Triazine-functionalized chitosan-encapsulated superparamagnetic nanoparticles as reusable and robust nanocarrier for glucoamylase immobilization

Triazine-functionalized chitosan-encapsulated superparamagnetic nanoparticles as reusable and robust nanocarrier for glucoamylase immobilization

Accepted Manuscript Title: Triazine-functionalized chitosan-encapsulated superparamagnetic nanoparticles as reusable and robust nanocarrier for glucoa...

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Accepted Manuscript Title: Triazine-functionalized chitosan-encapsulated superparamagnetic nanoparticles as reusable and robust nanocarrier for glucoamylase immobilization Authors: Mahkameh Amirbandeh, Asghar Taheri-Kafrani, Asieh Soozanipour, Cedric Gaillard PII: DOI: Reference:

S1369-703X(17)30204-8 http://dx.doi.org/doi:10.1016/j.bej.2017.08.001 BEJ 6757

To appear in:

Biochemical Engineering Journal

Received date: Revised date: Accepted date:

11-5-2017 19-7-2017 3-8-2017

Please cite this article as: Mahkameh Amirbandeh, Asghar Taheri-Kafrani, Asieh Soozanipour, Cedric Gaillard, Triazine-functionalized chitosanencapsulated superparamagnetic nanoparticles as reusable and robust nanocarrier for glucoamylase immobilization, Biochemical Engineering Journalhttp://dx.doi.org/10.1016/j.bej.2017.08.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Triazine-functionalized chitosan-encapsulated superparamagnetic nanoparticles as reusable and robust nanocarrier for glucoamylase immobilization

Mahkameh Amirbandeha, Asghar Taheri-Kafrania,*, Asieh Soozanipoura, Cedric Gaillardb

a

: Department of Biotechnology, Faculty of Advanced Sciences and Technologies,

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

: INRA – UR 1268 Unité Biopolymères, Interactions, Assemblages, Rue de la

Géraudière, BP 71627, 44316 Nantes Cedex 3, France.

* Corresponding Author

Email: [email protected] Tel:+9831 3793 43 46 Fax:+9831 3793 23 42

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Graphical Abstract

Highlights 

A novel nanocarrier based on functionalized chitosan-grafted MNPs was synthesized.



The covalent attachment of glucoamylase on this novel nanocarrier was investigated.



Immobilized glucoamylase showed improved catalytic activity and great reusability.



This biocatalyst exhibited excellent performance at broader temperatures and pHs.



The immobilized enzyme has a wide prospect for its applications in bio-industries.

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Abstract Aspergillus niger glucoamylase (GLA) was covalently immobilized on 1,3,5-triazinefunctionalized chitosan coated superparamagnetic nanoparticles (MNPCh-CC). The morphology, structure and properties of functionalized nanocomposite were investigated, as well as GLA immobilization process, through different analytical tools. Different experimental parameters such as optimum temperature, pH, reaction time and enzyme concentration were studied for free and immobilized enzyme. The GLA immobilized on nanocarrier exhibited excellent catalytic activity at pH 4.5 and 60 °C. Notably, the immobilized GLA showed quite impressive stability, even after 10 reaction cycles, it could still retain about 70% of the initial activity. The results showed that immobilization process couldn’t significantly inhibit enzyme-substrate interaction and subsequently retained its effective catalytic activity. The substantial improvement of reactivity, reusability, and stability of this biocatalyst system may confer it a wider range of applications in industrial processes.

Keywords: Glucoamylase; Magnetic nanoparticles; Immobilization; Chitosan; Cyanuric chloride; Reusability.

