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Activated alginate-montmorillonite beads as an efficient carrier for pectinase immobilization
International Journal of Biological Macromolecules 137 (2019) 253–260
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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Activated alginate-montmorillonite beads as an efficient carrier for pectinase immobilization Maryam Mohammadi a,b, Maryam Khakbaz Heshmati b, Khashayar Sarabandi c, Maryam Fathi d, Loong-Tak Lim e, Hamed Hamishehkar f,⁎ a
Food and Drug Safety Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Department of Food Science and Technology, Faculty of Agriculture, University of Tabriz, Tabriz, Iran Faculty of Food Science & Technology, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran d Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran e Department of Food Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada f Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran b c
a r t i c l e
i n f o
Article history: Received 30 April 2019 Received in revised form 27 June 2019 Accepted 28 June 2019 Available online 29 June 2019 Keywords: Alginate Montmorillonite clay Immobilization
a b s t r a c t Pectic compounds are responsible for turbidity in many juices. The elimination of these compounds, such as using pectinase can improve the appearance and storage stability of the products. In this study, the covalent immobilization of pectinase from Aspergillus aculeatus was studied on alginate-montmorillonite beads. The prepared beads were characterized by FT-IR and SEM. The immobilization procedure did not affect the optimal temperature (40 °C) of pectinase for achieving the maximum activity but the optimal pH changed was reduced from 5.5 to 5.0. A significant decrease in Michaelis constant (Km) value was observed after immobilization, indicating the affinity of enzyme to the substrate has been enhanced after immobilization, although the thermal stabilities of both forms of enzymes were comparable. After 6 cycles reusing of immobilized enzyme, its initial activity was remained about 53%. Finally, the immobilized pectinase was applied for the clarification of pineapple juice, showing that the immobilized enzyme is promising for use in the fruit juice industry. © 2019 Published by Elsevier B.V.
1. Introduction Pectinases (E.C.3.2.1.15) depolymerize and deesterifiy pectic compounds through breaking α-1,4-glycosidic linkages between two galacturonic residues [1]. These enzymes have important applications in clarification of fruit juices to reduce cloudiness and apply an advantageous influence in improving the quality, yield and filterability of fruit juices. Also, they have favorable applications in textile, paper/pulp, and waste water treatment industries [2]. In spite of their beneficial properties (high selectivity, mild operational conditions and ecofriendly process), industrial applications are still limited because of their high cost, low stability under operational pH and temperature, difficulties in enzyme recycling and reusing in continuous process [3]. Enzyme immobilization can potentially address these issues. This method could provide many advantages, including i) improve the stability of enzyme against the environment alteration ii) facilitate the enzyme activity in high concentration of the substrate, iii) the possibility of reusability of enzyme to apply in industrial process, and iv) the ⁎ Corresponding author. E-mail address: [email protected] (H. Hamishehkar).
https://doi.org/10.1016/j.ijbiomac.2019.06.236 0141-8130/© 2019 Published by Elsevier B.V.
possibility of easily separation of products from immobilized enzyme as compared to free enzyme [4]. Enzyme immobilization typically involves: (i) covalent immobilization of enzyme to a carrier via crosslinking agents such as glutaraldehyde; (ii) physical adsorption of enzyme molecules on a carrier; and (iii) entrapment or encapsulation of enzyme in polymers [5]. Among these methods, covalent immobilization is most efficient in preventing the enzyme from releasing from the carrier. In contrary, the physical adsorption such as electrostatic and ionic interactions is based on weaker chemical binding, disrupting under unfavorable ionic conditions. Also, in entrapment methods, the leaching of enzyme from support pores is very possible and consequently the reusability of support is decreased [6]. However, covalent immobilization may decrease the enzyme activity due to stearic hindrance and created modifications in the three-dimensional configuration of the enzyme [7]. Thus, the choice of suitable support is critical. To date, the immobilization of pectinases have been investigated onto different carrier such as magnetic corn starch microspheres [8], Florisil and nanosilica supports [9], chitosan-tethered silica [10], alginate beads [11], sodium alginate/graphene oxide [5], magnetite nanoparticles [12], chitosan-coated chitin [13], macroporous resin coated with chitosan [14], chitosan magnetic nanoparticles [1], amino-modified
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magnetic nanoparticle Fe3O4@SiO2–NH2 carrier [15], polyethyleneimine-coated pulp fiber [16], AOT-Fe3O4 nanoparticles [16] nylon-6 activated using glutaraldehyde [17], and so on. Sodium alginate (SA) is a natural accruing biopolymer obtained from marine algae which can create biocompatible and stable hydrogels when cross-linked with divalent metal ions. It has many applications in food and biotechnological industries as a gelling, stabilizing and thickening agent, as well as a for enzyme and cell immobilization [18]. While they are cost-effective, biocompatible, highly porous, and resistant to microbial contamination, alginate beads have low mechanical and thermal stability and tend to swell or crush if dissolved in nonisotonic media. The incorporation of nanofillers to the biopolymer matrix may enhance some of its properties such as its mechanical and thermal features [19]. For example, montmorillonite (MMT) fillers have been applied in various nanocomposite systems, because of beneficial features such as their high surface area, high ion exchange and high adsorption ability [20]. Despite these beneficial properties of MMT as nanofiller substance, studies related to its application in enzyme immobilization for food application are limited. Moreover, entrapping enzymes in beads tends to be inefficient due to leakage. Deng et al. immobilized the pectinase into calcium alginate microspheres for fruit juice. The immobilized enzyme lost more than 65.0% residual activity on its Sixth cycle [21]. In another study, the pectinase immobilized into calcium alginate as support lost 91% of its initial activity after seven times of reuses. de Oliveira et al. [22] studied on immobilization of pectinase from Aspergillus aculeatus in alginate beads [11]. The thermal stability results showed the immobilized enzyme was not stable at temperatures above 50 °C due to its weak mechanical strength [22]. To overcome this limitation, the covalent immobilization of pectinase can be performed by activating of carboxyl groups of SA with crosslinking agents [23]. Researchers have shown that immobilization of pectinase via covalent linkage on to amino and carboxylic acid functionalized nanosilica [24] and amino (-NH2) functionalized magnetic silica [25] can result in enhanced stability and activity of the enzyme. In the present paper, we developed a method to immobilize pectinase through covalent attachment through glutaraldehyde crosslinker (GA) on a hybrid structure of alginate-MMT bead support. The beads and immobilized enzyme were investigated by Fourier transform infrared (FT-IR) spectroscopy, while surface modifications of the support induced the immobilization process were explored by scanning electron microscopy (SEM). Immobilization parameters, such as crosslinking time, GA concentration and the amount of enzyme to alginatemontmorillonite (alginate-MMT) beads, were optimized to maximize the enzyme yield and activity, and the characteristic of immobilized enzyme such as kinetic parameters, thermal stabilities and reusability capability for degradation of pectin biopolymer were compared to the free enzyme. Finally, the immobilized pectinase was used for clarification of pineapple juice. 2. Methods and materials 2.1. Materials Dinitrosalicylic acid (DNS), Coomassie brilliant blue, ammonium solution (NH3, 25%), sodium acetate and D-galacturonic acid were purchased from Merck Chemicals Co. (Darmstadt, Germany). Pectinase from Aspergillus aculeatus, pectin from apple, sodium potassium tartarate, SA and GA were obtained from Merck (St. Louis, MO, USA). MMT was purchase from Iranian Chemical Co. (Mashhad, Iran). 2.2. Methods 2.2.1. Activation of MMT Activation of MMT was done according to method which reported previously by Dincer et al. [26]. According to this method, the activation of MMT was done with 1 M sulfuric acid at 80 °C for 2 h under refluxing
