Preparation of a stable and robust nanobiocatalyst by efficiently immobilizing of pectinase onto cyanuric chloride-functionalized chitosan grafted magnetic nanoparticles

Accepted Manuscript Preparation of a stable and robust nanobiocatalyst by efficiently immobilizing of pectinase onto cyanuric chloride-functionalized chitosan grafted magnetic nanoparticles Asieh Soozanipour, Asghar Taheri-Kafrani, Masoumeh Barkhori, Mahmoud Nasrollahzadeh PII: DOI: Reference:

S0021-9797(18)31250-5 https://doi.org/10.1016/j.jcis.2018.10.053 YJCIS 24208

To appear in:

Journal of Colloid and Interface Science

Received Date: Revised Date: Accepted Date:

7 August 2018 12 October 2018 18 October 2018

Please cite this article as: A. Soozanipour, A. Taheri-Kafrani, M. Barkhori, M. Nasrollahzadeh, Preparation of a stable and robust nanobiocatalyst by efficiently immobilizing of pectinase onto cyanuric chloride-functionalized chitosan grafted magnetic nanoparticles, Journal of Colloid and Interface Science (2018), doi: https://doi.org/ 10.1016/j.jcis.2018.10.053

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Preparation of a stable and robust nanobiocatalyst by efficiently immobilizing of pectinase onto cyanuric chloride-functionalized chitosan grafted magnetic nanoparticles

Asieh Soozanipour a, Asghar Taheri-Kafrani a,*, Masoumeh Barkhori a, Mahmoud Nasrollahzadeh b

a

Department of Biotechnology, Faculty of Advanced Sciences and Technologies,

University of Isfahan, Isfahan, 81746-73441, Iran. b

Department of Chemistry, Faculty of Science, University of Qom, Qom 3716146611,

Iran.

* To whom correspondence should be addressed: Tel: +98 31 37 93 4346 Fax: +98 31 37 93 23 42 E-mail address: [email protected]

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Abstract In industrial processes, effective degradation of polygalacturonic acid using immobilized pectinase is preferred over free one due to its stability and efficient functional reuses. Pectinase was covalently conjugated to the surface of cyanuric chloride functionalized chitosan encapsulated magnetite nanoparticles. The results obtained of various analytical tools and biochemical studies demonstrated successful synthesis and immobilization processes, high immobilization efficiency and loading capacity. The circular dichroism (CD) results of free and immobilized pectinase revealed the partial decreases in the α-helices and β-sheets, and marginal increases in the unordered elements contents of pectinase upon the immobilization onto Fe3O4@Ch-CC nanoparticles, along with stability improvement. The immobilized pectinase was retained about 60% of its initial catalytic activity after 13 recycles at optimum conditions (40 ˚C, pH 4.5). The storage stability of pectinase was increased due to immobilization, after 75 days storage at 4 °C, the free and immobilized enzyme retained 43% and 74% of the initial activity, respectively. The immobilized pectinase showed higher storage stability and better performance at wider ranges of pH and temperature, compared to free pectinase. Keywords: Pectinase immobilization; Cyanuric chloride; Chitosan; Magnetic nanoparticles; Reusability; Conformational stability.

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1. Introduction Over the last few decades, the significant advances in green technology have testified the important role of using bio-components in the development of industrial processes. The employment of enzymes as an alternative to chemical catalysts in many areas promotes green processes due to their unique properties including mild reaction condition, biodegradability, high specificity, low toxicity, and catalytic efficiency [1, 2]. Currently pectinases are widely applied in a number of industrial processes, such as fruit juice extraction and clarification, textile and plant fiber processing, tea, coffee and oil extraction, paper making, treatment of industrial wastewater, containing pectin materials [3, 4]. Despite perfect catalytic properties of pectinases, their industrial applications are limited due to the drawbacks regarding the lack of long-term stability under process conditions, as well as the fact that the difficulties associated with recycling should be resolved before their implementation at industrial scale. There have been many approaches to increase the potential for enzyme reuse and also improve the enzyme stability: enzyme immobilization, enzyme engineering, and medium engineering [5, 6]. Immobilization of enzyme onto various supports has much potential in bioapplications because it provides a facile separation of the reaction media for repeated use and also minimizes the enzyme contamination of the products [7-11]. Although the immobilization of enzymes may cause positive or negative alterations on structure and physicochemical properties of the enzyme surroundings, in many cases it can be a key agent for improving stability, selectivity and tolerance inhibitors compared to the free enzymes. However, one of the drawbacks of immobilization process is mass transfer limitations imposed by some matrixes. Developing nanoscaled inorganic carriers would be an effective approach for overcoming the substrate diffusion limitation [12-17]. Moreover, immobilization process can improve catalytic 3

