1. Introduction

Magnetic iron oxide nanoparticles (Fe3O4 MNPs) have been used in various fields owing to their unique properties including large specific surface area and simple separation with magnetic field [1]. Because of the high surface energy, the pristine Fe3O4 (magnetite) nanoparticles are generally unstable and tend to aggregate easily, which strongly affects their dispersion in aqueous medium. In addition, Fe3O4 nanoparticles are highly susceptible to oxidation to γ-Fe2O3 nanoparticles in the presence of oxygen [2]. To overcome such limitations, various surface modification methods have been developed to modify the surface of pristine Fe3O4 nanoparticles via loading of other chemicals or biological materials during or after the synthesis process to improve the dispersibility, stability, biocompatibility and 1

biodegradability for specific purposes [3,4]. With proper surface modification, these desirable properties of Fe3O4 nanoparticles could be improved, and the oxidation process from Fe3O4 nanoparticles to γ-Fe2O3 nanoparticles could be greatly prevented [4-6]. Surface modification such as immobilization of biomolecules has been demonstrated as a promising approach to address these issues. The use of tannic acid, one of the most wellknown plant-derived polyphenol, has been reported in the construction of multifunctional coatings in material-independent surface chemistry. Tannic acid functionalized substrates do not present observable cytotoxicity [7]. The identification of new polyphenol coating precursors is of great importance because the immense structural diversity of this family of natural compounds can lead to coatings with novel chemical and biological properties [8]. Immobilization of enzymes on solid supports can enhance enzyme stability and proteolysis efficiency as well as facilitating separation and recovery for reuse [9-11]. One of commonly used method for immobilization of enzymes on magnetic nanoparticles is covalent bonding on bare or modified magnetic nanoparticles with the formation of covalent bonds between the chemical moieties on the particles and on the enzymes. This method is often associated with some reduced enzyme activity, but strong binding of enzyme on magnetic nanoparticles can be accomplished with the prevention of immobilized enzymes leakage [9]. Trypsin (EC 3.4.21.4) has been commonly used in biological research during proteomics experiments. Trypsin, which specifically cleaves peptide bonds on the C-terminal group of lysine or arginine, is a pancreatic serine endoprotease, and is traditionally used for protein digestion [12]. It is also used for numerous biotechnological processes such as protein primary structure analysis, in the breakdown of casein in milk for baby food, and in the development of cell and tissue culture protocols [13-15]. The use of immobilized trypsin for cleavage of proteins has several advantages compared to its soluble form [16]. For instance, immobilized trypsin reactors have been integrated into separation systems such as reversed-phase liquid chromatography or capillary electrophoresis, mass spectrometry analysis, for proteome studies [17]. Furthermore, immobilized trypsin derivatives have been used for continuous hydrolysis of casein [18], affinity chromatography [19], cutaneous dressing [20] and many different applications. Enzymatic hydrolysis can be carried out under milder reaction conditions than chemical processes. Generally, the side reactions do not occur and the nutritional value of the protein source do not decrease in the enzymatic processes. Furthermore, enzymes are specific to the substrate and produce protein hydrolysates which have better quality in terms of chemical and nutritional characteristics [21,22]. Casein is the main protein in milk, and various bioactive peptides have been identified from casein hydrolysates [23]. The aim of this work is to covalently immobilize trypsin on tannic acid (TA) coated Fe3O4 nanoparticles and to investigate the digestion of casein from bovine milk. Firstly, the magnetite nanoparticles were prepared via solvothermal method. The solvothermal method can yield control over the particle size with a carefully adjustment of the involved parameters, thus the homogeneous particles distribution can be obtained [4,24]. Secondly, tannic acid was coated on magnetite nanoparticles and trypsin (TR) was covalently immobilized on tannic acid modified magnetite nanoparticles. Finally, the enzymatic digestion of casein by the immobilized and the free trypsin was investigated to determine whether more peptide fragments could be obtained by immobilized trypsin in terms of accurate protein identification.

