Amino functionalization of carboxymethyl cellulose for ...

International Journal of Biological Macromolecules 114 (2018) 1018–1025

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Amino functionalization of carboxymethyl cellulose for efficient immobilization of urease Fatma S. Alatawi a, M. Monier b,⁎, Nadia H. Elsayed c,d a

Biochemistry Department, Faculty of Science, University of Tabuk, Tabuk 71421, Saudi Arabia Chemistry Department, Faculty of Science, Mansoura University, Mansoura, Egypt Department of Chemistry, Faculty of Science, University of Tabuk, Tabuk 71421, KSA d Department of Polymers and Pigments, National Research Center, Dokki, Cairo 12311, Egypt b c

a r t i c l e

i n f o

Article history: Received 26 December 2017 Received in revised form 14 March 2018 Accepted 22 March 2018 Available online 24 March 2018 Keywords: Carboxymethyl cellulose Polyacrylamide Hoffmann degradation Urease

a b s t r a c t Jack bean urease (EC.3.5.1.5) was effectively immobilized onto amino functionalized epichlorohydrin crosslinked carboxymethyl cellulose (ECH-CMC) beads that were fabricated by graft co-polymerization of polyacrylamide (PAm) onto ECH-CMC beads in presence of potassium persulfate (KPS)/thiourea (TU) combined redox initiator followed by Hoffmann degradation. The progress of the synthesis along with immobilization processes was investigated by FTIR spectra. Also, the morphological structures of the beads before and after urease immobilizations were examined using SEM. Immobilization efficiency and immobilized urease amounts were estimated as a function of the amino functionalization degrees. The effects of pH and temperature on urease activity were studied. The results showed that after immobilization the optimum pH and temperature displayed higher shifts to 8 and 45 °C, respectively, which reveal a higher structural stability upon immobilization performance. Moreover, the kinetic studies indicated that the urea hydrolysis reaction, which catalyzed by urease enzyme displayed a good fit with Michaelis–Menten equation, and the kinetic parameters Km and vm were estimated to be 14 ± 0.7 mM and 2 ± 0.2 μmol NH3/min·mg immobilized urease, respectively. Furthermore, the immobilized urease maintained approximately 88% of its initial activity after the 10th reuse cycle. © 2018 Elsevier B.V. All rights reserved.

1. Introduction In various pharmaceutical, biomedical and biological applications that incorporate biochemical reactions, enzymes play a vital role in controlling the overall processes [1,2]. Usually, sub-ambient conditions are strongly needed during storage and manufacturing of enzymes in order to avoid drastic stereochemical and conformational alternations that are likely to occur within the structure of the enzyme protein if exposed to imperfect conditions over long periods of time. Thus, maintaining the protein-enzyme structure at room temperature is considered a major challenge in the fields of enzymes-based biomedical and pharmaceutical industries, which necessitated the urgent need to search for new techniques and methods to improve the enzyme stability [3–5]. For many years, enzyme immobilization has been considered as one of the most significant techniques in the fields of biotechnology and enzyme-based biomedical industries due to its advantages such as storage

⁎ Corresponding author at: Chemistry Department, Faculty of Science, Mansoura University, 35516, Egypt. E-mail addresses: [email protected], [email protected], (M. Monier), [email protected]. (N.H. Elsayed).

https://doi.org/10.1016/j.ijbiomac.2018.03.142 0141-8130/© 2018 Elsevier B.V. All rights reserved.

stability and reusability, which of course doesn't compare with the free enzymes [6,7]. Different common techniques such as adsorption, entrapment and covalent binding are extensively employed for immobilization of various types of enzymes onto the appropriate solid support materials [8,9]. However, as a result of the strong irreversible bond formation between the enzyme molecules and the solid support material, covalent binding provides a considerably effective and durable immobilization method [10]. Besides the great importance of urea in fertilizers manufacturing and agricultural industries, it also has a significant biological and biomedical role as the main product of protein metabolism and association with urinary tract and kidney diseases [11]. The chemical degradation of urea into carbon dioxide and ammonia is considered of a great importance for patients in renal failure [12–14]. This process could be catalyzed using the nickel-containing enzyme urease (urea aminohydrolase, EC 3.5.1.5) and that is why immobilized urease is usually utilized in hemodialysis machine for blood cleaning [15,16]. In many previous studies, a number of polymeric supports derived from polysaccharides such as cellulose, chitosan, and alginate were prepared and employed for immobilization of different enzymes [16–22]. Among these polysaccharides, carboxymethyl cellulose (CMC) is a

