Covalent immobilization of trypsin on polyvinyl alcohol-coated magnetic nanoparticles activated with glutaraldehyde

Journal Pre-proof Covalent immobilization of trypsin on polyvinyl alcohol-coated magnetic nanoparticles activated with glutaraldehyde Selmihan Sahin, Ismail Ozmen

PII:

S0731-7085(19)32961-9

DOI:

https://doi.org/10.1016/j.jpba.2020.113195

Reference:

PBA 113195

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received Date:

6 December 2019

Revised Date:

16 February 2020

Accepted Date:

19 February 2020

Please cite this article as: Sahin S, Ozmen I, Covalent immobilization of trypsin on polyvinyl alcohol-coated magnetic nanoparticles activated with glutaraldehyde, Journal of Pharmaceutical and Biomedical Analysis (2020), doi: https://doi.org/10.1016/j.jpba.2020.113195

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Covalent immobilization of trypsin on polyvinyl alcohol-coated magnetic nanoparticles activated with glutaraldehyde

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Selmihan Sahin, Ismail Ozmen* Suleyman Demirel University, Arts and Sciences Faculty, Department of Chemistry, Cunur,

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Isparta, 32260, Turkey.

*Corresponding author.

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E-mail address: [email protected] (I. Ozmen)

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Highlights

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Graphical abstract

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Trypsin immobilized on polyvinyl alcohol coated MNPs with high activity recovery and protein coupling yield. The immobilization of trypsin on polyvinyl alcohol coated MNPs improved its stability and reusability. The immobilized trypsin digested cyt c to the small peptide fragments. The immobilized trypsin have good operational stability at wider pH and temperature.

ABSTRACT Magnetic nanoparticles were coated with polyvinyl alcohol and activated with glutaraldehyde for trypsin immobilization. The prepared magnetic nanoparticles were characterized by transmission electron microscopy, fourier transform infrared spectroscopy, thermal gravimetric analysis, zeta potential meter and vibrating sample magnetometer. Free and immobilized trypsin showed optimum activity at pH 6.0, 30°C and pH 7.0, 40°C, respectively. Immobilized trypsin was more stable than the free enzyme at 40°C. After immobilization, Km of the

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immobilized trypsin increased, however, Vmax value was almost the same with free trypsin. According to the results, the immobilized trypsin retained 50% of its initial activity, whereas free trypsin retained 19% of its initial activity after 12-days at 4°C. Immobilized trypsin

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sustained 56% of its initial activity after eight times of successive reuse. The performance of the immobilized trypsin was evaluated by digestion of cytochrome c. The peptide fragments in

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digest solution were determined by using MALDI-TOF mass spectrometry. Immobilized

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trypsin showed effective proteolytic activity in shorter time (15 min) than free trypsin (24 h). Hence, immobilized trypsin on the polyvinyl alcohol coated magnetic nanoparticles could be

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promising biocatalyst for large-scale proteomics studies and practical applications.

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Keywords: Trypsin; immobilization; magnetic nanoparticles; polyvinyl alcohol; digestion;

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cytochrome C

1. Introduction Trypsin (Try, E.C.3.2.21.4) is an enzyme in hydrolyses group selectively catalyze the hydrolysis of peptide bonds between arginine and lysine amino acid residues of proteins. It is used in several practical applications in the food industry such as proteolysis of milk casein for baby foods, extraction spices and aromas from vegetables, stabilization of beer and production of hypoallergenic foods. In recent years, it has also gained great attention in proteomic studies for protein digestion and organic synthesis. However, using of free Try in these applications

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has limitations such as difficult to recovery it from reaction systems, needed long times, low sensitivity, inefficient digestion and rapidly auto-digestion resulting in

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interfering fragments and decreasing reaction efficiency through the process [1, 2].

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Immobilization is useful to overcome these limitations by improving the stability, activity and reusability of the enzyme. Also, immobilized enzymes can be easily removed from the reaction

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medium which provides automated control to the reaction systems [3] .

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Key points in enzyme immobilization are the support material and immobilization method [4]. Magnetic nanoparticles (MNPs) are considered as an ideal support because of advantages such as simple preparation, strong magnetic response, small size and minimized diffusional

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limitations, large surface area and high enzyme loading capacity [5]. In addition, immobilized enzymes on MNPs can be effectively separated from the reaction medium by an external

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magnet that is a much easier way compared with other materials [2]. However, naked MNPs

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are very unstable and easily oxidized in air [6]. They can be protected against oxidation and functionalized for enzyme immobilization by coating surface with chemically active polymers [7] or with compounds including amino (-NH2), hydroxide (-OH), carboxylic acid (-COOH) and phosphate groups [6, 8]. Previously, Wang et al [9] have improved digestion efficiency of Try with its immobilization on polyaniline-coated nano-Fe3O4/carbon nanotube composite. They attributed to these result to the high surface area-to-volume ratio of nanoparticles, which

can increase interaction between Try and the substrates (proteins). Also, auto-digestion of Try was reduced after immobilization and thus, efficiency of digestion was increased [9]. Cabrera et al [10] prepared immobilized Try on polyaniline-coated magnetic diatomite nanoparticles with improved properties in terms of activity and stability. Zdarta et al [1] reported two different systems by immobilizing Try on magnetite-lignin and magnetite-chitin hybrid supports. They indicated that using magnetite hybrid materials for enzyme immobilization provides higher stability, more effective proteolytic activity in peptide digestion and reusability. Kim and Lee