performance of enzyme due to decreasing aggregation, producing favorable microenvironment, and enhancing the rigidification of the 3D structure of enzyme which might protect it against autoproteolysis and denaturing agents [18, 19]. So far, various enzymes were immobilized onto functionalized magnetic nanoparticles [19-22]. The advantages of magnetic nanoparticles over traditional supports are related to their high specific surface area, chemically modifiable surface, easy and low-cost synthesis, low cytotoxicity, and also significant magnetic responsiveness [23-25]. So, immobilization of pectinase on nanocarriers, especially magnetic nanoparticles, has received much attention in recent studies [26-32]. The enzymes can be conjugated to the solids by interactions ranging from reversible physical methods to irreversible cross-linking and stable covalent bonds. However, one type of technique is not ideal for all enzymes; it depends on nature of the enzyme structure [33, 34]. Among available immobilization techniques, physical bonding is too weak, and leaching of the enzyme can take place fast by minor changes in pH, ionic strength or temperature [35]. While covalent binding is a superior method for large-scale industrial application, because it can provide a powerful bond between the enzyme and support. Besides, it enhances stability, improves reusability, and prevents enzyme leakage from the support in the reaction media, [36, 37]. Since bare magnetic nanoparticles cannot provide good interaction with enzymes owing to the shortage of appropriate surface functional groups, nanoparticles surfaces should be covered by suitable biocompatible compounds to provide conditions for covalent attachment of enzymes [38, 39]. Until now, different biocompatible compounds, such as silica, chitosan, alginate, dextran and polymers have been successfully used for functionalization of magnetic nanoparticles to fabricate the immobilized enzyme with high activity and stability [40-44]. 4

Chitosan, an abundant natural polymer, is a biocompatible, hydrophilic, and biodegradable polysaccharide having preferable structural feasibilities for chemical and mechanical modification to acquire novel features and functions in diverse areas of biotechnology. However, the poor mechanical properties of chitosan may increase autolysis of the enzyme, and subsequently the enzyme structure will be destroyed during retentive recycling [43, 45]. Thus it is proposed that the Fe3O4/chitosan nanoparticles can be activated by trichlorotriazine (TCT) for covalent attachment of enzyme which is favorable for wide practical applications. TCT has been expressed as a considerable type of linker module for biomolecules and medicines immobilization, due to its economical, chemoselective reactivity and biocompatibility attributes. Nevertheless, it is rarely used for functionalization of nanocomposites [46, 47]. Based on these aspects, the present study is concentrated on the fabrication of a suitable nanocarrier for covalent immobilization of pectinase to improve catalytic properties, loading capacity, reusability, and stability of the immobilized enzyme, compared to the native one. Encouraged by the unrivaled properties and many bio- and industrial applications of nanocomposites supported enzymes in diverse areas, herein, we explore triazine-functionalized chitosan-encapsulated superparamagnetic nanoparticles for high loading capacity, catalytic activity, stability and reusability of the immobilized pectinase, which can be extended to the immobilization of other enzymes. This nanocarrier can greatly increase the possibility of continues utilization of pectinase in industrial processes.

2. Experimental section 2.1. Materials

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Pectinase (EC 3.2.1.15 from Aspergillus niger, aqueous glycerol solution, ≥5 units/mg protein), Polygalacturonic acid, D-(+)-Galacturonic acid, and chitosan were purchased from SigmaAldrich Chemical Co. The protein assay standard was bovine serum albumin (BSA), obtained from Bio-Rad (USA). DNS (3,5-dinitrosalicylic acid) was prepared from Fluka. Ammonium hydroxide (25% wt NH4OH), iron (III) chloride hexahydrate (FeCl3.6H2O), iron (II) chloride tetrahydrate (FeCl2.4H2O), cyanuric chloride (CC), n,n-diisopropylethylamine (DIPEA), potassium sodium tartrate (KNaC4H4O6.4H2O), and Coomassie brilliant blue G250 were prepared from Merck Chemical Co. All solutions were prepared with double-distilled water. The sodium acetate buffer (50 mM) was used as a buffer for preparing enzyme solutions. All other reagents were of analytical grade. 2.2. Instrumental Analysis The morphology, average particle size, and size distribution of the nanoparticles were determined by transmission electron microscopy (TEM) using Philips CM10 HT-100 KV under 50 nm scale. The surface modification of nanocarrier and pectinase immobilization were confirmed by Fourier transform infrared spectroscopy (FTIR) using JASCO 6300 spectrophotometer employing potassium bromide (KBr) pellet method in the range of 400-4000 cm-1. The magnetic properties of the synthesized nanoparticles were measured on alternating gradient force magnetometer from Meghnatis Daghigh Kavir Co. Thermo-gravimetric analysis (TGA) was performed for powdered samples with a heating rate of 10 °C/min from room temperature to 650 °C under a high purity nitrogen atmosphere. Energy electron loss spectroscopy (EELS) analysis was performed with a Gatan Imaging Filter (GIF 2001, Gatan, Sunnyvale CA) equipped with a 1k x 1k x 12 bit Multiscan CCD camera. Circular dichroism (CD) spectra were recorded on a CD6 dichrograph (Jobin Yvon, Longjumeau, France).