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1. Introduction Enzymes are important biocatalysts with variety applications in the food, paper, agriculture, leather, textile and chemical industries [1]. The enzyme technology is more attractive than traditional organic synthesis, since having specificity and low toxicity properties, and also product raw materials includes of biomolecules, which cannot be constructed, degraded or modified under convenient conditions [2, 3]. Contrary to all these advantages, the practical industrial applications of enzymes in different processes are often confined by the undesirable operation and storage stability, high sensibility to the environmental conditions, large incubation time and the difficulty recycling [4, 5]. Over the few years, scientists have provided different techniques such as protein engineering, chemical modification, addition of additive and immobilization that make easy enzymatic practical applications. Among them, the novel developments in immobilized enzymes can offer an effective way to improve their functional properties, stabilization, separation, and facilitating reutilization. [6, 7]. A successful immobilization depended on suitable matrix selection and mode of attachment. Nowadays, different inorganic nanomaterials have attracted much attention for enzyme immobilization, due to their particular properties such as high specific surface area for the attachment of enzymes, special chemical combination and no health hazard and also providing a shorter diffusional way for the substrates [8-17]. Magnetite nanoparticles have extensively applied for enzyme stabilization. The most importance of nanoparticles is simple separation of immobilized enzyme from mixture reaction using an external magnet and consequently reusability. Whereas they have inert surface and cannot directly attached with enzymes by

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covalent binding, magnetite nanoparticles should be coated by protective polymers to provide active functional groups for enzyme immobilization [18-21]. Chitosan, a natural polymer, is safe, non-hazard, and having many amine, hydroxyl, and hydroxymethyl groups that can be strongly interacted with enzymes. Thus chitosan is a suitable candidate for immobilization of enzymes [22, 23]. Glucoamylase (EC 3.2.1.3) (α-1,4-glucan-glucano hydrolase), an industrial enzyme, is wildly used for different industries such as food and beverage industries, textile, detersive, brewing, pharmaceutical and paper-making industries [24, 25]. Nevertheless, practical industrial applications of glucoamylases (GLA) are often limited by their low operational stability, short life cycle and non-recovery of the reaction systems. To overcome the above mentioned drawbacks, immobilization of glucoamylase on various natural and synthetic supports is considered to be an impressive and effective strategy to improve the stability of enzyme and its easy separation from the reaction systems and recycling for reuse. Up to now, immobilization of glucoamylase on various solid supports has been examined [26-31]. The enzyme easily washed out from carrier in physical immobilization, resulting in glucoamylase slightly operational stability [32-35], but covalent binding between enzyme and the support, can stabilize glucoamylase and avoid from its leakage [36-41]. Encouraged by the unique properties and many applications of magnetic nanoparticles, herein we wish to report a simple covalent immobilization procedure, high loading capacity, and high catalytic activity, thermal stability and easily reusability of the immobilized GLA, which can be extended to the immobilization of other industrial enzymes.

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2. Experimental section 2.1. Materials Glucoamylase (EC 3.2.1.3 from Aspergillus niger), starch, glucose and chitosan were purchased from Sigma-Aldrich 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.

2.2. Apparatus Fourier transform infrared (FTIR) spectra were recorded on a JASCO 6300 spectrophotometer. The transmission electron microscopy (TEM) was carried out on a Philips CM10 HT-100 KV transmission electron microscope. Atomic force microscopy (AFM) images were acquired using a Park Scientific Instrument Autoprobe CP (Sunnyvale, CA). The magnetic properties of nanoparticles were measured by vibrating sample magnetometer (VSM, Meghnatis Daghigh Kavir Co, Iran). Activity of enzyme was measured utilizing UV–Vis, Carry-500 double beam spectrophotometer (USA).

2.3. Synthesize of chitosan grafted superparamagnetic nanoparticle Although, numerous chemical methods can be used to synthesize magnetic nanoparticles [4246], the co-precipitation technique is probably the simplest and most efficient chemical pathway, which was used in this study [47]. For surface modification of magnetic nanoparticles and 6

synthesize chitosan grafted magnetic nanoparticles (MNPCh), 2.0 g dry MNPs were added to 0.25 g chitosan that was dissolved in 50 mL of acetic acid solution (1%, v/v). The Fe3O4 nanoparticles were homogeneously dispersed in the chitosan solution with vigorously stirring for 30 minute. Finally, chitosan coated MNPs were obtained by adding 50 mL of NaOH (1.0 M) solution to the mixture. Then, the MNPChs were separated from the reaction mixture by an external permanent magnet, washed with mili-Q water several times, and dried under vacuum at 60 °C.

