Article pubs.acs.org/IECR
Glucose Oxidase Immobilization on Guar Gum−Gelatin DualTemplated Silica Hybrid Xerogel Vandana Singh* and Devendra Singh Department of Chemistry, University of Allahabad, Allahabad 211002, Uttar Pradesh, India S Supporting Information *
ABSTRACT: Hybrid xerogel (H5) crafted from guar gum−gelatin dual-templated polymerization of tetramethoxysilane (TMOS) behaved as an effective carrier support for glucose oxidase (GOX). H5 has been characterized using Fourier transform spectroscopy (FTIR), X-ray diffraction (XRD), Field emission scanning electron microscopy (FESEM), BET surface area studies, and Zeta potential and particle size distribution analysis. Under optimized conditions, H5−GOX showed a much higher bioactivity (20.73 U mg−1) than free GOX (9.26 U mg−1). The kinetic parameters of free GOX (KM = 13.988 mg mL−1, Vmax = 1.785 μmole mL−1 min−1) and H5−GOX (KM = 5.403 mg mL−1, Vmax = 1.75 μmole mL−1 min−1) revealed that immobilization could enhance the substrate affinity of GOX, although it did not alter the enzymatic reaction rate. H5−GOX retained ∼96% of its initial bioactivity with 15 days of storage as a dry solid at room temperature. Recycling of H5−GOX showed only marginal loss in bioactivity up to six cycles.
1. INTRODUCTION Immobilization is an effective technique for reducing the cost of enzymatic biotransformations.1−3 The immobilized enzymes can be easily recycled in continuous automated processes; nevertheless, the immobilization can also alter the stability, activity, specificity, and selectivity of an enzyme. Among all the known methods of enzyme immobilization,4 adsorption5 is most attractive as it does not alter the intrinsic nature of an immobilized enzyme. In general, porous materials can be useful in enzyme immobilization6 because they offer large surface area for enzyme loading. Heterogeneous biocatalytic transformations have been very frequently carried out on silica or alumina support for their high surface area and thermal and chemical stability.7 Enzyme immobilization by adsorption is however associated with a serious problem of enzyme leakage that warrants new materials to achieve maximum bioactivity with minimum enzyme leakage. There has been recent interest in using nanomaterials8,9 and biomimetic silica supports10,11 for enzyme immobilization. Magnetic hollow mesoporous silica nanospheres are known for ultrafast immobilization of enzymes.12 Entrapment of urease in silica nanocomposites derived from tetraethoxysilane13 is also reported. A robust biocatalyst for glucose oxidation has been fabricated by immobilizing glucose oxidase in mesoporous cagelike FDU-1 silica.14 The mesoporous silica such as MCM-type15 and BBA-type material have been widely used for enzyme immobilization due to their large pore volume and controllable pore size, but the surfactants16 used for templating such materials may have a detrimental effect on the environment. Mimicking living organisms, many sol−gel composites of silica have been designed.16 Most of these studies involve a single templating biomolecule, either a polysaccharide17 or a protein.18 However, there are few reports on polysaccharide− protein dual templation, where gelatin has been used as a protein template with alginic acid19 and gum acacia20 as polysaccharide components in two separate studies. It has been © 2014 American Chemical Society
shown that gelatin activates the silica formation by forming hybrid aggregates, while polysaccharides are responsible for controlling the silica microstructure.19 These studies have accorded the previous models of biosilicification and established that multiple templating may be a suitable approach for obtaining complex porous silica architectures. In our recent paper,20 a silver nanoparticle-incorporated gelatin−gum acacia nanocompositional hybrid material was used to immobilize diastase α-amylase. Though a number of carrier matrices are known21−25 for maintaining the activity of enzymes under the extreme conditions, the concept of biosilification using a polysaccharide−protein dual template is of interest as these templating molecules are eco-friendly and do not involve any environmental implications in designing enzyme support materials. Guar gum is a commercially available abundant polysaccharide having a β(1→4)-linked-D-mannopyranosyl backbone