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Reversible immobilization of glucoamylase onto metal–ligand functionalized magnetic FeSBA-15
Biochemical Engineering Journal 68 (2012) 159–166
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Reversible immobilization of glucoamylase onto metal–ligand functionalized magnetic FeSBA-15 Guanghui Zhao a , Jianzhi Wang a , Yanfeng Li a,∗ , Huayu Huang b , Xia Chen a a State Key Laboratory of Applied Organic Chemistry, Institute of Biochemical Engineering & Environmental Technology, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China b Department of Science, Nanjing Agricultural University, Nanjing 210095, China
a r t i c l e
i n f o
Article history: Received 5 November 2011 Received in revised form 19 March 2012 Accepted 19 April 2012 Available online 25 April 2012 Keywords: Biocatalysis Affinity Immobilization Glucoamylase ATRP Regeneration
a b s t r a c t Magnetic SBA-15 (FeSBA-15) was prepared via wet impregnation, calcination and reduction, and p(glycidylmethacrylate) (PGMA) was grafted on the surface of FeSBA-15 using surface-initiated atom transfer radical polymerization (SI-ATRP) for a prescribed time. The epoxy groups of the PGMA were reacted with Cu(II) metal–ligand complex (i.e., imidazole or iminodiacetic acid) to form metal–chelate brush. Subsequently, the functionalized FeSBA-15 as a regenerated support was used for enzyme immobilization. Glucoamylase was immobilized as a model enzyme on the regenerated supports through metal-ion affinity interactions. The quality of glucoamylase immobilized on the regenerated supports is defined by determining of the enzyme activity, thermal stability, and reusability. The results indicate that the metal–chelate brushes offer an efficient route to immobilize enzymes via metal-ion affinity interactions. The applicability of the regenerated supports in the current study is relevant for the conjugation of other enzymes beyond glucoamylase. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Enzyme immobilization may permit fast separation of the enzyme from the reaction mixture, reuse of the enzyme, simplification of the reactor design, and improvement of their stability or tolerability [1]. Up to now, many different scaffolds and supports with a range of functionality, morphology, and physical properties have been studied for the immobilization of enzymes such as silica [2], polysaccharide [3], polymer nanofibers [4], hydrogels [5], and nanoparticles [6]. In particular, introduction of physical functionality, such as magnetic [7], thermosensitive [8], and regenerated properties [9], into nanomaterials is desirable for enzyme immobilization. Nonetheless, assembling multifunctional support nanomaterials are infrequently reported. Here our target is to synthesize nanomaterials for enzyme immobilization with combined magnetic and regenerated properties. To make this design feasible, a base material with a sophisticated structure is needed to carry the multifunctional entities. Mesoporous molecular sieves with longrange ordered porous structures, such as the SBA series, are ideal template materials for assembling these dual-targeting carriers and
∗ Corresponding author. Tel.: +86 931 8912528; fax: +86 931 8912113. E-mail addresses: [email protected], [email protected] (Y. Li). 1369-703X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2012.04.009
many reports have already described the use of these materials for enzyme immobilization [10]. Polymer brushes offer certain advantages over other materials as they are covalently anchored to the substrate providing excellent mechanical stability and present a high surface area template with functionality controllable by monomer type and brush length [11]. Among the various chemical functional groups that can be integrated into polymers, the epoxy group is particularly attractive because of their reactive pendant oxirane rings, which provides a large number of subsequent reactions, offering a variety of opportunity for chemical modification of the parent polymers [12]. Several methods have been developed to prepare polymercoatings on magnetite nanoparticles such as physical adsorption of polymers, emulsion polymerization in the presence of nanoparticles, and the so-called “grafting to” and “grafting from” methods [13]. Silane chemistry has been applied to metal oxide surfaces to promote metal–metal and metal–polymer adhesion. Previous literatures [14] have demonstrated the formation of polysiloxanes on the surface of magnetite via alkylalkoxylianes self-assembly. Atom transfer radical polymerization (ATRP) is a recently developed “living” or “controlled” radical polymerization method [15], which does not require stringent experimental conditions. Therefore, tethering of polymer brushes covalently onto a solid substrate by ATRP is a versatile and effective method of altering the surface properties of the material [16]. And surface-initiated ATRP allows
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the preparation of well-defined polymer brushed with dormant chain ends on various types of substrates [17]. More recently, Xu et al. [18] reported the covalent immobilization of glucose oxidase on well-defined PGMA–Si(111) hybrids via surface-initiated ATRP. This research aims at utilizing the controlled growth of polymer brushes on FeSBA-15 to develop regenerated supports with magnetic property. First, magnetic iron particles were impregnated into the SBA-15 cages by thermal decomposition, followed by reduction. Subsequently, the use of ATRP to grow PGMA from initiators bound to magnetic FeSBA-15 surface affords relatively fine control over polymer molecular weight, which allows large increases in capacity. PGMA brushes are particularly attractive because they can be functionalized to exploit a number of affinity interactions. Finally, functionalization of PGMA with imidazole (or iminodiacetic acid) results in enzyme binding via metal-ion affinity interactions. In order to compare the effectiveness of different functionalized brushes as scaffolds for enzymes, quantitative data on binding chemistry, kinetics of binding, and amount bound need to be examined in the context of the activity of immobilized enzyme.
The FeSBA-15–PGMA@Imidazole (or FeSBA-15–PGMA@IDA) support was activated by Cu2+ activation procedure. Cu2+ activation procedure is as followings: Cu(II) ion was chelated with the functional groups of the FeSBA-15–PGMA@Imidazole (or FeSBA-15–PGMA@IDA) support. A 5 mg mL−1 solution of Cu(II) ions was prepared from sulfate salts in distilled water. The FeSBA-15–PGMA@Imidazole (or FeSBA-15–PGMA@IDA) support was suspended in the Cu(II) ions solution at room temperature for 6 h. The support was taken out, washed several times with acetate buffer solution (50 mM, pH 5.5) and submerged into glucoamylase buffer solution (pH 5.5, 50 mM acetate buffer). The immobilization process was carried out at 30 ◦ C in a shaking air bath for 2 h. Finally, the support was taken out, thoroughly rinsed with acetate buffer (50 mM, pH 5.5). The amount of immobilized protein on the support was determined as described above. The amount of protein in the enzyme solution and in the washed solution was determined by the Bradford method using bovine serum albumin as a standard [19], and the amount of protein bound on the carriers was calculated from the formula:
2. Materials and methods
Protein bound (mg g−1 ) =
2.1. Enzymes and reagents Glucoamylase (exo-1, 4-a-d-glucosidase, EC3.2.1.3 from Aspergillus niger 10 U mg−1 ) was purchased from Yixing Enzyme Preparation Company (China); mesoporous SBA-15 silica molecular sieves were purchased from Changchun Jilin University High-Tech. Co. Ltd. (Jilin, China); glycidyl methacrylate (GMA) was obtained from Ciba Specialty Chemicals (China) Ltd., Guangzhou; 3-aminopropyltriethoxysilane (APTES) was purchased from Wuhan University Silicone New Material Co. Ltd. (China); 2bromo-2-methylpropionyl bromide was purchased from Aldrich. All other chemicals were of analytical grade, and used as received. 2.2. Preparation of metal–ligand functionalized magnetic FeSBA-15 First, magnetic iron particles were impregnated into the SBA-15 cages by thermal decomposition, followed by reduction. Subsequently, the use of ATRP to grow PGMA from initiators bound to magnetic FeSBA-15 surface affords relatively fine control over polymer molecular weight, which allows large increases in capacity. PGMA brushes are particularly attractive because they can be functionalized to exploit a number of affinity interactions. Finally, PGMA brushes are functionalized by imidazole or iminodiacetic acid. Details of the preparation were thoroughly described in supplementary material. 2.3. Enzyme immobilization Enzyme immobilizations on different functionalized FeSBA-15 were performed in following ways. Due to the active epoxy groups in the FeSBA-15–PGMA nanocomposites, glucoamylase immobilization was carried out by the treatment of the enzyme solution with the FeSBA-15–PGMA directly. 1.0 g of FeSBA-15–PGMA nanocomposites was equilibrated in 50 ml acetate buffer solution (50 mM, pH 5.5) for 24 h. It was then transferred to the same fresh buffer (50 ml) containing glucoamylase (0.1 g). The immobilization process was carried out at 30 ◦ C in a shaking air bath for 12 h. After this, the immobilized glucoamylase was recovered by magnetic separation, thoroughly rinsed with acetate buffer solution (50 mM, pH 5.5) two times to remove unbound glucoamylase. The washed solution was collected to assay the amount of residual enzyme. The resulting immobilized glucoamylase was held at 4 ◦ C prior to use.
