Design and synthesis of a novel nanocomposite based on magnetic dopamine nanoparticles for purification of α-amylase from the bovine milk

In this paper, a novel nanocomposite based on magnetic nanoparticles decorated by dopamine were reported. Three modified magnetic nanocomposites by dopamine were offered with different type of linkers. The mentioned magnetic nanocomposites were applied to separate α-amylase protein from fresh bovine milk. All of the magnetic nanocomposites were characterized and investigated by using Fourier-transform infrared spectroscopy, energy-dispersive X-ray spectroscopy, field-emission scanning microscope, X-ray diffraction pattern, and vibrating-sample magnetometer analyses. To investigate the purifying application, sodium dodecyl sulfate polyacrylamide gel electrophoresis, one-dimensional isoelectric focusing gel electrophoresis, and alpha-amylase activity assay were employed. With paying attention to factors such as yield of purification and concentration of separated protein by each of magnetic nanocomposite, it could be concluded that the length of linkers played an important role in α-amylase protein separation. According to the results, the best separation and purification of α-amylase protein with 49.83% recovery and 40.11-fold purification efficiency was related to longest length linker, 1,4-butanediol diglycidyl ether, because of considerable conjugation with nanocomposite. Also, docking calculation has shown that the binding energy is − 1.697 kcal/mol and ΔG = − 6.844 kcal/mol which result that the interaction process between dopamine and α-amylase protein is spontaneous.

, immobilized metal affinity chromatography 7 , and size exclusion chromatography 8 to separate proteins. On the other side, electrophoresis methods are another procedures that can be applied to separate amino acids, and proteins 9 . Recently, magnetic nanocomposites have been highlighted as novel purification materials to apply in different contexts such as energy conversion 10,11 , catalysts 12,13 , enzyme and biomacromolecules separation 14 . Investigations of magnetic nanoparticles in biomedical fields such as tissue engineering 15,16 , detection of virus 17 , and cancer biomarkers 18 , are exclusively developed. As well as, scientists have focused in separation and adsorption of momentous and particular biomacromolecules such as DNA and proteins 19 from complexes of their sources by magnetic-based nanocomposites. Separating and adsorbing of proteins can be the result of magnetic nanoparticles potency to interact with target biomacromolecule on their surface 20 . Controlling the size of nanoparticles by synthesized procedures is an important factor which must be considered for purification applications. The surface modification with organic or inorganic compounds such as metals 21 , and biomolecules 20 make the nanocomposites able to create positive or negative charges on their surface to make interaction with proteins. Fe 3 O 4 magnetic nanoparticles (Fe 3 O 4 MNPs) with owning advanced features like high surface area and low toxicity are applied in separation of biomacromolecules 22 , hyperthermia of cancer therapy 23,24 , and catalytic agent in chemical reactions 25,26 . Fe 3 O 4 MNPs are able to be modified like other magnetic nanoparticles by different kinds of chemical functional agent 27 . Based on previous studies about purification aspects, modified Fe 3 O 4 MNPs are applied to separate heme proteins 28 , and α-amylase proteins 29 . In chemistry and biological contexts, enzymes have been used to start and continue reactions as a biological catalysts. These catalysts have more benefits in comparison to chemical catalysts. α-amylase (α-1,4-glucan-4-glucanohydrolase) proteins as biological catalysts received much attention in enzymatic reactions because of their abilities to hydrolyze starch 30 . This type of protein with three-dimensional structure is able to play an important role in food industry, textile, clinical, medicinal and chemistry fields 31 .
In this research study, three novel magnetic nanocomposite based on modifying the surface of Fe 3 O 4 MNPs are designed and synthesized with different shells such as tetraethyl orthosilicate (TEOS), (3-chloropropyl)trimethoxysilane (CPTMS), (3-aminopropyl)trimethoxysilane (APTMS), epichlorohydrin (ECH), 1,4-butanediol diglycidyl ether (BDDE), and dopamine (DA) (Fig. 1). These magnetic nanocomposites with different length and type of linkers are applied to separate α-amylase protein from fresh bovine milk. Obtained results from the separation of mentioned proteins are revealed that the length and type of linkers must be considered as important factors for protein purification. In addition to these descriptions, docking calculations have shown that the binding energy is − 1.697 (kcal/mol) and ΔG = − 6.844 (kcal/mol) which indicate that interaction process between dopamine (DA) and α-amylase protein is spontaneous.  Preparation of magnetic Fe 3 O 4 @SiO 2 @APTMS@ECH@DA nanocomposite. To coat DA shell on the obtained core-shell structure, in brief, 0.54 g of Fe 3 O 4 @SiO 2 @APTMS@ECH powder was dispersed in ethanol for 10 min. Afterwards, 1.75 g of DA was added to the mixture solution. Next, the mixture was refluxed for 18 h. Finally, the obtained product was separated using an external magnet and washed three times with distilled water to remove unreacted substances and the drying process was conducted at 60 °C for overnight (see supplementary information file, Fig. S7). Fourier-transform infrared spectroscopy. The spectra were recorded using Fourier-transform infrared (FT-IR) spectrometer (Shimadzu FT-8400 s model, Japan) to characterize the formation of new functional groups in each synthesis step. 0.1-1.0% of each sample was well mixed into 200-250 mg of fine KBr powder for preparation of sample pellets. Considering the spectral resolution (4 cm −1 ) and a determined frequency range (400-4000 cm −1 ), each spectrum was taken at room temperature and the average number of scans was between 6 and 18 32 .