2. Experimental

2.1. Materials 2

1

Ferric chloride hexahydrate (FeCl3·6H2O, >99%), sodium acetate, di-sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium dodecyl sulphate, sodium hydroxide (≥97%), ethanol (>99.2%) were obtained from Merck (Germany). Ethylene glycol (EG) (99%, C2H6O2) and ammonium hydrogen carbonate were provided from Tekkim (Turkey). Trypsin from bovine pancreas, Nα-Benzoyl-DL-arginine 4-nitroanilide hydrochloride (BApNA, 98%), albumin from bovine serum (BSA; 98%, agarose gel electrophoresis), casein from bovine milk, Bradford Reagent, benzaimidine, 4-nitroaniline (p– nitroaniline), Tetramethylethylenediamine (TEMED), ammonium persulfate (APS),

sodium

dodecyl

sulfate

(SDS),

β-merkaptoetanol

(99.0%),

Tris(hydroxymethyl)aminomethane, acetic acid, methanol (>99.8%) were purchased from Sigma-Aldrich (USA). PageRuler™ Unstained Low Range Protein Ladder (Protein Gel Electrophoresis) was obtained from Thermo Scientific™ (USA). All chemicals were of analytical grade and used without further purification. All aqueous solutions were prepared with ultrapure water (18 MΩ·cm).

2.2. Synthesis of magnetite nanoparticles and its modification with tannic acid

The magnetite nanoparticles (Fe3O4) were synthesized by a solvothermal method [25,26]. FeCl3· 6H2O (4.440 g) and NaAc (14.40 g) were first dissolved in ethylene glycol (160 mL) and the mixture was stirred at 200 rpm for 1 h. Afterward, the solution was transferred into a 50 mL Teflon-lined stainless-steel autoclave. The autoclave was sealed and heated at 200°C and maintained for 8 h. Then, it was allowed to cool to room temperature. The magnetite nanoparticles were washed with deionized water three times and ethanol once and separated by magnetic decantation. Finally, the magnetite nanoparticles were dried in vacuum at 70 °C for 12 h. The synthesized magnetite nanoparticles (1.5040 g) were dissolved in 60 mL of deionized water at 40 °C for 1 h. Then, tannic acid (0.7127 g) was dissolved in 30 mL of deionized water and the solution was added drop wise into the magnetite nanoparticles dispersion. The mixture was stirred at 200 rpm at 40 °C for 2 h. The tannic acid modified magnetite nanoparticles were separated by magnetic decantation. The obtained product was washed with 3

deionized water thrice and ethanol once and separated with a magnet. Then, the product was dried in vacuum at 70 °C for 12 h [27].

2.3. Trypsin immobilization and digestion of casein

0.5030 g of tannic acid modified magnetite nanoparticles (Fe3O4-TA) were stirred in the 3 mL of sodium hydroxide solution (0.1 M, pH 9.4) at 200 rpm for 1 h at room temperature to obtain the quinone groups on the tannic acid modified magnetite nanoparticles by using the pH dependent oxidation reaction of polyphenols. Then, tannic acid modified magnetite nanoparticles were separated magnetically. 10 mg/mL trypsin solution (containing 6 mg/mL benzamidine in 0.1 M, pH 7.5 sodium phosphate buffer solution, PBS) was added into base treated modified magnetite nanoparticles. The mixture was incubated by stirring at 200 rpm at 4 °C for 3 h. The product was then separated magnetically, and washed with the same buffer solution for three times. The immobilized trypsin was stored at 4 °C until use. The efficiency of protein digestion by free and immobilized trypsin (Fe3O4-TA-TR) was evaluated by using casein protein as described elsewhere [28]. Standard stock casein solution (1 mg/mL) was prepared in NH4HCO3 buffer solution (0.1 M, pH 8.0). Prior to the digestion, casein was denatured in a water bath at 95 °C for 15 min to increase efficiency of protein cleaving [29]. 900 µL immobilized trypsin (1 mg/mL) in NH4HCO3 buffer solution (0.1 M, pH 8.0) was transferred into a 1.5 mL Eppendorf tube and then 100 µL of denaturated casein solution was added to the same tube. Then the mixture solution was incubated at 37 °C for 1 h, followed by the magnetically separation using a magnet. The liquid phase was examined by LC–MS/MS system. Similarly, 875 µL free trypsin (0.2 mg/mL) in NH4HCO3 buffer solution (0.1 M, pH 8.0) was transferred into a 1.5 mL Eppendorf tube and 100 µL of denaturated casein solution was added to the same tube. After the mixture solution was incubated at 37 °C for 13 h, digestion was stopped with the addition of 15 µL of acetic acid. Similarly, the liquid phase was examined by LC–MS/MS system.