(Ci − Cf )V W
(1)
where protein bound is the amount of bound enzyme onto superparamagnetic Fe3 O4 @Clays nanocomposites (mg g−1 ), Ci and Cf the concentrations of the enzyme protein initial and final in the reaction medium (mg mL−1 ), V the volume of the reaction medium (mL) and W is the weight of the carriers (g). All data used in this formula are the average of triplicate experiments. 2.4. Enzyme activity The reaction rate of the free and immobilized glucoamylase preparations was determined according to the method reported by Nelson [20], with only minor modification. In the standard conditions, using soluble starch as the substrate, which composed of 0.5 mL of 10 wt.% soluble starch gelatinized in water and 2.5 mL acetate buffer solution (50 mM, pH 5.5). The reaction was started by addition of 0.5 mL free glucoamylase (0.236 mg mL−1 ) or 0.1 g of immobilized glucoamylase. The mixture was incubated at 55 ◦ C under reciprocal agitation at 120 strokes per minute. After 15 min of reaction, agitation was stopped, and then the reaction was terminated by adding 5 mL of NaOH solution (0.1 M). The glucose content was determined using the DNS method [21]. The amount of glucose was obtained from the calibration curve and used in the calculation of enzyme activity. All activity measurement experiments were carried out three times, and the relative standard deviation is less than 1.0%. One unit of glucoamylase activity is defined as the amount of enzyme that produces 1.0 mmol of glucose from dissolubility starch per minute under the assay conditions. The relative activity (%) was the ratio between the activity of every sample and the maximum activity of sample. All experiments of activity measurement were carried out at least three times and the experimental error was less than 3%. 2.5. Characterization FT-IR spectra were obtained in transmission mode on a Fouriertransform infrared spectrophotometer (American Nicolet Corp. Model 170-SX) using the KBr pellet technique. Transmission electron microscopy (TEM, FEI Tecnai G20) was obtained to elucidate the dimensions and the structural details of the nanoparticles. TEM specimens were made by placing a drop of the nanoparticle suspension on a carbon-coated copper grid. Magnetization measurements were performed on a vibrating sample magnetometry (VSM, LAKESHORE-7304, USA) at room temperature. X-ray photoelectron
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Fig. 1. TEM images of superparamagnetic SBA-15 (a), FeSBA-15 (b) and FeSBA-15–PGMA (c).
spectroscopy (XPS) measurements were carried out on a VG ESCALAB 210 photoelectron spectrometer (VG Scientific Co.). 3. Results and discussion 3.1. Fabrication and characterization of metal–ligand functionalized magnetic FeSBA-15 There are a number of published papers on reversible enzyme immobilization, e.g., thiol-disulfide exchange of proteins containing surface thiol groups [22,23], immobilized metal chelate supports [24,25], and ionic exchange resins [26,27], but there are some problems arising in previous reports. The important problems were solved in this work: (1) magnetic FeSBA-15 mesoporous silica was chosen as the template because it is an ideal host material for magnetic nanoparticles large enough to possess a sufficient magnetization for magnetic separation. There is no researcher chose this nanomaterial for enzyme immobilization; (2) some researchers grafted PGMA on the outer surfaces of carrier for covalent immobilization of enzyme, but the carrier cannot be regenerated [24]. A number of researchers used monomer containing metal–ligand residues to fabricate regenerated carrier via free radical polymerization. However, the technology of free radical polymerization cannot control brush length of polymer, and the nanoparticles could be aggregated after free radical polymerization, which should affect the performance of immobilizing enzymes. Therefore, polymer brushes were covalently grated onto the surface of FeSBA-15 via ATRP in this study. And surface-initiated ATRP allows the preparation of well-defined polymer brushed with dormant chain ends on various types of substrates. However, the monomer containing metal–ligand residues could not be polymerized via ATRP technology. Therefore, firstly PGMA brushes are grated onto the surface of FeSBA-15 via ATRP in this study. Subsequently, PGMA brushes were functionalized by imidazole or iminodiacetic acid. Finally, functionalization of PGMA with imidazole (or iminodiacetic acid) results in enzyme binding via metal-ion affinity interactions. To our best knowledge, there is no similar study. 3.1.1. Preparation of magnetic FeSBA-15 The use of ordered mesoporous silica SBA-15 (Fig. 1a) as a template for the synthesis of iron oxide–silica nanocomposites has been demonstrated previously [28]. In this work, magnetic SBA15 mesoporous silica was chosen as the template because it is an ideal host material for magnetic nanoparticles large enough to possess a sufficient magnetization for magnetic separation. However, blockage may be caused in the 2D hexagonal porous structure of SBA-15 by impregnation of iron nanoparticles. This may limit the use of these composite materials for immobilization of enzymes
via the porous structure [29]. This can be overcome to some extent through the use of polymer chains grafted on the outer surfaces of magnetic FeSBA-15 and binding of multilayers of enzymes to these polymers. Magnetic FeSBA-15 was prepared via wet impregnation, calcination and reduction. As revealed by transmission electron microscopy (TEM), the obtained magnetite particles are deposited inside the inner cavities of SBA-15 (Fig. 1b). 3.1.2. Preparation of magnetic FeSBA-15–PGMA Diffusion limitation leads to slow replenishment of the consumed reactants and accumulation of the products around catalytic sites in the microspheric polymer materials, so that the efficiency of the biocatalyst is decreased. The immobilization enzymes on the outer surfaces of inorganic supports may overcome these disadvantages [30]. Despite their potential, the major disadvantage of inorganic supports is their low binding capacity relative to microspheric polymer materials [31]. Form this point of view, the use of polymer encapsulated the outer surfaces of inorganic supports and binding of multilayer of enzyme only on the surface of these polymers microsphere, that can reduce the diffusion limitation problem of immobilized enzyme system [32]. The chemical process to fabricate the supporting material is thoroughly described in the experimental section, and it is visually summarized schematically in Fig. 2. Upon silanization, the existence of amine group is more convincingly represented by the C–N, N–H signal (1334 cm−1 and 1564 cm−1 ) from the corresponding IR spectra (Fig. 3b). Prior to polymerization, the initiator must be immobilized to the surface. The ATRP initiator on the FeSBA-15 surface was prepared via amidation of amino-FeSBA-15 with 2bromoisobutyryl bromide. The conversion of the amino groups was estimated to be ∼31% based on the weight increase of the particles. The reaction of amino groups with 2-bromoisobutyryl bromide is also an efficient reaction with high conversion [33]. Therefore almost all the amino groups in the surface layer should be converted to Br-containing groups. In this work, the method of addition of Cu(II) complex (CuBr2 ) was chosen to control the concentration of the deactivating Cu(II) complex during the surface-initiated ATRP process on the FeSBA-15–Br surface. A study of thickness changes with time as determined by TEM demonstrated that surface-initiated polymerization only occurred on surfaces with the initiators present. TEM images show the FeSBA-15–PGMA arrays at an ATRP time of 5 h, where 20–30 nm coating is visible on the surface of FeSBA15–PGMA. For PGMA, this layer is on average 25 nm thick (Fig. 1c). 3.1.3. Functionalization of nanostructured FeSBA-15–PGMA An important feature of the FeSBA-15–PGMA surface is the preservation of the reactive epoxide groups during the ATRP
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Fig. 2. Schematic illustration of the synthesis and the chemistry used for the immobilization of glucoamylase on three brush systems.