with α-D-galactose units at branch points that are glycosidically linked to the 6-position of every second mannose unit.26 Gelatin is a fibrous animal protein with poor mechanical and thermal stability.27 It is derived by controlled hydrolysis of collagen and is a waste in animal slaughtering process. In principle, guar gum can interact with ε-amino groups of lysine or hydroxylysine of gelatin through secondary interactions. As a result, an entirely different conformational state is expected for these biomolecules when they are together present in a solution state. Thus, their combined templating effect may be interesting in developing exciting carrier supports for enzyme immobilization. Guar gum has been rarely used for enzyme immobilization except in a few reports, for example, agarose−guar gum membranes for the immobilization of invertase.28 Received: Revised: Accepted: Published: 3854
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hybrid hydrogels (Ba to Be) thus obtained were finally washed with water and dried. 2.4.3. Synthesis of Guar Gum−Gelatin−Silica Hybrids (“C” Series Hybrids). For synthesizing guar gum−gelatin dualtemplated hybrids, 10 mg of gelatin, 1.5 mL of TMOS, and 1.5 mL of MeOH were added to aqueous guar gum solutions of a definite pH (containing 100 mg of guar gum in 15 mL of distilled water). pH of distilled water was adjusted using the required amount of acid or alkali. These reaction mixtures were stirred for 7 h at 30 °C with a magnetic stirrer. The hydrogels thus obtained were washed well with water and dried under reduced pressure. The gelatin amount was then varied (5−30 mg) while using 1.5 mL of TMOS and 1.5 mL of MeOH with guar gum aqueous solutions (containing 100 mg of guar gum in 15 mL of distilled water that has been preadjusted at pH 5.5). The obtained hybrid hydrogels were washed with distilled water and dried to obtain hybrid samples (Cda to Cde). 2.4.4. Calcination of Hybrids. An optimum “C” Series hybrid sample (Cdb that has been renamed as H1) was calcined at different temperatures ranging from 200 to 700 °C for 2 h at each temperature to obtain hybrid xerogels (H2−H7). 2.4.5. Control Silica. TMOS (1.5 mL) and MeOH (1.5 mL) were added to 15 mL of distilled water that was preadjusted at pH 5.5. The mixture was stirred for 6 h at 30 °C with a magnetic stirrer to obtain a silica hydrogel that was washed with water and dried. The dried hydrogel on calcination at 500 °C for 2 h furnished the control silica (Cs). 2.5. Determination of pHzpc. For calculating pHzpc, the zeta potential measurements were performed at different pH values ranging from pH 2 to pH 9 at 25 °C using water as the dispersant. 2.6. Immobilization of Glucose Oxidase. The most suitable pH for the GOX loading was pH 5.6 (trial experiments not shown). H5 (200 mg) was kept in contact with 5 mL of glucose oxidase solution for 24 h in a orbital shaker set at 4 °C and 100 rpm. The enzyme-loaded hybrid (H5−GOX) was separated by centrifugation. In order to remove adhered GOX (if any), H5−GOX was washed thrice with 20 mL of 0.02 M phosphate buffer solution. The resulting filtrate was collected for determining the activity of the residual GOX in solution. 2.7. Activity of Immobilized Glucose Oxidase. The enzyme bioactivity was assessed in terms of D-(+)-glucose oxidation. Glucose oxidation forms gluconolactone in the first stage, which subsequently hydrolyzes to gluconic acid on aeration. The oxidation was assayed by titrating the released Dgluconic acid with 0.1 M NaOH. The calculation of the enzymatic activity was based on the amount of NaOH (0.1 M) consumed for neutralizing the D-gluconic acid liberated from a given quantity of glucose at 30 °C and pH 5.6. A definite amount of enzyme (either 200 mg of H5−GOX or 10 mg of solid GOX) was added to the D-glucose solution (0.7 g of D-glucose dissolved in 25 mL of 0.02 M phosphate buffer solution of pH 5.6). The solution was air ventilated for 15 min at 30 °C. To this reaction mixture, 20 mL of 0.1 M NaOH was added to neutralize the formed gluconic acid. The unspent NaOH in the reaction mixture was titrated with 0.1 M HCl. The volume of the consumed HCl was Vs (mL). The control experiment was performed identically but in the absence of the enzyme. The volume of 0.1 M HCl required in the control experiment is expressed as V0 (mL).