2.4. Characterization

The phase identification of the magnetite nanoparticles was investigated by X-ray diffraction (XRD) (PANalytical, Empyrean, Netherlands). The morphology of magnetite nanoparticles were examined by Philips XL30 SFEG scanning electron microscope (SEM). Magnetic properties of the prepared samples were studied using vibrating sample magnetometer (VSM LakeShore-7407, USA) at room temperature. FTIR spectra were recorded on Shimadzu UATR Two instrument (Japan) over the wavenumber range of 4000–400 cm-1. TGA analysis was performed on SII SEIKO thermogravimetric analyzer (Japan) under N2 flow, with the heating rate of 10 ºC/min, and temperature ranged from 25 to 1000 ºC. LC–MS/MS was carried out AB SCIEX 4000QTRAP equipped with ESI probe. Sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS–PAGE) was performed using a BIO-RAD MiniPROTEAN Tetra cell electrophoresis chamber apparatus.

2.5. LC/ MS-MS procedure

The peptides released from the digestion of casein by trypsin were separated on A Spark UHPLC system. After the samples preparation, measurements were carried out according to similar studies with minor modification [30,31]. Buffer A (0.1% formic acid in water) and buffer B (0.1% formic acid in acetonitrile) were used as mobile phases for gradient separation 4

by adjusting the flow rate to 0.5 µL/min and the injection volume to 20 µL. The gradient separation of peptides was carried out on Phenomenex Synergi Fusion-RP C18 (4.6µm x150mm, 5µm particle) column and the column temperature was set to 40 °C. For casein digestion, a gradient separation was carried out by linearly increasing buffer B from 5 % to 35 % over 30 min, then from 35 % to 100 % within 20 min, and kept constant for 10 min, while linearly decreasing buffer A from 95 % to 65 % over 30 min and then 0 % within the following 20 min.

2.6. SDS-PAGE study

SDS–PAGE was carried out for the investigation of digestion of casein (6 mg/mL) using immobilized trypsin, as described elsewhere [32]. For SDS-PAGE anal ysis, 20 mg immobilized trypsin was added into 1 mL casein solution (6 mg/mL in 0.1 M NH4HCO3). The mixture containing immobilized trypsin was kept at 37 °C. 30 µL aliquot was taken at 10 min intervals and transferred into an Eppendorf tube. Then, 70 µL SDS-PAGE loading buffer (1 mL Tris-HCl buffer (1 M), 1.6 mL glycerol (20%), 0.2 mL SDS (10%), 0.4 mL βmercaptoethanol and 8 mL deionized water, pH 6.8) was added to the same tube. Also, pure casein protein was studied according to the same procedure. Prior to the electrophoresis, samples were denatured at 95 °C in a water bath for 3 min to increase protein denaturation [29]. The samples (10 µL) were loaded on a SDS-PAGE gel which was prepared using a Mini-PROTEIN (Starter kit for hand casting 10% TGX Stain-Free polyacrylamide gels, includes acrylamide solutions and buffers). Then, the gel was placed in a BIO-RAD Mini-PROTEAN Tetra cell electrophoresis chamber apparatus. Electrophoresis was carried out at 200 V for 25 min at room temperature. Afterward, gel was treated with a solution containing 50% methanol and 10% acetic acid for 1 h [33] to fix the peptides on gel, followed by staining with a dye solution containing 17% ammonium sulfate, 0.0651% Coomassie G250, 34% methanol and 3% phosphoric acid for 1 h. Finally, gel was destained with 10% acetic acid solution overnight.

2.7. Statistical analysis

All experimental measurements were carried out in triplicate. The results were expressed as the mean value (standard deviation (SD) is ±3%).