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2.3. Synthesis and characterization of cyanuric chloride/chitosan coated Fe3O4 nanoparticles Magnetic nanoparticles were easily synthesized by a simple chemical co-precipitation method in basic media according to the literature [48]. To functionalize magnetic nanoparticles (MNPs) by chitosan, 2.0 g of dry MNPs were added to 0.25 g chitosan that was dissolved in 50 mL of acetic acid solution (1%, v/v). The MNPs were homogeneously dispersed in the chitosan solution with vigorously stirring for 30 minute. Finally, chitosan coated Fe 3O4 nanoparticles (Fe3O4 @Ch) were obtained by adding 50 mL of NaOH (1 M) solution to the mixture. The nanocomposites were washed with deionized water several times until the pH reached to 7.0 and subsequently dried under vacuum. Finally, the Fe3O4@Ch nanocomposites were cross-linked by cyanuric chloride: the Fe3O4@Ch powder (100 mg) suspended in anhydrous THF (10 ml) in sonicator for 20 min at 0 °C. The solution of cyanuric chloride (18 mg, 0.1 mmol) and N, N-diisopropylethylamine (DIPEA) (100 μL) dissolved in THF (30 mL), was added to the suspension, and the mixture was sonicated under N2 atmosphere at 0 °C for 5 h. The product (Fe3O4@Ch-CC) was magnetically isolated from the mixture by an external magnet, washed with THF for several times to remove unreacted substrates and byproducts, and dried under vacuum at 40 °C. 2.4 Covalent immobilization of pectinase on functionalized nanocarrier The pectinase immobilization on the surface of functionalized magnetic nanocarrier was carried out as fallowing: 5 mg of triazine-functionalized chitosan-encapsulated superparamagnetic nanoparticles (Fe3O4@Ch-CC) were dispersed in 2 mL sodium citrate buffer (50 mM, pH 4.5). Then various amounts of 5 mg/mL pectinase stoke solution were mixed with the nanocarrier suspension and shaken at room temperature for 8 h. The pectinase immobilized on nanocarrier was collected by an external permanent magnet, and the non-covalent attached enzymes were removed by washing the mixture three times with buffer. For measuring the mount of pectinase

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immobilized on the nanocarrier, the content of pectinase in the initial pectinase solution and collected washing solution were determined by Bradford protein assay, using bovine serum albumin as standard protein [49]. 2.5. Circular dichroism Analysis CD spectra were recorded on a CD6 dichrograph (Jobin Yvon, Longjumeau, France), using a scan time of 1 nm.s-1, 0.2 nm resolution, 10 accumulations, and 1.0 nm bandwidth. All measurements were carried out at 298 K with thermostatically controlled cell holders. The instruments were calibrated with ammonium d-10-camphor sulfonic acid. The data were expressed as molar ellipticity [θ] (with units of deg.cm2.dmol-1), which is defined as [θ] = 100 θobs/cl, where θobs is the observed ellipticity in degrees, c is the molar concentration (mol.L-1), and l is the path length of the light in cm. Far-UV CD spectra were scanned between 260 and 190 nm at 0.1 nm intervals. Samples containing immobilized pectinase were prepared by using an appropriate aliquot of the freshly prepared Fe3O4 @Ch-CC/Pectinase in a 2 ml vial addition of 50 mM sodium acetate buffer pH 4.5, and vortexing the particles to get a clear colloidal suspension. CD spectra of the free and immobilized pectinase were collected in buffer, using rectangular quartz glass cuvettes with 0.1 cm optical path length. The blank control for the free enzyme solution was a 50 mM sodium acetate buffer and for the immobilized enzyme an appropriate suspension of unloaded Fe3O4@Ch-CC nanoparticles in buffer. The averaged blank spectrum was subtracted from the averaged sample spectrum to get the corrected lineshape. 2.6. Catalytic activity of free and immobilized pectinase The activity of pectinase was determined by measuring the amount of reducing sugar (galacturonic acid equivalent) production from the enzymatic hydrolysis of polygalacturonic acid, using 3,5-dinitrosalicylic acid (DNS) reagent [50]. The solution containing 1 mL enzyme