F.S. Alatawi et al. / International Journal of Biological Macromolecules 114 (2018) 1018–1025

water-soluble cellulose derivative, which is not only employed as an additive in both pharmaceutical and food industries but also implemented in fabrication of various support materials for efficient immobilization of several enzymes such as lactase, α-amylase, isoamylase, polyphenol oxidase and Lipase [23–29]. Here in this work, amino-functionalized cross-linked CMC beads were developed and employed as a polymeric support for covalent immobilization of urease. The chemical structure of the functionalized CMC beads before and after urease immobilization was investigated using FTIR spectra and SEM. Also, the immobilization efficiency and retained activity were investigated as a function of the essential parameters such as functionalization degree, temperature, and pH. 2. Materials and methods 2.1. Materials Carboxymethyl cellulose sodium salt (CMC) (MW = 200 kDa, and 90% substitution degree), epichlorohydrin (ECH), acrylamide (Am), potassium persulfate (KPS), thiourea (TU), glutaraldehyde and urease (Jack bean Canavalia ensiformis, MW 480 kDa, ∼60 U/mg) were purchased from Sigma Aldrich. All other chemicals and reagents were used as received. 2.2. Preparation of ECH cross-linked CMC (ECH-CMC) beads The ECH cross-linked CMC microsphere beads were prepared in accordance with previous reports [30,31]. Briefly, 1 g of sodium CMC was mixed with 15 mL 10% (w/v) sodium hydroxide solution and stirred continuously till complete dissolution. In a separate flask, the dispersion medium, which composed of 60 mL liquid paraffin, 0.5 g Spain 80, 1 mL carbon tetrachloride and 2 mL butyl alcohol were mixed and stirred till homogeneous mixture was obtained. Both CMC solution and dispersion medium were mixed in a reactor with stirring at 700 rpm. After 10 min and during the stirring, 2 mL of the cross-linker ECH was injected with the reaction mixture and stirring was continued for 12 h at about 30 °C. The obtained microsphere beads were then filtered and rinsed with ethanol, acetone and distilled water before finally collected in Petri dish and storing in an oven at 40 °C for 24 h. 2.3. Graft copolymerization of polyacrylamide (PAm) onto ECH-CMC The graft copolymerization was carried out via free radical initiation under a nitrogen atmosphere. Table 1 collects the formulation codes and Am amounts that were utilized for the preparation of different ECHCMC grafted PAm (ECM-g-PAm) samples with various grafting percentages (GP) values. ECH-CMC microsphere beads (1 g) were mixed with 50 mL double distilled water under moderate nitrogen bubbling rate (~10 cm3/min) with constant stirring. 5 mL acidified aqueous solution containing KPS/TU combined redox initiator (21 mM) was injected with the reaction mixture and stirring continued for 10 min at approximately 50 °C. 10 mL Am aqueous solution with definite concentrations was added to the reaction mixture, and the reaction continued for approximately 3 h. The grafted ECM-g-PAm beads with different GP values were separated from the reaction medium then rinsed with ethanol and distilled water to extract the PAm homo-polymer and finally dried in an oven at 40 °C. GP values were estimated according to the following Eq. (1) [16].