Functionalization of Fe
Energy-dispersive X-ray spectroscopy. The elemental composition of sample was identified by energydispersive X-ray (EDX) device (SAMx model, France) with the accelerating voltage of 20 kV, 10 s live time, and using ultrathin window detector.
Field-emission scanning microscopy. Using the field-emission scanning microscope (FE-SEM) (ZEISS-Sigma VP model, Germany), the Morphology, structure and size of samples were characterized, operating at a 15 kV. Each sample was mounted with double side carbon tape on stainless steel stub, and gold sputter-coating technique was performed (Agar Sputter Coater model, Agar Scientific, England). Besides, the images were taken with a determined scan rate (30 ns/pixel) 32 .
Vibrating-sample magnetometer. Vibrating-sample magnetometer (VSM) was used to evaluate the saturation magnetization value (LBKFB model magnetic kavir, Iran). All the hysteresis loop curves were determined using an applied magnetic field from − 15,000 to + 15,000 Oe.
Isolation and removal of cream and casein from bovine milk. First, fresh bovine milk was made from a local dairy and transferred to the lab. To separate the cream from the milk, the milk was centrifuged at 6000 rcf, for 15 min at 37 °C. Subsequently, to separate casein from the milk, the pH of obtained skim milk was reduced to 5 by hydrochloric acid solution on ice to precipitate casein. Then, the obtained solution was centrifuged at 6000 rcf, for 10 min at 10 °C 33 , and the pH of the supernatant was finally brought to 7 by sodium hydroxide solution. The obtained casein-free skim milk (CFSM) was used to continue the process.
Isolation of α-amylase from the bovine milk by synthesized magnetic nanocomposites. α-Amylase isolation by synthesized magnetic nanocomposites, was performed according to the method  34,35 , preparing buffer A (50 mL of 0.05 M phosphate buffer containing 50 mM NaCl was made and its pH was adjusted to 7.8) and elution buffer B (50 mL of 0.05 M phosphate buffer containing 0.3 M NaCl was made and its pH was adjusted to 7.8). In the next step, 300 mg of each synthesized magnetic nanocomposite was mixed with 2.5 mL of buffer A and then 0.2 mL of CFSM was added and the solution was mechanically stirred for 30 min. Subsequently, each magnetic nanocomposite was separated using an external magnet from the suspension and washed three times by 2.5 mL of buffer A to remove unbound proteins. Following that, each isolated magnetic nanocomposite was stirred for 10 min with 1 mL of buffer B to separate α-amylase from their surface. Then, using a magnet, the solution containing α-amylase was removed from each nanocomposite and used for the rest of the steps.
α-Amylase activity assay. α-Amylase activity assay was performed according to the method described by Zakowski et al. On this basis, 0.2 mL of CFSM was mixed with 0.3 mL of phosphate buffer (0.05 M) and 0.5 mL of starch solution (1% w/v) as a substrate, at 37 °C for 10 min. Then, to stop the reaction, 1 mL of DNS solution (1% w/v) was added and the solution was heated in a water bath for 5 min. Subsequently, 0.33 mL of potassium sodium tartrate solution (4% w/v) was added and cooled rapidly in ice. Finally, the absorbance of the resulting solution was measured at 540 nm and the maltose concentration was determined using a standard curve 5 .