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[11] reported immobilization of Try on glutaraldehyde-pre-activated chitosan nonwoven. The obtained immobilized Try had a lower pH stability than free trypsin, but an excellent thermal stability. Aslani et al [6] improved thermal and pH stability of Try with immobilization on the

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Fe3O4@SiO2-NH2. Atacan et al [12] prepared pillar[5]arene immobilized Try and evaluated its microwave-assisted digestion of cytochrome C (Cyt c). They showed a high efficient and fast

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microwave-assisted digestion of Cyt c with pillar[5]arene immobilized Try by using MALDI-

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TOF mass spectrometry (MALDI-TOF MS) for 15 second. Azevedo et al [13] immobilized Try onto magnetic-chitosan composite to detect antinutrients in aquafeeds. Even though there are many researches on immobilization of Try on the magnetic nanoparticles

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and different supports, there are hardly any reports on the immobilization of Try on the polyvinyl alcohol (PVA) based support. Caramori et al [3] immobilized Try on PVA

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glutaraldehyde/polyaniline composite and showed that it has good enzyme loading capacity and

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better properties such as pH profile, thermal stability and storage stability than free one. In this study, PVA polymer was chosen for coating surface of MNPs due to its easy availability, hydrophilic character and high concentration of reactive hydroxyl groups which are capable chemical reaction [3]. Try was covalently immobilized on glutaraldehyde activated PVA coated-MNPs (PMNPs-GA). The optimum conditions for activity and thermal stability, storage

stability and reusability of the immobilized Try were investigated. Also, digestion of cyt c with the immobilized Try was evaluated and monitored by MALDI-TOF MS. 2. Materials and methods 2.1. Materials Polyvinyl alcohol (PVA), Benzoyl-L-arginine ethyl ester (BAEE), benzamidine HCl, glutaraldehyde (GA) and Cytochrome C (cyt c) were obtained from Merck. Try from bovine pancreas was purchased from Amresco Corporation. Other chemicals and reagents were

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analytical grade unless otherwise stated. 2.2.Intrumentation

Fourier transform infrared spectroscopy (FT-IR) analyses were carried out with a of Perkin

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Elmer Frontier Fourier transformation infrared spectrometer. Thermal gravimetric analysis (T.G.A) were performed on a Perkin–Elmer (Beaconsfield, BucksHP91QA, England) thermal

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analyzer under N2 atmosphere with a heating from 25 to 800°C. Morphology analysis was

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performed on a transmission electron microscopy (T.EM ). MALDI-TOF MS analysis was performed with an Ultraflextreme MALDI-TOF, Bruker Daltonics, United states. MS were obtained in the positive reflection mode. Matrix solution was a saturated solution of α-cyano-

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4-hydroxycinnamic acid (HCCA) in 30:70 (v/v) acetonitrile/water containing 0.1% trifluoroacetic acid. The instrument parameters were: number of shots, 4000; acceleration

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potential, 20 kV; UV laser, 336 nm. MALDI-TOF MS results were obtained by using Bruker

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FlexAnalysis software. Zeta potential measurements were conducted with a ZetaSizer instrument by using 0.1 wt.% of PMNPs-GA suspension in the pH range between pH 4.0-11.0. Three measurements were performed for each sample at room temperature. Vibrating sample magnetometer (VSM) was used to obtain magnetization curves (M-H) of MNPs and PMNPs and the measurements was performed at room temperature.

2.3. Preparation of PVA coated MNPs Preparation of MNPs has been previously described [14]. To coat surface of MNPs with PVA, 3% w/v of PVA solution was prepared in water at 90 °C. 1 g of MNPs was added to the PVA solution and stirred at room temperature and 150 rpm for 20 h. The final ratio of MNPs:PVA was 1:3. PMNPs were collected by centrifugation at 8000 rpm for 10 min. After that, they were washed three times with distilled water and dried at 50°C. 2.4. Covalent immobilization of Try on PMNPs

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Try was immobilized on aldehyde-functionalized PMNPs with covalent Schiff base linkage and these particles were named as PMNPs-GA-Try. For this, 25 mg of PMNPs were activated with %2.5 w/v of GA (in 0.1 M H2SO4) and washed with distilled water three times. Then,

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activated PMNPs were mixed with Try solution containing 0.25 mg/mL benzamidine and incubated at 25°C for a time with gentle shaking. Immobilization conditions such as time (1 h

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and 3 h), pH (4.0-11.0) and enzyme amount (0.375 mg-3 mg) were optimized. Finally, the