Grafting percentage ðGP Þ ¼

A−B 100 B

1019

2.4. Amino functionalization of the grafted ECM-g-PAm beads The amino functionalization of the grafted ECM-g-PAm samples was carried out using Hofmann degradation in accordance with previous articles [32,33]. In the beginning, the grafted ECM-g-PAm beads (1 g) were allowed to swell by immersing in distilled water for approximately 12 h. Then 20 mL 15% (wt/v) sodium hydroxide solution was added, and the reaction flask was cooled in ice bath for 30 min before adding 5 mL NaOCl aqueous solution. The reaction was later completed under magnetic stirring in the ice bath for 12 h. Back acid-base titration was utilized for estimation of the amination degree as explained in the following. Upon compellation of the degradation reaction, a definite weight of each of the obtained beads was immersed in a known volume of standard HCl solution for 6 h. Then the beads were extracted, and the supernatant HCl solution was titrated against standard sodium hydroxide solution to estimate the residual unreacted HCl content. 2.5. Immobilization of urease The previously prepared amino-functionalized beads (CM) with different amination degrees were first activated by stirring 1 g of the desired beads in 30 mL glutaraldehyde solution (10% (v/v)) for approximately 5 h. The activated beads were then extracted and rinsed with double distilled water. For urease immobilization, 20 mL aqueous urease enzyme solutions with concentration ranged between 1 and 5 mg/mL were prepared in a phosphate buffer adjusted at pH 7.0 and temperature 5 °C. The activated CM beads were then added to the urease solution with definite concentration, and the mixture was equilibrated on a shaker at 5 °C. After 10 h, the beads loaded with the urease enzyme were removed and rinsed with distilled water. The chemical modifications of the cross-linked CMC beads along with activation and urease covalent couplings are schematically displayed in Scheme 1. In order to estimate the amount of urease immobilized on the modified CM beads and immobilization efficiency, modified Pierce BCA protein assay kit (ThermoFisher Scientific) was employed for quantitative measurement of urease protein concentration within initial urease solution and supernatant solution. The immobilized urease amount (mg/g) and immobilization efficiency were calculated by Eqs. (2) and (3) [2,34]. Immoblized urease amount ðmg=gÞ ¼

ðmi −ms Þ W

ð2Þ

where mi and ms are the amounts of urease protein in both initial solution and supernatant washing solution, respectively. W is the weight of the beads. Immobilized efficiency ð%Þ ¼

mi −ms mi

100

ð3Þ

2.6. Urease activity assay As previously reported by Zhou et al. [34], urease activity was investigated by studying its capability to stimulate the urea hydrolysis according to the following reaction:

ð1Þ

where A and B are the mass of ECM-g-PAm and ECH-CMC respectively. The three prepared ECM-g-PAm samples with GP values 55, 96 and 147% were identified as CM1, CM2, and CM3, respectively.

The working solution in case of free urease composed of 3 mL 0.1 M urea solution mixed with 3 mL, 0.3 mg/L aqueous urease solution at pH 7. For Immobilized enzyme, the studied immobilized urease CM

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2.8. Effects of pH and temperature

Table 1 Synthesis of ECM-g-PAm under various conditions. Formulation code

Am (mol/L)

GP (%)

Amination degree (mmol g−1)

CM1 CM2 CM3

0.75 1.50 2.25

55 96 147

4 ± 0.4 6 ± 0.7 8±1

beads were mixed with 3 mL 0.1 M urea solution and to which 3 mL phosphate buffer solution at pH 7 was added. The enzymatic hydrolysis of the urea substrate was performed for 5 min, and then Nessler's reagent was added to determine the amount of liberated ammonia spectrophotometrically (Perkin–Elmer Bio UV–visible) at 460 nm. As previously reported, one unit of urease activity (U) catalyzes the release of 1 μmol ammonia/min under assay conditions [6,34,35]. Eq. (4) was employed to determine the relative activity [33].

2.9. Kinetic studies The kinetic parameters of the urease-catalyzed degradation of urea were estimated according to Lineweaver–Burk technique and Michaelis–Menten kinetics Eq. (5) [10].

1 Km 1 1 ¼ þ v0 vmax ½S vmax

ð5Þ

Relative activity ð%Þ ¼

Activity 100 Maximum activity

The activities of both free and immobilized urease were examined under different pH values by adjusting the pH of the working solutions using suitable phosphate buffers with pH values ranged between 4 and 9. Moreover, in order to investigate the influence of the temperature, the urease activity assay for both free and immobilized urease was performed at pH 7 under different temperatures changed from 5 to 60 °C.

ð4Þ

The maximum activity is considered 100% in case of either free or immobilized urease experiments.

where vo is the rate of urea hydrolysis, [S] is the molar concentration of urea, vmax is the maximum reaction rate, and Km is the Michaelis constant (mol/ L). For this purpose, the enzymatic hydrolysis of urea was performed at different initial substrate urea concentrations changed between 0.002 and 0.1 M in presence of both free and immobilized urease.