Measurement of total protein concentration. The protein amount of the samples was measured by biophotometer (Eppendorf) at 595 nm, according to the Bradford assay using bovine serum albumin (BSA) as standard 36 . Interpret the result of SDS-PAGE analysis using quantity one software. The gel was scanned using a calibrated densitometer and analyzed by quantity one 1-D analysis software (Bio-Red, v4.6.3). As a result of this analysis, the relative quantity and peak density (peak OD) for each band were obtained and the α-amylase purification efficiency was calculated for all nanocomposites.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE
One-dimensional isoelectric focusing (1D-IEF) gel electrophoresis. To determine the purified α-amylase isoelectric point, 1D-IEF in slab gel was performed. This technique also confirmed the purity of the purified enzymes. According to the protocol, 10 mL of acrylamide gel solution containing a mixture of 5.5 g of urea (9.1 M final), 2 mL 10% v/v Triton X-100 (2% v/v final), 2 mL Milli-Q water, 1.35 mL acrylamide stock solution (30% w/v acrylamide, 1.6% w/v bisacrylamide; treated with amberlite), 0.2 mL ampholytes pH 5-8, 0.2 mL ampholytes pH 7-9 and 0.1 mL ampholytes pH 3.5-10 was prepared. Then, to initiate polymerization, 20 μL of APS (10% w/v) and 10 μL TEMED were added. Finally, the gel was poured using electrophoresis tools. 20 mM NaOH solution was used as anodic buffer and 20 mM H 3 PO 4 solution was used as cathodic buffer. Subsequently, 3 μL of marker (IEF standards, Bio-Rad) and 8 μL of each sample were loaded into the wells and the gel was run under 350 V for 10 h. After complete the run, the gel was stained with colloidal Coomassie brilliant blue (R-250) staining solution overnight, then de-stained with acetic acid solution (1% v/v) for 1 h 38 .

Result and discussion
Three magnetic nanocomposites were fabricated by modifying the surface of Fe 3 O 4 MNPs with different linkers and shells (Fig. 1). The mentioned magnetic nanocomposites were characterized using various analysis methods (FT-IR, EDX, FE-SEM, XRD, VSM analyses). FT-IR analysis was used to indicate the formation of new functional groups, EDX analysis to characterize structural composition, XRD pattern to indicate the crystalline phase of Fe 3 O 4 MNPs, and VSM analysis to evaluate the magnetic properties and saturation magnetization value; which are discussed, respectively. Apart from these analysis methods, to evaluate these magnetic nanocomposites for separation of α-amylase protein from fresh bovine milk, other analysis methods were applied such as SDS-PAGE analysis to determine weight molecular and purity of separated proteins, and 1D-IEF analysis to determine isoelectric point. Also, the molecular modeling and docking study were conducted too.

Characterization of synthesized magnetic nanocomposites. FT-IR analysis. As illustrated in
FT-IR spectrum of Fe 3 O 4 MNPs (Fig. 2a), an absorption band at around 578 cm −1 and a broadband at 3400 cm −1 are related to the stretching vibration modes of Fe-O and presented hydroxyl groups on the surface of nanoparticle 24,39,40 . Coating the silica shell on the surface of Fe 3 O 4 core are accompanied by appearance of new functional groups (Fig. 2b). As could be seen, three absorption bands around 478 cm −1 , 800 cm −1 , and 1100 cm −1 can be attributed to the bending, symmetric, and asymmetric stretching vibration modes of Si-O-Si 41 . In addition to this, two absorption bands around 1632 cm −1 and 3200-3600 cm −1 (3413 cm −1 ) are assigned as stretching vibration mode of O-H, and O-H stretching vibration mode of Si-OH 42 . In continue, the presence of APTMS shell as second layer is characterized by observing new absorption bands. As indicated in Fig. 2c 45 . Following that, in Fig. 2d, small absorption band around 755 cm −1 can determine CH 2 -Cl functional group in the ECH structure 46 . Two broad absorption bands around 1098 cm −1 and 1064 cm −1 can be related to the epoxy group of ECH 47 . The FT-IR spectrum of magnetic Fe 3 O 4 @SiO 2 @APTMS@ECH@DA nanocomposite is indicated in Fig. 2e. The presence of three absorption bands around 1121 cm −1 , 1490 cm −1 , and 1616 cm −1 are assigned as aliphatic and aromatic C-H bending and N-H bonding vibration modes, respectively. Also, an absorption band around 2924 cm −1 can be ascribed to C-H stretching vibration mode of aromatic group 48 . Besides, a broad absorption band around 3350 cm −1 can be related to the presence of OH groups of dopamine 49 .

EDX analysis and FE-SEM imaging.