2.4. Preparation of trichlorotriazine-functionalized MNPCh The MNPCh powder (100 mg) suspended in anhydrous THF (10 ml) in sonicator for 20 min at 0 °

C. The solution of trichlorotriazine (cyanuric chloride, CC) (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 was collected by a magnet, washed with anhydrous THF several times, and dried under vacuum.

2.5. Enzyme immobilization In a round bottom flask, 5 mg of triazine functionalized MNPChs (MNPCh-CC) were dispersed in 3 mL of acetate buffer (50 mM, pH 4.5). Then, various volumes of the glucoamylase solution with a concentration of 4 mg/mL were added into the suspension. The mixture was shaken at room temperature for different times (2-10 h). After these times the immobilized glucoamylase (MNPCh-CC/GLA) were separated from free GLA by magnetic separation, and washed three times with acetate buffer (50 mM, pH 4.5). The amount of the enzyme immobilized

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on MNPCh-CC was determined by measuring the initial and final concentration of GLA in the immobilization medium using the Bradford protein assay [48]. In this method, the amount of protein in the supernatant was determined colorimetrically (595 nm) with the Bio-Rad protein assay reagent concentrate using BSA as the standard protein. The amount of bound enzymes onto MNPCh-CC was calculated from Eq. (1):

where ci and cs are the concentrations of glucoamylase initially used for reaction, and the unbound glucoamylase collected in each purification cycle, respectively.

2.6. Enzyme activity assay

Enzyme activity was measured by calculating the rate of the reducing sugars production by using 3,5-dinitrosalicylic acid reagent [49]. For preparing reaction mixture, substrate ,starch (3% w/v) dissolved in 50 mM Acetate buffer (pH 4.5), was added to appropriate volume of either free and immobilized enzyme and then incubated at 60◦C for 15 min under shaking condition. Immobilized glucoamylases were collected from reaction mixture by magnet and the reaction was terminated by adding 2 ml DNS solution and the mixture was put into boiling water for 5 minutes. The absorbance of reducing sugar released in the mixture was determined at 520 nm after the temperature of the mixture reached to room temperature. All experiments were carried out three times and observed that experimental error is less than 3%. One unit (IU) of glucoamylase activity was defined as the amount of enzyme that catalyzed 1µmol of glucose per minute. The activity recovery of the immobilized enzyme is calculated from equation 2:

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where R is the activity recovery of the immobilized enzyme (%), A is the activity of the immobilized enzyme (U), and A0 is the activity of the free enzyme in solution before immobilization (U).

2.7. Optimum conditions, reusability and storage stability of immobilized glucoamylase The optimum conditions for the maximum activity of free and immobilized enzyme were evaluated at determined ranges of temperature (30-70 ◦C) and pH (3.5 - 7.5). The effect of temperature on the activities of the enzymes was investigated at pH 4.5 and also effect of pH was measured at 60 ◦C according to assay method as described above. In all experiments, to achieve the correct evaluation between activities of both free and immobilized enzymes, the amount of free and immobilized GLA used for the reaction were adjusted to be equivalent. The reusability of immobilized glucoamylase was measured by incubating the immobilized glucoamylase at the same conditions. After the end of each cycle reaction, the immobilized glucoamylase was separated from reaction mixture by magnet and washed with acetate buffer (50 mM, pH 4.5). For starting next enzymatic reaction cycle the fresh substrate solution added to immobilized enzyme container, then the activity assay test was repeated frequently until up where the reagent was indicative of enzyme activity. The specific activity determined in the first run was taken as 100%. The storage stability of free and immobilized glucoamylase at 4 ◦C in 50 mM acetate buffer, pH 4.5, for 105 days was determined by calculating the residual activity in enzyme samples every 15 days intervals.