process. In principle, all nucleophilic groups, such as, NH2 , SH, OH, and COOH groups, will react readily and irreversibly with the epoxy groups in subsequent surface functionalization [24,34]. Thus, the reaction between the epoxy group of GMA units and the imino group of imidazole (or IDA) was used to introduce the imidazole (or IDA) on the PGMA brushes, as shown in Fig. 2. The FT-IR spectra (Fig. 3d and e) of FeSBA-15–PGMA@Imidazole and FeSBA15–PGMA@IDA indicated that the modification of FeSBA-15–PGMA with imidazole (or IDA) was successful.
3.2. Immobilization of glucoamylase onto PGMA, imidazole and IDA brush Adsorption through ion-exchange, hydrogen bonding and hydrophobic interaction might be relatively a mild protein immobilization method, since the non-covalent bonding has been widely applied in protein separation and refolding processes. It was also well known that multipoint interaction may serve to enhance the structural stability of enzymes. The glucoamylase was immobilized
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Fig. 3. The IR spectra of FeSBA-15 (a), FeSBA-15–NH2 (b), FeSBA-15–PGMA (c), FeSBA-15–PGMA@Imidazole (d) and FeSBA-15–PGMA@IDA (e).
via covalent or non-covalent interactions on PGMA, imidazole and IDA brush. The FeSBA-15–PGMA with the ATRP reaction time 5 h exhibits an optimal selectivity equivalent for the immobilization of glucoamylase and is thus used for all immobilization experiments (data not shown). In order to achieve high biological activities or binding capacities of the immobilized glucoamylase, three different immobilization strategies which were shown schematically in Fig. 2 were discussed in the current study. In the immobilization strategy I, glucoamylase was immobilized on the FeSBA-15–PGMA via epoxy groups, which are inherently present in the support after polymerization. Taking advantage of metal–chelate properties, in the immobilization strategy II and III, Cu2+ ions was coordinated to the imidazole (or IDA) ligand and the enzyme was bound FeSBA15–PGMA@Imidazole and FeSBA-15–PGMA@IDA via Cu2+ ions. In the immobilization strategy I, the theoretical content of epoxy groups, calculated on the basis of the PGMA content in the FeSBA-15–PGMA was 5.21 mmol g−1 . The content of epoxy groups on the surface of FeSBA-15–PGMA sample determined by the pyridine–HCl method [35], and differs from the theoretical value (3.72 mmol g−1 ). Because some of the epoxy groups usually remain in bulk structure of FeSBA-15–PGMA, it is not accessible for subsequent reactions or for analytical determinations. FeSBA-15–PGMA with active epoxy groups were prepared and used for glucoamylase immobilization by covalent reaction via the amino groups of the glucoamylase and the epoxy groups of FeSBA-15–PGMA under mild condition (Fig. 2). The amounts of bound glucoamylase are 10.6 mg g−1 on FeSBA-15–PGMA. In the immobilizing strategy II and III, the amount of bound glucoamylase is related to the density of ligands on the surface of the FeSBA-15–PGMA. A high density of chelator provides high capacity and therefore high load ability. The content of functional groups on the FeSBA-15–PGMA was examined by elemental analysis (Vario EL III, German) as shown in Table 1. The imidazole groups content of FeSBA-15–PGMA@Imidazole reaches 2.46 mmol g−1 , which is higher than IDA groups content of FeSBA15–PGMA@IDA (0.96 mmol g−1 ). To evaluate the efficiency of immobilization via imidazole, IDA groups in comparison with that using epoxy groups, three types of brushes were used for immobilization of glucoamylase. The binding capacity of glucoamylase immobilized via imidazole groups was found to be twice as high as the capacity of this enzyme bound using epoxy groups (Table 1).
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Several reports described new supports for glucoamylase immobilization [36–39], and the amount of the immobilized enzyme to 1.0 g of supports was 4.95–20.6 mg. In the present work, the capacity of the former was approximately 21.6 mg g−1 while it was 10.6 mg g−1 for the epoxy groups and for 17.3 mg g−1 the IDA groups. All these immobilization studies published in the literature have been performed under different conditions and different organism was used for glucoamylase source. Therefore, it is almost impossible to compare these immobilization results. Interestingly, glucoamylase immobilized on FeSBA-15–PGMA (epoxy groups) for 12 h exhibited only 52.8% of activity compared to that achieved only after 60 min using FeSBA-15–PGMA@Imidazole (imidazole groups), and 75.6% of activity compared to that achieved only after 60 min using FeSBA-15–PGMA@IDA (IDA groups) (data not shown). The results confirmed that immobilization via imidazole or IDA groups are faster than that using epoxy group and other research reported before in the literature: 2.5 h [40], and 9 h [41]. Compared with these supports, the FeSBA-15–PGMA@Imidazole (imidazole groups) prepared in this study had a fast immobilization progress, such a short the immobilization time showed that there are two advantages to FeSBA-15–PGMA@Imidazole (imidazole groups) used for glucoamylase immobilization: (1) the shorter the immobilization time is, the less activity loss is. (2) The shorter the immobilization time is, the lower cost is [42]. The results can be explained by the fact that all these functional groups (imidazole or IDA groups) are exposed at the FeSBA-15 surface because of their hydrophilic character, while most of the epoxides are buried within the polymer and inaccessible for immobilization. Hence, FeSBA-15–PGMA@Imidazole having imidazole groups is an excellent carrier for immobilization of enzymes. 3.3. Effect of pH and temperature on the catalytic activity The change in optimum pH depends on the charge of the enzyme and/or of the water insoluble matrix. This change is useful in understanding the structure–function relationship of enzyme and to compare the activity of free and immobilized enzyme as a function of pH. The effect of pH on the activities of free and immobilized glucoamylase samples was investigated in the pH range of 1.5–8 at 55 ◦ C, and the results are given in Fig. 4. It was observed that the optimum pH of free enzyme was 3.5, while the operable pH for the immobilized glucoamylase was extended between pH 2.5 and 5.5. This shift may depend on the immobilization method as well as the basic character of the support material. Furthermore, the pH profiles of the immobilized glucoamylase preparations are broader than that of the free enzyme (Fig. 4), which meant that the immobilization methods preserved the enzyme activity in a wider pH range. Interestingly, the glucoamylase immobilized on FeSBA15–PGMA@Imidazole and FeSBA-15–PGMA@IDA supports exhibit higher activity than other supports [43] under the acidic condition (pH = 1.5), which could be explained by the weak-base of imidazole and ionized IDA groups, protected it from unfolding and prevented the conformation transition of the enzyme at acidic condition. Effect of temperature on the relative activity of free and immobilized glucoamylase is shown in Fig. 4. The immobilized enzyme showed an optimum reaction temperature between 55 and 65 ◦ C, that is near the literature value of 65 ◦ C [43], whereas free enzyme had an optimum temperature about 55 ◦ C. These results could probably be attributed to the stabilization of glucoamylase molecules resulting from multipoint attachment of the enzyme molecules on the surface of the FeSBA-15 by covalent or chelate bonds. The multipoint bond formation between support and glucoamylase molecules could reduce the conformational flexibility and may result in higher activation energy for the molecule to reorganize the proper conformation for the binding to substrate [44,45]. In addition, the results suggested that the immobilized glucoamylase
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Table 1 Content of the element N, functional group and protein loading capacity. Content
FeSBA-15–PGMA
FeSBA-15–PGMA@Imidazole
N content (mmol g−1 ) Functional group content (mmol g−1 ) Theoretical content (mmol g−1 ) Protein loading (mg g−1 )
0.26 3.72 (Epoxy groups) 5.21 (Epoxy groups) 10.6
5.19 2.46 (Imidazole functional group) 5.21 (Imidazole functional group) 21.6
FeSBA-15–PGMA@IDA 1.22 0.96 (IDA functional group) 5.21 (IDA functional group) 17.3
Fig. 5. Thermal stabilities of glucoamylase immobilized on the three supports in comparison to free glucoamylase.