conditions. The slurry was centrifuged and washed with deionized water for several time to neutral the precipitate. Then, the neutral slurry was filtered through a glass fiber and dried overnight at 55 °C before use. 2.2.2. Preparation of alginate-MMT beads To prepare alginate-MMT beads, MMT was dispersed in distilled water under continuous stirring for 1 h followed by added to SA suspension (3% w/v). The alginate and MMT suspension was mixed together and stirred to form a homogenous mixture. The mixture was then injected by a syringe pump in a beaker containing CaCl2 solution (5% w/v), and allowed to crosslink for 2 h at ambient temperature. 2.2.3. Crosslinking of alginate–MMT beads by glutaraldehyde To incorporate new aldehyde groups, beads were rinsed in 2.5% (v/ v) GA solution under gentle stirring for 2 h. After passing this time to eliminate the excess glutaraldehyde, the beads surface was washed comprehensively with deionized water and kept at 4 °C in phosphate buffer until further analysis (0.01 M, pH 7.0). 2.2.4. Immobilization of pectinase on cross-linked alginate–MMT beads Cross-linked beads (50 mg) were contacted with 2 mL of pectinase (19 IU/mL) in acetate buffer (0.l M, pH 5.0) in a shaker incubator for 4 h at 4 °C. The upper solution was analyzed for the unloaded enzyme. After washing with acetate buffer (0.l M, pH 5.0), the beads were kept at the refrigerated temperature for further use. 2.3. Characterization of alginate-MMT beads The FT-IR spectra of alginate-MMT beads, cross-linked alginate– MMT beads, and pectinase immobilized on cross-linked alginate–MMT were measured with a FT-IR spectrometer in the range of 4000–400 cm−1 (Tensor 27 spectrometer, Bruker, Germany). Also, surface morphologies of freeze-dried alginate-MMT beads (Christ α 1–4, SciQuip, Germany) were examined using SEM (EM 3200, KYKY, China). 2.4. Enzyme assay 2.4.1. Protein content and pectinase activity assays The bounded enzyme was measured according to the Bradford method [27], using bovine serum albumin (10–1000 μg·mL) as a standard. The difference between the protein content of the initial stock and the protein content of the washing solutions was considered as immobilization yield. 2.4.2. Determination of enzyme activity The enzyme activity was estimated as described previously [28]. In this method, the reaction solution was incubated for 20 min at 40 °C in a shaker incubator. The enzymatic reaction was stopped by adding 1 mL of DNSA reagent followed heating the reaction media in boiling water bath for 10 min. Finally, before cooling the solution in ice bath, 0.333 mL of sodium potassium tartrate was added to fix the obtained color. Optical density of the solution was measured at 575 nm by the UV spectrophotometer (Jasco V-630, Japan). The amount of liberated reducing sugar was calculated as D-galacturonic acid equivalent using a calibration curve. The sodium acetate buffer (0.l M, pH 5.0) containing 0.5% pectin was considered as the blank sample. To determine the pectinolytic activity of the immobilized enzyme, 50 mg of enzyme loaded alginate-MMT beads were incubated with pectin (0.5% w/v in 0.1 M sodium acetate buffer, pH 5.0) for 20 min at 40 °C in shaker incubator. The required amount of pectinase to liberate 1 μmol of Dgalacturonic acid from pectin per time unit at optimum conditions for achieving the maximum activity of enzyme was considered as one unit of pectinase activity (U).
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2.5. Effects of pH and temperature
percentage acid according to the following formula:
The effects of buffer pH on the free and immobilized pectinase activity were evaluated by calculating the pectinase activity in different buffer solutions (0.1 M) (pH 3.0–5.50 acetate buffers, pH 6.0–8.0 phosphate buffers) at 40 °C. The effects of temperature on the free and immobilized pectinase activity were studied by calculating the activities at various temperatures (30–60 °C) at pH 5.
Percentage acid ¼
2.6. Thermal stability experiments The stability of the free and immobilized pectinase under thermal condition was measured by their incubation in acetate buffer (0.l M, pH 5.0), in the absence of substrate at various temperatures (30–60 °C) for specified intervals. Samples were removed at determined time intervals and their activities were measured using the Miller method [28]. 2.7. Determination of kinetic parameters Kinetic parameters (Vmax, Km, kcat and kcat/Km) of free and immobilized pectinase were estimated from Lineviwer-Burk curve by measuring the initial reaction rates corresponding to different concentrations of pectin (2–10 mg·mL) under the optimal condition for achieving the maximum activity for both forms of the enzyme in accordance with Section 2.4.2. 2.8. Reusability studies The reusability of immobilized pectinase was calculated by using frequent cycles of degradation of 0.5% w/v apple pectin in sodium acetate buffer (0.l M, pH 5.0) at 40 °C using spectrophotometer. After one reaction cycle, immobilized enzyme was separated, washed with acetate buffer (0.1 M, pH 5.0) and to start a new reaction cycle a new substrate solution was added for six repetitive cycles. 2.9. Application of immobilized enzyme in pineapple juice clarification 2.9.1. Clarification of pineapple juice The immobilized pectinase was evaluated for the clarification of pineapple juice. The fresh pineapple juice was obtained and centrifuged to precipitate coarse particles. The upper solution was used for the juice clarification process. The same amount of both forms of enzymes (38 IU) was mixed with 1 mL of pineapple juice and incubated for 3 h at 40 °C. The increase in %T at 650 nm was measured for every 0.5 h using a spectrophotometer [29]. 2.9.2. Determination of total sugars and reducing sugars The total sugars amount of pineapple juice was calculated according to method presented by [30]. In short, 0.5 mL of the diluted sample was mixed with 1 mL of phenol solution (2%) followed by the rapid addition of sulfuric acid (98%, 2.5 mL). After passing 10 min for color development and 30 min cooling in ice bath, the absorbance of sugar sample was read at 490 nm. Different concentrations of glucose:fructose:galactose (50–250 μg·mL) were applied as standard sugar sample. Reducing sugars content of pineapple juice was determined as described by [28]. 2.9.3. Determination of pH, total titratable acidity, color and viscosity of pineapple juice before and after clarification The pH of pineapple juice was determined using pH meter (Metrohm, 827 pH lab, Switzerland). The total acid value was determined by titrating 10 mL of diluted juice with 0.1 N NaOH, using phenolphthalein (1%) as an indicator. The acidity value was calculated as citric acid (0.0064 acid factor) equivalent [31]. Results expressed are in
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Titr acid factor 100 10 ðmL juiceÞ
To determine the color value of pineapple juice before and after addition of free and immobilized pectinase, equal volume of ethanol was added to juice and mixed for 30.0 min. After filtration and remove the coarse particles, the absorbance of samples was measured at 430 nm with spectrophotometer. Distilled water was considered as a reference [21]. Viscosity was measured using Oswald viscometer (Pisco, India) at constant room temperature. 2.9.4. Pectin test To examine the existence of pectin in pineapple juice before and after clarification, samples were added to twice the volume of acidified ethanol. Then samples were mixed upside down three times, stood 15.0 min. The formation of gel indicates that the juice contains pectin [21]. 2.10. Statistical analysis Statistical analysis was carried out by one-way ANOVA with Duncan significant difference test to compare between treatments at the significant level of 5% (P b 0.05) (SPSS, version 16.0, Chicago, IL, USA). 3. Results and discussion 3.1. Characterization of alginate-MMT beads and immobilized pectinase onto beads surface Fig. 1 shows the surface modifications of sodium alginate-MMT beads before and after pectinase immobilization. Before immobilization, the bead surface has noticeable roughness and the dispersed MMT particles are discernable on the surface (Fig. 1a, b). The roughness of bead surface increased after enzyme immobilization, with the immobilized enzyme appeared as white entities (Fig. 1c). Similar changes in surface morphology were reported by [32] after enzyme immobilization (procerain B) with glutaraldehyde on glutaraldehyde-activated chitosan beads. By comparing the SA (Fig. 2a) and MMT (Fig. 2b) FT-IR spectrum with alginate-MMT beads spectrum (Fig. 2c), both characteristic bands of sodium alginate and MMT were detected. The peaks at around 1647 and 1460 cm−1 were due to the stretching vibrations of carboxyl groups (asymmetric and symmetric, respectively), which are characteristics of the alginate polymer [33]. The peak at 1034 cm−1 was due to Si\\O stretching vibration, and those at 915, 878 and 798 cm−1 could be correlated to AlAlOH, AlFeOH, and AlMgOH of MMT [33]. For the Alginate-MMT beads activated by GA (Fig. 2d), a absorbance at 1424 cm−1 disappeared and the intensity of the characteristics bands of the alginate (asymmetric and symmetric stretching vibration of COO groups) decreased, indicating the successful activation of carboxyl groups of the alginate beads by GA [34]. Spectrum for the enzymeimmobilized sample exhibited a peak around 1423 cm−1. Moreover, the carboxyl stretching bands of alginate were shifted (Fig. 2e), confirming the successful attachment of pectinase onto alginate-MMT beads using GA. 3.2. Development of MMT-alginate beads and pectinase immobilization Considering the low mechanical and thermal stabilities of alginate beads that restrict their industrial applications, the MMT nanofiller was used in this study to improve the alginate beads drawbacks. To archive the successful immobilization process, the effects of initial enzyme amounts and cross-linker concentrations were investigated. Among the various initial pectinase concentration tested (38, 72, and 152 IU), the 38 IU of enzyme was found to be the most optimal, and therefore was
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Fig. 1. SEM images of alginate-montmorillonite (MMT) beads before (a, b) and after immobilization (c).