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resulting PMNPs-GA-Try were washed with K-phosphate buffer 25 mM pH 6.0 containing 1 M NaCl to desorb those enzyme molecules that were bound by electrostatic interactions, and then washed with K-phosphate buffer 25 mM pH 6.0. The remaining activity of the PMNPs-

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GA-Try was subsequently assayed in the optimum conditions. Amount of immobilized Try was determined with the Bradford by measuring protein

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concentration in solutions. The protein coupling yield (Y) and the activity recovery (E) were

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calculated as follows:

Y=100x[(P0-P1-P2)/P0], E=100x(A1/A0) Where P0 is the initial protein amount of the initial enzyme solution and P1 and P2 are protein

amount of the final enzyme solutions and the washing solutions, respectively; A1 and A0 are the activity of the immobilized and free Try, respectively. 2.5. Activity assay of free and immobilized Try

Activity of free and immobilized Try was measured by the modified method of Bergmeyer et al [15]. For free Try activity, 0.25 mM Nα-benzoyl-L-arginine ethyl ester (BAEE) in 25 mM K-phosphate buffer was used as substrate. 0.2 mL Try solution was added to 3.0 mL of the substrate solution and the increase in absorbance of the mixture at 253 nm was measured during 1 min in optimum conditions. For determination of immobilized Try activity, 25 mg of PMNPs-GA-Try was used. To obtaine same reaction volume, distilled water (0.2 mL) was added to the aforementioned

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reaction mixture. After this, it was incubated at 25ºC, 180 rpm for 1 min and then immobilized enzyme was collected with centrifugation. The absorbance at 253 nm was measured in the supernatant spectrophotometrically.

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One unit of Try activity was defined as the amount of Try required to release of 1 μmol Nα-

2.6. Characterization of immobilized Try

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benzoyl-L-arginine from BAEE per minute under the assay conditions [15].

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The activities of free and immobilized Try were measured at different pHs ranging from 5.0 to 9.0 at 25°C for determination of the optimum pH. The buffers were 25 mM citrate (pH 5.0), K-phosphate (pH 6.0 and 7.0) and sodium carbonate (pH 8.0 and 9.0). To determine the

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optimum temperature of free and immobilized Try, the activities of both enzymes were determined at different temperatures ranging from 20°C to 60°C. The relative activity (%) was

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sample.

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calculated by the percentage of the remaining activity compared with the initial activity for each

To determine the kinetic parameters (Km and Vmax) of free and immobilized Try, the

enzymatic activity was measured at optimum conditions with increasing concentration of BAEE (between 1 mM-20 mM). The Km and Vmax parameters for free and the immobilized Try were calculated from Lineweaver−Burk.

Thermal stability was evaluated by incubating free and immobilized Try in a water bath at 40°C for different time intervals (0-180 min). Samples were withdrawn and subjected to activity assay for remaining activity. The initial activity was considered as 100%. For storage stability studies, free and immobilized Trys were kept in buffer at 4°C and the remaining activity of free and immobilized Try was measured at certain intervals. The reusability was determined by repeated utilization of immobilized Try to catalyze the hydrolysis of BAEE. After reaction, the PMNPs-GA-Try was removed from the reaction

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mixture by magnetic separation and washed with 25 mM K-phosphate buffer (pH 7.0). The original activity of the PMNPs-GA-Try was considered as 100%. 2.7. Try digestion of cyt c

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Cyt c was used as model protein. To obtain 1 mg/mL of cyt c solution, 10 mg of cyt c was dissolved in 10 mL of 50 mM NH4HCO3 (pH 8.2) without any previous treatment. For

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immobilized Try digestion, 25 mg of PMNPs-GA-Try was added into each tube containing 50

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μg of cyt c. After incubation at 37 °C, 110 rpm for 15 min, the PMNPs-GA-Try was collected by magnetic separation, and the supernatant was collected and further analyzed with MALDITOF MS.

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For free Try digestion, free Try was added into cyt c solution (1:50 wt ratio of Try to protein) and then incubated at 37 °C, 110 rpm for 24 h. The enzymatic digestion was stopped with

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addition of acetic acid and the mixture was further analyzed by using MALDI-TOF MS.

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2.8. Statistical analysis

All measurements were done in triplicate, and data were reported as mean ± standard

deviation (SD).

3. Results and discussion 3.1. Characterization of the PMNPs To provide covalent immobilization of enzymes, surface of MNPs should be modified with different methods to produce different reactive groups such as -NH2, -OH and -COOH [7, 14, 16]. In this study, surface of MNPs was coated with PVA containing reactive -OH groups providing a surface modification for enzyme immobilization. The transmission electron microscopy image in Fig. 1 indicates that the coating of MNPs

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surface by adsorption of PVA is successful and that PMNPs have particle size around 244 nm. The chemical structure of MNPs, PMNPs and PMNPs-GA, PMNPs-GA-Try and Try was studied by FTIR (Fig. 2). For MNPs (Fig. 2a), peaks at 585 cm−1 corresponded to the Fe-O bond

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[14, 17]. In the PMNPs spectrum (Fig. 2b), the additional bands at 2922 cm−1 corresponded to C–H stretching, at 1397 and 1322 cm−1 corresponded to C–C stretching vibrations and at 1082

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cm−1is attributed to M–O–C (M=Fe) bond that were obtained with the PVA coating.