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2.8. Determination of kinetic parameters The activity of free and immobilized enzyme in different concentrations of the substrate (1-40 mg/ml) can get the Michaelis-Menton constant (Km) and apparent maximum velocity (νmax) by Lineweaver-Burk plots. The temperature affiliation of the rate constant (under the passivation temperature) of an enzyme-catalyzed reaction can be explained by the Arrhenius equation 3:

Where A is the pre-exponential agent, k is the rate constant, R is the gas constant, Ea is the activation energy, and T is the absolute temperature in Kelvin unit. The activation energy of catalysis for both the free and immobilized glucoamylase (Ea) was determined from the slope that obtained from Arrhenius plot (of ln k against 1/T).

3. Results and discussion 3.1. Characterization of synthesized nanocarrier and immobilization process A schematic illustration of the step-by-step approach used for the preparation of the CCfunctionalized chitosan-encapsulated magnetic nanoparticles for the immobilization of GLA is shown in Scheme 1. The covalent attachment of glucoamylase on synthesized nanocomposite via nucleophile groups of cyanuric chloride and amino residues of the GLA can promote partial conformational changes on the immobilized GLA.

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Scheme 1. Schematic representation of the method for covalently immobilization of glucoamylase on functionalized chitosan-encapsulated magnetic nanoparticles.

The immobilization yield of glucoamylase onto MNPCh-CC was estimated about 85% at optimum conditions, by measuring the initial and final concentration of glucoamylase in the immobilization medium using the Bradford protein assay. The glucoamylase loading amount on the MNPCh-CC was about 195 mg of enzyme per nanocarriers gram. Presence of surface functional groups and binding of GLA onto MNPCh-CC were investigated by FTIR spectroscopy. Fig. 1 showed the FTIR spectra of MNP, MNPCh, MNPCh-CC, MNPCh-CC/GLA, and Chitosan. As it shown in Fig. 1a, the characteristic absorption bonds at about 580 and 3420 cm-1 were assigned to the vibration of the Fe-O bonds and the –OH groups, respectively, that demonstrated the existence of MNP components. In the FTIR spectrum of MNPCh and free chitosan, the –C-O stretching vibration at 1420 cm-1 and N-H bending vibration at 1595 cm−1 were appeared, confirming the presence of chitosan shell on MNPs (Fig. 1b and Fig. 1e). Furthermore, in comparison with Fig. 1b, Fig. 1c showed the new bands at about 1450 and 1527 cm-1 that can be attributed to the stretching vibrations of C-N and C=N bonds, respectively, indicating the presence of triazine units on the nanocarrier surface. After immobilization of glucoamylase, the bands at 3410–3450 cm−1 (the characteristic signal of the 11

stretching vibration of amine groups), the C-N stretching vibration at 1392 cm−1 and the symmetric and asymmetric stretching vibrations of C-O at 1457 and 1645 cm−1 (the characteristic signal of amide carbonyl (CO-NH) groups), demonstrated the successful immobilization of glucoamylase on functionalized nanoparticles.

Figure 1. The FTIR spectra of (a) MNP, (b) MNPCh, (c) MNPCh-CC, (d) MNPCh-CC/GLA and (e) Chitosan.

The transmission electron microscopy (TEM) provides two-dimensional projections of the solid structure and information on particle size and morphology of nanocarrier for enzyme immobilization. Fig. 2 shows the representative TEM image of the MNPCh-CC and confirmed the nanometer dimensions of this nanocomposite. As it shown in this figure, the nanoparticles are spherical with average diameter of 10 nm. The small dimensions of nanoparticles provided larger specific surface to volume ratio and subsequently higher efficient enzyme immobilization. 12

Figure 2. TEM image of Triazine-functionalized chitosan-encapsulated superparamagnetic nanoparticles.

