the enhanced stability of the constructed HNFs. The ability of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs to remove moxifloxacin was also assessed. The antibiotic was removed through adsorption and biodegradation mechanisms. The toxicity of degradation products against two G\u003csup\u003e+\u003c/sup\u003e and two G bacteria was remarkably lower than parent fluoroquinolone.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article (and its Supplementary Information files).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKhashayar Vojdanitalab: Conceptualization, Methodology, Investigation, and Writing - Original Draft. Hossein Jafari: Data curation, Writing- Original draft preparation, Visualization, and Software. Somayeh Mojtabavi\u003cem\u003e:\u003c/em\u003e Formal analysis, Writing - Review \u0026amp; Editing, and Conceptualization. Mahtab Shokri: Visualization, Investigation, and Methodology. Hoda Jahandar: Software, Validation, and Investigation. Mohammad Ali Faramarzi: Supervision, Conceptualization, Methodology, Resources, Data Curation, Writing - Review \u0026amp; Editing, Project administration, and Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by grant number 1400-2-104-54845\u0026nbsp;from\u0026nbsp;Biotechnology Research Center\u003cem\u003e,\u003c/em\u003e Tehran University of Medical Sciences, Tehran, Iran, to M.A.F.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKovalenko, G. \u0026amp; Perminova, L. 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Water Research \u003cb\u003e46\u003c/b\u003e, 5575\u0026ndash;5582 (2012).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Bioremoval, Enzyme immobilization, Hybrid nanoflowers, Laccase, Moxifloxacin","lastPublishedDoi":"10.21203/rs.3.rs-1351173/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1351173/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOrganic-inorganic hybrid nanoflowers (HNFs) have been synthesized by soft biomineralization procedures and are mainly used in biocatalysis and biosensing. Previously-reported methods for the synthesis of HNFs have so far required a 3-day incubation, bath sonication, or shear stress tension, which possibly introduces damage to the organic component. In this study, a novel method for instant fabrication of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs was developed without using harsh conditions. The prepared HNFs were assembled instantly by the \u0026ldquo;concentrated method,\u0026rdquo; which resulted in the fast growth of the flower-shaped nanostructures by a higher collision rate of the primary nucleation sites. The obtained results indicated that catalytic efficiency and enzymatic activity of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs were 113% and 110%, respectively, compared to the free enzyme. Also, the stability of the immobilized enzyme was enhanced by 400% in basic pH values. The activity of laccase@Co\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;HNFs declined to 50% of the initial value after 10 reusability cycles, indicating successful immobilization of the enzyme. Structural studies revealed a 32% increase in the α-helix content after hybridization with cobalt phosphate, which improved the activity and stability of the immobilized laccase. Furthermore, the fabricated HNFs exhibited a considerable ability to remove moxifloxacin as an emerging pollutant. The antibiotic (10 mg/L) was removed by 24% and 75% after 24 h through adsorption and biodegradation mechanisms, respectively. This study introduces a new method for synthesizing HNFs, which could be used for the instant fabrication of efficient biocatalysts, biosensors, and adsorbents to be potentially employed in industrial, biomedical, and environmental applications.\u003c/p\u003e","manuscriptTitle":"Instantaneous synthesis of organic-inorganic laccase-cobalt phosphate hybrid nanoflowers: Structural and biocatalytic characterization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-02-22 21:06:29","doi":"10.21203/rs.3.rs-1351173/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-05-04T06:48:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-03-02T13:54:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"09be3970-714e-466f-baea-5a3f7690d91a","date":"2022-02-19T20:51:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-02-19T20:11:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-02-19T19:57:15+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2022-02-19T18:00:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-02-19T17:16:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2022-02-11T17:49:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"31c049cf-9e73-4b66-a926-b6fde8d04507","owner":[],"postedDate":"February 22nd, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-05-25T04:59:07+00:00","versionOfRecord":[],"versionCreatedAt":"2022-02-22 21:06:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1351173","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1351173","identity":"rs-1351173","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}