Fig. 4. Effect of temperature and pH on enzyme activities.
possessed better pH and temperature stability than the free one. Similar behavior has been also observed in the case of an immobilized d-hydantoinase as well as an immobilized glucoamylase [46,47]. 3.4. Stability and reusability of immobilized glucoamylase The stability of an enzyme is critical to its practical applications. Fig. 5 shows the stability of glucoamylase immobilized on FeSBA-15–PGMA, FeSBA-15–PGMA@Imidazole and FeSBA15–PGMA@IDA supports over 6 h of testing at 57 ◦ C in acetate buffer solution (50 mM, pH 5.5). The preparations exhibited a similar trend, whereas the immobilized glucoamylase decreased less and more slowly than the free one. The catalytic activity of immobilized glucoamylase retained their initial activity of about 65–68% after 6 h. The lower stability of glucoamylase had also been reported by Carpio et al. [48], the catalytic activity of immobilized glucoamylase only retained their initial activity of about 40–50% after 6 h. In sharp contrast, the free glucoamylase only retained their initial
activity of about 43% after 6 h in this study. These results demonstrated that the thermal stability of immobilized glucoamylase was much better than the free one. This could be explained by the carriers enhancing the enzyme rigidity, protected it from unfolding and prevented the conformation transition of the enzyme at high temperature. These results indicated that the immobilization procedures had considerably improved the thermal stability. It is important to investigate the performance of immobilized enzymes during recycled use for potential industrial applications. In the study, glucoamylases immobilized on FeSBA-15–PGMA, FeSBA-15–PGMA@Imidazole and FeSBA-15–PGMA@IDA supports were recycled for the hydrolyzation of starch. The reaction was performed for 15 min at 55 ◦ C, after which the spent immobilized glucoamylases were recovered by magnetic separation, washed three times with acetate buffer solution (50 mM, pH 5.5), and then used again for a fresh reaction. The assay conditions remained the same as described above. The variation of activity of the immobilized glucoamylase after multiple reuses is showed in Fig. 6. It was observed that the residual activity of the immobilized enzymes is 67.14% for FeSBA-15–PGMA, 65.12% for FeSBA-15–PGMA@Imidazole and 67.84% for FeSBA-15–PGMA@IDA after the 10th reuse. Obviously, the results indicated that the immobilized glucoamylases significantly increases its operational stability. 3.5. The regeneration of supports The immobilized enzyme also could loss its activity completely after several times of reuse. Therefore, the regeneration of supports at the end of the life of the immobilized enzyme, which can effectively reduce the cost in industry applications, is very essential. In order to show the reusability of the functional FeSBA-15–PGMA supports; adsorption–desorption cycle of glucoamylase was repeated three times by using the same supports. The regeneration of supports in the report is carried out in three steps; firstly, desorptions of adsorbed glucoamylase from
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concluded that the functionalizable supports provide an economical, efficient and reversible system for enzyme immobilization. Acknowledgements The authors thank the financial supports from the National Natural Science Foundation of China (No. 21074049), the Opening Foundation of State Key Laboratory of Applied Organic Chemistry (SKLAOC-2009-35), the Fundamental Research Funds for the Central Universities (lzujbky-2012-75), and the Opening Foundation of State Key Laboratory of High Performance Civil Engineering Materials (2012–2013). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bej.2012.04.009. References
Fig. 6. Reuse of glucoamylase immobilized on the three supports.
FeSBA-15–PGMA@Imidazole and FeSBA-15–PGMA@IDA supports were carried out in a batch system. The immobilized enzyme preparations were placed within the desorption medium containing 1.0 M EDTA at room temperature for 5 h as described above and were then repeatedly used in adsorption/desorption cycle of glucoamylase. Subsequently, the supports were activated with Cu2+ and finally immobilization of the enzyme in a usual procedure is carried out (Fig. 2). Many works about reversible immobilization of glucoamylases on different carriers are cited in the literature. Bayramo˘glu et al. [49], prepared a nanofibrous polymer grafted magnetic poly(GMAMMA)-g-MAA beads for reversible immobilization of trypsin. After six-repeated adsorption–desorption cycles, the adsorption capacity of the magnetic beads was decreased 8%. Subsequently, this researcher team reversibly immobilized catalase on fibrous polymer grafted and metal chelated chitosan membrane. At the end of the sixth cycles, catalase immobilization capacity of the CH-gpoly(IA) membrane was decreased about 7% [50]. Erdem Yavuz reported poly(vinylbenzylchloride) beads grafted with polymer brushes carrying hydrazine ligand for reversible invertase immobilization. The sixth adsorption–desorption cycle of invertase, the amount of immobilized enzyme was about 3.6% lower than that of the first use [51]. In this study, the immobilization capacities of the regenerated supports did not significantly change during the following three regeneration cycles. These results showed that Cu2+ ions incorporated FeSBA-15–PGMA@Imidazole and FeSBA-15–PGMA@IDA supports can be repeatedly used in enzyme immobilization without detectable losses in their initial adsorption capacities.