used in further analyses (Table 1). Further increasing in the initial amount of enzyme, decreases the immobilization yield and consequently enzyme activity. Among many cross-linking and activating agents are used to enzyme immobilization, GA is preferred because it is cost effective, being easily available and the ability to activate the numerous hydroxyl and carboxyl group of sodium alginate and make the enzyme–support cross-linking. Excessive concentration of GA may lead to unwanted cross-linking between enzyme molecules, leading to aggregation and loss of enzymatic activity [35]. Thus, the optimum concentration of GA is critical. To optimize the immobilization procedure, cross-linking parameters (cross-linker concentration and cross-linking
time) were investigated. Results from the preliminary experiments showed that the optimal concentration of GA and cross-linking time were 2.5% v/v and 2 h, respectively. Under these conditions, the maximum immobilization yield of pectinase (85%) was achieved. At lower GA concentrations, reduced immobilization yield and lower enzyme activity were observed. 3.3. Enzyme assay 3.3.1. Effect of pH on the free and immobilized pectinase activity Enzyme activity is significantly affected by reaction pH due to the substrate connecting to enzyme active site and catalytic process are often sensitive to the charge distribution on both the external environment and enzyme molecules [2]. Fig. 3 shows the obtained activity of free and immobilized enzymes influenced by different pH levels. The optimal pH values for free and immobilized enzymes were 5.5 and 5.0, respectively. In both enzymes, the activity reduced with increasing pH, but the immobilized enzyme showed higher stability under acidic conditions (pH 3, 4 and 4.6), as compared with the free enzyme. The covalent bonds created between enzyme-enzyme and enzyme-carriers might have contributed to the increased pH stability for the immobilized enzyme. These results are in agreement with those reported by Dai et al. who reported a shift of optimum pH to a lower value for covalently immobilized pectinase onto beads composed of sodium alginate/graphene oxide [5]. In our previous finding, the optimum pH of immobilized inulinase on glutathione stabilized gold magnetic nanoparticles was shifted from 5.0 to 4.5 and the activity of immobilized enzyme acidic pH values was higher than of free form [36]. 3.3.2. Effect of temperature on pectinase activity and stability Thermal stability is important factor for industrial applications of enzyme, which can be improved through immobilization. The effect of different temperatures on activities of free and immobilized pectinase were summarized in Fig. 4. The optimum temperature for both free and immobilized enzymes was 40 °C. Further increasing in temperature leads to declines in activity for both forms of pectinase. The thermal stability of free and immobilized pectinases was determined based on the activity remained after incubation the enzymes at different temperatures for 120 min (Fig. 5a, b). As can be seen from pictures, the immobilized pectinase showed higher thermal stability in the range of 30–50 °C compared to the free enzyme. However, both enzymes
Table 1 The effect of enzyme initial amount on immobilization yield and enzyme activity.
Fig. 2. FTIR spectra of a) alginate beads; b) MMT; c) alginate-MMT beads; d) alginate-MMT beads activated using glutaraldehyde; and e) pectinase immobilized on alginate-MMT beads.
Enzyme concentration (IU)
Immobilization yield (%)
Enzyme activity (μmol·min)
38 72 152
85 ± 2.5 65 ± 2.0 50 ± 2.1
0.45 ± 0.018 0.4 ± 0.015 0.38 ± 0.02
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Fig. 3. Effects of pH on free and immobilized pectinase activity.
showed similar thermal stability at 55 and 60 °C. These observations indicated that the support did not confer thermal protection at elevated temperatures. Similarly, de Oliveira et al. [22] evaluated the thermal stability of pectinase entrapment in alginate beads. They observed that the elevated temperature such as 60 °C had the destructive effect on the enzyme activity. In the present study, both forms of enzyme retained approximately 40% of original activity at 60 °C after 120 min. 3.3.3. Kinetic parameters Km and Vmax are related to partitioning and diffusional effects of substrate. Km value represents the enzyme affinity for used substrate where higher values are indicator of lower affinity. Vmax value describes the maximum enzymatic reaction rate when the all active sites of the enzyme molecules are occupied with the substrate [37]. The value of kcat (turnover number) is used to compare the catalytic efficiency of the free and immobilized enzymes. It typically defined units of product molecules produced per unit of enzyme molecules per unit of time. kcat/Km ratio shows the catalytic efficiency of immobilization process [36]. Fig. 6 represents the Lineweaver-Burk curve obtained by measuring the initial reaction rates corresponding to varying amounts of pectin as a substrate (0.2 to 1.0 mg·mL). Kinetic parameters values for the free and immobilized pectinase are shown in Table 2. According to obtained results, the enzyme affinity for used substrate has increased after immobilization. This behavior could be attributed to the stabilization of enzymes in their active structure and increased availability of the active sites. Similar observations on decreased Km value after immobilization have been reported by other researchers for immobilized pectinase [9]
and amylase [38]. On the other hand, the Vmax of immobilized enzyme decreased slightly compared to free form, which could be attributed to the decreased enzyme conformational flexibility, hampering substrate binding to the active site of the enzyme. Decreases in Vmax values after immobilization have been reported for different immobilized enzymes such as pectinase [11], Candida rugosa lipase [39] and amylase [40], all in alginate beads as support. In contrast, the increase in Vmax value upon immobilization has been previously reported by other authors
Fig. 4. Effects of temperature on free and immobilized pectinase activity.
Fig. 6. Lineweaver Burk curve of free and immobilized pectinase.
Fig. 5. Thermal stability of free (a) and immobilized pectinase (b).
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Table 2 Kinetic parameters of free and immobilized pectinase. Enzyme form
Km (mg·mL)
Vmax (μmol min−1 mL−1)
kcat (min−1)
kcat/Km (min−1 mg−1 mL)
Free pectinase Immobilized pectinase
11.49a 6.06b
2.93a 1.73b
18,312.5a 10,812.5b
1593.7b 1784.24a
Values correspond to the average of three tests. Different letters (a–b) indicate significant differences (p b 0.05).
[41,42]. The kcat of immobilized pectinase decreased compared to free enzyme. A decrease in kcat after immobilizing process may be due to the created structural changes in enzyme active site resulting in covalent immobilization that restrict substrate accessibility to immobilized pectinase due to steric hindrance. The kcat/Km value of the immobilized pectinase was identified to be higher than that of its free enzyme. This result demonstrated that catalytic efficiency increased after immobilization. This increase in catalytic efficiency is commonly observed in enzyme immobilization [22,43].