According to the EDX spectrum of magnetic Fe 3 O 4 @SiO 2 @CPTMS@DA nanocomposite (Fig. 3a), the presence of two iron peaks can be related to the magnetic Fe 3 O 4 cores. Silicon, carbon, and oxygen peaks can confirm coating inorganic TEOS, CPTMS shells. Also, the presence of carbon and nitrogen peaks can be attributed to the coated DA structure. On the other side, given the FE-SEM imaging from magnetic Fe 3 O 4 @SiO 2 MNPs and magnetic Fe 3 O 4 @SiO 2 @CPTMS@DA nanocomposite ( Fig. 3b-d), the sphere morphology with almost uniform structure is observed. As well as, in comparison to the average size of Fe 3 O 4 @ SiO 2 MNPs (40-43 nm), the size of magnetic Fe 3 O 4 @SiO 2 @CPTMS@DA nanocomposite has increased from 155 to 250 nm; which is due to the surface functionalization process with different shells.
VSM analysis. In general, magnetic susceptibility and saturation magnetization value of magnetic-based nanostructures can be determined by vibrating-sample magnetometer analysis. It has been indicated that different factors including core size, shell thickness, interparticle and intraparticle interactions, and iron-group crystalline structure can impact on magnetic properties 51 . As could be seen in Fig. 5a,b, the saturation magnetization value of bare Fe 3 O 4 MNPs before surface modifying is 76.20 emu/g (Fig. 5a); while this factor for magnetic Fe 3 O 4 @ SiO 2 @CPTMS@DA nanocomposite has decreased to 20.77 emu/g (Fig. 5b) (3) nanocomposite, which has the longest linker, showed the highest efficiency in purification of α-amylase from bovine milk, which increased the specific activity of α-amylase by 40-fold; while, magnetic Fe 3 O 4 @SiO 2 @CPTMS@DA (1) and Fe 3 O 4 @SiO 2 @APTMS@ECH@DA (2) nanocomposites showed an approximately 30-fold increase. In explaining the cause, it can be said that the longer and more flexible and available linker for immobilizing the ligand in the structure of magnetic Fe 3 O 4 @ SiO 2 @APTMS@BDDE@DA (3) nanocomposite, gives the ligand more access to α-amylase. Figure 6a compares the specific activity of α-amylase in purified by magnetic Fe 3 O 4 @SiO 2 @CPTMS@DA (1), Fe 3 O 4 @SiO 2 @APTMS@ ECH@DA (2), and Fe 3 O 4 @SiO 2 @APTMS@BDDE@DA (3) nanocomposites. Also, Table 1 shows that the best and most efficient result for the separation and purification of α-amylase from the casein-free skim milk (CFSM),

SDS-PAGE assay results.
The results of the SDS-PAGE and single-band observation on the gel, without the presence of any significant impurities, indicate that all three magnetic nanocomposites bind specifically to α-amylase and do not bind to other proteins. As indicated in Fig. 7a, CFSM has many proteins that are observed in multiple bands in lane 1. After purification of α-amylase from CFSM, a major band of approximately 58 kDa molecular weight (MW) is obtained in lines 3, 4 and 5 on the gel. As can be seen in Fig. 7b, the α-amylase purified by the three magnetic nanocomposites including Fe 3 O 4 @SiO 2 @CPTMS@DA (1), Fe 3 O 4 @SiO 2 @APTMS@ECH@DA (2), and Fe 3 O 4 @SiO 2 @APTMS@BDDE@DA (3), has an isoelectric point (PI) of about 6.5-6.8. IEF analysis in addition to determining the pI of the isolated enzymes, confirms their SDS-PAGE results in terms of purity. It should be noted that in both Fig. 7a,b, the band derived from the α-amylase purified by magnetic Fe 3 O 4 @SiO 2 @ APTMS@BDDE@DA (3) nanocomposite, is sharper than the other two bands, indicating that the purification of α-amylase from the bovine milk using magnetic Fe 3 O 4 @SiO 2 @APTMS@BDDE@DA (3) nanocomposite, is more effective and impressive. To complete the results, the purification efficiency of α-amylase by three magnetic Fe 3 O 4 @SiO 2 @CPTMS@DA (1), Fe 3 O 4 @SiO 2 @APTMS@ECH@DA (2) and Fe 3 O 4 @SiO 2 @APTMS@BDDE@DA (3) nanocomposites was calculated based on semi-quantitative analysis with quantity one software in addition to the specific activity calculations. Figure 7c shows the bands selected for analysis in the software (see supplementary information file, Figs. S10, S11). Also, Table 2 shows the peak density (peak OD) and relative quantity of each band calculated by the software. Based on the results shown in Table 3, the purification efficiency of α-amylase was obtained for all three nanocomposites. As determined, magnetic Fe 3 O 4 @SiO 2 @APTMS@BDDE@DA (3) nanocomposite has the highest value with 67.7% purification efficiency (see supplementary information file, Fig. S12).