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(appropriate amount of either free or immobilized pectinase) and 1 mL substrate (polygalacturonic acid 1% w/v) in 50 mM sodium acetate buffer pH 4.5, was incubated at 50 °C for 30 min. Subsequently, immobilized pectinases were separated by a magnet, and the reactions were stopped with the addition of 2 mL DNS reagent. The final step was started by heating the solution in boiling water for 10 min and terminated by cooling at room temperature. After that, the resultant of enzyme activity was measured based on the amount of produced reducing sugar and its absorbance at 530 nm. The released galacturonic acid concentration was determined by reference to the standard curve. All activity experiments were carried out at least three times and the experimental error was less than 3%. One unit (IU) of pectinase activity was defined as the amount of enzyme that produced 1µmol of galacturonic acid from dissolubility of polygalacturonic acid per minute. 2.7. Effects of environmental conditions on the catalytic activity of free and immobilized pectinase The pectinase activity was investigated at various temperature and pH values. For assessment of pectinase activity as a function of temperature to reach the optimum temperature, the equivalent content of free and immobilized enzyme was incubated in a variety of temperatures ranging from 40 to 80 °C at pH 4.5, according to standard assay conditions mentioned in section 2.5. The optimum pH of both free and immobilized pectinase were determined by measuring the activity of enzymes at various pH values (3.5, 4.5, 5.5, 6.5, and 7.5) at 50 °C under standard assay conditions, as mentioned in section 2.5. 2.8. Determination of storage stability and reusability of the immobilized pectinase The free and immobilized pectinase were kept in 50 mM sodium acetate buffer at optimal pH and 4 °C to evaluate detect the storage stability during 75 days. The residual activity was

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measured at regular intervals of time and the activity determined on the first day was taken as 100% of the activity. The reusability experiments were done by repeated using of immobilized pectinase for hydrolysis of polygalacturonic acid at different temperatures, pH 4.5. After completion of each activity stage, pectinase on MNPs was separated by magnetization and was thoroughly washed with buffer. They were then reused by dispersing them in a fresh substrate solution. The activity obtained at each round was compared with the first run defined as the control to calculate the relative activity. 2.9. Kinetic parameter and determination of diffusional limitation The kinetic parameters of free and immobilized pectinase were determined by measuring the initial reaction velocity of pectinase catalyzed degradation of polygalacturonic acid at various concentrations of substrate (1–15 mg/mL). The Michaelis–Menten constant (Km) and apparent maximum velocity (υmax) were calculated from four linear transformations of Michaelis–Menten equation [51]. Also diffusional limitation of pectinase was determined in terms of effectiveness factor (

at

various substrate concentration from 1 to 10 mg/mL, using following equation (1):

(1)

Where νimmobilized enzyme and νfree enzyme are the reaction rates catalysed by free and immobilized enzyme with same concentrations under identical conditions, respectively.

3. Results and discussion 3.1. Synthesis and characterization of support and immobilization process Magnetic nanoparticles were synthesized according to the co-precipitation method and were modified with chitosan and cyanuric chloride (Fig. 1). The pH values lower than 5.5 result in the

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protonation of amino groups of chitosan (positive charge). The high density of hydroxyl groups on MNP surfaces can interact with positively charged amino groups of chitosan through electrostatic interactions. The chitosan can prevent the degradation and aggregation of Fe3O4 nanoparticles. Besides, the surface amino groups of Fe3O4@Ch nanocarriers were activated for enzyme attachment using cyanuric chloride under a nucleophilic substitution chemical reaction. Finally, pectinase was covalently bounded on the nanocarrier to produce Fe3O4@ChCC/Pectinase (Fig. 1).

Figure 1. Synthesis of triazine-functionalized chitosan-encapsulated magnetic nanoparticles (Fe3O4@Ch-CC) and the immobilization of pectinase.

The amount of pectinase immobilized onto nanocarrier was calculated using Bradford protein assay. The pectinase loading amount on the surface of Fe3O4@Ch-CC nanocarrier was about 188 mg of enzyme per nanocarrier gram at optimum conditions, by measuring the initial and final 11

concentration of pectinase in the immobilization medium, using the Bradford protein assay. It should be considered that all immobilized enzymes may not be active, as the immobilization procedure can cause conformational changes in enzymes and consequently some of the immobilized enzymes may lose their activity. Therefore, it is important to estimate the amount of active enzymes immobilized onto functionalized nanocarrier, by measuring the activity of both immobilized and unbound enzymes and comparison with the activity of the equivalent free enzymes in solution (before immobilization). The results are presented in Table 1. As shown in this table, the immobilization process leads to decreased enzyme activity relative to the equivalent free enzyme activity in solution. The difference between immobilized protein (using Bradford assay) and immobilized active enzyme (calculated from the activity assay) indicated that the Bradford method determined only the immobilized protein content and it would not be able to determine how many enzymes are in active forms after immobilization. The results obtained demonstrated that only about 60% of the immobilized pectinase onto Fe3O4@Ch-CC were active (Table 1).