3.3.4. Reusability studies The reusability of an immobilized enzyme is an essential aspect for its commercial/industrial justification [36]. As depicted in Fig. 7, the immobilized pectinase preserved more than 53% of original activity after 6 cycles of reuse. This observed decrease in the immobilized pectinase activity may be due to the detachment of the enzyme from the sodium alginate-MMT beads caused by mechanical damages during washing process between runs and/or denaturation of enzyme during reuse assay [44]. It should be noted that the activities determined from the reusability test was higher than those reported by other researchers who immobilized the enzyme in alginate beads via noncovalent methods. In a previous reported research, the immobilized cellulose lost 90% of initial activity in the fourth cycle of reuses [45]. In the literature which has been reported by Anwar et al. the entrapment protease in calcium alginate lost its complete activity during the fourth cycle of reuse [46]. They justified that this decrease in enzyme activity may be due to either the washing of beads at the end of each cycle resulting in the release of the enzyme from the pores of alginate beads or changes in beads structure due to repeated reuse. It is possible that the combination of alginate with appropriate fillers such as MMT diminishes the pore size of beads and consequently decreases the enzyme leakage from alginate beads in entrapment technique. Also, the results achieved in the present study are comparable to those reported enzyme immobilizations in another supports via covalent bonding. Fang et al. [15] immobilized pectinase onto Fe3O4@SiO2–NH2 support. Their results showed that 64.4% of the initial activity was recovered after 7 cycle reuses [47]. Mardani et al., immobilized α-amylase on chitosanmontmorillonite nanocomposite beads via covalent binding. Their
results showed that the immobilized enzyme lost 53% of initial activity after reusing 5 cycles [48]. 3.4. Clarification of pineapple fruit juice The turbidity of fruit juices is predominantly due to the presence of starchy, protein and pectic constituents [29]. Table 3 shows the clarification values of juice treated with free and immobilized pectinase as compared to untreated pineapple juice. During the 2 h clarification treatment, the rate of increase transmittance by free pectinase was slightly higher than the immobilized pectinase. As the treatment progressed, the rate of turbidity reduction was proceeded by the immobilized enzyme, attributable to the higher stability of immobilized enzyme as compared to the free form at acidic condition. This result represents that the light transmittance of juice treated with both free pectinase and pectinase-immobilized alginate-MMT beads was significantly (p b 0.05) higher than control juice and immobilization could be promising for the clarification of fruit juices. The other physicochemical indexes of pineapple juice before and after clarification have been mentioned in Table 4. According to obtained results, after clarification using both free and immobilized pectinase, pH value shifted significantly toward acidic pH values (p b 0.05). Also, there was slight increase in the percentage acid of both free and immobilized enzymes treated juice as compared to control juice. This behavior can be justified due to the action of pectinase on pectic substrates and the release of organic acids, mainly galacturonic acid [22]. The reducing sugars content present in the clarified juice was found to be more than approximately 20% that of untreated Juice due to the transformation of pectin into soluble D-galacturonic acid. These results are consistent with those reported by [49]. In case of viscosity value, there was reduction by 40% of viscosity in clarified pineapple juice as compared untreated juice. The decrease in viscosity after the treatment of juices by pectinases was documented by other researchers [50,51]. The pectin tests result showed that both free and immobilized pectinase treatments had no pectin. It was confirmed that the treatment of free and immobilized enzyme could effectively degrade the pectin in pineapple juice. 4. Conclusion In the present work, pectinase from Aspergillus aculeatus was successfully immobilized on Alginate-MMT beads via covalent
Table 3 Clarification of pineapple juices by free and immobilized pectinase.
Fig. 7. Reusability of pectinase immobilized on alginate-MMT beads.
Time (h)
Control (%T650)
Free enzyme (%T650)
Immobilized enzyme (%T650)
0.5 1 1.5 2 2.5 3
51.8 ± 0.3b 52.0 ± 0.32b 52.6 ± 0.29b 52.2 ± 0.3b 51.7 ± 0.31b 51.5 ± 0.33c
88.0 ± 0.31a 90.0 ± 0.27a 91.8 ± 0.2a 93.2 ± 0.25a 90.8 ± 0.3a 88.0 ± 0.3b
85.8 ± 0.2a 88.2 ± 0.25a 90.0 ± 0.3a 93.1 ± 0.27a 93.8 ± 0.31a 95.0 ± 0.2a
Values correspond to the average of three tests. Different letters (a–c) indicate significant differences (p b 0.05).
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Table 4 The physicochemical indexes of pineapple juice before and after clarification. Physicochemical indexes
Pineapple juice
Pectinase treatment
Immobilized pectinase treatment
pH Acid value (%) Total sugar (mg·mL) Reducing sugar (mg·mL) Color (420 nm) Viscosity (Pa·s) Pectin
3.40 ± 0.02a 0.520 ± 0.003a 15.600 ± 0.03a 9.732 ± 0.04b 0.435 ± 0.009a 1.750 ± 0.003a Have
3.1 ± 0.03b 0.521 ± 0.002a 15.325 ± 0.03a 11.620 ± 0.02a 0.100 ± 0.008b 1.065 ± 0.004b None
3.09 ± 0.03b 0.522 ± 0.001a 15.535 ± 0.04a 11.750 ± 0.03a 0.085 ± 0.007b 1.050 ± 0.003b None
Values correspond to the average of three tests. Different letters (a–b) indicate significant differences (p b 0.05).
immobilization. The immobilized pectinase showed higher activity compared to the free enzyme in extreme pH conditions. Also, the Km value showed that the affinity of enzyme to the substrate increased after the immobilization process. The immobilized pectinase showed enhanced stability and reusability for pectin hydrolysis, retaining more than 53% of activity after 6 consecutive cycles. Preliminary study showed that the immobilized pectinate could be used to clarify pineapple juice, demonstrating its potential for fruit juice processing in the food industry. Declaration of Competing Interest The authors have no conflicts of interest to declare. Acknowledgment The authors would like to acknowledge from Drug Applied Research Center, Tabriz University of Medical Sciences for financial support. References [1] U.V. Sojitra, S.S. Nadar, V.K. Rathod, Immobilization of pectinase onto chitosan magnetic nanoparticles by macromolecular cross-linker, Carbohydr. Polym. 157 (2017) 677–685. [2] U.V. Sojitra, S.S. Nadar, V.K. Rathod, A magnetic tri-enzyme nanobiocatalyst for fruit juice clarification, Food Chem. 213 (2016) 296–305. [3] A.A. Kadam, J. Jang, S.C. Jee, J.S. Sung, D.S. Lee, Chitosan-functionalized supermagnetic halloysite nanotubes for covalent laccase immobilization, Carbohydr. Polym. 194 (2018) 208–216. [4] I. Benucci, K. Liburdi, I. Cacciotti, C. Lombardelli, M. Zappino, F. Nanni, M. Esti, Chitosan/clay nanocomposite films as supports for enzyme immobilization: an innovative green approach for winemaking applications, Food Hydrocoll. 74 (2018) 124–131. [5] X.Y. Dai, L.M. Kong, X.L. Wang, Q. Zhu, K. Chen, T. Zhou, Preparation, characterization and catalytic behavior of pectinase covalently immobilized onto sodium alginate/ graphene oxide composite beads, Food Chem. 253 (2018) 185–193. [6] H.U. Rehman, M.A. Nawaz, A. Aman, A.H. Baloch, S.A.U. Qader, Immobilization of pectinase from Bacillus licheniformis KIBGE-IB21 on chitosan beads for continuous degradation of pectin polymers, Biocatal. Agric. Biotechnol. 3 (4) (2014) 282–287. [7] W. Sheng, Y. Xi, L. Zhang, T. Ye, X. Zhao, Enhanced activity and stability of papain by covalent immobilization on porous magnetic nanoparticles, Int. J. Biol. Macromol. 114 (2018) 143–148. [8] B. Wang, F. Cheng, Y. Lu, W. Ge, M. Zhang, B. Yue, Immobilization of pectinase from Penicillium oxalicum F67 onto magnetic cornstarch microspheres: characterization and application in juice production, J. Mol. Catal. B Enzym. 97 (2013) 137–143. [9] D. Alagöz, S.S. Tükel, D. Yildirim, Immobilization of pectinase on silica-based supports: impacts of particle size and spacer arm on the activity, Int. J. Biol. Macromol. 87 (2016) 426–432. [10] Z. Lei, S. Bi, H. Yang, Chitosan-tethered the silica particle from a layer-by-layer approach for pectinase immobilization, Food Chem. 104 (2) (2007) 577–584. [11] H.U. Rehman, A. Aman, A. Silipo, S.A.U. Qader, A. Molinaro, A. Ansari, Degradation of complex carbohydrate: immobilization of pectinase from Bacillus licheniformis KIBGE-IB21 using calcium alginate as a support, Food Chem. 139 (1–4) (2013) 1081–1086. [12] L. Mosafa, M. Shahedi, M. Moghadam, Magnetite nanoparticles immobilized pectinase: preparation, characterization and application for the fruit juices clarification, J. Chin. Chem. Soc. 61 (3) (2014) 329–336. [13] H.L. Ramirez, L. Gõmez Brizuela, J. Úbeda Iranzo, M. Arevalo-Villena, A.I. Briones Pérez, Pectinase immobilization on a chitosan-coated chitin support, J. Food Process Eng. 39 (1) (2016) 97–104. [14] K. Liu, G. Zhao, B. He, L. Chen, L. Huang, Immobilization of pectinase and lipase on macroporous resin coated with chitosan for treatment of whitewater from papermaking, Bioresour. Technol. 123 (2012) 616–619.