Atomic force microscopy (AFM) is the technique able to visualize biomolecules at the singlemolecule level with sub-nanometre accuracy in liquid. AFM was used to determine the morphological changes occurring on the surface of functionalized MNPChs in the absence and presence of enzyme. As it shown in Fig. 3, the modified MNPs surface appeared fairly smooth (Fig. 3a) before, but became quite rough after glucoamylase attachment that emphasized the coverage of the glucoamylae on the surface of modified MNPs (Fig. 3b).

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Figure 3. The 3D AFM images of (a) modified MNPs and (b) glucoamylase immobilized on functionalized nanocarrier.

The magnetic properties of MNP, MNPCh, and MNPCh-CC/GLA were investigated using VSM analysis at room temperature. The magnetizations versus magnetic field are presented in Fig. 4. The results showed that the saturation magnetization (Ms) values of MNP, MNPCh and MNPChCC/GLA were 66.2, 49.6 and 33.5 emu/g, respectively. The gradual decline in magnetic response implied an increase of thickness of the shell layer on the MNPs surface along with the functionalization procedure. Accordingly, the amount of Ms for MNPCh-CC/GLA was found to 14

be considerably lower than MNPCh and MNPCh-CC due to the coating of magnetic nanoparticles with chitosan and functionalization of chitosan-grafted MNPs. When comparing the Ms values for the MNPs and conjugated MNPs, it should be considered that the conjugation decreases the Ms value caused by the added mass due to surface modifications and functionalization of the nanocarrier. In addition, the magnetic separability of prepared MNPChCC/GLA was tested in an aqueous solution by placing an external magnet close to the glass bottle. The suspended particles could be easily collected by the magnet within 1 min. The results confirmed the superparamagnetic behavior of MNPCh-CC/GLA which is useful for simply separation of immobilized glucoamylase using magnetic field.

Figure 4. Room temperature magnetization curves of (a) MNP, (b) MNPCh, and (c) MNPCh-CC/GLA.

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3.2. Optimization of immobilization conditions and activity assay Glucoamylase was covalently immobilized on MNPCh-CC at 50 mM acetate buffer, pH 4.5, at room temperature. The yield of immobilization was explained by the amounts of enzyme immobilized on the nanocarriers of uniform mass, which was measured by the Bradford method. The effects of incubation time on the amount of immobilized enzyme showed that the amount of immobilized GLA was increased by increasing time incubation until 6 h and decreased slightly afterwards. The most of enzyme loaded in suitable time (6 h) on MNPCh-CC were about 195 mg of enzyme per nanocarriers gram (Fig. 5). It seems that most of functional groups on the surface of MNPCh-CC were blocked by GLA until 6 h and afterwards, the amount of immobilized GLA decreased due to increasing steric hindrance on the surface of MNPCh-CC and lack of available functional groups.

Figure 5. The yield of glucoamylase immobilized onto MNPCh-CC surface. 16

The optimum reaction conditions were fined as pH 4.5 and a temperature of 60 ◦C for immobilized glucoamylase, and pH 5.5 and a temperature of 50 ◦C for free glucoamylase (Fig. 6).As it shown in Fig 6a, the activity of free and immobilized GLA was assayed at different pHs in the range of 3.5-7.5. Two types of enzymes were found to exhibit quite similar trend as the pH varied. As shown in Fig. 6a, the free and immobilized enzymes showed a remarkable increasing activity at pH 5.5 and 4.5, respectively (optimum pHs). However, the activity of both enzymes decreased slightly as the pH changed from optimum pH. The activity recovery of the immobilized GLA on MNPCh-CC was about 92%, at optimum pH and 60 °C. The activity of free and immobilized GLA was assayed at different temperatures in the range of 30-70 ◦C. The MNPCh-CC/GLA showed similar temperature–activity trend with free GLA as shown in Fig. 6b. The results showed that the activity recovery of the immobilized GLA on the MNPCh-CC could reach about 97 % of free enzyme activity at optimum temperature and pH 4.5. By increasing the temperature from 30 to 60 ◦C, the activity of immobilized enzymes increased accordingly, activity values for temperature above 60 ◦C decreased, which might be due to conformational changes of enzyme or its denaturation at higher temperatures. The changes in enzyme activity can be depended on activity site microenvironment as well as 3D conformation, synergistic effect between enzyme and matrix. Beside high temperatures can affect on the rate of enzymatic reaction and provides higher reaction rate, covalent immobilization is an ideal way to obtain the high thermal stability of enzymes and protect them from denaturation. The variation of the activity at various amount of pH maybe resulted from the specifications of the nature of enzyme/nanoparticles binding.