Present communication describes the utilization of this versatile seed gum in designing a suitable carrier matrix for immobilization of glucose oxidase enzyme, where the concept of guar gum−gelatin dual templation has been used. Glucose oxidase has been selected for the present study because of its importance as an analytical tool in medical and environmental monitoring applications.29,30 It is a globular dimeric protein31 that catalyzes the oxidation of glucose (to D-glucono-1,5lactone and hydrogen peroxide) using molecular oxygen as electron acceptor. Glucose oxidase is composed of two identical subunits, each of which folds in two domains: one binds to glucose, while other binds noncovalently to cofactor flavine adenine dinucleotide (FAD).32
2. EXPERIMENTAL SECTION 2.1. Materials and Reagents. Tetramethoxysilane (98% TMOS; Merck, Germany) was used as silica precursor. Guar gum (Merck) was used as supplied. Analytical grade of glucose oxidase, D-glucose, NaOH, HCl, and phosphate salts (Merck, India) were used. Gelatin, phenolphthalein, and oxalic acid (Qualigens, India) were used. 2.2. Characterization. X-ray diffraction (Cu Kα source) of the powdered samples was carried out on a XRD Pananalytical X-Pert Pro X-ray powder diffractometer. IR was done by forming KBr pellets through a Fourier transform infrared (FTIR) spectrophotometer, JASCO FTIR, within the spectral range of 400−4000 cm−1 and resolution of 4 cm.−1 Field emission scanning electron microscopy (FESEM) was used to observe microscopic morphology on a FEI ESEM QUANTA 200 instrument with an accelerating voltage of 15 kV. To avoid charging, the samples were coated with gold. Temperature treatment of the hybrids was carried out in N2 using an electric muffle furnace (Metrex Scientific Instruments (P) Ltd., New Delhi, India). After calcination, the materials were left inside the muffle furnace for cooling to room temperature. A WT Classic BET surface area analyzer (WAKO, India) was used for surface area determination (at 77 K). Prior to gas adsorption, all the samples were degassed for 4 h at 533 K. Zeta potential and particle size measurements were carried out on Malvern Zetasizer Ver 6.20 using water as dispersant at 25 °C. 2.3. Enzyme Stock Solution. Glucose oxidase stock (GOX) solution containing 46 U of enzyme per mL was prepared by dissolving 100 mg of solid GOX (activity 230 U mg−1) in 50 mL of phosphate buffer of pH 5.6. 2.4. Material Synthesis. Percent enzyme loading and the bioactivity of the enzyme loaded hybrids were chosen as criteria for the material design. The best performance sample (H5− GOX) was used to optimize the oxidation of D-(+)-glucose. 2.4.1. Synthesis of Guar Gum−Silica Hybrids (“A” Series Hybrids). Guar gum-templated hybrids were synthesized by adding 1.5 mL of TMOS and 1.5 mL of MeOH to the guar gum solutions of known concentrations (containing 100−200 mg of guar gum in 15 mL of distilled water of pH 7). These reaction mixtures were stirred at 30 °C for 7 h with a magnetic stirrer. The hybrid hydrogels thus obtained were washed well with water and dried to obtain the hybrid samples (Aa to Ac). 2.4.2. Synthesis of Gelatin−Silica Hybrids (“B” Series Hybrids). Gelatin-templated hybrids were synthesized by adding 1.5 mL of TMOS and 1.5 mL of MeOH to gelatin solutions of known concentrations (containing 5−20 mg of gelatin in 15 mL of water of pH 7). These reaction mixtures were stirred at 30 °C for 6−7 h with a magnetic stirrer. The 3855
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as controls. Series A and Series B hybrids syntheses involved the templating of guar gum and gelatin, respectively (Table S1, Supporting Information). The size of the sol particles and cross-linking therein is dependent upon the reaction pH and “r” ratio (H2O:TMOS). In the present study, the value of “r” was fixed at 10:1. The criterion for the selection of this ratio was based on our past experience with similar materials and on our trial experiments for this study. It was noticed that the addition of MeOH as cosolvent was necessary for homogenizing the reaction mixture, and the equimolar ratio of MeOH and TMOS was most suited (variation of TMOS and MeOH with respect to the templates is not shown) for triggering the gelation in the optimum time, which was 6−7 h in the present study. Beyond this ratio, the gelation time was either much prolonged or no gel was formed. The overall criteria for the synthesis optimization were the gelation time and bioactivity of the immobilized enzyme at the hydrogels. For optimizing the guar gum amount in the “A” Series