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Activated alginate-montmorillonite beads as an efficient carrier for pectinase immobilization Maryam Mohammadi a,b, Maryam Khakbaz Heshmati b, Khashayar Sarabandi c, Maryam Fathi d, Loong-Tak Lim e, Hamed Hamishehkar f,⁎ a
Food and Drug Safety Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Department of Food Science and Technology, Faculty of Agriculture, University of Tabriz, Tabriz, Iran Faculty of Food Science & Technology, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran d Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran e Department of Food Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada f Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran b c
a r t i c l e
i n f o
Article history: Received 30 April 2019 Received in revised form 27 June 2019 Accepted 28 June 2019 Available online 29 June 2019 Keywords: Alginate Montmorillonite clay Immobilization
a b s t r a c t Pectic compounds are responsible for turbidity in many juices. The elimination of these compounds, such as using pectinase can improve the appearance and storage stability of the products. In this study, the covalent immobilization of pectinase from Aspergillus aculeatus was studied on alginate-montmorillonite beads. The prepared beads were characterized by FT-IR and SEM. The immobilization procedure did not affect the optimal temperature (40 °C) of pectinase for achieving the maximum activity but the optimal pH changed was reduced from 5.5 to 5.0. A significant decrease in Michaelis constant (Km) value was observed after immobilization, indicating the affinity of enzyme to the substrate has been enhanced after immobilization, although the thermal stabilities of both forms of enzymes were comparable. After 6 cycles reusing of immobilized enzyme, its initial activity was remained about 53%. Finally, the immobilized pectinase was applied for the clarification of pineapple juice, showing that the immobilized enzyme is promising for use in the fruit juice industry. © 2019 Published by Elsevier B.V.
1. Introduction Pectinases (E.C.3.2.1.15) depolymerize and deesterifiy pectic compounds through breaking α-1,4-glycosidic linkages between two galacturonic residues [1]. These enzymes have important applications in clarification of fruit juices to reduce cloudiness and apply an advantageous influence in improving the quality, yield and filterability of fruit juices. Also, they have favorable applications in textile, paper/pulp, and waste water treatment industries [2]. In spite of their beneficial properties (high selectivity, mild operational conditions and ecofriendly process), industrial applications are still limited because of their high cost, low stability under operational pH and temperature, difficulties in enzyme recycling and reusing in continuous process [3]. Enzyme immobilization can potentially address these issues. This method could provide many advantages, including i) improve the stability of enzyme against the environment alteration ii) facilitate the enzyme activity in high concentration of the substrate, iii) the possibility of reusability of enzyme to apply in industrial process, and iv) the ⁎ Corresponding author. E-mail address: [email protected] (H. Hamishehkar).
https://doi.org/10.1016/j.ijbiomac.2019.06.236 0141-8130/© 2019 Published by Elsevier B.V.
possibility of easily separation of products from immobilized enzyme as compared to free enzyme [4]. Enzyme immobilization typically involves: (i) covalent immobilization of enzyme to a carrier via crosslinking agents such as glutaraldehyde; (ii) physical adsorption of enzyme molecules on a carrier; and (iii) entrapment or encapsulation of enzyme in polymers [5]. Among these methods, covalent immobilization is most efficient in preventing the enzyme from releasing from the carrier. In contrary, the physical adsorption such as electrostatic and ionic interactions is based on weaker chemical binding, disrupting under unfavorable ionic conditions. Also, in entrapment methods, the leaching of enzyme from support pores is very possible and consequently the reusability of support is decreased [6]. However, covalent immobilization may decrease the enzyme activity due to stearic hindrance and created modifications in the three-dimensional configuration of the enzyme [7]. Thus, the choice of suitable support is critical. To date, the immobilization of pectinases have been investigated onto different carrier such as magnetic corn starch microspheres [8], Florisil and nanosilica supports [9], chitosan-tethered silica [10], alginate beads [11], sodium alginate/graphene oxide [5], magnetite nanoparticles [12], chitosan-coated chitin [13], macroporous resin coated with chitosan [14], chitosan magnetic nanoparticles [1], amino-modified
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magnetic nanoparticle Fe3O4@SiO2–NH2 carrier [15], polyethyleneimine-coated pulp fiber [16], AOT-Fe3O4 nanoparticles [16] nylon-6 activated using glutaraldehyde [17], and so on. Sodium alginate (SA) is a natural accruing biopolymer obtained from marine algae which can create biocompatible and stable hydrogels when cross-linked with divalent metal ions. It has many applications in food and biotechnological industries as a gelling, stabilizing and thickening agent, as well as a for enzyme and cell immobilization [18]. While they are cost-effective, biocompatible, highly porous, and resistant to microbial contamination, alginate beads have low mechanical and thermal stability and tend to swell or crush if dissolved in nonisotonic media. The incorporation of nanofillers to the biopolymer matrix may enhance some of its properties such as its mechanical and thermal features [19]. For example, montmorillonite (MMT) fillers have been applied in various nanocomposite systems, because of beneficial features such as their high surface area, high ion exchange and high adsorption ability [20]. Despite these beneficial properties of MMT as nanofiller substance, studies related to its application in enzyme immobilization for food application are limited. Moreover, entrapping enzymes in beads tends to be inefficient due to leakage. Deng et al. immobilized the pectinase into calcium alginate microspheres for fruit juice. The immobilized enzyme lost more than 65.0% residual activity on its Sixth cycle [21]. In another study, the pectinase immobilized into calcium alginate as support lost 91% of its initial activity after seven times of reuses. de Oliveira et al. [22] studied on immobilization of pectinase from Aspergillus aculeatus in alginate beads [11]. The thermal stability results showed the immobilized enzyme was not stable at temperatures above 50 °C due to its weak mechanical strength [22]. To overcome this limitation, the covalent immobilization of pectinase can be performed by activating of carboxyl groups of SA with crosslinking agents [23]. Researchers have shown that immobilization of pectinase via covalent linkage on to amino and carboxylic acid functionalized nanosilica [24] and amino (-NH2) functionalized magnetic silica [25] can result in enhanced stability and activity of the enzyme. In the present paper, we developed a method to immobilize pectinase through covalent attachment through glutaraldehyde crosslinker (GA) on a hybrid structure of alginate-MMT bead support. The beads and immobilized enzyme were investigated by Fourier transform infrared (FT-IR) spectroscopy, while surface modifications of the support induced the immobilization process were explored by scanning electron microscopy (SEM). Immobilization parameters, such as crosslinking time, GA concentration and the amount of enzyme to alginatemontmorillonite (alginate-MMT) beads, were optimized to maximize the enzyme yield and activity, and the characteristic of immobilized enzyme such as kinetic parameters, thermal stabilities and reusability capability for degradation of pectin biopolymer were compared to the free enzyme. Finally, the immobilized pectinase was used for clarification of pineapple juice. 2. Methods and materials 2.1. Materials Dinitrosalicylic acid (DNS), Coomassie brilliant blue, ammonium solution (NH3, 25%), sodium acetate and D-galacturonic acid were purchased from Merck Chemicals Co. (Darmstadt, Germany). Pectinase from Aspergillus aculeatus, pectin from apple, sodium potassium tartarate, SA and GA were obtained from Merck (St. Louis, MO, USA). MMT was purchase from Iranian Chemical Co. (Mashhad, Iran). 2.2. Methods 2.2.1. Activation of MMT Activation of MMT was done according to method which reported previously by Dincer et al. [26]. According to this method, the activation of MMT was done with 1 M sulfuric acid at 80 °C for 2 h under refluxing