4. Conclusions In summary, this work demonstrates an effective and convenient strategy to obtain functionalizable and metal–chelate surfaces for enzyme immobilization. Glucoamylase was found to interact with functionalizable supports more rapidly compared to FeSBA-15–PGMA to form the metal–chelate complex. The amount of glucoamylase uptake was found to be the highest for Cu2+ -doped FeSBA-15–PGMA@Imidazole, while FeSBA-15–PGMA exhibited the lowest amount of glucoamylase uptake. After regeneration of supports, the biocatalyst also reserved its original reusability. It can be
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Reversible immobilization of glucoamylase onto metal–ligand functionalized magnetic FeSBA-15 Guanghui Zhao a , Jianzhi Wang a , Yanfeng Li a,∗ , Huayu Huang b , Xia Chen a a State Key Laboratory of Applied Organic Chemistry, Institute of Biochemical Engineering & Environmental Technology, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China b Department of Science, Nanjing Agricultural University, Nanjing 210095, China
a r t i c l e
i n f o
Article history: Received 5 November 2011 Received in revised form 19 March 2012 Accepted 19 April 2012 Available online 25 April 2012 Keywords: Biocatalysis Affinity Immobilization Glucoamylase ATRP Regeneration
a b s t r a c t Magnetic SBA-15 (FeSBA-15) was prepared via wet impregnation, calcination and reduction, and p(glycidylmethacrylate) (PGMA) was grafted on the surface of FeSBA-15 using surface-initiated atom transfer radical polymerization (SI-ATRP) for a prescribed time. The epoxy groups of the PGMA were reacted with Cu(II) metal–ligand complex (i.e., imidazole or iminodiacetic acid) to form metal–chelate brush. Subsequently, the functionalized FeSBA-15 as a regenerated support was used for enzyme immobilization. Glucoamylase was immobilized as a model enzyme on the regenerated supports through metal-ion affinity interactions. The quality of glucoamylase immobilized on the regenerated supports is defined by determining of the enzyme activity, thermal stability, and reusability. The results indicate that the metal–chelate brushes offer an efficient route to immobilize enzymes via metal-ion affinity interactions. The applicability of the regenerated supports in the current study is relevant for the conjugation of other enzymes beyond glucoamylase. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Enzyme immobilization may permit fast separation of the enzyme from the reaction mixture, reuse of the enzyme, simplification of the reactor design, and improvement of their stability or tolerability [1]. Up to now, many different scaffolds and supports with a range of functionality, morphology, and physical properties have been studied for the immobilization of enzymes such as silica [2], polysaccharide [3], polymer nanofibers [4], hydrogels [5], and nanoparticles [6]. In particular, introduction of physical functionality, such as magnetic [7], thermosensitive [8], and regenerated properties [9], into nanomaterials is desirable for enzyme immobilization. Nonetheless, assembling multifunctional support nanomaterials are infrequently reported. Here our target is to synthesize nanomaterials for enzyme immobilization with combined magnetic and regenerated properties. To make this design feasible, a base material with a sophisticated structure is needed to carry the multifunctional entities. Mesoporous molecular sieves with longrange ordered porous structures, such as the SBA series, are ideal template materials for assembling these dual-targeting carriers and
∗ Corresponding author. Tel.: +86 931 8912528; fax: +86 931 8912113. E-mail addresses: [email protected], [email protected] (Y. Li). 1369-703X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2012.04.009
many reports have already described the use of these materials for enzyme immobilization [10]. Polymer brushes offer certain advantages over other materials as they are covalently anchored to the substrate providing excellent mechanical stability and present a high surface area template with functionality controllable by monomer type and brush length [11]. Among the various chemical functional groups that can be integrated into polymers, the epoxy group is particularly attractive because of their reactive pendant oxirane rings, which provides a large number of subsequent reactions, offering a variety of opportunity for chemical modification of the parent polymers [12]. Several methods have been developed to prepare polymercoatings on magnetite nanoparticles such as physical adsorption of polymers, emulsion polymerization in the presence of nanoparticles, and the so-called “grafting to” and “grafting from” methods [13]. Silane chemistry has been applied to metal oxide surfaces to promote metal–metal and metal–polymer adhesion. Previous literatures [14] have demonstrated the formation of polysiloxanes on the surface of magnetite via alkylalkoxylianes self-assembly. Atom transfer radical polymerization (ATRP) is a recently developed “living” or “controlled” radical polymerization method [15], which does not require stringent experimental conditions. Therefore, tethering of polymer brushes covalently onto a solid substrate by ATRP is a versatile and effective method of altering the surface properties of the material [16]. And surface-initiated ATRP allows
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the preparation of well-defined polymer brushed with dormant chain ends on various types of substrates [17]. More recently, Xu et al. [18] reported the covalent immobilization of glucose oxidase on well-defined PGMA–Si(111) hybrids via surface-initiated ATRP. This research aims at utilizing the controlled growth of polymer brushes on FeSBA-15 to develop regenerated supports with magnetic property. First, magnetic iron particles were impregnated into the SBA-15 cages by thermal decomposition, followed by reduction. Subsequently, the use of ATRP to grow PGMA from initiators bound to magnetic FeSBA-15 surface affords relatively fine control over polymer molecular weight, which allows large increases in capacity. PGMA brushes are particularly attractive because they can be functionalized to exploit a number of affinity interactions. Finally, functionalization of PGMA with imidazole (or iminodiacetic acid) results in enzyme binding via metal-ion affinity interactions. In order to compare the effectiveness of different functionalized brushes as scaffolds for enzymes, quantitative data on binding chemistry, kinetics of binding, and amount bound need to be examined in the context of the activity of immobilized enzyme.
The FeSBA-15–PGMA@Imidazole (or FeSBA-15–PGMA@IDA) support was activated by Cu2+ activation procedure. Cu2+ activation procedure is as followings: Cu(II) ion was chelated with the functional groups of the FeSBA-15–PGMA@Imidazole (or FeSBA-15–PGMA@IDA) support. A 5 mg mL−1 solution of Cu(II) ions was prepared from sulfate salts in distilled water. The FeSBA-15–PGMA@Imidazole (or FeSBA-15–PGMA@IDA) support was suspended in the Cu(II) ions solution at room temperature for 6 h. The support was taken out, washed several times with acetate buffer solution (50 mM, pH 5.5) and submerged into glucoamylase buffer solution (pH 5.5, 50 mM acetate buffer). The immobilization process was carried out at 30 ◦ C in a shaking air bath for 2 h. Finally, the support was taken out, thoroughly rinsed with acetate buffer (50 mM, pH 5.5). The amount of immobilized protein on the support was determined as described above. The amount of protein in the enzyme solution and in the washed solution was determined by the Bradford method using bovine serum albumin as a standard [19], and the amount of protein bound on the carriers was calculated from the formula:
2. Materials and methods
Protein bound (mg g−1 ) =
2.1. Enzymes and reagents Glucoamylase (exo-1, 4-a-d-glucosidase, EC3.2.1.3 from Aspergillus niger 10 U mg−1 ) was purchased from Yixing Enzyme Preparation Company (China); mesoporous SBA-15 silica molecular sieves were purchased from Changchun Jilin University High-Tech. Co. Ltd. (Jilin, China); glycidyl methacrylate (GMA) was obtained from Ciba Specialty Chemicals (China) Ltd., Guangzhou; 3-aminopropyltriethoxysilane (APTES) was purchased from Wuhan University Silicone New Material Co. Ltd. (China); 2bromo-2-methylpropionyl bromide was purchased from Aldrich. All other chemicals were of analytical grade, and used as received. 2.2. Preparation of metal–ligand functionalized magnetic FeSBA-15 First, magnetic iron particles were impregnated into the SBA-15 cages by thermal decomposition, followed by reduction. Subsequently, the use of ATRP to grow PGMA from initiators bound to magnetic FeSBA-15 surface affords relatively fine control over polymer molecular weight, which allows large increases in capacity. PGMA brushes are particularly attractive because they can be functionalized to exploit a number of affinity interactions. Finally, PGMA brushes are functionalized by imidazole or iminodiacetic acid. Details of the preparation were thoroughly described in supplementary material. 2.3. Enzyme immobilization Enzyme immobilizations on different functionalized FeSBA-15 were performed in following ways. Due to the active epoxy groups in the FeSBA-15–PGMA nanocomposites, glucoamylase immobilization was carried out by the treatment of the enzyme solution with the FeSBA-15–PGMA directly. 1.0 g of FeSBA-15–PGMA nanocomposites was equilibrated in 50 ml acetate buffer solution (50 mM, pH 5.5) for 24 h. It was then transferred to the same fresh buffer (50 ml) containing glucoamylase (0.1 g). The immobilization process was carried out at 30 ◦ C in a shaking air bath for 12 h. After this, the immobilized glucoamylase was recovered by magnetic separation, thoroughly rinsed with acetate buffer solution (50 mM, pH 5.5) two times to remove unbound glucoamylase. The washed solution was collected to assay the amount of residual enzyme. The resulting immobilized glucoamylase was held at 4 ◦ C prior to use.