hybrids, a known weight of guar gum (100−200 mg) was dissolved in 15 mL of H2O to which 1.5 mL of TMOS and 1.5 mL of MeOH were added, and the pH of the reaction mixture was adjusted at pH 7. The gelation time for these materials was ∼7 h. The hydrogels thus obtained were washed well with water and dried. In a preliminary experiment, dried hybrid gels after GOX loading were assessed for their bioactivities as described in Section 2.7. Similarly, control gelatin−silica hybrids (“B” Series) were also synthesized by using varying amounts of gelatin (5−20 mg) but in the absence of guar gum, while keeping the other conditions same as that of “A” Series hybrids. For up to 7.5 mg of gelatin, the gelation time of “B” Series hybrids did not vary from that of polysaccharide−silica hybrids, that is, 7 h. However, further increase in gelatin weight reduced the gelation time by one hour. This decrease in gelation time with an increase in gelatin content can be attributed to the operation of the secondary interactions between the polysaccharide and gelatin. These hybrid samples were also screened as immobilization support for GOX. Although sample Bb showed the highest activity among the “B” Series hybrids, Bc gelled in relatively lesser time. Thus, for optimizing the synthesis of guar gum−gelatin dual-templated hybrids (“C” Series), 100 mg of guar gum and 10 mg of gelatin were used, while keeping the rest of the variables fixed (15 mL of H2O, 1.5 mL of TMOS, and 1.5 mL of MeOH). Under this optimized condition, the reaction mixture was set at various pH values ranging from pH 4 to 8 as pH is an important parameter that decides the final microstructure of the hybrid material. In the present study, the hybrid formation has been studied in the pH range from pH 4 to 8 as the gelation was much delayed when pH < 4 was used. At pH > 4, deprotonated silanols are involved in condensation because the isoelectric point of silica ranges between pH 1 and 3. The hydrolysis rate of siloxane bonds increased by over 3 orders of magnitude between pH 4 and 7 as compared to pH < 4. Gelling time for the studied pH range was ∼7 h. The most appropriate pH for the hybrid synthesis (in terms of bioactivity of the immobilized GOX) was found to be pH 5.5 (Table S1, Supporting Information) at which the gelatin amount was varied again while keeping other process parameters same (100 mg of guar gum, 1.5 mL of TMOS, and 1.5 mL of MeOH in 15 mL H2O). The optimum sample of guar gum−gelatin−silica hybrids (Cdb, which has been renamed H1) was obtained by using 100 mg of guar gum, 7.5 mg of gelatin, 15 mL of water, 1.5 mL of TMOS, 1.5 mL of MeOH, and pH 5.5 (Table S1, Supporting Information).
The activity unit (U) of GOX is defined as the amount of enzyme required to oxidize 1 μmol of β-D-(+)-glucose to Dgluconic acid and H2O2 per minute at pH 5.6 at 30 °C. Then the activities of GOX were calculated by the following expression Eznyme Activity (U ) =
V0 − Vs × f × C × 1000 15
(1)
where the dilution factor, f, = 1; C, the concentration of HCl, = 0.1 M. Each measurement was repeated thrice, and results given are the average of three readings. The % loading of the enzyme was calculated as below. %Enzyme Loading =
Utotal − Uremain × 100 Utotal
( 2)
where Utotal is the activity of total free enzyme that was loaded on H5, and Uremain is the activity of GOX in the centrifugate after H5−Enz was removed. The bioactivity of the H5−GOX was monitored at various pH values and temperatures in comparison to free enzyme activity. To obtain the enzyme solutions of different pH, 10 mg of solid free glucose oxidase was dissolved in 25 mL of phosphate buffer solutions of different pH that ranged from pH 4 to 8.33 Similarly, 10 mg of free GOX was added to 25 mL of 0.02 M phosphate buffer (NaH2PO4−Na2HPO4 solution of pH 5.6 containing 0.7 g of glucose). The solution was ventilated by air for 15 min at different temperature (25−60 °C) in different sets of experiments. The reaction mixtures were quenched by the addition of 20 mL of 0.1 M NaOH. The bioactivities of the reaction mixtures were calculated as described above. Identical studies were performed using 200 mg of H5−GOX (containing 10 mg of GOX) instead of 10 mg of solid GOX. 2.8. Stability of the Immobilized Enzyme. For evaluating the shelf life of the immobilized enzyme, the enzyme impregnated hybrid was stored as a dry solid for 15 days at room temperature (30 °C). The free enzyme solution (in phosphate buffer) was also stored for the same time period at 30 °C. The bioactivities of the stored enzymes were determined as described in Section 2.7.