conditions. The slurry was centrifuged and washed with deionized water for several time to neutral the precipitate. Then, the neutral slurry was filtered through a glass fiber and dried overnight at 55 °C before use. 2.2.2. Preparation of alginate-MMT beads To prepare alginate-MMT beads, MMT was dispersed in distilled water under continuous stirring for 1 h followed by added to SA suspension (3% w/v). The alginate and MMT suspension was mixed together and stirred to form a homogenous mixture. The mixture was then injected by a syringe pump in a beaker containing CaCl2 solution (5% w/v), and allowed to crosslink for 2 h at ambient temperature. 2.2.3. Crosslinking of alginate–MMT beads by glutaraldehyde To incorporate new aldehyde groups, beads were rinsed in 2.5% (v/ v) GA solution under gentle stirring for 2 h. After passing this time to eliminate the excess glutaraldehyde, the beads surface was washed comprehensively with deionized water and kept at 4 °C in phosphate buffer until further analysis (0.01 M, pH 7.0). 2.2.4. Immobilization of pectinase on cross-linked alginate–MMT beads Cross-linked beads (50 mg) were contacted with 2 mL of pectinase (19 IU/mL) in acetate buffer (0.l M, pH 5.0) in a shaker incubator for 4 h at 4 °C. The upper solution was analyzed for the unloaded enzyme. After washing with acetate buffer (0.l M, pH 5.0), the beads were kept at the refrigerated temperature for further use. 2.3. Characterization of alginate-MMT beads The FT-IR spectra of alginate-MMT beads, cross-linked alginate– MMT beads, and pectinase immobilized on cross-linked alginate–MMT were measured with a FT-IR spectrometer in the range of 4000–400 cm−1 (Tensor 27 spectrometer, Bruker, Germany). Also, surface morphologies of freeze-dried alginate-MMT beads (Christ α 1–4, SciQuip, Germany) were examined using SEM (EM 3200, KYKY, China). 2.4. Enzyme assay 2.4.1. Protein content and pectinase activity assays The bounded enzyme was measured according to the Bradford method [27], using bovine serum albumin (10–1000 μg·mL) as a standard. The difference between the protein content of the initial stock and the protein content of the washing solutions was considered as immobilization yield. 2.4.2. Determination of enzyme activity The enzyme activity was estimated as described previously [28]. In this method, the reaction solution was incubated for 20 min at 40 °C in a shaker incubator. The enzymatic reaction was stopped by adding 1 mL of DNSA reagent followed heating the reaction media in boiling water bath for 10 min. Finally, before cooling the solution in ice bath, 0.333 mL of sodium potassium tartrate was added to fix the obtained color. Optical density of the solution was measured at 575 nm by the UV spectrophotometer (Jasco V-630, Japan). The amount of liberated reducing sugar was calculated as D-galacturonic acid equivalent using a calibration curve. The sodium acetate buffer (0.l M, pH 5.0) containing 0.5% pectin was considered as the blank sample. To determine the pectinolytic activity of the immobilized enzyme, 50 mg of enzyme loaded alginate-MMT beads were incubated with pectin (0.5% w/v in 0.1 M sodium acetate buffer, pH 5.0) for 20 min at 40 °C in shaker incubator. The required amount of pectinase to liberate 1 μmol of Dgalacturonic acid from pectin per time unit at optimum conditions for achieving the maximum activity of enzyme was considered as one unit of pectinase activity (U).
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2.5. Effects of pH and temperature
percentage acid according to the following formula:
The effects of buffer pH on the free and immobilized pectinase activity were evaluated by calculating the pectinase activity in different buffer solutions (0.1 M) (pH 3.0–5.50 acetate buffers, pH 6.0–8.0 phosphate buffers) at 40 °C. The effects of temperature on the free and immobilized pectinase activity were studied by calculating the activities at various temperatures (30–60 °C) at pH 5.
Percentage acid ¼
2.6. Thermal stability experiments The stability of the free and immobilized pectinase under thermal condition was measured by their incubation in acetate buffer (0.l M, pH 5.0), in the absence of substrate at various temperatures (30–60 °C) for specified intervals. Samples were removed at determined time intervals and their activities were measured using the Miller method [28]. 2.7. Determination of kinetic parameters Kinetic parameters (Vmax, Km, kcat and kcat/Km) of free and immobilized pectinase were estimated from Lineviwer-Burk curve by measuring the initial reaction rates corresponding to different concentrations of pectin (2–10 mg·mL) under the optimal condition for achieving the maximum activity for both forms of the enzyme in accordance with Section 2.4.2. 2.8. Reusability studies The reusability of immobilized pectinase was calculated by using frequent cycles of degradation of 0.5% w/v apple pectin in sodium acetate buffer (0.l M, pH 5.0) at 40 °C using spectrophotometer. After one reaction cycle, immobilized enzyme was separated, washed with acetate buffer (0.1 M, pH 5.0) and to start a new reaction cycle a new substrate solution was added for six repetitive cycles. 2.9. Application of immobilized enzyme in pineapple juice clarification 2.9.1. Clarification of pineapple juice The immobilized pectinase was evaluated for the clarification of pineapple juice. The fresh pineapple juice was obtained and centrifuged to precipitate coarse particles. The upper solution was used for the juice clarification process. The same amount of both forms of enzymes (38 IU) was mixed with 1 mL of pineapple juice and incubated for 3 h at 40 °C. The increase in %T at 650 nm was measured for every 0.5 h using a spectrophotometer [29]. 2.9.2. Determination of total sugars and reducing sugars The total sugars amount of pineapple juice was calculated according to method presented by [30]. In short, 0.5 mL of the diluted sample was mixed with 1 mL of phenol solution (2%) followed by the rapid addition of sulfuric acid (98%, 2.5 mL). After passing 10 min for color development and 30 min cooling in ice bath, the absorbance of sugar sample was read at 490 nm. Different concentrations of glucose:fructose:galactose (50–250 μg·mL) were applied as standard sugar sample. Reducing sugars content of pineapple juice was determined as described by [28]. 2.9.3. Determination of pH, total titratable acidity, color and viscosity of pineapple juice before and after clarification The pH of pineapple juice was determined using pH meter (Metrohm, 827 pH lab, Switzerland). The total acid value was determined by titrating 10 mL of diluted juice with 0.1 N NaOH, using phenolphthalein (1%) as an indicator. The acidity value was calculated as citric acid (0.0064 acid factor) equivalent [31]. Results expressed are in
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Titr acid factor 100 10 ðmL juiceÞ
To determine the color value of pineapple juice before and after addition of free and immobilized pectinase, equal volume of ethanol was added to juice and mixed for 30.0 min. After filtration and remove the coarse particles, the absorbance of samples was measured at 430 nm with spectrophotometer. Distilled water was considered as a reference [21]. Viscosity was measured using Oswald viscometer (Pisco, India) at constant room temperature. 2.9.4. Pectin test To examine the existence of pectin in pineapple juice before and after clarification, samples were added to twice the volume of acidified ethanol. Then samples were mixed upside down three times, stood 15.0 min. The formation of gel indicates that the juice contains pectin [21]. 2.10. Statistical analysis Statistical analysis was carried out by one-way ANOVA with Duncan significant difference test to compare between treatments at the significant level of 5% (P b 0.05) (SPSS, version 16.0, Chicago, IL, USA). 3. Results and discussion 3.1. Characterization of alginate-MMT beads and immobilized pectinase onto beads surface Fig. 1 shows the surface modifications of sodium alginate-MMT beads before and after pectinase immobilization. Before immobilization, the bead surface has noticeable roughness and the dispersed MMT particles are discernable on the surface (Fig. 1a, b). The roughness of bead surface increased after enzyme immobilization, with the immobilized enzyme appeared as white entities (Fig. 1c). Similar changes in surface morphology were reported by [32] after enzyme immobilization (procerain B) with glutaraldehyde on glutaraldehyde-activated chitosan beads. By comparing the SA (Fig. 2a) and MMT (Fig. 2b) FT-IR spectrum with alginate-MMT beads spectrum (Fig. 2c), both characteristic bands of sodium alginate and MMT were detected. The peaks at around 1647 and 1460 cm−1 were due to the stretching vibrations of carboxyl groups (asymmetric and symmetric, respectively), which are characteristics of the alginate polymer [33]. The peak at 1034 cm−1 was due to Si\\O stretching vibration, and those at 915, 878 and 798 cm−1 could be correlated to AlAlOH, AlFeOH, and AlMgOH of MMT [33]. For the Alginate-MMT beads activated by GA (Fig. 2d), a absorbance at 1424 cm−1 disappeared and the intensity of the characteristics bands of the alginate (asymmetric and symmetric stretching vibration of COO groups) decreased, indicating the successful activation of carboxyl groups of the alginate beads by GA [34]. Spectrum for the enzymeimmobilized sample exhibited a peak around 1423 cm−1. Moreover, the carboxyl stretching bands of alginate were shifted (Fig. 2e), confirming the successful attachment of pectinase onto alginate-MMT beads using GA. 3.2. Development of MMT-alginate beads and pectinase immobilization Considering the low mechanical and thermal stabilities of alginate beads that restrict their industrial applications, the MMT nanofiller was used in this study to improve the alginate beads drawbacks. To archive the successful immobilization process, the effects of initial enzyme amounts and cross-linker concentrations were investigated. Among the various initial pectinase concentration tested (38, 72, and 152 IU), the 38 IU of enzyme was found to be the most optimal, and therefore was
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Fig. 1. SEM images of alginate-montmorillonite (MMT) beads before (a, b) and after immobilization (c).