Table 1: Maximal loading of pectinase on Fe3O4@Ch-CC. Added enzyme Protein (mg/g carrier) 436

Activity (U/g carrier) (A) 2414

Unbounded enzyme Protein (mg/g carrier) 248

Activity (U/g carrier) (B) 804

Immobilized enzyme Protein (mg/g carrier) 189

Activity (U/g carrier) (I) 688

Specific activity (U/mg protein)

Immobilized yield = I/(A-B)%

3.64

59.6

Moreover, the activity recovery of the immobilized enzyme was calculated from equation (2):

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where AR is the activity recovery of the immobilized enzyme (%), A is the activity of the immobilized enzyme (U), and A0 is the total activity of free enzyme in the solution before immobilization (U). The activity recovery of the immobilized pectinase on Fe 3O4 @Ch-CC nanocarrier could reach 29 (± 0.3) % of free enzyme activity at optimum condition (60 °C, pH 4.5). The synthesis of magnetic nanoparticles and deposition of chitosan network on their surface were confirmed with FTIR spectra. Fig. 2a shows the FTIR spectrum of free pectinase, triazinefunctionalized chitosan-encapsulated magnetic nanoparticles (Fe3O4@Ch-CC), the immobilized pectinase onto Fe3O4@Ch-CC nanocarrier (Fe3O4@Ch-CC/Pectinase), and chitosan. As shown in Fig. 2a (II), the characteristic wavenumbers of 580 cm−1 and 3420 cm−1 which corresponded to Fe-O and -OH groups respectively, demonstrated the existence of magnetic nanoparticle and chitosan components. The absorption peaks of chitosan at 1595 cm−1 (N–H bending vibration) and 1420 cm−1 (C–O stretching vibration) in the FTIR spectrum of free chitosan (Fig. 2a (IV)) was also appeared in the spectrum of Fe3O4@Ch-CC (Fig. 2a (II)), demonstrating the successful coating of chitosan on the surface of MNPs. Furthermore, the absorption peaks of 1020-1250 cm−1, 820-850 cm−1 and 1527 cm−1 in Figs. 2a (II and III) resulted from the C-N, C-Cl and C=N bonds, respectively, confirmed the presence of chitosan and cyanuric chloride on the nanocarrier surfaces.

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Figure 2. (a) The FTIR spectra of (I) free pectinase, (II) Fe3O4@Ch-CC, (III) Fe3O4@ChCC/Pectinase, (IV) chitosan. (b) The FTIR spectra of (I) fresh immobilized pectinase, and (II) fifth cycle reused immobilized pectinase.

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Successful attachment of pectinase molecules onto the Fe3O4@Ch-CC was verified by a comparison of the FTIR spectra of free and immobilized pectinase (Figs. 2a (I and III)). The successful attachment of pectinase can be concluded from the occurrence of obvious bands at 2920, 1390, 1500-1600 and 1600-1700 cm−1, which are associated with C-Haliph, C-N stretching vibration, C=O stretching vibrations (Amide I band), and N—H bending vibrations (Amide II bands) of the amino groups of the enzyme, respectively. Characteristic bands found in the infrared spectra of proteins and polypeptides include the Amide I and Amide II. These arise from the amide bonds that link the amino acids. The absorption associated with the Amide I band leads to stretching vibrations of the C=O bond of the amide, absorption associated with the Amide II band leads primarily to bending vibrations of the N—H bond. Because both the C=O and the N—H bonds are involved in the hydrogen bonding that takes place between the different elements of secondary structure, the locations of both the Amide I and Amide II bands are sensitive to the secondary structure content of a protein. Also, the FTIR spectrum of 5th run reused immobilized pectinase and fresh immobilized pectinase (Fig. 2b I and II) indicated that the secondary structure of the immobilized enzyme (1600-1700 cm-1) has slight shift toward upper wavenumbers after 5 times recycling (Fig. 2b II). The results demonstrated a slight conformational changes of enzyme due to reuses. The slight structural changes of enzyme may be due to changes in the microenvironment of the immobilized enzyme, caused by unbounded substrate or products around the immobilized enzyme. To measure the amount of pectinase immobilized on modified MNPs, the TGA analysis was performed. Fig. 3 displays comparative weight loss of chitosan-coated MNPs and pectinase immobilized on modified MNPs. For both samples, the small mass loss at temperatures below 15

200 °C was attributed to the evaporation of water or organic solvent physically absorbed on the nanocarriers, while the major weight loss above 200 °C was observed due to thermal decomposition of organic molecules. The content of pectinase immobilized onto functionalized nanocarrier was estimated about 16% from the weight loss difference between Fe3O4 @Ch-CC and Fe3O4@Ch-CC/pectinase. The mass ratio of immobilized enzyme is calculated from equation (3):

where

is mass ratio of coating of organic layer on nanoparticles (mg/g), and X is weight loss

percentage related to coating organic layer observed in TGA curves. Therefore, the results of TGA analysis showed that the amount of enzyme binding capacity was about 190 (mg/g), which was in consistent with the results of the Bradford protein assay (188 mg of enzyme per nanocarriers gram).