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After activation with GA, the new small band located at 1740 cm−1 was assigned to the carbonyl group (C=O) (Fig. 2c). When compared Fig. 2d and 2e, the characteristic peaks of Try at 1406, 1454 and 2931 cm−1 (Fig. 2e) also appeared in the spectrum of PMNPs-GA-Try (Fig.

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2d). Characteristic peaks at 1562 cm−1 and 1542 cm−1 were attributed to the vibration of amide I (C=O stretching) band and the amide II (NH bending), respectively [18] (Fig. S1). The

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appearance of all these peaks indicated that Try was immobilized onto PMNPs-GA

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successfully.

TGA curves of the MNPs and PMNPs are shown in Fig. 3. The weight percentage of PVA

on MNPs was calculated as about 5.3 % with the difference of the weight percentage of MNPs and PMNPs at 800 °C. Zeta potential of the PMNPs-GA at different pHs (between 4.0-9.0) was determined (Fig. S2). The zeta potentials of PMNPs-GA negatively decreased with the increase in pH until a

plateau obtained around pH 10.0 region. This could be attributed to PMNPs-GA had more negatively charged surface with the increase of pH. Because pI value of Try was 10.5, it is cationic at below this pH value. Thus, the electrostatic interaction between negative the PMNPs-GA and positive charged Try increased with the decrease in the negative zeta potential of the PMNPs-GA. This situation could be improved first adsorption and then covalent immobilization of Try on the PMNPs-GA in relatively uniform orientation [19]. Magnetic properties of the MNPs and PMNPs were determined from the M-H curves prepared

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by using VSM. The remanence and coercive force values of MNPs and PMNPs were very small and negligible (Fig S3). While the remanence (σr) is 0.3 emu/g and the coercivity (Hc) is 4 Oe for the MNPs, the remanence (σr) is 0.7 emu/g and the coercivity (Hc) is 14 Oe for the PMNPs.

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According to the result, it could be said that PMNPs could be easily dispersed in the solutions providing better interaction between substrate and the immobilized enzyme. Also, immobilized

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Try could be easily removed from the solution for effective reusability in practical applications.

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The saturation magnetizations (Ms) was determined as 56.25 emu/g and 53.12 emu/g for MNPs and PMNPs, respectively. This decrease could be attributed to increase in the mass of nanoparticles after coating with PVA [7, 10].

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3.2. Covalent immobilization of Try to PMNPs activated with GA Two-step process was attempted for covalent immobilization of Try on PMNPs. The surface

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of MNPs was coated with PVA, functionalized with GA, and used to immobilize enzyme

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through a covalent Schiff base linkage. Different immobilization conditions were examined such as time, pH, and amount of enzyme. Activity recovery (%) and protein coupling yield (%) of the process was assessed to determine the optimum conditions for immobilization of Try and its activity.

3.3. Effect of reaction time on the immobilization of Try To determine the effect of reaction time on Try immobilization, the study was performed for 1 h and 3 h. The protein coupling yield (%) increased with longer immobilization time and was determined as 52% and 79% at 1 h and 3 h, respectively (Table 1). However, the activity recovery (%) was decreased from 99% to 30% with incubation time and the higher activity was obtained for 1 h incubation. This may be related with steric hindrance of enzyme molecules causing difficult interaction between substrate with enzyme active site [5]. Hence, all

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immobilization studies were performed for 1 h. According to the Table 2, the protein coupling yield and activity recovery results of this study can comparable with the other studies [4, 20-22]. Try immobilized on PMNPs-GA showed high

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activity recovery. This could be attributed to the hydrophilic character of PVA resulted to better dispersibility of the PMNPs in water solution. Thus, the interaction PMNPs-GA with substrate

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could be improved. Also, the use of PMNPs-GA for Try immobilization provided high protein

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coupling yield compared to other reported support materials because of its large surface and the high amount of reactive groups on its surface [20]. 3.4. Effect of pH on Try immobilization

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The pH of the incubation reaction has effect on the conformation of the enzyme, the state of the support’s surface and functional groups[19]. Therefore, pH of the enzyme solution is an

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important factor for immobilization of enzyme through the amino group on supports.The

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optimum pH for immobilization of Try to the GA activated-PMNPs was investigated in a pH range between 4.0-11.0. The optimal pH for immobilization of Try was pH 6.0 (Fig. 4). Overall, the protein coupling yield was increased with increasing pH while the relative activity of immobilized Try decreased with pH higher than 6.0. GA activated-supports provide immobilization of proteins terminated amino group (reactive at these pH ranges) even at acidic-neutral pH values (6.0-8.0). At basic pH, lysine is active for

immobilization but carbonyl groups of GA are not. Hence, immobilization of Try at pH 6.0 on the GA activated-PMNPs will occur mainly via the most reactive and exposed amine groups (very likely, the terminal amino group). Try lost most of its activity below and above pH 6.0, (section 3.6). This means that it is easily inactivated at these pH values since the immobilization of Try can’t be favorable. Furthermore, pI value of Try was 10.5 and thus, it becomes cationic below this pH value while it is negatively charged at pH 11.0 [19]. The high protein coupling yield at alkaline pHs may be a result of favoring the lysine group in this pH range where