used in further analyses (Table 1). Further increasing in the initial amount of enzyme, decreases the immobilization yield and consequently enzyme activity. Among many cross-linking and activating agents are used to enzyme immobilization, GA is preferred because it is cost effective, being easily available and the ability to activate the numerous hydroxyl and carboxyl group of sodium alginate and make the enzyme–support cross-linking. Excessive concentration of GA may lead to unwanted cross-linking between enzyme molecules, leading to aggregation and loss of enzymatic activity [35]. Thus, the optimum concentration of GA is critical. To optimize the immobilization procedure, cross-linking parameters (cross-linker concentration and cross-linking
time) were investigated. Results from the preliminary experiments showed that the optimal concentration of GA and cross-linking time were 2.5% v/v and 2 h, respectively. Under these conditions, the maximum immobilization yield of pectinase (85%) was achieved. At lower GA concentrations, reduced immobilization yield and lower enzyme activity were observed. 3.3. Enzyme assay 3.3.1. Effect of pH on the free and immobilized pectinase activity Enzyme activity is significantly affected by reaction pH due to the substrate connecting to enzyme active site and catalytic process are often sensitive to the charge distribution on both the external environment and enzyme molecules [2]. Fig. 3 shows the obtained activity of free and immobilized enzymes influenced by different pH levels. The optimal pH values for free and immobilized enzymes were 5.5 and 5.0, respectively. In both enzymes, the activity reduced with increasing pH, but the immobilized enzyme showed higher stability under acidic conditions (pH 3, 4 and 4.6), as compared with the free enzyme. The covalent bonds created between enzyme-enzyme and enzyme-carriers might have contributed to the increased pH stability for the immobilized enzyme. These results are in agreement with those reported by Dai et al. who reported a shift of optimum pH to a lower value for covalently immobilized pectinase onto beads composed of sodium alginate/graphene oxide [5]. In our previous finding, the optimum pH of immobilized inulinase on glutathione stabilized gold magnetic nanoparticles was shifted from 5.0 to 4.5 and the activity of immobilized enzyme acidic pH values was higher than of free form [36]. 3.3.2. Effect of temperature on pectinase activity and stability Thermal stability is important factor for industrial applications of enzyme, which can be improved through immobilization. The effect of different temperatures on activities of free and immobilized pectinase were summarized in Fig. 4. The optimum temperature for both free and immobilized enzymes was 40 °C. Further increasing in temperature leads to declines in activity for both forms of pectinase. The thermal stability of free and immobilized pectinases was determined based on the activity remained after incubation the enzymes at different temperatures for 120 min (Fig. 5a, b). As can be seen from pictures, the immobilized pectinase showed higher thermal stability in the range of 30–50 °C compared to the free enzyme. However, both enzymes
Table 1 The effect of enzyme initial amount on immobilization yield and enzyme activity.
Fig. 2. FTIR spectra of a) alginate beads; b) MMT; c) alginate-MMT beads; d) alginate-MMT beads activated using glutaraldehyde; and e) pectinase immobilized on alginate-MMT beads.
Enzyme concentration (IU)
Immobilization yield (%)
Enzyme activity (μmol·min)
38 72 152
85 ± 2.5 65 ± 2.0 50 ± 2.1
0.45 ± 0.018 0.4 ± 0.015 0.38 ± 0.02
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Fig. 3. Effects of pH on free and immobilized pectinase activity.
showed similar thermal stability at 55 and 60 °C. These observations indicated that the support did not confer thermal protection at elevated temperatures. Similarly, de Oliveira et al. [22] evaluated the thermal stability of pectinase entrapment in alginate beads. They observed that the elevated temperature such as 60 °C had the destructive effect on the enzyme activity. In the present study, both forms of enzyme retained approximately 40% of original activity at 60 °C after 120 min. 3.3.3. Kinetic parameters Km and Vmax are related to partitioning and diffusional effects of substrate. Km value represents the enzyme affinity for used substrate where higher values are indicator of lower affinity. Vmax value describes the maximum enzymatic reaction rate when the all active sites of the enzyme molecules are occupied with the substrate [37]. The value of kcat (turnover number) is used to compare the catalytic efficiency of the free and immobilized enzymes. It typically defined units of product molecules produced per unit of enzyme molecules per unit of time. kcat/Km ratio shows the catalytic efficiency of immobilization process [36]. Fig. 6 represents the Lineweaver-Burk curve obtained by measuring the initial reaction rates corresponding to varying amounts of pectin as a substrate (0.2 to 1.0 mg·mL). Kinetic parameters values for the free and immobilized pectinase are shown in Table 2. According to obtained results, the enzyme affinity for used substrate has increased after immobilization. This behavior could be attributed to the stabilization of enzymes in their active structure and increased availability of the active sites. Similar observations on decreased Km value after immobilization have been reported by other researchers for immobilized pectinase [9]
and amylase [38]. On the other hand, the Vmax of immobilized enzyme decreased slightly compared to free form, which could be attributed to the decreased enzyme conformational flexibility, hampering substrate binding to the active site of the enzyme. Decreases in Vmax values after immobilization have been reported for different immobilized enzymes such as pectinase [11], Candida rugosa lipase [39] and amylase [40], all in alginate beads as support. In contrast, the increase in Vmax value upon immobilization has been previously reported by other authors
Fig. 4. Effects of temperature on free and immobilized pectinase activity.
Fig. 6. Lineweaver Burk curve of free and immobilized pectinase.
Fig. 5. Thermal stability of free (a) and immobilized pectinase (b).
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Table 2 Kinetic parameters of free and immobilized pectinase. Enzyme form
Km (mg·mL)
Vmax (μmol min−1 mL−1)
kcat (min−1)
kcat/Km (min−1 mg−1 mL)
Free pectinase Immobilized pectinase
11.49a 6.06b
2.93a 1.73b
18,312.5a 10,812.5b
1593.7b 1784.24a
Values correspond to the average of three tests. Different letters (a–b) indicate significant differences (p b 0.05).
[41,42]. The kcat of immobilized pectinase decreased compared to free enzyme. A decrease in kcat after immobilizing process may be due to the created structural changes in enzyme active site resulting in covalent immobilization that restrict substrate accessibility to immobilized pectinase due to steric hindrance. The kcat/Km value of the immobilized pectinase was identified to be higher than that of its free enzyme. This result demonstrated that catalytic efficiency increased after immobilization. This increase in catalytic efficiency is commonly observed in enzyme immobilization [22,43].