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Figure 3. TGA spectra of Fe3O4@Ch-CC and Fe3O4@Ch-CC/Pectinase

One of the important features of MNPs is the protection of magnetism properties during the modification and immobilization of enzymes. The saturation magnetizations versus magnetic field were investigated for pure Fe3O4, Fe3O4@Ch-CC and Fe3O4@Ch-CC/Pectinase nanocomposites using VSM analysis at room temperature. As shown in Fig 4, the saturation magnetization (Ms) values of Fe3O4, Fe3O4@Ch-CC, and Fe3O4@Ch-CC/Pectinase were 65.4, 58.2 and 46.4 emu/g, respectively. The slow decrement in magnetic responsiveness caused by functionalization, surface modifications and enzyme conjugation onto the nanocarrier.

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Figure 4. (a) Magnetic hysteresis loops of pure Fe3O4, Fe3O4@Ch-CC, and Fe3O4 @ChCC/Pectinase at 300 K. (b) Enlarged view of (a) between -400 and +400 Oe and (c) nanocarrier dispersion in water before (left) and after (right) magnetic separation.

The obtained results can prove the functionalization of nanocomposites as well as enzyme immobilization due to the changes in Ms values. The as-prepared Fe3O4@Ch-CC/Pectinase not 18

only dispersed quickly in a buffer solution but also separated simply via an external permanent magnet nearby the glass bottle within 60 sec (Fig 4c), due to the superparamagnetic behavior of nanocomposite respond well to its magnetic field. Furthermore, no hysteresis in the magnetization was observed and remanence magnetization (Mr) was close to zero in magnetization versus applied magnetic field curves (M–H loop). Transmission electron Microscopy (TEM) determines the morphology and size of the assynthesized nanocarrier. The TEM image of Fe3O4@Ch-CC is shown in Fig. 5a (in the scale of 50 nm) and confirmed the spherical shape and nanometer dimensions of nanoparticles. Besides, the average diameter of the synthesized nanocarrier was less than 10 nm. The larger specific surface to volume ratio and subsequently higher efficient pectinase immobilization can be achieved from small dimensions of nanoparticles. The spatially resolved electron energy-loss spectroscopy (EELS) in the scanning transmission electron microscopy mode was collected. One of the most important applications of EELS as a suitable chemical analysis is investigation of the oxidation state of synthesized magnetic nanoparticles recognize precisely the presence of two types of iron oxides [47]. The EELS spectra of iron oxide nanoparticles was presented in Figs. 5b, 5c, and 5d. In principle, both low and high energy area of the spectrum can be observed including the oxygen K- and the iron L23ionization edges in the high-energy part and also the zero loss peak and the plasmon structure in the low-energy part (Figs. 5b and 5c). The first EELS spectrum could be acquired by placing the probe at the edge of the nanoparticle. The second EELS spectrum was acquired subsequently by moving the probe to the particle center within a few seconds. The both spectra was taken 5 second. The EELS analysis of the Fe 2p L2,3 edge, in the region of 706–726 eV, may confirm the assignment of a mixture of Fe (II) and Fe (III) as the peaks are very close to each other (Fig. 5d).

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Figure 5. (a) TEM images of triazine-functionalized chitosan-encapsulated superparamagnetic nanoparticles. (b and c) Typical EELS spectrum measured from a large iron oxide nanoparticle. The energy windows used for the background subtraction under the O-K- and Fe- L23-edges. (d) Assignment of a mixture of Fe (II) and Fe (III) nanoparticles. The immobilization process may cause conformational changes in 2D structure of enzyme which was determined by CD spectroscopy. As shown in Fig 6, the far-UV CD spectra of both free and immobilized pectinase showed a dominance of typical β-sheet structure with a broad negative minimum around 215 nm. The CD spectrum of the free enzyme is nearly identical to the CD spectrum of the immobilized enzyme. The solution spectrum of pectinase showed a mean residue

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ellipticity of about −10460 deg.cm2.dmol−1 at 216 nm, while the value for the immobilized enzyme was very similar within 5% (about −9940 deg.cm2.dmol−1). The results of deconvolution of the CD spectra and calculation of -helix and -structure was judged using Chou and Fasman method [52] to give the closest assessment of the -helix content to what can be assessed from in the analysis of pectinase structure defined by crystallographic measurements obtained from protein data bank (PDB). Contents of secondary structure elements of free and immobilized pectinase evaluated by deconvolution of their CD spectra are presented in Table 2. The results revealed the partial decreases in the α-helices and β- sheets, and marginal increases in the unordered elements contents of pectinase upon the immobilization onto Fe 3O4@Ch-CC nanoparticles. This can be due the fact that some of the functional groups in enzyme structure are involved in the covalent linkages to the nanoparticles surface, the strength of hydrogen bonds among the functional groups of enzyme molecule decreases, leading to the changes in the enzyme molecule structure.

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Figure 6. Far-UV CD spectra of free and immobilized pectinase onto Fe3O4@Ch-CC nanoparticles in 50 mM sodium acetate buffer, pH 4.5 at 298 K

Table 2. Free and immobilized pectinase sequence secondary structure based on Chou and Fasman method implemented by Dicroprot 2000 software.