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immobilization of Try may occur via lysine group in addition to terminal amino group. These results also compatible with the Zeta potential results (Section 3.1). 3.5. Effect of enzyme amount on the immobilization of Try

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The immobilization of Try was carried out at pH 6.0 and 25 °C, and the amount of Try in the immobilization mixture was increased from 0.375 to 3 mg of Try by adding different

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volumes of 3 mg/mL of initial Try solution (Fig. 5).

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Although immobilized Try amount increased with increasing initial amount of Try added, protein coupling yield decreased and reached a saturation level at 0.75 mg Try. Conversely, specific activity of immobilized Try increased with the Try concentration ranging from 0.375

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mg to 0.75 mg, but that it decreased with increasing Try concentration. Hence, the optimal Try amount for immobilization was determined to be 0.75 mg in the immobilization mixture. When

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used 0.375 mg of Try, the immobilized amount of enzyme was not enough to show high activity.

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When the immobilization mixture contained 1.5 and 3 mg of the amount of Try, the specific activity of immobilized enzyme was decreased. At these situations, even if the protein coupling yield was almost same, the amount of immobilized enzyme was increased. Because of these, diffusion limitation and changing at enzyme conformation could be occur as well as some active sites might be hidden, which led to decrease in activity when used 3 mg of Try [4, 7].

3.6. Effect of pH and temperature on free and immobilized Try activity The effect of pH on the relative activity of the free and PMNPs-GA-Try was studied in a pH range pH 5.0–9.0 at 25°C (Fig. 6a). The optimal pH for free and immobilized Try was 6.0 and 7.0, respectively. The shift observed towards pH 7.0 with the immobilized Try could be associated with ionizable groups in the enzyme and the support material [23]. Immobilized enzyme showed greater activity than free one in a wider pH range. Similar results were found by Atacan and Ozacar [8] for immobilized Try on the tannic acid modified Fe3O4 and by

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Tuzmen et al [23] on dye attached magnetic beads. In spite of that, Kim and Lee [11] reported that Try from Porcine Pancreas covalently immobilized on Chitosan nonwoven showed lower pH stability than free Try.

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Fig. 6b shows the effect of temperature on the activity of free and immobilized Try. The optimum activity was observed at 40°C for the immobilized Try and 30°C for free enzyme. The

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observed increase at optimum temperature after immobilization could be related with increase

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in the rigidity of immobilized enzyme [6]. Immobilized Try showed higher activity than the free enzyme in the range 35–40°C. This phenomenon is associated with the conformational stability provided by the immobilization and the reduction in autocatalysis [6, 8].

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3.7. Determination of kinetic parameters

The kinetic parameters have been determined by performing activity assay at optimized

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conditions, using different BAEE concentrations. Km and Vmax parameters calculated from the

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data using the Lineweaver–Burk plots (Fig. 7). The kinetic parameters of free Try and immobilized Try on the PMNPs-GA were given in Table 3. For free Try, the Km and Vmax were calculated as 9 mM and 12.8 µM/min, respectively

whereas for immobilized Try on the PMNPs-GA, Km and Vmax were determined as 10 mM and 12.3 µM/min, respectively. There are slight difference between the Km and Vmax values of free and the immobilized Try. The Km value of immobilized Try was found to be higher than that of

free one. This means that there is a decrease in the enzyme-substrate affinity after immobilization. Lower affinity could be explained by steric hindrance and possible diffusional limitation of substrate [24]. Similar results were obtained in previous studies [24, 25]. On the other hand, the difference between free Try and the immobilized Try could be negligible attributed to that immobilization had no significant effect on the integrity of the active site in the enzyme [26]. Also, it could be said that immobilized Try on the PMNPs-GA protected its enzymatic properties and Try immobilized on the surface on the support with good orientation

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and a minimum distortion resulted from immobilization. 3.8. Thermal stability of free and immobilized Try

Stability of free and immobilized Try was investigated at 40 °C at different time intervals

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(Fig. 8). After 180 min, the residual activity of immobilized Try was 16% and 9% of the initial activity for free Try. These results highlight the stability provided by immobilization of Try as

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a result of multipoint interaction with the support [3, 4, 6]. Aslani et al [6] was determined that

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the retained activity of immobilized Try on Fe3O4@SiO2 –NH2 and free Try was 73% and 55% of initial activity, respectively, at 40 °C after 30 min. Try immobilized on polyaniline-coated magnetic diatomite nanoparticles retained almost 25% of its initial activity at 45 °C after 120

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min which was similar with free Try [10]. However, the thermal stability of Try increased with immobilization on chitosan nonwoven and immobilized Try sustained almost all activity at

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65°C after 60 min [11]. The immobilized Try on PMNPs-GA showed about two-times higher