(Ci − Cf )V W
(1)
where protein bound is the amount of bound enzyme onto superparamagnetic Fe3 O4 @Clays nanocomposites (mg g−1 ), Ci and Cf the concentrations of the enzyme protein initial and final in the reaction medium (mg mL−1 ), V the volume of the reaction medium (mL) and W is the weight of the carriers (g). All data used in this formula are the average of triplicate experiments. 2.4. Enzyme activity The reaction rate of the free and immobilized glucoamylase preparations was determined according to the method reported by Nelson [20], with only minor modification. In the standard conditions, using soluble starch as the substrate, which composed of 0.5 mL of 10 wt.% soluble starch gelatinized in water and 2.5 mL acetate buffer solution (50 mM, pH 5.5). The reaction was started by addition of 0.5 mL free glucoamylase (0.236 mg mL−1 ) or 0.1 g of immobilized glucoamylase. The mixture was incubated at 55 ◦ C under reciprocal agitation at 120 strokes per minute. After 15 min of reaction, agitation was stopped, and then the reaction was terminated by adding 5 mL of NaOH solution (0.1 M). The glucose content was determined using the DNS method [21]. The amount of glucose was obtained from the calibration curve and used in the calculation of enzyme activity. All activity measurement experiments were carried out three times, and the relative standard deviation is less than 1.0%. One unit of glucoamylase activity is defined as the amount of enzyme that produces 1.0 mmol of glucose from dissolubility starch per minute under the assay conditions. The relative activity (%) was the ratio between the activity of every sample and the maximum activity of sample. All experiments of activity measurement were carried out at least three times and the experimental error was less than 3%. 2.5. Characterization FT-IR spectra were obtained in transmission mode on a Fouriertransform infrared spectrophotometer (American Nicolet Corp. Model 170-SX) using the KBr pellet technique. Transmission electron microscopy (TEM, FEI Tecnai G20) was obtained to elucidate the dimensions and the structural details of the nanoparticles. TEM specimens were made by placing a drop of the nanoparticle suspension on a carbon-coated copper grid. Magnetization measurements were performed on a vibrating sample magnetometry (VSM, LAKESHORE-7304, USA) at room temperature. X-ray photoelectron
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Fig. 1. TEM images of superparamagnetic SBA-15 (a), FeSBA-15 (b) and FeSBA-15–PGMA (c).
spectroscopy (XPS) measurements were carried out on a VG ESCALAB 210 photoelectron spectrometer (VG Scientific Co.). 3. Results and discussion 3.1. Fabrication and characterization of metal–ligand functionalized magnetic FeSBA-15 There are a number of published papers on reversible enzyme immobilization, e.g., thiol-disulfide exchange of proteins containing surface thiol groups [22,23], immobilized metal chelate supports [24,25], and ionic exchange resins [26,27], but there are some problems arising in previous reports. The important problems were solved in this work: (1) magnetic FeSBA-15 mesoporous silica was chosen as the template because it is an ideal host material for magnetic nanoparticles large enough to possess a sufficient magnetization for magnetic separation. There is no researcher chose this nanomaterial for enzyme immobilization; (2) some researchers grafted PGMA on the outer surfaces of carrier for covalent immobilization of enzyme, but the carrier cannot be regenerated [24]. A number of researchers used monomer containing metal–ligand residues to fabricate regenerated carrier via free radical polymerization. However, the technology of free radical polymerization cannot control brush length of polymer, and the nanoparticles could be aggregated after free radical polymerization, which should affect the performance of immobilizing enzymes. Therefore, polymer brushes were covalently grated onto the surface of FeSBA-15 via ATRP in this study. And surface-initiated ATRP allows the preparation of well-defined polymer brushed with dormant chain ends on various types of substrates. However, the monomer containing metal–ligand residues could not be polymerized via ATRP technology. Therefore, firstly PGMA brushes are grated onto the surface of FeSBA-15 via ATRP in this study. Subsequently, PGMA brushes were functionalized by imidazole or iminodiacetic acid. Finally, functionalization of PGMA with imidazole (or iminodiacetic acid) results in enzyme binding via metal-ion affinity interactions. To our best knowledge, there is no similar study. 3.1.1. Preparation of magnetic FeSBA-15 The use of ordered mesoporous silica SBA-15 (Fig. 1a) as a template for the synthesis of iron oxide–silica nanocomposites has been demonstrated previously [28]. In this work, magnetic SBA15 mesoporous silica was chosen as the template because it is an ideal host material for magnetic nanoparticles large enough to possess a sufficient magnetization for magnetic separation. However, blockage may be caused in the 2D hexagonal porous structure of SBA-15 by impregnation of iron nanoparticles. This may limit the use of these composite materials for immobilization of enzymes
via the porous structure [29]. This can be overcome to some extent through the use of polymer chains grafted on the outer surfaces of magnetic FeSBA-15 and binding of multilayers of enzymes to these polymers. Magnetic FeSBA-15 was prepared via wet impregnation, calcination and reduction. As revealed by transmission electron microscopy (TEM), the obtained magnetite particles are deposited inside the inner cavities of SBA-15 (Fig. 1b). 3.1.2. Preparation of magnetic FeSBA-15–PGMA Diffusion limitation leads to slow replenishment of the consumed reactants and accumulation of the products around catalytic sites in the microspheric polymer materials, so that the efficiency of the biocatalyst is decreased. The immobilization enzymes on the outer surfaces of inorganic supports may overcome these disadvantages [30]. Despite their potential, the major disadvantage of inorganic supports is their low binding capacity relative to microspheric polymer materials [31]. Form this point of view, the use of polymer encapsulated the outer surfaces of inorganic supports and binding of multilayer of enzyme only on the surface of these polymers microsphere, that can reduce the diffusion limitation problem of immobilized enzyme system [32]. The chemical process to fabricate the supporting material is thoroughly described in the experimental section, and it is visually summarized schematically in Fig. 2. Upon silanization, the existence of amine group is more convincingly represented by the C–N, N–H signal (1334 cm−1 and 1564 cm−1 ) from the corresponding IR spectra (Fig. 3b). Prior to polymerization, the initiator must be immobilized to the surface. The ATRP initiator on the FeSBA-15 surface was prepared via amidation of amino-FeSBA-15 with 2bromoisobutyryl bromide. The conversion of the amino groups was estimated to be ∼31% based on the weight increase of the particles. The reaction of amino groups with 2-bromoisobutyryl bromide is also an efficient reaction with high conversion [33]. Therefore almost all the amino groups in the surface layer should be converted to Br-containing groups. In this work, the method of addition of Cu(II) complex (CuBr2 ) was chosen to control the concentration of the deactivating Cu(II) complex during the surface-initiated ATRP process on the FeSBA-15–Br surface. A study of thickness changes with time as determined by TEM demonstrated that surface-initiated polymerization only occurred on surfaces with the initiators present. TEM images show the FeSBA-15–PGMA arrays at an ATRP time of 5 h, where 20–30 nm coating is visible on the surface of FeSBA15–PGMA. For PGMA, this layer is on average 25 nm thick (Fig. 1c). 3.1.3. Functionalization of nanostructured FeSBA-15–PGMA An important feature of the FeSBA-15–PGMA surface is the preservation of the reactive epoxide groups during the ATRP
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Fig. 2. Schematic illustration of the synthesis and the chemistry used for the immobilization of glucoamylase on three brush systems.