2.7. Characterization 2.10. Reusability and thermal stability The attenuated total reflection Fourier transform infrared spectra (ATR-FTIR) (Perkin–Elmer, USA) was utilized to investigate all modification steps that had been performed on the CMC starting from ECH cross-linking passing with PAm grafting and Hoffmann degradation ending with the urease coupling via covalent immobilization. The morphological appearance of the prepared ECH-CMC, ECM-gPAm before and upon Hoffmann degradation as well as the urease immobilized beads were all visualized using scanning electron microscope (SEM) (FEI Company, The Netherlands). The studied beads were coated with a thin layer of gold, and imaging was performed by 20 kV potentials with magnification range 1000–20,000.

In order to examine the reusability of the immobilized urease, the beads loaded with urease were implemented in the working solution, and the reaction was performed at pH 7 under the optimum temperature for 10 min. Then the beads were extracted, rinsed in the phosphate buffer and reused again in a new working solution. This process was repeated for 10 times and after each time the activity was determined. In addition, the thermal stability of both free and immobilized urease was examined by storing both types at room temperature for one week. Within predetermined time intervals, a sample was taken to measure the retained activities.

Scheme 1. Immobilization of urease.


1021

3. Results and discussions 3.1. Characterization

Fig. 1. FT-IR spectra of ECH-CMC (a), ECM-g-PAm (CM2) (GP = 96%) (b), CM2 after Hofmann degradation reaction (c), and urease immobilized beads (d).

FTIR spectra of the cross-linked ECH-CMC beads, ECM-g-PAm before and after amination via Hoffmann's degradation as well as the urease immobilized active beads are displayed in Fig. 1. The spectrum of the ECH-CMC beads (Fig. 1a) displayed the well-known CMC peaks at 1420 and 1610 cm−1 that are related to symmetric and asymmetric stretching vibrations of the carboxylate anion, respectively. In addition, the observed bands at 2930 and 3430 cm−1 could be assigned to the aliphatic C\\H and O\\H bonds, respectively [36,37]. Moreover, the observed peaks at 1330 and 1260 cm−1 could be related to the stretching vibration of C\\O\\C and C\\C, which are formed during the interaction between the CMC and ECH cross-linker [37]. The spectrum of ECM-g-PAm (CM2) with GP = 96% (Fig. 1b) showed the appearance of a new characteristic band at approximately 1660 cm−1, which assigned to the amidic C_O stretching vibration of the grafted PAm chains. Upon the performance of the Hoffmann degradation, this amide C_O peaks exhibited an obvious intensity decrease (Fig. 1c), which indicate the successful amination reaction. Furthermore, the urease coupling onto the glutaraldehyde activated beads was revealed by the appearance of peaks at 3190, 1650 and 1608 cm−1 (Fig. 1d), which confirm the insertion of the protein macromolecules onto the modified polymeric support. The SEM images of the native cross-linked ECH-CMC, ECM-g-PAm (CM2) before and after Hoffmann's degradation along with the urease immobilized beads were presented in Fig. 2. As can be noticed, the surface morphology of the investigated beads exhibited obvious changes during the performance of each step. In the beginning, the cross-linked

Fig. 2. SEM of ECH-CMC (a), ECM-g-PAm (CM2) (GP = 96%) (b), CM2 after Hofmann degradation reaction (c), and urease immobilized beads (d).

1022


efficiency was obviously raised from 56 ± 1% to 96 ± 2% by increasing the GP values. This can be explained as a result of the high availability of active aldehyde units through which the urease macromolecules are able to link. Furthermore, the retained activity of the immobilized urease was significantly reduced from 89 ± 2% to 67 ± 1% by increasing the functionalization degree. These observations could be explained as in the following; by increasing the amount of immobilized urease on the support beads, the protein-protein interaction may takes place due to the overcrowding of the inserted protein macromolecules, which will subsequently limit the urease-urea interaction. Additionally, increasing the functionalization degree may cause multipoint attachment between the urease protein and the active aldehyde units on the activated beads support; this may slightly alter the conformation of the macromolecular structure of urease and consequently, decrease the retained activity. 3.4. Effect of pH and temperature Fig. 3. Urease immobilization on the ECM-g-PAm (from CM2).