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activity than free one at the end of the 180 min at 40 °C. 3.9. Storage stability and reusability of immobilized Try Storage stability and reusability of immobilized enzymes are important parameters for its

practical application, especially in terms of economical point. Both free and immobilized Try were kept at 4°C for 12-days and the residual activity measurements was determined at different time intervals. The free enzyme maintained around 19% of its initial activity after 12-days,

whereas the immobilized Try maintained around 50% of its initial activity (Fig. 9a). Hence, immobilized enzyme showed higher storage stability than free enzyme as also reported by Atacan and Ozacar [8]. This is due to the multipoint interaction between the enzyme and the support that provide higher stability [8]. The reusability of immobilized Try was determined eight times in a fresh reaction mixture containing 25 mM K-phosphate buffer (pH 7.0) (Fig. 9b). Immobilized Try retained 56% of its initial activity after eight uses.

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3.10. Digestion of cyt c with immobilized Try To explore the practical application of immobilized Try, the efficiency of digestion of cyt c by free and immobilized Try was investigated. Cyt c is a globular small protein (MW 12355

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Da) without disulfide bonds and 21 cleavage sites by Try [27]. As shown in Fig. S4, the mass spectrum of intact cyt c confirmed the molecular weight of the cyt c as 12357 Da. Fig. 10a and

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Fig. 10b shows the mass spectrum of cyt c after digestion with free enzyme and immobilized

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Try, respectively. Although both MS spectra were similar, exclusive peaks below 1500 Da were observed in the spectrum of free Try, probably related with auto-digestion of Try [22]. Furthermore, higher molecular weights peptides were obtained after digestion with

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immobilized Try. This could be due to steric limitations as results of immobilization. Similar results were obtained by Rocha et al [22] and Sun et al [28]. The digestion time was significantly

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shortened from 24 h for digestion in-solution to 15 min for the digestion based on the

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immobilized Try. The results showed that immobilized Try on PMNPs-GA offers efficient proteolysis with more accelerated reaction rate. This could be explained with properties of PMNPs such as high surface area for protein binding and water dispersibility providing more efficient interaction of Try with the substrate. Sun et al [29] reported that Try immobilized on Cu(II) and Zn(II) ions treated o metal affinity magnetic nanoparticles (IMANs) showed faster and more efficient BSA-digestion than in-solution digestion. The results of this study

demonstrate that PMNPs-GA-Try can be used for faster and more efficient digestion of proteins in practical applications. 4. Conclusions The PVA coated MNPs were successfully prepared and activated with GA for covalent immobilization of Try. The optimum immobilization conditions have been determined as immobilization for 1 h with 0. 75 mg of Try in mixture at pH 6.0 with results of high protein loading capacity (79%) and the activity recovery (99%). In comparison with the free Try,

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immobilized Try on the PMNPs-GA exhibited higher operational stability at wider pH and temperature range and also, higher thermal and storage stability. Immobilized Try was retained 56% of its initial activity after eight times repeated uses. Furthermore, immobilized Try on the

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PMNPs-GA digested cyt c to the small peptide fragments in very short time (15 min) which were quite similar with the fragments digested by free Try for 24 h. According to all these

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results, it can be said that PMNPs-GA is promising support for immobilization of Try. Try

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immobilized on the PMNPs-GA can be possibly used in proteomics studies and attractive for other practical applications of Try in biomedical applications. Besides the activation of PMNPs’surface with GA, it can also be activated with different crosslinkers containing

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different functional groups to improve thermal and storage stability of immobilized Try. Therefore, future work will focus on improving stability of enzyme by using different

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crosslinkers.

Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References

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[1] J. Zdarta, K. Antecka, A. Jedrzak, K. Synoradzki, M. Luczak, T. Jesionowski, Biopolymers conjugated with magnetite as support materials for trypsin immobilization and protein digestion, Colloids Surf B Biointerfaces 169 (2018) 118-125. [2] K. Atacan, B. Cakiroglu, M. Ozacar, Covalent immobilization of trypsin onto modified magnetite nanoparticles and its application for casein digestion, Int J Biol Macromol 97 (2017) 148-155. [3] S.S. Caramori, F.N. de Faria, M.P. Viana, K.F. Fernandes, L.B. Carvalho, Trypsin immobilization on discs of polyvinyl alcohol glutaraldehyde/polyaniline composite, Materials Science and Engineering: C 31(2) (2011) 252-257. [4] J. Liu, Y. Liu, D. Jin, M. Meng, Y. Jiang, L. Ni, Z. Liu, Immobilization of trypsin onto large-pore mesoporous silica and optimization enzyme activity via response surface methodology, Solid State Sciences 89 (2019) 15-24. [5] J. Feng, S. Yu, J. Li, T. Mo, P. Li, Enhancement of the catalytic activity and stability of immobilized aminoacylase using modified magnetic Fe 3 O 4 nanoparticles, Chemical Engineering Journal 286 (2016) 216-222. [6] E. Aslani, A. Abri, M. Pazhang, Immobilization of trypsin onto Fe3O4@SiO2 -NH2 and study of its activity and stability, Colloids Surf B Biointerfaces 170 (2018) 553-562. [7] S. Sahin, I. Ozmen, Determination of optimum conditions for glucose-6-phosphate dehydrogenase immobilization on chitosan-coated magnetic nanoparticles and its characterization, Journal of Molecular Catalysis B: Enzymatic 133 (2016) S25-S33. [8] K. Atacan, M. Ozacar, Characterization and immobilization of trypsin on tannic acid modified Fe3O4 nanoparticles, Colloids Surf B Biointerfaces 128 (2015) 227-236. [9] S. Wang, H. Bao, P. Yang, G. Chen, Immobilization of trypsin in polyaniline-coated nanoFe3O4/carbon nanotube composite for protein digestion, Anal Chim Acta 612(2) (2008) 1829. [10] M.P. Cabrera, T.F.d. Fonseca, R.V.B.d. Souza, C.R.D.d. Assis, J. Quispe Marcatoma, J.d.C. Maciel, D.F.M. Neri, F. Soria, L.B.d. Carvalho, Polyaniline-coated magnetic diatomite nanoparticles as a matrix for immobilizing enzymes, Applied Surface Science 457 (2018) 2129. [11] J.S. Kim, S. Lee, Immobilization of Trypsin from Porcine Pancreas onto Chitosan Nonwoven by Covalent Bonding, Polymers (Basel) 11(9) (2019). [12] K. Atacan, A.N. Kursunlu, M. Ozmen, Preparation of pillar[5]arene immobilized trypsin and its application in microwave-assisted digestion of Cytochrome c, Mater Sci Eng C Mater Biol Appl 94 (2019) 886-893. [13] R.D.S. Azevedo, I.P.G. Amaral, A.C.M. Ferreira, T.S. Esposito, R.S. Bezerra, Use of fish trypsin immobilized onto magnetic-chitosan composite as a new tool to detect antinutrients in aquafeeds, Food Chem 257 (2018) 302-309. [14] A.H.A. Al-Dhrub, S. Sahin, I. Ozmen, E. Tunca, M. Bulbul, Immobilization and characterization of human carbonic anhydrase I on amine functionalized magnetic nanoparticles, Process Biochemistry 57 (2017) 95-104. [15] Bergmeyer H.U., Gawehn K., Grassi M., Methods of enzymatic analysis., 2nd ed., New York, 1974. [16] P. Shinde, M. Musameh, Y. Gao, A.J. Robinson, I.L. Kyratzis, Immobilization and stabilization of alcohol dehydrogenase on polyvinyl alcohol fibre, Biotechnol Rep (Amst) 19 (2018) e00260. [17] X.Y. Wang, X.P. Jiang, Y. Li, S. Zeng, Y.W. Zhang, Preparation Fe3O4@chitosan magnetic particles for covalent immobilization of lipase from Thermomyces lanuginosus, Int J Biol Macromol 75 (2015) 44-50.

Jo

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-p

ro of

[18] H. Wu, Y. Liang, J. Shi, X. Wang, D. Yang, Z. Jiang, Enhanced stability of catalase covalently immobilized on functionalized titania submicrospheres, Materials Science and Engineering C 33(3) (2013) 1438-45. [19] G. Peng, C. Zhao, B. Liu, F. Ye, H. Jiang, Immobilized trypsin onto chitosan modified monodisperse microspheres: A different way for improving carrier's surface biocompatibility, Applied Surface Science 258(15) (2012) 5543-5552. [20] L.Y. Jun, N.M. Mubarak, L.S. Yon, C.H. Bing, M. Khalid, P. Jagadish, E.C. Abdullah, Immobilization of Peroxidase on Functionalized MWCNTs-Buckypaper/Polyvinyl alcohol Nanocomposite Membrane, Scientific Reports 9(1) (2019) 2215. [21] C. Daglioglu, F. Zihnioglu, Covalent immobilization of trypsin on glutaraldehydeactivated silica for protein fragmentation, Artif Cells Blood Substit Immobil Biotechnol 40(6) (2012) 378-84. [22] C. Rocha, M.P. Gonçalves, J.A. Teixeira, Immobilization of trypsin on spent grains for whey protein hydrolysis, Process Biochemistry 46(2) (2011) 505-511. [23] N. Tüzmen, T. Kalburcu, A. Denizli, α-Amylase immobilization onto dye attached magnetic beads: Optimization and characterization, Journal of Molecular Catalysis B: Enzymatic 78 (2012) 16-23. [24] G. Bayramoglu, V.C. Ozalp, M.Y. Arica, Magnetic Polymeric Beads Functionalized with Different Mixed-Mode Ligands for Reversible Immobilization of Trypsin, Industrial & Engineering Chemistry Research 53(1) (2013) 132-140. [25] X. Liu, X. Zhu, M.A. Camara, Q. Qu, Y. Shan, L. Yang, Surface modification with highlyhomogeneous porous silica layer for enzyme immobilization in capillary enzyme microreactors, Talanta 197 (2019) 539-547. [26] S. Narayan Patel, V. Singh, M. Sharma, R.S. Sangwan, N.K. Singhal, S.P. Singh, Development of a thermo-stable and recyclable magnetic nanobiocatalyst for bioprocessing of fruit processing residues and D-allulose synthesis, Bioresour Technol 247 (2018) 633-639. [27] Y. Cao, L. Wen, F. Svec, T. Tan, Y. Lv, Magnetic AuNP@Fe 3 O 4 nanoparticles as reusable carriers for reversible enzyme immobilization, Chemical Engineering Journal 286 (2016) 272-281. [28] X. Sun, X. Cai, R.Q. Wang, J. Xiao, Immobilized trypsin on hydrophobic cellulose decorated nanoparticles shows good stability and reusability for protein digestion, Anal Biochem 477 (2015) 21-7. [29] J. Sun, H. Ma, Y. Liu, Y. Su, W. Xia, Y. Yang, Improved preparation of immobilized trypsin on superparamagnetic nanoparticles decorated with metal ions, Colloids and Surfaces A: Physicochemical and Engineering Aspects 414 (2012) 190-197.