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Figure 6. The effects of (a) pH and (b) temperature on the activity of glucoamylase immobilized on MNPCh-CC nanocarrier.

3.3. Reusability and storage stability of immobilized glucoamylase The ability of native enzymes for reusing is one of major limitations for their applications in continues reactions. The immobilized glucoamylase on magnetic nonocarriers can be easily separate from the reaction mixture using an external magnetic field and can be introduced to new

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reaction. In this regard, the reusability of MNPCh-CC/GLA was investigated over a ten reaction cycle at different pHs in the range of 3.5-7.5 at 60 ◦C and also at different temperatures of 30-70 ◦

C at pH 4.5. The specific activity of the first run was set to 100%. The efficiency of the final

product (glucose) was used to measure the catalytic activities of the fictitious nanosystems. As shown in Fig. 7a, among different pHs, the higher relative activity was observed at pH of 4.5 and after 10 recycles, the MNPCh-CC/GLA could still have a high level of about 68% of the initial activity. At different investigated temperatures, the immobilized glucoamylase showed the best relative activity at 60 ◦C, and 69% of its initial activity was retained after 10 reaction cycles. (Fig. 7b). The decreases in enzymatic activity during repeated use can be related to repetitive encountering of substrate to the active site of immobilized enzyme and following by weakening of binding strength between the matrix and the immobilized glucoamylase that cause of leaching of enzyme in recycling experiments and consequently loss of activity. Furthermore, the recurrent encountering of substrate with the active site of immobilized enzyme causes its distortion and leads to loss of activity. To evaluate the storage stability of free and immobilized glucoamylase, the reaction mixtures were kept on the same condition (pH 4.5, 4 ◦C, 50 mM acetate buffer) for different storage time. The catalytic activity measured after any 15 day and the results represented the improvement of storage stability due to immobilization. As it shown in Fig 8, the immobilized glucoamylase could still retained 64% of its initial activity after 105 day, however, the free one could retained 20% of the initial activity at the same condition. According to these results, the activity of enzyme was usually declined after a long time. Therefore, immobilization significantly prevented this event and improved the storage stability of enzyme.

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Figure 7. The reusability of the immobilized enzyme on MNPCh-CC nanocarrier at different (a) pHs and (b) temperatures. 20

Figure 8. The storage stability of free and immobilized glucoamylase on MNPCh-CC nanocarrier versus incubation time at 4 ᵒC.

3.4. Kinetic parameters One way to evaluate the success of immobilization process is to measure the Michaelis-Menton constant (Km) and maximum velocity (νmax). Kinetic parameters for free and immobilized glucoamylase was calculated by Lineweaver-Burk plots of the rates of reducing sugar (glucose equivalent) at different concentrations of starch (1-30 mg/ml). The obtained results are shown in Table 1. As it shown in this table, the value of Km for immobilized enzyme was higher than free enzyme. Also, the νmax value of immobilized enzyme decreased due to immobilization. This 21

could be due to the fact that the conformational changes in 3D structure of glucoamylase and steric influences eventuating from limitation of the availability of substrate to the active site are affected because of immobilization. The dependence of the rate constant on temperature (below the inactivation temperature) of an enzyme-catalyzed reaction can be represented by the Arrhenius equation. The values of activation energy (Ea) were quite low and ranged from 6.7 kJ/mol for GLA immobilized on MNPCh-CC to the maximum of 8.5 kJ/mol for the free GLA (Table 1). In summary, the values of Ea for the immobilized enzymes are smaller than that with free enzyme, implying that the immobilized enzymes are less temperature sensitive. Furthermore, the decrease in activation energy of the enzyme due to immobilization confirms that there is a mass transfer control for the immobilized GLA rather than kinetic control.