ECH-CMC displayed a smooth regular surface (Fig. 2a), which became rough and irregular upon the insertion of the grafted PAm chains (Fig. 2b). After the amination reaction, the surface was slightly corroded as a result of the Hofmann degradation treatment (Fig. 2c) but the beads still maintain its integrity. Fig. 2d indicated a comparatively rough and porous morphology, which could be explained as a result of urease protein aggregation via covalent coupling with the active points created on the beads surface. 3.2. Urease immobilization The estimated immobilized urease amounts as well as the immobilization efficiency that calculated using Eqs. (1) and (2), respectively, had been investigated under different urease concentrations using CM2 beads and the results were presented in Fig. 3. As can be noticed, by increasing the urease concentration, the amount of immobilized urease was significantly increased while the immobilization efficiency was dramatically reduced. At low urease concentrations, the protein macromolecules will completely linked to the available active sites existed on the glutaraldehyde activated CM2 beads. By increasing the urease concentration throughout the immobilization solution, the amounts of the immobilized urease will gradually increase till all the active sites are fully occupied with the urease macromolecules. The excess urease will not bind to the beads and lost in the washing supernatant solution, which may illustrate the reason of immobilization efficiency lowering by increasing the urease concentration throughout the immobilization solution. The measurements indicated that 1 g of the investigated CM2 beads take up to 143 ± 3 mg urease. 3.3. Effect of functionalization degree The activation of the prepared ECM-g-PAm was performed through the conversion of the grafted amide groups into \\NH2 groups via Hoffmann's degradation followed by treatment with glutaraldehyde. Thus, we will account for the functionalization degree in terms of the GP values. The influence of the functionalization degree on the retained activity along with immobilization efficiency was studied, and the obtained results are collected in Table 2. As expected the immobilization

It is well-known that the immediate microenvironment can control the behavior of the enzyme protein macromolecule [33]. The immobilized enzymes use to exhibit its maximum activity at pH values, not the same as in case of free enzymes in solution. In case of the immobilized enzyme, the optimum pH usually depends upon the charge of the solid support that is very close to the enzyme active sites. Since enzyme activity is strongly influenced by the pH value, it is necessary to investigate the influence of varying the pH of the reaction medium on the activity of both free and immobilized enzyme. For these reasons, the hydrolysis of urea catalyzed by either free or immobilized urease was examined under pH range from 4 to 9 at 35 °C. As can be noticed in Fig. 4, free and immobilized urease (on CM2) displayed the optimum pH values at 7 and 8, respectively. These results revealed the variation of the H+ and OH− distribution within the immediate vicinity of the immobilized urease and bulk of the working solution considering the anionic nature of the CMC based polymeric support. Similar shifts were previously reported [33,38,39]. In order to account for the influence of temperature on the activity of both free and immobilized urease, the activity assay was performed under the temperature range from 5 to 60 °C and the results are displayed in Fig. 5. Both free and immobilized urease by CM2 displayed the highest activities at approximately 35 and 45 °C, respectively. Moreover, the immobilized urease exhibited a comparatively broader profile under high temperatures, which confirm the higher thermal stability. This can be explained as a result of the multi-point attachments that covalently link urease protein to the support beads, which without doubt will restrict the conformational alternations upon raising the temperature [10,33]. 3.5. Kinetic studies The well-known kinetic parameters related to an enzymatic reaction such as maximum rate (vm) and Michaelis constant (Km) can be evaluated by detecting the variation of the reaction rate under different substrate concentrations. The Km value can be taken as a measurement for the affinity between enzyme and substrate. Higher Km value reveals a low enzyme-substrate affinity. In addition, vm value indicates the theoretical maximum rate of the enzymatic reaction. Fig. 6a presents the variation of the initial rate of the urease-catalyzed urea hydrolysis reaction as a function of urea concentration using both free and immobilized

Table 2 Comparison of immobilization capacity and activity of urease onto modified ECM-g-PAm beads. Beads

GP (%)

Immobilized urease (mg/g)