3. RESULTS AND DISCUSSION The microstructure of a sol−gel polysilicate material depends upon its synthetic process parameters. The presence of templates has catalytic and steric influences on the condensation polymerization of silica sols. Polysaccharides and proteins possess special functionalities that maintain secondary interactions with silica silanols during the course of hydrolysis and condensation reactions of silica alkoxides. Such interactions modify the relative rate of hydrolysis and condensation of silica precursor sols and control the overall ultrastructure and texture of the hybrid hydrogel that gets decided at the time of gelation, and all the subsequent steps, that is, aging, drying, and densification, depend upon the gel’s ultrastucture. Although the presence of a polysaccharide or a protein modifies the hydrolysis and condensation mechanism, the collective use of these biomolecules may have a special impact on the gel’s nature as they mutually affect each others’ conformational states in solution. In the present study, to optimize the synthetic conditions of guar gum−gelatin dualtemplated silica hybrids (“C” Series hybrids), singly templated hybrid samples, Series “A” and Series “B”, were first synthesized 3856
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(asymmetric Si−O−Si stretching), 949 cm−1 (Si−OH stretching), 798 cm−1 (symmetric S−O−Si stretching), 571 cm−1, 464 cm−1 (oxygen ring breathing mode), and 1639 cm−1 (O−H bending).34 The IR spectrum of the hybrid gel (H5) showed absorption peaks characteristic of a silica matrix (463 cm−1, 797 cm−1, 1087 cm−1) and guar gum (3464 cm−1 (O−H stretching overlapped with guar gum hydroxyl), 2937 cm−1, (CH2 stretching), 2890 cm−1 (CH stretching), and 1639 cm−1 (O− H bending)). In the FTIR spectrum of H5−GOX (Figure 1), no significant change was witnessed except shifting of the silanol peak to a shorter wavenumber (3454 cm−1). This shift indicated that the surface silanol of siloxane domains are involved in enzyme immobilization. As no new peak is visible in H5−GOX, no formal bonding between GOX and H5 is indicated. It is concluded that the immobilization is absolutely physical in nature. Other silica-related peaks in H5−GOX are observed at 464, 798, and 1087 cm−1. 3.1.2. Field Emission Electron Microscopy. The surface morphologies of H5 and H5−GOX are significantly different as evident from their FESEM images (Figure 2). H5 shows smooth surface-scattered bulk particles of different sizes, while H5−GOX has a lamellar structure where the deposition of the enzyme is observed as a clogged hybrid surface. 3.1.3. X-ray Diffraction Studies. X-ray diffraction patterns of H5 and H5−GOX are shown in Figure 3. Both of them show similar amorphous humps at nearly the same diffraction angle centers (2θ 22.76° and 2θ 22.81°, respectively). The diffraction angle centers of H5 and H5−GOX are slightly shifted from the diffraction angle of the control silica (2θ 23.98°) (XRD not shown). Involvement of physical forces in enzyme uptake is indicated by similar XRD patterns of H5 and H5−GOX (see the FTIR study, Figure 1). 3.1.4. BET Surface Area and Particle Size Distribution. Templating has marginally increased the surface area of the hybrid gel. The surface area of the calcined sample (35.76 m2 g−1) was significantly higher than the surface area of the uncalcined sample (19.04 m2 g−1). This difference may be attributed to the increased porosity of the hybrid when organics are lost during the calcination process.35 The surface area of H5 showed a marginal increase after the enzyme uptake (37.95 m2 g−1) as now the surface area of the immobilized enzyme is also included, but after the enzyme loading, the increase in surface area was not substantial. It can be assumed that the enzyme sits inside a porous network structure of hybrid silica, and the surface of the enzyme is now included in the total surface area of the hybrid. Identically synthesized control silica had a surface area of 16.92 m2 g−1 that reflects the effect of templating on the hybrid’s microstructure. Particle size distribution in H5 is shown in Figure 4, where silica particles showed wide polydispersity (122 nm to 1.1 μm) as was also affirmed by the FESEM image of H5. 3.2. Enzyme Immobilization. A total of 100 mg of H5 was used to immobilize 10 mg of solid GOX. The hybrid matrix was efficient in enzyme uptake as ∼89% of the loaded GOX was adsorbed by H5. 3.3. Effect of pH on Enzyme Bioactivity. The pH optimum of the studied enzymatic reaction was determined by varying the pH of the assay reaction mixtures using the phosphate buffers (Figure 5A). The residual enzyme activity was determined as described earlier. The enzyme activity was sensitive to pH values both in the free and immobilized states, and the immobilization did not change the pH response of the enzyme (Figure 5A). In the present study, the pH optimum for