3. Results and discussions

3.1. Immobilization of trypsin on tannic acid modified Fe3O4

Methods of immobilization can affect enzyme activity when the coupling point is situated near the enzyme active site. For high-molecular-weight enzymes such as trypsin, introduction of an appropriate spacer often endows the enzyme with more flexibility. This can significantly increase the activity of the enzyme, due to reduction of steric hindrance compared to the immobilized enzyme without the spacer. In this study, the unbound trypsin determination was carried out using Bradford method for enzyme content on tannic acid coated magnetite 5

nanoparticles (Fe3O4-TA) [34]. The covalently bound trypsin amount was found to be 91% of trypsin in the initial solution. The binding process can be rationally regulated with specific functional groups [4]. In addition, low-cost and efficient immobilization without using toxic chemicals was carried out by oxidizing the tannic acid coated on the Fe3O4 through pH increment. Upon the oxidization of polyphenols, the quinones moieties are formed on TA. Quinones is a cyclic organic compound group containing two carbonyl groups. As mentioned in Table 1, there are reports describing that ketones and aldehydes, which have carbonyl groups, can react with amine containing substances under mild conditions. The obtained carbonyl groups with the formation of quinones and amine containing trypsin can react in the room temperature to yield Schiff-base product with a pH sensitive covalent bonds [35]. Therefore, working condition must be chosen above pH 6 to prevent the hydrolysis of Schiffbase bonds. Optimum pH of immobilized trypsin found in this study (pH 9) rules out the possibility of the covalent bonds. This functional coating not only provides a facile strategy for the covalent immobilization of biomolecules under mild reaction conditions, but can also be adapted for a wide range of materials without the need for surface pretreatment [7,36]. The process of covalent immobilization was shown in Scheme 1.

3.2. The studies of activity, stability of free and immobilized trypsin

Immobilized enzymes usually show better thermal and pH stability with easier separation, and can be reused, which appear suitable for practical applications. Immobilized trypsin showed a good reusability than free trypsin since the immobilization process utilized somewhat strong covalent binding in this study. All studies of free and immobilized trypsin on activity were given in Supplementary data (Fig S1-S6). In Figure S1, Nα-Benzoyl-DL-arginine 4nitroanilide hydrochloride (BApNA) hydrolysis showed maximum activity for free form of the trypsin at pH 7.5, but the immobilized trypsin demonstrated its maximum activity at pH 9.0. These results were expected because the number of positively charged groups of enzyme linked with the amino groups to the carrier decreases after immobilization [27]. Also, this pH value avoids the hydrolysis of Schiff base covalent bonds owing to the medium pH. In general, the optimum pH values of animal-origin trypsin are in the range of pH 6.0 to 9.0 [37, 38]. The maximum activity was observed at 45 °C for both forms of enzyme (Fig. S2). The value of optimum temperature for these and similar study was 45 °C [39]. Immobilized form of the enzyme was more stable at higher temperatures than free form. The immobilized enzyme was found to be more thermostable than the free enzyme at 55 °C (Fig. S3). Higher optimum temperature might be resulting from the improved physical properties of the enzyme after immobilization by multipoint covalent attachment on the magnetic nanoparticles. Stability during storage is highly important parameter, which should be taken into account in systems, which use immobilized form of enzymes. Both free and immobilized trypsin were stored at 4 °C under the same conditions and the activity measurements were carried out for a 120-day period (Fig. S4). Effect of substrat (BApNA) concentration changes on enzymatic activity was carried out by changing the BApNA concentration from 0.5 to 3 mM. The kinetic fitting for BApNA hydrolysis reaction was calculated according to the Michaelis–Menten equation. Michaelis constant (Km) and the maximum reaction velocity (Vmax) of free and immobilized forms of the enzymes were obtained from Lineweaver-Burk plots (1/V versus 1/S). The immobilized trypsin (12.1 mM) possessed a higher Michaelis constant than the free trypsin (5.1 mM) (Fig. S5), which indicates the structural changes on the enzyme after covalent immobilization. Furthermore, the relative activity of immobilized trypsin decreased to 59% of its initial activity after 8 cycles of reuse. The stable covalent bonds between trypsin and Fe3O4-TA might be the reason for this decent reusability of immobilized trypsin (Fig. S6). Our results showed that immobilized form of the trypsin by covalent bonds provided a decent 6

stability on the Fe3O4-TA surface. All these results demonstrate that the tannic acid modified Fe3O4 are good supports for trypsin immobilization and the process can find more biological applications.