Free pectinase Fe3O4@Ch-CC/Pectinase Crystallography

Helix 4.8(±0.4) 4.2(±0.3) 5.0

Sheet 47.3(±1.2) 44.6(±1.7) 52.0

Other 47.9(±1.3) 51.2(±1.6) 43.0

3.2. Comparison of the catalytic activity of the free and immobilized pectinase at different environmental conditions The immobilization of pectinase on functionalized nanocarriers could change the enzyme conformation and subsequently its catalytic activity at different temperatures and pHs. In fact, the alteration in enzyme activity can be relied on active site microenvironment as well as 3D structure and the synergistic effect of support and enzyme. As clearly observed in Fig. 7a, the optimum temperature for free and immobilized pectinase was at 40 °C. Increasing the temperature in the reaction media has fairly various effects on the activity of enzymes, but the activity of the immobilized pectinase in all studied temperatures was more than free enzyme. At temperatures higher than 40 °C, the enzyme inactivation can be due to unfolding and denaturation of the enzyme. The higher activity of immobilized pectinase in comparison with free one is due to decreases in conformational changes and increases in enzyme rigidity upon immobilization. The catalytic activity of free and immobilized pectinase at different pHs is presented in Fig. 7b. As shown in this figure, both free and immobilized pectinase illustrated better activity at acidic pHs and exhibited good adaptability to environmental acidity related to basic conditions, while 22

the optimum pH was estimated at pH 4.5. Furthermore, the expanding of pH stability for immobilized pectinase may be depended on the nature characteristics of the nanocarrier and also the charge changes of the enzyme after immobilization. Thus, the free enzyme is less active at pH values differ from the optimal pH.

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Figure 7. The effects of (a) temperature and (b) pH on the activity of pectinase immobilized on Fe3O4@Ch-CC nanocarrier and (c) Activity of the immobilized pectinase on Fe 3O4@Ch-CC nanocarrier during 13 cycles of reusing at different temperatures.

3.3. Reusability and storage stability of pectinase When considering enzyme performance, the activity, selectivity or specificity of enzymes are important causes of using enzymes in different fields, while the lack of separation and reuse of them are critical points in the development of enzyme applications. The significant benefit of enzyme immobilization is the capability of separation to reuse the enzyme and thereby reducing the manufacturing costs of the enzymes in industrial applications. Furthermore, magnetic nanocarriers can be considered as appropriate matrixs for enzyme immobilization to improve the recoverability and reusability of the biocatalysts. Besides, it can provide an excellent catalytic nanoenvironment to overcome diffusion problems, especially when using macromolecules as substrate. The reusability of immobilized pectinases was carried out at different temperatures of 30, 40, 50 and 60 °C (at pH 4.5), recovered by magnetization, washed and then assayed again for 13 consecutive cycles (Fig. 7c). The pectinase activity at first run was set to 100%, and the evaluation of the catalytic activity of nanobiocatalyst was done using the yield of the final target product. As shown in Fig. 4c, among different investigated temperatures, higher relative activity was observed at 40 °C and about 60% of the initial activity of Fe3O4 @Ch-CC/Pectinase retained after 13 recycles. Some reasons including enzyme denaturation due to temperature and hydrodynamic stress, and leaching of the enzyme from nanocarriers during incubation were the main causes for the activity reduction of the immobilized pectinase after several times reuses.

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The storage stability of both free and immobilized pectinase was determined by incubating biocatalysts in 50 mM acetate buffer, pH 4.5, at 4 ˚C during 75 days. After 75 days, only 43% of the initial activity of free pectinase remained while the immobilized pectinase lost 26% of its initial activity (Fig. 8). The storage stability of pectinase was significantly improved due to immobilization. The results may be depended on the minimization of structural changes and conformational stability of immobilized enzyme due to immobilization process which prevents possible distortion effects on the active site of pectinase. Furthermore, enzymes can be aggregated in long-term storage, and this could greatly reduce the enzyme activity, while, immobilization of enzymes, particularly covalent attachment, leading the enzymes to disperse on the surface of nanocarriers and inhibit their aggregation.

Figure 8. The storage stability of free and immobilized pectinase on Fe3O4@Ch-CC nanocarrier at 4 ᵒC.