The significant advantage of dual templation in synthesizing the immobilization support is evident from Table S1 of the Supporting Information. The maximum bioactivities achieved for polysaccharide and protein singly templated materials (“A” and “B” Series hybrids) were 9.29 U mg−1 and 8.16 U mg−1, respectively, in comparison to 18.13 U mg−1 that was observed for the optimum dual-templated hybrid material (H1). H1 has been calcined at a definite temperature (ranging from 200 to 700 °C) in a nitrogen atmosphere to further tailor its properties. Among the calcined materials, H5 (H1 calcined at 500 °C) had optimum activity (20.73 U mg−1). Activity of the GOX immobilized control silica (Table S1, Supporting Information) was much lower (12.07 Umg−1), which clearly indicate the advantage of the templation. H5 and enzyme immobilized H5 (H5−GOX) were characterized using FTIR, FESEM, BET, and XRD. pHzpc of H5 has been determined to be 2.6. For its computation, zeta potential values (at 25 °C) of the hybrids dispersion in water were plotted with respect to pH values (pH 2−9) that were used for their measurement and pH that corresponded to zero zeta potential value was considered as pHzpc of the material. Oxidation of glucose has been optimized using H5−GOX, and the kinetic parameters were derived to understand the immobilization behavior of this material. The percentage loading of GOX at the hybrid sample (H5) was calculated (see eq 1) to be 89%. 3.1. Characterization. 3.1.1. Fourier Transform Infrared Spectroscopy. Silica incorporation in the hybrid was confirmed by locating silica-related peaks in the infrared spectrum of the hybrid (Figure 1). In the control silica (spectrum not shown), the peaks are observed at 3453 cm−1 (SiO−H), 1087 cm−1
Figure 1. FTIR spectra of H5 (A) and H5−GOX (B). 3857
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Figure 2. FESEM image of H5 (A) and H5−GOX (B).
4.2. Thus, the immobilization in the present study cannot be explained by electrostatic interactions. The physical forces such as hydrogen bonding and van der Waals interactions may be responsible for the immobilization. Physical adsorption has also been indicated by the IR and XRD studies. The enzyme activity declined (for both free and immobilized enzymes) as the measurement pH was increased. The activity of the immobilized enzyme is higher as compared to the free enzyme in the studied pH range. 3.4. Effect of Temperature on Enzyme Bioactivity. The temperature effect on glucose oxidation was studied using the free and immobilized enzyme (H5−GOX) (Figure 5B). The immobilized enzyme can be used at a relatively lower temperature (40 °C) to achieve the optimum bioactivity in comparison to the free enzyme (45 °C). A sharp decrease in enzyme activity with an increase in temperature greater than 55 °C indicates enzyme denaturation. 3.5. Hydrolysis and Kinetic Parameters. The kinetic parameters for the free and immobilized GOX have been determined at pH 5.6 at 40 °C. The relation between substrate concentration and rate of enzymatic reaction is described by the Michaelis−Menten (MM) equation, which can be represented into a linear form as the Lineweaver−Burk equation for computation of Vmax (the maximum rate of reaction in μmole mL−1 min−1) and KM (Michaelis constant in mg mL−1) values. Usually KM of an immobilized enzyme is not the same as that of the free enzyme. This difference may be due to diffusion limiations,36 steric effects,37 and ionic strength.38 Kinetic parameters, the Michaelis constant (KM), and the maximum enzyme activity (Vmax) have been determined using the Lineweaver plots (Figure 6). The kinetic parameters of the free (KM = 13.988 mg mL−1, Vmax = 1.785 μmole mL−1 min−1) and immobilized (KM = 5.403 mg mL−1, Vmax = 1.75 μmole mL−1 min−1) enzymes revealed that the immobilization led to a 2.6 fold increase in the enzyme substrate affinity. This change indicated that the immobilization led to a favorable change in the GOX conformation or decreased the enzyme inhibition. Vmax values of the free and immobilized enzyme are almost the same, which indicated that the immobilization has no adverse affect on the rate of the enzymatic reaction. The kinetic parameters indicated that although the immobilization has increased the enzyme−substrate affinity, that is, enzyme
Figure 3. XRD of H5 (A) and H5−GOX (B).