3.3. X-ray diffraction analysis

X-ray diffraction pattern of pure Fe3O4 is presented in Fig. 1(A). XRD pattern of Fe3O4 powder showed that all of the obtained diffraction peaks are well matched with the cubic structure of Fe3O4 inverse spinel phase (ICDD card no. 98-018-3971) with Fd3m space group. Fig. 1(A) shows that there are seven visible diffraction peaks, which can be indexed to the structure of the solvothermally synthesized Fe3O4. A broad and diffuse band was appeared at around 2θ=35.35 in Fe3O4 [40,41]. XRD results indicated highly pure Fe3O4 was synthesized with the excellent crystallinity. The results also demonstrated that a single cubic magnetite phase was obtained in the products.

3.4. Thermogravimetric analysis

As Fig. 1(B) shows, the additional quantitative evidence of the structure of Fe3O4, Fe3O4-TA and Fe3O4-TA-TR was revealed by thermal analysis. In the case of Fe3O4 MNPs (Fig. 1(B)(a)), the weight loss of 6.04% is due to the physically adsorbed water molecules on magnetite nanoparticle surface. The weight loss of Fe3O4-TA (Fig. 1(B)(b)) was found to be about 12% at broad temperature range of 0–1000 °C. Such an intense TA presence can imply that tannic acid is not only attached to the magnetite surface, but it is also connected to the other TA molecules through physical interactions by forming anchored nanoparticles. The mass loss at the temperature range of 500- 600 °C corresponds to the degradation of the main chains of TA [42].Thus, this temperature range reflects the thermal stability of the macromolecule skeleton. For the Fe3O4-TA-TR (Fig. 1(B)(c)), the complete decomposition temperature value was between 700 and 800 °C. Also, the Fe3O4-TA-TR underwent a weight loss of approximately 34% of its initial mass when heated till 1000 °C. Also, it can be concluded that Fe3O4-TA and Fe3O4-TA-TR demonstrated the same thermal decomposition profile except that Fe3O4-TA-TR underwent sharper mass loss phases, which evidently imply the intense trypsin presence.

3.5. FTIR analysis

Fig. 1(C) presents the FTIR spectra of Fe3O4, Fe3O4-TA and Fe3O4-TA-TR. In the spectra, the onset of the Fe-O bands in the region below 800 cm−1 are prominently observed [42]. The shape and position of both signals centered near 548 and 636 cm−1 are consistent with Fe-O stretches in the inverse spinel structure of Fe3O4 (Fig. 1(C)(a)). The signals in the middle region of the spectra (1700–1000 cm−1) are related with the organic content at the surface of magnetite because of tannic acid modification in Fig. 1(C)(b). The absorption bands between 1600 and 1400 cm−1 are related to aromatic –C=C– bonds. The peaks at 1397 and 1064 cm−1 7

in the spectrum of Fe3O4-TA belong to phenol groups [27]. After the functionalization with tannic acid, two obvious bands appearing at 2885 and 2987 cm−1 are associated with C–H stretching vibrations. After trypsin immobilization, the changes in the spectrum of Fe3O4-TATR are observed between 1000 and 1700 cm−1 in Fig. 1(C)(c). Particularly, the FTIR spectrum exhibits a strong peak corresponding to the (C=N) at 1628 cm−1, which proves the formation of Schiff base. Also, the peak at 1532 cm−1 originates from the in-plane N-H bending of trypsin [43]. The results found in this study are comparable with those studies available in the literature. The significantly similar data from the literature concerning FTIR Schiff-base stretching bands were given in Table 1 [43-47]. Therefore, the immobilization procedure enables the utilization of immobilized enzyme for the practical applications.

3.6. Magnetic measurements

Fig. 1(D) shows the magnetic hysteresis curves of the Fe3O4, Fe3O4-TA and Fe3O4-TA-TR measured at 300 K. The saturation magnetization value of Fe3O4 was larger than those of the saturation magnetization of Fe3O4-TA and Fe3O4-TA-TR. From Fig. 1(D)(a), the saturation magnetization of the Fe3O4 is about 63.5 emu/g at room temperature [25]. After tannic acid modification on the Fe3O4, the saturation magnetization of Fe3O4-TA is about 61 emu/g, which is illustrated in Fig 1(D)(b). The saturation magnetization value of Fe3O4-TA-TR is about 39 emu/g Fig 1(D)(c). The results indicate that the saturation magnetization decreases after modification with tannic acid and trypsin immobilization. Especially, the substantial decrease after immobilization implies the large amount of trypsin immobilized on the modified surface, which indicates the superior enzyme binding property of the procedure.