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Figure legends

Fig. 1. Transmission electron microscopy images of polyvinyl alcohol (PVA) coated magnetic

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nanoparticles (MNPs).

Fig. 2. FTIR spectra of (a) magnetic nanoparticles (MNPs), (b) polyvinyl alcohol coated magnetic nanoparticles (PMNPs), (c) polyvinyl alcohol coated magnetic nanoparticles activated with glutaraldehyde (PMNPs-GA), (d) polyvinyl alcohol coated magnetic

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nanoparticles-glutaraldehyde-Try (PMNPs-GA-Try) and (e) Trypsin.

Fig. 3. Thermal gravimetric analysis of (a) magnetic nanoparticles (MNPs) and (b) polyvinyl

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alcohol coated magnetic nanoparticles (PMNPs).

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Fig. 4. Effect of pH on immobilization of Try on the polyvinyl alcohol coated magnetic

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nanoparticles-glutaraldehyde (PMNPs-GA). Immobilization conditions: 25 mM K-phosphate

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buffer at 25 oC, 110 rpm for 1 h. Each point represents the mean of three experiments ± S.D.

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Fig. 5. Effect of Try amount on immobilization of Try on the polyvinyl alcohol coated magnetic

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nanoparticles-glutaraldehyde (PMNPs-GA). Immobilization conditions: 25 mM K-phosphate buffer at 25 oC, pH 6.0 and 110 rpm for 1 h. Each point represents the mean of three

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experiments ± S.D.

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Fig. 6. The effect of pH (a) (Reaction conditions: 25 mM K-phosphate buffer at 25 oC) and the temperature (b) (Reaction conditions: 25 mM K-phosphate buffer at pH 6.0 for free enzyme,

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the mean of three experiments ± S.D.

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pH 7.0 for immobilized enzyme) on the free and immobilized Trypsin. Each point represents

Fig. 7. Lineweaver–Burk plots for free (a) and immobilized Try (b). Each point represents the mean of three experiments ± S.D.

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Fig. 8. Thermal stability of free and immobilized Try at 40 °C in the 25 mM K-phosphate buffer

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of three experiments ± S.D.

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at pH 6.0 for free enzyme and pH 7.0 for immobilized enzyme. Each point represents the mean

Fig. 9. The storage stability and reusability of polyvinyl alcohol coated magnetic nanoparticlesglutaraldehyde-Try (PMNPs-GA-Try). Conditions: 4 oC in the 25 mM K-phosphate buffer at pH 6.0 for free enzyme and pH 7.0 for immobilized enzyme. Each point represents the mean of

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three experiments ± S.D.

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Fig. 10. MALDI-TOF mass spectra of peptides resulting from the digestion of cyt c by using (a) immobilized Try for 15 min and (b) free Try for 24 h in 50 mM ammonium bicarbonate

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buffer, pH 8.2.

Table 1 Effect of reaction time on the immobilization of Try. Reaction conditions: 25 mM K-phosphate buffer (pH 6.0) at 25 oC, 110 rpm. Immobilization time (h) 1 3

Activity recovery (%) 99±2 30±6

Protein coupling yield (%) 52±6 79±8

Table 2

studies.

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Comparison of the protein coupling yield and activity recovery results of this study and other

Activity recovery (%)

Reference

-

[21]

68.9

46

[22]

-

65

[4]

81

-

[20]

79

99

In this study

Protein coupling Supports

Enzim yield (%) Try

Glyoxyl-spent grain

Try

Large-pore mesoporous silica

Try Peroxidase Try

lP

MWCNTs-Buckypaper/Polyvinyl alcohol Nanocomposite Membrane PMNPs-GA

63

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glycol

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Silica-GA-polyethylene 5000

Table 3

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Free Try

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Kinetic parameters of free and the immobilized Try.

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Immobilized Try

Km (mM)

Vmax(µM/min)

9

12.8

10

12.3