process. In principle, all nucleophilic groups, such as, NH2 , SH, OH, and COOH groups, will react readily and irreversibly with the epoxy groups in subsequent surface functionalization [24,34]. Thus, the reaction between the epoxy group of GMA units and the imino group of imidazole (or IDA) was used to introduce the imidazole (or IDA) on the PGMA brushes, as shown in Fig. 2. The FT-IR spectra (Fig. 3d and e) of FeSBA-15–PGMA@Imidazole and FeSBA15–PGMA@IDA indicated that the modification of FeSBA-15–PGMA with imidazole (or IDA) was successful.
3.2. Immobilization of glucoamylase onto PGMA, imidazole and IDA brush Adsorption through ion-exchange, hydrogen bonding and hydrophobic interaction might be relatively a mild protein immobilization method, since the non-covalent bonding has been widely applied in protein separation and refolding processes. It was also well known that multipoint interaction may serve to enhance the structural stability of enzymes. The glucoamylase was immobilized
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Fig. 3. The IR spectra of FeSBA-15 (a), FeSBA-15–NH2 (b), FeSBA-15–PGMA (c), FeSBA-15–PGMA@Imidazole (d) and FeSBA-15–PGMA@IDA (e).
via covalent or non-covalent interactions on PGMA, imidazole and IDA brush. The FeSBA-15–PGMA with the ATRP reaction time 5 h exhibits an optimal selectivity equivalent for the immobilization of glucoamylase and is thus used for all immobilization experiments (data not shown). In order to achieve high biological activities or binding capacities of the immobilized glucoamylase, three different immobilization strategies which were shown schematically in Fig. 2 were discussed in the current study. In the immobilization strategy I, glucoamylase was immobilized on the FeSBA-15–PGMA via epoxy groups, which are inherently present in the support after polymerization. Taking advantage of metal–chelate properties, in the immobilization strategy II and III, Cu2+ ions was coordinated to the imidazole (or IDA) ligand and the enzyme was bound FeSBA15–PGMA@Imidazole and FeSBA-15–PGMA@IDA via Cu2+ ions. In the immobilization strategy I, the theoretical content of epoxy groups, calculated on the basis of the PGMA content in the FeSBA-15–PGMA was 5.21 mmol g−1 . The content of epoxy groups on the surface of FeSBA-15–PGMA sample determined by the pyridine–HCl method [35], and differs from the theoretical value (3.72 mmol g−1 ). Because some of the epoxy groups usually remain in bulk structure of FeSBA-15–PGMA, it is not accessible for subsequent reactions or for analytical determinations. FeSBA-15–PGMA with active epoxy groups were prepared and used for glucoamylase immobilization by covalent reaction via the amino groups of the glucoamylase and the epoxy groups of FeSBA-15–PGMA under mild condition (Fig. 2). The amounts of bound glucoamylase are 10.6 mg g−1 on FeSBA-15–PGMA. In the immobilizing strategy II and III, the amount of bound glucoamylase is related to the density of ligands on the surface of the FeSBA-15–PGMA. A high density of chelator provides high capacity and therefore high load ability. The content of functional groups on the FeSBA-15–PGMA was examined by elemental analysis (Vario EL III, German) as shown in Table 1. The imidazole groups content of FeSBA-15–PGMA@Imidazole reaches 2.46 mmol g−1 , which is higher than IDA groups content of FeSBA15–PGMA@IDA (0.96 mmol g−1 ). To evaluate the efficiency of immobilization via imidazole, IDA groups in comparison with that using epoxy groups, three types of brushes were used for immobilization of glucoamylase. The binding capacity of glucoamylase immobilized via imidazole groups was found to be twice as high as the capacity of this enzyme bound using epoxy groups (Table 1).
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Several reports described new supports for glucoamylase immobilization [36–39], and the amount of the immobilized enzyme to 1.0 g of supports was 4.95–20.6 mg. In the present work, the capacity of the former was approximately 21.6 mg g−1 while it was 10.6 mg g−1 for the epoxy groups and for 17.3 mg g−1 the IDA groups. All these immobilization studies published in the literature have been performed under different conditions and different organism was used for glucoamylase source. Therefore, it is almost impossible to compare these immobilization results. Interestingly, glucoamylase immobilized on FeSBA-15–PGMA (epoxy groups) for 12 h exhibited only 52.8% of activity compared to that achieved only after 60 min using FeSBA-15–PGMA@Imidazole (imidazole groups), and 75.6% of activity compared to that achieved only after 60 min using FeSBA-15–PGMA@IDA (IDA groups) (data not shown). The results confirmed that immobilization via imidazole or IDA groups are faster than that using epoxy group and other research reported before in the literature: 2.5 h [40], and 9 h [41]. Compared with these supports, the FeSBA-15–PGMA@Imidazole (imidazole groups) prepared in this study had a fast immobilization progress, such a short the immobilization time showed that there are two advantages to FeSBA-15–PGMA@Imidazole (imidazole groups) used for glucoamylase immobilization: (1) the shorter the immobilization time is, the less activity loss is. (2) The shorter the immobilization time is, the lower cost is [42]. The results can be explained by the fact that all these functional groups (imidazole or IDA groups) are exposed at the FeSBA-15 surface because of their hydrophilic character, while most of the epoxides are buried within the polymer and inaccessible for immobilization. Hence, FeSBA-15–PGMA@Imidazole having imidazole groups is an excellent carrier for immobilization of enzymes. 3.3. Effect of pH and temperature on the catalytic activity The change in optimum pH depends on the charge of the enzyme and/or of the water insoluble matrix. This change is useful in understanding the structure–function relationship of enzyme and to compare the activity of free and immobilized enzyme as a function of pH. The effect of pH on the activities of free and immobilized glucoamylase samples was investigated in the pH range of 1.5–8 at 55 ◦ C, and the results are given in Fig. 4. It was observed that the optimum pH of free enzyme was 3.5, while the operable pH for the immobilized glucoamylase was extended between pH 2.5 and 5.5. This shift may depend on the immobilization method as well as the basic character of the support material. Furthermore, the pH profiles of the immobilized glucoamylase preparations are broader than that of the free enzyme (Fig. 4), which meant that the immobilization methods preserved the enzyme activity in a wider pH range. Interestingly, the glucoamylase immobilized on FeSBA15–PGMA@Imidazole and FeSBA-15–PGMA@IDA supports exhibit higher activity than other supports [43] under the acidic condition (pH = 1.5), which could be explained by the weak-base of imidazole and ionized IDA groups, protected it from unfolding and prevented the conformation transition of the enzyme at acidic condition. Effect of temperature on the relative activity of free and immobilized glucoamylase is shown in Fig. 4. The immobilized enzyme showed an optimum reaction temperature between 55 and 65 ◦ C, that is near the literature value of 65 ◦ C [43], whereas free enzyme had an optimum temperature about 55 ◦ C. These results could probably be attributed to the stabilization of glucoamylase molecules resulting from multipoint attachment of the enzyme molecules on the surface of the FeSBA-15 by covalent or chelate bonds. The multipoint bond formation between support and glucoamylase molecules could reduce the conformational flexibility and may result in higher activation energy for the molecule to reorganize the proper conformation for the binding to substrate [44,45]. In addition, the results suggested that the immobilized glucoamylase
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Table 1 Content of the element N, functional group and protein loading capacity. Content
FeSBA-15–PGMA
FeSBA-15–PGMA@Imidazole
N content (mmol g−1 ) Functional group content (mmol g−1 ) Theoretical content (mmol g−1 ) Protein loading (mg g−1 )
0.26 3.72 (Epoxy groups) 5.21 (Epoxy groups) 10.6
5.19 2.46 (Imidazole functional group) 5.21 (Imidazole functional group) 21.6
FeSBA-15–PGMA@IDA 1.22 0.96 (IDA functional group) 5.21 (IDA functional group) 17.3
Fig. 5. Thermal stabilities of glucoamylase immobilized on the three supports in comparison to free glucoamylase.