3.3.4. Reusability studies The reusability of an immobilized enzyme is an essential aspect for its commercial/industrial justification [36]. As depicted in Fig. 7, the immobilized pectinase preserved more than 53% of original activity after 6 cycles of reuse. This observed decrease in the immobilized pectinase activity may be due to the detachment of the enzyme from the sodium alginate-MMT beads caused by mechanical damages during washing process between runs and/or denaturation of enzyme during reuse assay [44]. It should be noted that the activities determined from the reusability test was higher than those reported by other researchers who immobilized the enzyme in alginate beads via noncovalent methods. In a previous reported research, the immobilized cellulose lost 90% of initial activity in the fourth cycle of reuses [45]. In the literature which has been reported by Anwar et al. the entrapment protease in calcium alginate lost its complete activity during the fourth cycle of reuse [46]. They justified that this decrease in enzyme activity may be due to either the washing of beads at the end of each cycle resulting in the release of the enzyme from the pores of alginate beads or changes in beads structure due to repeated reuse. It is possible that the combination of alginate with appropriate fillers such as MMT diminishes the pore size of beads and consequently decreases the enzyme leakage from alginate beads in entrapment technique. Also, the results achieved in the present study are comparable to those reported enzyme immobilizations in another supports via covalent bonding. Fang et al. [15] immobilized pectinase onto Fe3O4@SiO2–NH2 support. Their results showed that 64.4% of the initial activity was recovered after 7 cycle reuses [47]. Mardani et al., immobilized α-amylase on chitosanmontmorillonite nanocomposite beads via covalent binding. Their
results showed that the immobilized enzyme lost 53% of initial activity after reusing 5 cycles [48]. 3.4. Clarification of pineapple fruit juice The turbidity of fruit juices is predominantly due to the presence of starchy, protein and pectic constituents [29]. Table 3 shows the clarification values of juice treated with free and immobilized pectinase as compared to untreated pineapple juice. During the 2 h clarification treatment, the rate of increase transmittance by free pectinase was slightly higher than the immobilized pectinase. As the treatment progressed, the rate of turbidity reduction was proceeded by the immobilized enzyme, attributable to the higher stability of immobilized enzyme as compared to the free form at acidic condition. This result represents that the light transmittance of juice treated with both free pectinase and pectinase-immobilized alginate-MMT beads was significantly (p b 0.05) higher than control juice and immobilization could be promising for the clarification of fruit juices. The other physicochemical indexes of pineapple juice before and after clarification have been mentioned in Table 4. According to obtained results, after clarification using both free and immobilized pectinase, pH value shifted significantly toward acidic pH values (p b 0.05). Also, there was slight increase in the percentage acid of both free and immobilized enzymes treated juice as compared to control juice. This behavior can be justified due to the action of pectinase on pectic substrates and the release of organic acids, mainly galacturonic acid [22]. The reducing sugars content present in the clarified juice was found to be more than approximately 20% that of untreated Juice due to the transformation of pectin into soluble D-galacturonic acid. These results are consistent with those reported by [49]. In case of viscosity value, there was reduction by 40% of viscosity in clarified pineapple juice as compared untreated juice. The decrease in viscosity after the treatment of juices by pectinases was documented by other researchers [50,51]. The pectin tests result showed that both free and immobilized pectinase treatments had no pectin. It was confirmed that the treatment of free and immobilized enzyme could effectively degrade the pectin in pineapple juice. 4. Conclusion In the present work, pectinase from Aspergillus aculeatus was successfully immobilized on Alginate-MMT beads via covalent
Table 3 Clarification of pineapple juices by free and immobilized pectinase.
Fig. 7. Reusability of pectinase immobilized on alginate-MMT beads.
Time (h)
Control (%T650)
Free enzyme (%T650)
Immobilized enzyme (%T650)
0.5 1 1.5 2 2.5 3
51.8 ± 0.3b 52.0 ± 0.32b 52.6 ± 0.29b 52.2 ± 0.3b 51.7 ± 0.31b 51.5 ± 0.33c
88.0 ± 0.31a 90.0 ± 0.27a 91.8 ± 0.2a 93.2 ± 0.25a 90.8 ± 0.3a 88.0 ± 0.3b
85.8 ± 0.2a 88.2 ± 0.25a 90.0 ± 0.3a 93.1 ± 0.27a 93.8 ± 0.31a 95.0 ± 0.2a
Values correspond to the average of three tests. Different letters (a–c) indicate significant differences (p b 0.05).
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Table 4 The physicochemical indexes of pineapple juice before and after clarification. Physicochemical indexes
Pineapple juice
Pectinase treatment
Immobilized pectinase treatment
pH Acid value (%) Total sugar (mg·mL) Reducing sugar (mg·mL) Color (420 nm) Viscosity (Pa·s) Pectin
3.40 ± 0.02a 0.520 ± 0.003a 15.600 ± 0.03a 9.732 ± 0.04b 0.435 ± 0.009a 1.750 ± 0.003a Have
3.1 ± 0.03b 0.521 ± 0.002a 15.325 ± 0.03a 11.620 ± 0.02a 0.100 ± 0.008b 1.065 ± 0.004b None
3.09 ± 0.03b 0.522 ± 0.001a 15.535 ± 0.04a 11.750 ± 0.03a 0.085 ± 0.007b 1.050 ± 0.003b None
Values correspond to the average of three tests. Different letters (a–b) indicate significant differences (p b 0.05).
immobilization. The immobilized pectinase showed higher activity compared to the free enzyme in extreme pH conditions. Also, the Km value showed that the affinity of enzyme to the substrate increased after the immobilization process. The immobilized pectinase showed enhanced stability and reusability for pectin hydrolysis, retaining more than 53% of activity after 6 consecutive cycles. Preliminary study showed that the immobilized pectinate could be used to clarify pineapple juice, demonstrating its potential for fruit juice processing in the food industry. Declaration of Competing Interest The authors have no conflicts of interest to declare. Acknowledgment The authors would like to acknowledge from Drug Applied Research Center, Tabriz University of Medical Sciences for financial support. References [1] U.V. Sojitra, S.S. Nadar, V.K. Rathod, Immobilization of pectinase onto chitosan magnetic nanoparticles by macromolecular cross-linker, Carbohydr. Polym. 157 (2017) 677–685. [2] U.V. Sojitra, S.S. Nadar, V.K. Rathod, A magnetic tri-enzyme nanobiocatalyst for fruit juice clarification, Food Chem. 213 (2016) 296–305. [3] A.A. Kadam, J. Jang, S.C. Jee, J.S. Sung, D.S. Lee, Chitosan-functionalized supermagnetic halloysite nanotubes for covalent laccase immobilization, Carbohydr. Polym. 194 (2018) 208–216. [4] I. Benucci, K. Liburdi, I. Cacciotti, C. Lombardelli, M. Zappino, F. Nanni, M. Esti, Chitosan/clay nanocomposite films as supports for enzyme immobilization: an innovative green approach for winemaking applications, Food Hydrocoll. 74 (2018) 124–131. [5] X.Y. Dai, L.M. Kong, X.L. Wang, Q. Zhu, K. Chen, T. Zhou, Preparation, characterization and catalytic behavior of pectinase covalently immobilized onto sodium alginate/ graphene oxide composite beads, Food Chem. 253 (2018) 185–193. [6] H.U. Rehman, M.A. Nawaz, A. Aman, A.H. Baloch, S.A.U. Qader, Immobilization of pectinase from Bacillus licheniformis KIBGE-IB21 on chitosan beads for continuous degradation of pectin polymers, Biocatal. Agric. Biotechnol. 3 (4) (2014) 282–287. [7] W. Sheng, Y. Xi, L. Zhang, T. Ye, X. Zhao, Enhanced activity and stability of papain by covalent immobilization on porous magnetic nanoparticles, Int. J. Biol. Macromol. 114 (2018) 143–148. [8] B. Wang, F. Cheng, Y. Lu, W. Ge, M. Zhang, B. Yue, Immobilization of pectinase from Penicillium oxalicum F67 onto magnetic cornstarch microspheres: characterization and application in juice production, J. Mol. Catal. B Enzym. 97 (2013) 137–143. [9] D. Alagöz, S.S. Tükel, D. Yildirim, Immobilization of pectinase on silica-based supports: impacts of particle size and spacer arm on the activity, Int. J. Biol. Macromol. 87 (2016) 426–432. [10] Z. Lei, S. Bi, H. Yang, Chitosan-tethered the silica particle from a layer-by-layer approach for pectinase immobilization, Food Chem. 104 (2) (2007) 577–584. [11] H.U. Rehman, A. Aman, A. Silipo, S.A.U. Qader, A. Molinaro, A. Ansari, Degradation of complex carbohydrate: immobilization of pectinase from Bacillus licheniformis KIBGE-IB21 using calcium alginate as a support, Food Chem. 139 (1–4) (2013) 1081–1086. [12] L. Mosafa, M. Shahedi, M. Moghadam, Magnetite nanoparticles immobilized pectinase: preparation, characterization and application for the fruit juices clarification, J. Chin. Chem. Soc. 61 (3) (2014) 329–336. [13] H.L. Ramirez, L. Gõmez Brizuela, J. Úbeda Iranzo, M. Arevalo-Villena, A.I. Briones Pérez, Pectinase immobilization on a chitosan-coated chitin support, J. Food Process Eng. 39 (1) (2016) 97–104. [14] K. Liu, G. Zhao, B. He, L. Chen, L. Huang, Immobilization of pectinase and lipase on macroporous resin coated with chitosan for treatment of whitewater from papermaking, Bioresour. Technol. 123 (2012) 616–619.
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