3.7. Scanning electron microscopy analysis

The morphologies of the Fe3O4, Fe3O4-TA and Fe3O4-TA-TR were characterized by SEM. Fig 2 (a) shows a typical low magnification SEM image of the Fe3O4. From Fig. 2 images, agglomerate structures of cauliflower-like magnetite can be observed. These agglomerate 8

nanoparticles can be considered with sizes approximately less than 500 nm resulting from magnetic interaction between the particles [48]. Also, the solvothermal method enables us to control over the particle size and shape for the specific applications. Furthermore, after TA modification, agglomerate nanoparticles decreases in size owing to the fact that TA is coated on nanoparticles by reducing the magnetic interaction by leading to the nanoparticle separating from the main agglomerate nanoparticles (Fig 2 (a)). Also, enzyme immobilization makes the Fe3O4-TA slightly bigger, which implies the trypsin binding (Fig 2 (a)).

3.8. LC-MS/MS

Trypsin specifically hydrolyzes peptide bonds at the carboxyl side of lysine and arginine amino acids. The efficiency of the free and immobilized trypsin for casein digestion were investigated by LC–MS/MS. The total ion LC–MS chromatograms of digested casein by free and immobilized trypsin were shown in Fig. 3(A) and 3(B). As can be seen at the higher time intervals, the smaller amount of peptides with high molecular weight was observed by immobilized trypsin compared to free trypsin, showing the efficient digestion of casein by immobilized trypsin. Also, more unrelated peaks were observed in the chromatogram of free trypsin, which result from the trypsin autohydrolysis. Free trypsin proceeds to hydrolyze peptide fragments of casein to peptides with low molecular weight as can be seen at the initial times. Therefore, more peptide fragments could be obtained by immobilizing trypsin on an appropriate support material without autohydrolysis, and improved protein digestion [28].

3.9. SDS-PAGE

The SDS-PAGE electropherogram for casein degradation is shown in Fig. 4. Casein contains three different proteins (α, , and κ). The major casein subunits may be distinguished by electrophoresis and the casein subunits vary primarily in molecular weight and isoelectric point. Casein is rich in proline residues without any disulfide bridges, which do not interact. As a result, it has relatively simple tertiary structure [49]. The major bands at the position with molecular weight about 30 kDa indicated these subunits of casein [50]. The band of the casein is clearly seen at 30 kDa in Line 2 and Line 6. As can be seen in the electropherogram, casein was cleaved by immobilized trypsin effectively with increasing time up to 30 min. When the digestion time increased to 30 min., the intensity of casein bands decreased and discolored [51]. The peptides formed during digestion were visible as some minor bands at the positions lower than the molecular weight between 20 and 10 kDa in Line 3, Line 4 and Line 5. The decent digestion of casein by immobilized trypsin may be attributed to its broad cleavage specificity and its stability on the biocompatible material [52]. Particularly, the degradation bands of casein are clearly demonstrated in this study. It can be concluded that hydrolysis of casein by immobilized trypsin was very effective.

4. Conclusions

In this study, we mainly focused on magnetic property of iron oxide for magnetic applications of immobilized enzymes. Fe3O4 was prepared by solvothermal method and was functionalized with tannic acid. Trypsin was immobilized on tannic acid-modified Fe3O4 by using facile, low-cost, and efficient covalent binding method. The hydrolysis products of casein formed in the presence of immobilized trypsin were confirmed successfully by using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The presented tannic acid modified Fe3O4 can also 9

be used as an appropriate support material for immobilization of various biological molecules such as antibody/antigen and other enzymes. The surface modification method improved the dispersibility, stability, biocompatibility of the magnetic nanoparticles for specific purposes. Due to the above mentioned advantages, as well as their long-term stability and simple fabrication, the trypsin immobilized magnetic nanoparticles can potentially simplify protein analyses and separation. Acknowledgments