Figure 4. Particle size distribution in H5.
the enzyme uptake was pH 5.6 as had been previously reported by other authors.32 The strength of the electrostatic interaction between the enzyme and the silica support may be considered very important in maintaining the overall activity of the enzyme. Using the zeta potential study, pHzpc of H5 has been determined to be pH 2.6. This indicated that surface charge of H5 is positive at pH < 2.6, neutral at pH 2.6, and negative at pH > 2.6. At the measurement pH (pH 5.6), the surface of H5 will be negative, and at the same time, the net charge of the GOX enzyme will also be negative as its isoelectric point (pI) is 3858
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Figure 5. (I) Effect of pH on the activities of the free (A) and immobilized GOX (B). (II) Effect of temperature on the activities of the free (A) and immobilized GOX (B).
The initial activity of the immobilized enzyme (20.73 U mg−1) remained almost same (19.86 U mg−1) even after 15 days of storage at room temperature (30 °C), while the bioactivity of the free enzyme stored in the phosphate buffer declined significantly (5.24 U mg−1) within 24 h, and the activity after 15 days was almost negligible. Thus, the immobilization presented a reasonable advantage in terms of shelf life of the enzyme. Not only was storage easier for the immobilized enzyme (as it did not require phosphate buffers for the storage), but 87% of its initial activity was retained up to 15 days of storage at 30 °C, which indicated that the immobilization enhanced the enzyme stability.
4. CONCLUSIONS Hybrid xerogel obtained from guar gum−gelatin dualtemplated polymerization of tetramethoxysilane (TMOS) proved very effective in immobilizing glucose oxidase. The immobilization did not affect the optimum pH for the enzyme bioactivity, but it did lower the optimum temperature for reaching the maximum activity. This material is attractive for glucose oxidase immobilization as this carrier support retained the enzyme activity in the immobilized state. Nevertheless, immobilization could enhance the room temperature storage ability of the enzyme. The material may be useful toward glucose sensor development.
Figure 6. Lineweaver kinetic graphs for the free enzyme (A) and immobilized enzyme (B).
substrate complex, is more easily formed; the dissociation rate of this complex remained the same as that of the free enzyme− substrate complex. It appears that the immobilization process has provided a structural stability to the enzyme that prevented an irreversible unfolding of the enzymatic protein and thus the substrate affinity. The material is attractive because immobilization usually reduces enzyme activity. 3.6. Recycling and Storage. The immobilized enzyme (H5−GOX) was recycled for six repeated cycles. In the first cycle, the activity loss for H5−GOX was only 2.92%. In second cycle, another loss of 13.75% takes place, and after this, the bioactivity of the recycled material did not change. It appears that the enzyme immobilized at less hindered sites of siloxane domains was lost in the first two cycles, while the deep-seated enzyme did not come out on recycling. The immobilized GOX retained 81% of its initial activity even in the sixth cycle (Figure 7), which is quite good bioactivity for the material to be exploited commercially. The free GOX was stored in phosphate buffer of pH 5.6 for 15 days at room temperature, while the immobilized enzyme (H5−GOX) was stored as a dry solid in the same environment.
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ASSOCIATED CONTENT
S Supporting Information *
Figure S1: FTIR of control silica. Figure S2: XRD of control silica. Figure S3: FESEM of control silica. Figure S4: pHzpc determination. Table S1: Synthesis optimization of hybrid. Activity measurement was completed using 200 mg of H5− GOX and 25 mL of 0.02 M phosphate buffer of pH 5.6 containing 0.7 g of glucose, and released gluconic acid was monitored by the titration method as described in Section 2.7). This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel. +918127598952. E-mail: singhvandanasingh@rediffmail. com. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank the Council of Scientific and Industrial Research (C.S.I.R.), New Delhi, India, for financial support to
Figure 7. Recycling of H5−GOX. 3859
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carryout the present work. The authors acknowledge the Indian Institute of Sciences, Banglore, for use of the FESEM facility and the National Centre of Experimental Mineralogy and Petrology, University of Allahabad, for use of the XRD facility. Use of the IR facility is acknowledged to Banaras Hindu Univeristy, Varanasi, India. Use of the zeta potential facility is acknowledged to Professor Kankan Bhattacharya, Indian Cultivation of Sciences, Kolkata, India.
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dx.doi.org/10.1021/ie402341c | Ind. Eng. Chem. Res. 2014, 53, 3854−3860