3.4. Kinetic parameters and determination of diffusional limitation

25

The Michaelis–Menten constant (Km) and apparent maximum velocity (υmax) are two important parameters for determining the characteristics of enzyme activity. To evaluate the effect of immobilization on the activity of pectinase, the kinetic parameters, Km and vmax, of the immobilized and free enzyme were compared. The kinetic parameters were calculated from four possible linear transformations of the Michaelis–Menten equation at various concentration of substrate from 0 to 15 mg/mL. The calculated values of Km, υmax and enzyme efficiency (υmax / Km) for both free and immobilized pectinase are presented in Table 3. As shown in this table, the immobilized pectinase has Km value slightly higher than the free enzyme and its vmax value was almost the same as free enzyme, based on the four linearization methods. Kinetics results showed that immobilization of pectinase caused slight changes in the affinity of enzyme to its substrate (polygalacturonic acid). An increase in Km could be probably due to conformational flexibility reduction caused by immobilization procedure. This led to lower accessibility of substrate to the active site of immobilized enzyme in comparison with free pectinase. Table 3. Kinetics parameters of free and immobilized enzymes calculated from four linear transformations of Michaelis–Menten equation. Enzyme

Free pectinase

Immobilized pectinase

Michaelis–Menten equation

Lineweaver–Burk plot (1/υ vs 1/S)

Eadie–Hofstee plot (υ vs υ/S)

Hanes–Woolf plot (S/υ vs S)

Scatchard Plot (υ/S vs υ)

Km (mg/mL)

1.99

2.204

2.67

2.43

υmax (U/mL)

0.284

0.297

0.315

0.304

υmax/ Km

0.142

0.134

0.117

0.125

Km (mg/mL)

2.44

2.75

2.815

2.98

υmax (U/mL)

0.289

0.301

0.310

0.312

υmax/ Km

0.118

0.109

0.110

0.105

The diffusion effect represents mass transfer of macromolecular substrates in catalyst as an important

factor

for

restricting

enzyme

activity after 26

immobilization.

With using

Polygalacturonic acid as substrate, the rate ratio ν immo/ν

free

(effectiveness factor) values were

0.837, 0.931, 0.952, 0.961, and 0.966 for 1 mg/mL, 2 mg/mL, 5 mg/mL, 7.5 mg/mL. 10 mg/mL respectively. The resultant of η values determined that with increasing concentrations of substrate, η value also increased. 3.5. Comparison with different supports Recently, various studies have been done on immobilization of pectinase onto magnetic supports. In the current work, pectinase was covalently immobilized on Fe 3O4@Ch-CC and the results were compared with some of the previous reports (Table 4). As shown in Table 4, the amount of loading enzyme on synthesized Fe3O4 @Ch-CC nanocomposite is relatively similar to other reported supports. Furthermore, a dramatically higher residual activity and durability were apperceived in compared to previous literatures indicating the more efficiency of this nanobiocatalyst. Consequently, the synthesized support in this study can be considered as a potential nanocarrier for practical applications in bio-industries as well as biosensing.

Table 4. Comparison of the properties of immobilized pectinase on various magnetic supports. Support

Immobilization technique

Optimum conditions pH

Temp.

Reusability times

activity(%)

Amount of pectinase loaded

Storage stability days

activity(%)

Ref.

(mg/g)

MNPs/SiO2/chitosan

Covalent

3.5

50

-

-

-

30

50

[28]

MNPs//SiO2/NH2

Covalent

8.5

55

7

64

45

30

80

[29]

MNPs/SiO2-CMCS

Covalent

4

55

10

68

220

40

50

[30]

MNPs/APTS/GA

Covalent

4

50

8

50

17.5

24

60

[26]

magnetic cornstarch

Adsorption

4

40

8

60

-

-

-

[31]

Covalent

4.5

40

13

60

195

75

74

This work

microspheres Fe3O4@Ch-CC

27

4. Conclusions With considering the increasing demands of stable nanobiocatalysts in diverse industries, the immobilization of enzymes can provide a new approach for this purpose. In the present study triazine-functionalized chitosan-encapsulated superparamagnetic nanoparticles were used for covalent immobilization of pectinase. The comparison of catalytic performance of the immobilized enzyme with free pectinase at various temperatures and pHs indicated increased activity of the immobilized pectinase. The Bradford protein assay demonstrated that the pectinase loading amount on the magnetite nanocarriers was about 198 mg of enzyme per nanocarriers gram and it was confirmed by TGA analysis. CD and FTIR analyses determined immobilization process led to detail changes in the enzyme structure. Furthermore, a dramatically higher residual activity and durability were apperceived in compared to previous literatures indicating more efficiency of this nanobiocatalyst (Table 4). Comparison of the Km and υmax values of free and immobilized pectinases illustrated that pectinase protected its specificity to substrate due to immobilization. The possibility of substrate purification could be readily provided by magnetic recoverability. The results of this study demonstrated that immobilization process improved enzymes stability and catalytic properties. All in all, the most important benefit of the immobilized pectinase in comparison with the free pectinase as well as other supports at previous literatures was the higher stability and reusability of the immobilized enzyme that can decrease industrial expenses. This suggested that pectinase immobilized on functionalized MNPs by covalent binding is suitable for practical application in food, chemical and pharmaceutical industries

Acknowledgement 28

The authors are grateful to the Center of Excellence of Research Council of the University of Isfahan for financial supports of this work.

29

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Graphical abstract

37