I.E.%

Urease activity U g−1 beads

Activity retention %

CM1 CM2 CM3

55 96 147

80 ± 1.5 143 ± 3 212 ± 4.2

56 ± 1 73 ± 1.2 96 ± 2

4318 ± 3 6789 ± 5 8661 ± 6

89 ± 2 78 ± 1 67 ± 1


1023

Fig. 4. Effect of pH on urease activity.

urease. The linear plot of 1 / vo (μmol of NH3/min·mg urease)−1 vs 1 / [urease] (M−1) for free and immobilized urease by CM2 (Fig. 6b) confirm the fit with Michaelis–Menten kinetics and provided an accurate estimation of both Km and vm values. As can be noticed, the obtained Km value in case of immobilized urease is 14 ± 0.7 mM, which is comparatively higher than that of free urease (11 ± 0.5 mM). On the other hand, immobilized urease displayed a lower vm value (2 ± 0.2 μmol NH3/min·mg urease) compared to that obtained by free urease (5 ± 0.2 μmol NH3/min·mg urease). In previous studies concerning urease immobilization by various solid supports, the kinetic parameters followed the same trend observed in the current study [40]. In all cases, Km displayed an increase accompanied by vm decrease upon immobilization whatever the type of the used solid support. These observations can be illustrated due to the diffusion limitation that may retard the urea-urease interaction after fixation of the enzyme protein on the solid support matrix or the conformational alternations that usually accompanying the covalent bonding of the macromolecule protein enzyme onto the polymeric solid support via multi-point attachment, in addition to the fact that enzyme immobilization will limit the free movement of the enzyme in the working solution and consequently, delay the substrate accessibility to the enzyme active sites [10]. All the aforementioned reasons may reasonably explain the observed lowering of vm and elevation of Km. However, in the current study, the ratio between the Km values related both

Fig. 5. Effect of temperature on urease activity.

Fig. 6. (a) Variation of initial hydrolysis rate of urea versus urease concentration. (b) Lineweaver–Burk plot of free and immobilized urease.

free and immobilized urease was approximately 1.3, which reveals that the urease-urea affinity is just slightly affected upon immobilization. 3.6. Reusability and thermal stability The reusability studies of the immobilized urease by CM2 were performed, and the results were presented in Fig. 7. As can be

Fig. 7. Operating stability of immobilized urease.

1024


References

Fig. 8. Thermal stability of free and immobilized urease.

observed the activity was slowly decreased, and the immobilized urease maintained approximately 88% of its initial activity upon the compellation of the 10th reuse cycle. Actually, it is well known that enzyme immobilization, particularly through covalent bonding is capable of maintaining the conformation of the three-dimensional structure of the protein macromolecules from being damaged or even extremely altered during the repeated performance of the enzyme [10]. In addition, another vital issue that may affect the utility of enzyme application is the retained activity during storage. For this reason, the thermal stabilities of free and immobilized urease were examined as a function of storage time at room temperature, and the results are shown in Fig. 8. It is clearly obvious that urease immobilization onto the modified solid support beads has enhanced the thermal stability. After 7 days, the immobilized urease by CM2 maintained approximately 75% of its initial activity, which considered promising results compared to the free urease which almost lost its activity under the same condition. 4. Conclusion In this work, urease enzyme was successfully immobilized via covalent binding onto amino functionalized epichlorohydrin (ECH) crosslinked carboxymethyl cellulose (CMC) polymeric solid support with various functionalization degrees. In the beginning, ECH-CMC beads were modified through graft copolymerization of polyacrylamide then amino-functionalized via Hoffmann degradation reaction and finally activated by glutaraldehyde before the performance of urease immobilization. The modification steps and immobilization process were confirmed using FTIR and SEM. The beads with the highest degree of functionalization were loaded with 212 ± 4.2 mg urease per one gram beads and retain approximately 68% of the enzyme activity. The immobilized urease displayed a higher thermal stability at relatively high temperatures compared to that exhibited by the free enzyme. Also, Reusability and storage stability were significantly improved. The immobilized urease maintained 88% of its initial activity after the 10th reuse cycle, and the residual activity was around 75% after storage for one week at room temperature. Acknowledgment The authors would like to acknowledge University of Tabuk for the financial support under research project number S 1438-0150.

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