Table 1. Kinetic parameters of the free and immobilized glucoamylase on MNPCh-CC. Free GLA

MNPCh-CC/GLA

Km (mM)

37

45.8

vmax (mM/min)

2.3

Ea (kJ/mol)

1.5

6.7

8.5

3.5. Comparison with various supports In current work, glucoamylase was covalently immobilized on MNPCh-CC. The major properties of the immobilized glucoamylase were compared with some of the previous reports (Table 2). As it shown in this table, the amount of enzyme loading on synthesized MNPCh-CC nanocomposite is higher than other reported supports. Additionally, a remarkably higher residual 22

activity and storage stability were observed in comparison with previous works indicating the efficiency of this nanocomposite for industrial applications. Chitosan is a biocompatible, hydrophilic, and biodegradable polysaccharide containing high density of charged amine, hydroxyl, and hydroxymethyl groups that can strongly interact with hydroxyl groups on MNPs and avoid the degradation and aggregation of MNPs. Also the co-immobilization of enzymes and very high concentrations of high-molecular-weight (MW) hydrophilic polymers such as chitosan can promote the generation of new hyperhydrophilic microenvironments surrounding every molecule of immobilized enzyme. This new hyperhydrophilic microenvironment can strongly improve the stability as well as the catalytic behavior of the immobilized enzyme. In addition to these advantages, the conjugation of MNPCh and cyanuric chloride provided conditions for covalent immobilization of GLA and caused highly retained activity of enzyme immobilized on the chitosan-coated MNPs.

Table 2. Comparison of properties of immobilized glucoamylase on various solid supports. Support

Immobilization technique

Optimum conditions

Reusability

pH

Temp

times

activity(%)

Amount of GLA loaded (mg/g)

Storage stability days

activity(%)

Ref.

Graphite / MNPs

Covalent

4

45

10

58

70

-

-

[40]

MNPs/chitosan

Ionic adsorption

2.5

65

10

68

12

6h

60

[29]

MNPs/COOH

Electrostatic interaction

5.5

30

8

40

80

25

70

[35]

MNCN/NH3

Covalent

4

55

10

60

45

-

-

[39]

Dextrin-Xanthan

Cross link

5

60

24

45

8.5

90

60

[30]

Bagasse cellulose

Covalent

3.5

65

15

65

-

-

-

[41]

Metal-chelated beads

Metal-affinity adsorption

4.5

65

10

55

104

60

38

[31]

MNPCh-CC

Covalent

4.5

60

10

70

195

105

64

This work

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4. Conclusions We prepared a suitable nanocomposite for glucoamylase immobilization with high loading capacity up to 85% using cyanuric chloride as a novel cross linking agent. Immobilization process enhanced the enzyme thermal stability and storage stability. Also, due to the magnetic properties of the synthesized nanocomposite, the immobilized GLA can be easily separated from reaction system and reuse for new enzymatic reaction. Moreover, the covalent attachment of GLA to synthesized MNPCh-CC was proved by different characterization techniques. The optimum temperature and pH for immobilized enzyme were obtained at 60 ◦C and pH 4.5, respectively. Finally we propound that these nanocomposite can be a suitable carrier for glucoamylase immobilization and would have a wide outlook for other industrial enzymes. Accordingly, it is suggested that GLA immobilized on functionalized MNPs by covalent binding is suitable for practical application in food, chemical and pharmaceutical industries.

Acknowledgements The financial supports of this work by the Research Councils of University of Isfahan are gratefully acknowledged.

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