Effect of incubation time on the adsorption of α-amylase to synthesized magnetic nanocomposites and their absorption capacity as nanocarrier. 300 mg of the nanocomposite was added to 2.5 mL of phosphate buffer and after dissolution, 0.2 mL of casein-free skim milk (CFSM) was added. The resulting mixture was incubated at room temperature for 5,10,15,20,25,30,35 and 40 min, after which the supernatant was separated from the nanocomposite by magnet and reacted with 0.5 mL of 1% w/v starch solution. At the end of each time period and after the supernatant was removed, α-amylase enzymatic activity was measured according to the procedure described in experimental section, the results of which are visible in Fig. 8a. According to the diagram, as the incubation time increased, α-amylase activity decreased, with no activity observed after 30 min. This is due to the increased adsorption of α-amylase to the surface of magnetic nanocomposites over time. 0.2 mL of casein-free skim milk (~ 0.57 mg protein) was stirred individually with 100, 200 and 300 mg of synthesized magnetic nanocomposites for 30 min at room temperature in phosphate buffer. The magnetic nanocomposites were then separated from the solution using a magnet and washed three times with phosphate buffer. Finally, α-amylase was eluted from the magnetic nanocomposites and total protein concentrations of the samples were measured. In addition, the adsorption capacity of all three magnetic nanocomposites was obtained by using following Eq. (1).  Table 4 and Fig. 8b. Accordingly, the capacity of the magnetic Fe 3 O 4 @ SiO 2 @APTMS@BDDE@DA nanocomposite is higher than the others, which can be due to the longer linker on the surface of the nanocomposite, resulting in improved BDDE ligand access to α-amylase.
(1) Adsoption capacity = An average amount of eluted α -amylase from nanocomposite µg An average amount of nanocomposite (mg) Comparison. In 2017, Farzi-Khajeh et al., synthesized nanocomposites with different components and lengths and used them to separate α-amylase from bovine milk. The highest purification efficiency (calculated by specific activity) with these nanocomposites was 49.66% and the maximum adsorption capacity was 0.466 ± 0.023 μg protein (α-amylase) per mg 34 . While in this present study, the highest purification efficiency, is 49.83% based on specific activity and 67.70% based on semi-quantitative analysis by quantity one software and as well, the maximum absorption capacity has improved to 0.661 ± 0.028.   In other word, water, other molecules and ions were removed from the PDB structure. Then optimization and minimization were performed on amylase by force field OPLS-3. The binding site was determined based on 1ppi. Glide was used the preparation box set to 25 × 25 × 25 Å and centered at the point with − 10, 45 and 25. The 3D structure of DA is drawn with Gauss View 5, then were optimized using the density function theory (DFT) method 54 . Using Beck's three-parameter hybrid function and the Lee-Yang-Parr nonlocal correlation function (B3LYP) and 6-31 + G * basis set 55,56 . Finally, the docking experiment was performed using the Maestro algorithm by Glide 57 . Docking calculation has shown that the binding energy is − 1.697 (kcal/mol). Free energy calculation was performed using the MM-GBSA method 58 . ΔG = − 6.844 (kcal/mol) shows that this process is spontaneous. In this calculation, the number of hydrogen bond acceptors is 2.5, the number of hydrogen bond donors is 4 and molecular weight is 153.180 (g/mol), subsequently, in this calculation Lipinski's rule is not violated 59 . Also, Predicted central nervous system activity for this structure is inactive (see supplementary information file, Fig. S14) 60 .

Conclusions
Nowadays, new and high-tech procedures have made a big progress in separation and purification methods. Magnetic nanocomposites are one of those procedures which are employed widely in separation technologies especially in purification of important macromolecules like proteins. In current research, a unique magnetic nanocomposite which decorated by dopamine biomolecules, was designed and synthesized. Three different types of linker with different length were used to decorate magnetic nanoparticles by dopamine. The mentioned nanocomposite was evaluated in separation of α-amylase protein from fresh bovine milk. Structure and  www.nature.com/scientificreports/ morphology of nanocomposite was characterized and investigated by using FT-IR, EDX, FE-SEM, XRD, and VSM analyses. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, one-dimensional isoelectric focusing gel electrophoresis and alpha-amylase activity assay were used to investigate yield of α-amylase protein purification. After evaluation of obtained results, it was clearly concluded that the length of linkers played an important role in α-amylase protein separation. The best separation and purification of α-amylase protein with 49.83% recovery and 40.11-fold purification efficiency was related to longest length linker, 1,4-butanediol diglycidyl ether, because of considerable conjugation with nanocomposite. Docking calculation has shown that the binding energy is − 1.697 kcal/mol and ΔG = − 6.844 kcal/mol which result that interaction process between DA and α-amylase protein is spontaneous.