Fig. 4. Effect of temperature and pH on enzyme activities.
possessed better pH and temperature stability than the free one. Similar behavior has been also observed in the case of an immobilized d-hydantoinase as well as an immobilized glucoamylase [46,47]. 3.4. Stability and reusability of immobilized glucoamylase The stability of an enzyme is critical to its practical applications. Fig. 5 shows the stability of glucoamylase immobilized on FeSBA-15–PGMA, FeSBA-15–PGMA@Imidazole and FeSBA15–PGMA@IDA supports over 6 h of testing at 57 ◦ C in acetate buffer solution (50 mM, pH 5.5). The preparations exhibited a similar trend, whereas the immobilized glucoamylase decreased less and more slowly than the free one. The catalytic activity of immobilized glucoamylase retained their initial activity of about 65–68% after 6 h. The lower stability of glucoamylase had also been reported by Carpio et al. [48], the catalytic activity of immobilized glucoamylase only retained their initial activity of about 40–50% after 6 h. In sharp contrast, the free glucoamylase only retained their initial
activity of about 43% after 6 h in this study. These results demonstrated that the thermal stability of immobilized glucoamylase was much better than the free one. This could be explained by the carriers enhancing the enzyme rigidity, protected it from unfolding and prevented the conformation transition of the enzyme at high temperature. These results indicated that the immobilization procedures had considerably improved the thermal stability. It is important to investigate the performance of immobilized enzymes during recycled use for potential industrial applications. In the study, glucoamylases immobilized on FeSBA-15–PGMA, FeSBA-15–PGMA@Imidazole and FeSBA-15–PGMA@IDA supports were recycled for the hydrolyzation of starch. The reaction was performed for 15 min at 55 ◦ C, after which the spent immobilized glucoamylases were recovered by magnetic separation, washed three times with acetate buffer solution (50 mM, pH 5.5), and then used again for a fresh reaction. The assay conditions remained the same as described above. The variation of activity of the immobilized glucoamylase after multiple reuses is showed in Fig. 6. It was observed that the residual activity of the immobilized enzymes is 67.14% for FeSBA-15–PGMA, 65.12% for FeSBA-15–PGMA@Imidazole and 67.84% for FeSBA-15–PGMA@IDA after the 10th reuse. Obviously, the results indicated that the immobilized glucoamylases significantly increases its operational stability. 3.5. The regeneration of supports The immobilized enzyme also could loss its activity completely after several times of reuse. Therefore, the regeneration of supports at the end of the life of the immobilized enzyme, which can effectively reduce the cost in industry applications, is very essential. In order to show the reusability of the functional FeSBA-15–PGMA supports; adsorption–desorption cycle of glucoamylase was repeated three times by using the same supports. The regeneration of supports in the report is carried out in three steps; firstly, desorptions of adsorbed glucoamylase from
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concluded that the functionalizable supports provide an economical, efficient and reversible system for enzyme immobilization. Acknowledgements The authors thank the financial supports from the National Natural Science Foundation of China (No. 21074049), the Opening Foundation of State Key Laboratory of Applied Organic Chemistry (SKLAOC-2009-35), the Fundamental Research Funds for the Central Universities (lzujbky-2012-75), and the Opening Foundation of State Key Laboratory of High Performance Civil Engineering Materials (2012–2013). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bej.2012.04.009. References
Fig. 6. Reuse of glucoamylase immobilized on the three supports.
FeSBA-15–PGMA@Imidazole and FeSBA-15–PGMA@IDA supports were carried out in a batch system. The immobilized enzyme preparations were placed within the desorption medium containing 1.0 M EDTA at room temperature for 5 h as described above and were then repeatedly used in adsorption/desorption cycle of glucoamylase. Subsequently, the supports were activated with Cu2+ and finally immobilization of the enzyme in a usual procedure is carried out (Fig. 2). Many works about reversible immobilization of glucoamylases on different carriers are cited in the literature. Bayramo˘glu et al. [49], prepared a nanofibrous polymer grafted magnetic poly(GMAMMA)-g-MAA beads for reversible immobilization of trypsin. After six-repeated adsorption–desorption cycles, the adsorption capacity of the magnetic beads was decreased 8%. Subsequently, this researcher team reversibly immobilized catalase on fibrous polymer grafted and metal chelated chitosan membrane. At the end of the sixth cycles, catalase immobilization capacity of the CH-gpoly(IA) membrane was decreased about 7% [50]. Erdem Yavuz reported poly(vinylbenzylchloride) beads grafted with polymer brushes carrying hydrazine ligand for reversible invertase immobilization. The sixth adsorption–desorption cycle of invertase, the amount of immobilized enzyme was about 3.6% lower than that of the first use [51]. In this study, the immobilization capacities of the regenerated supports did not significantly change during the following three regeneration cycles. These results showed that Cu2+ ions incorporated FeSBA-15–PGMA@Imidazole and FeSBA-15–PGMA@IDA supports can be repeatedly used in enzyme immobilization without detectable losses in their initial adsorption capacities.
4. Conclusions In summary, this work demonstrates an effective and convenient strategy to obtain functionalizable and metal–chelate surfaces for enzyme immobilization. Glucoamylase was found to interact with functionalizable supports more rapidly compared to FeSBA-15–PGMA to form the metal–chelate complex. The amount of glucoamylase uptake was found to be the highest for Cu2+ -doped FeSBA-15–PGMA@Imidazole, while FeSBA-15–PGMA exhibited the lowest amount of glucoamylase uptake. After regeneration of supports, the biocatalyst also reserved its original reusability. It can be
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