This investigation has been supported by the Scientific Research Projects Commission of Sakarya University (Project number: 2015-50-02-014). M.Ö. acknowledges the partial support of the TUBA. References [1] M. Cao, Z. Li, J. Wang, W. Ge, T. Yue, R. Li, V.L. Colvin, W.W. Yu,;1; Food related applications of magnetic iron oxide nanoparticles: enzyme immobilization, protein purification, and food analysis, Trends Food Sci. Techn. 27 (2012) 47−56. [2] S. Chen, Z. Xu, H. Dai, S. Zhang,;1; Facile synthesis and magnetic properties of monodisperse Fe3O4/silica nanocomposite microspheres with embedded structures via a direct solution-based route, J. Alloys Compd. 497 (2010) 221–227. [3] V.N. Jadhav, A.I. Prasad, A. Kumar, R. Mishra, S. Dhara, K.R. Babuc, C.L. Prajapat, N.L. Misra, R.S. Ningthoujam, B.N. Pandey, R.K. Vatsa,;1; Synthesis of oleic acid functionalized Fe3O4 magnetic nanoparticles and studying their interaction with tumor cells for potential hyperthermia applications, Colloid Surf. B Biointerfaces 108 (2013) 158–168. [4] J.K. Xu, F.F. Zhang, J.J. Sun, J. Sheng, F. Wang and M. Sun,;1; Bio and Nanomaterials Based on Fe3O4, Molecules 19 (2014) 21506-21528. [5] M. Slováková, M. Sedlák, B. Kˇríˇzková, R. Kupˇcík, R. Bulánek, L. Korecká,ˇC. Draˇsar, Z. Bílková,;1; Application of trypsin Fe3O4@SiO2 core/shell nanoparticles for protein digestion, Process Biochem. 50 (2015) 2088–2098. [6] Y.T. Zhu, X.Y. Ren, Y.M. Liu, Y. Wei, L.S. Qing, & X. Liao,;1;Covalent immobilization of porcine pancreatic lipase on carboxyl-activated magnetic nanoparticles: Characterization and application for enzymatic inhibition assays, Mater. Sci. Eng. C Mater. Biol. Appl. 38 (2014) 278–285. [7] K. Xiong, P. Qi, Y. Yang, X. Li, H. Qiu, X. Li, R. Shen, Q. Tu, Z. Yang, N. Huang,;1; Facile immobilization of vascular endothelial growth factor on a tannic acid-functionalized plasma-polymerized allylamine coating rich in quinone groups, RSC Adv. 6 (2016) 17188– 17195. [8] D.G. Barrett, T.S. Sileika, P.B. Messersmith,;1; Molecular diversity in phenolic and polyphenolic precursors of tannin-inspired nanocoatings, Chem. Commun. 50 (2014) 7265– 7268.

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Figure 1. XRD pattern of solvothermally synthesized Fe3O4 (A), TGA curves (B), FTIR spectra (C) and magnetization curves (D) of (a) Fe3O4, (b) Fe3O4–TA, (c) Fe3O4–TA– TR at 300 K.

Figure 2. SEM images of Fe3O4 (a), Fe3O4–TA (b), and Fe3O4–TA–TR (c).

Figure 3. The total ion LC-MS chromatograms of digested casein by free (A) and immobilized trypsin (B).

Figure 4. SDS-PAGE electropherogram demonstrating marker (line 1), Casein (line 2 and line 6), and peptides formed by the digestion of casein catalyzed by immobilized trypsin for 10 min (lane 3), for 20 min (line 4), and for 30 min (line 5) (M: Marker). 14

Scheme 1. The illustration of pH-induced oxidation of pyrogallol groups of tannic acid and subsequent binding reactions with the amines on enzyme. Table 1. Comparison of FTIR spectral data for Schiff base in this study and other studies. Support Materials

Compounds

Schiff base (C=N)

of Binding

Stretching

References

(wavenumber) APTES-MNPs

Salicylaldehyde

1635.5

[43]

Salicylaldehyde

2,2-dimethyl-

1630

[44]

salicylaldehyde

1610

[45]

histidine and

1635

[46]

dialdehyde

1625

[47]

Trypsin

1628

This study

propylendiamine

3-aminopropyltri ethoxysilane modified mesoporous silica Fe3O4@SiO2@APTMS

glutaraldehyde

Fe3O4-2-aminoethyl dihydrogen phosphate

Fe3O4-TA

(pH increment)

TDENDOFDOCTD

15

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