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Immobilized transglucosidase in biomimetic polymer–inorganic hybrid capsules for efficient conversion of maltose to isomaltooligosaccharides
Biochemical Engineering Journal 46 (2009) 186–192
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Immobilized transglucosidase in biomimetic polymer–inorganic hybrid capsules for efficient conversion of maltose to isomaltooligosaccharides Lei Zhang, Yanjun Jiang, Zhongyi Jiang ∗ , Xiaohui Sun, Jiafu Shi, Wei Cheng, Qianyun Sun Key Laboratory for Green Chemical Technology of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, 92 Weijin Road, Nankai District, Tianjin 300072, PR China
a r t i c l e
i n f o
Article history: Received 21 January 2009 Received in revised form 6 May 2009 Accepted 8 May 2009
Keywords: Enzyme Immobilized Biocatalyst preparation Kinetics Biomimetic hybrid capsule Isomaltooligosaccharide
a b s t r a c t Isomaltooligosaccharides (IMOs) are relatively new functional food ingredients which have great potential to improve the quality of many foods due to their low calories, no cariogenicity and safety for diabetics. To convert maltose to IMOs efficiently, ␣-transglucosidase was immobilized in a kind of alginate–chitosan–calcium phosphate hybrid capsules (Alg–Chi–CaP), which were prepared through a facile bio-inspired mineralization process. The surface morphology of Alg–Chi–CaP capsule and alginate–chitosan capsule (Alg–Chi) was characterized by scanning electron microscopy (SEM). Due to the presence of inorganic shell, immobilization efficiency of transglucosidase in Alg–Chi–CaP capsules was higher than that in Alg–Chi capsules. The optimal temperature (60 ◦ C) and pH (6.0) value for enzymatic conversion catalyzed by transglucosidase immobilized Alg–Chi–CaP capsules were identical to those catalyzed by free transglucosidase. As compared to the free enzyme, transglucosidase in Alg–Chi–CaP capsules exhibited significantly higher recycling stability and storage stability in a broader temperature and pH range. © 2009 Elsevier B.V. All rights reserved.
1. Introduction In recent years, considerable R&D efforts have been devoted to oligosaccharide engineering with the focus on exploring the function of oligosaccharides for mammalian metabolic process [1–3]. Oligosaccharides are relatively new functional food ingredients which have great potential to improve the quality of many foods. Various oligosaccharides [4], including isomaltooligosaccharides (IMOs) [5], fructooligosaccharide [6] and soybean oligosaccharides [7], have been widely used for food or feed additives [8] and scaffold [9,10] due to their potential advantages such as bifidusstimulating activity [11], low calorific value [12] and low cariogenic properties [13] etc. IMOs, comparing with other oligosaccharides, have received peculiar attention since they are very stable in acid solution, relatively low in price and have extensive sources [4]. Commercially available IMO is defined as saccharides that have 40% ␣-(1-6) glucosidic linkages among the total linkages. IMOs with the degrees of polymerization (DP) ranging from 2 to 6 are produced from corn starch by serial reactions of starch with ␣-amylase and -amylase and transglucosidase [14]. ␣-transglucosidase from Aspergillus niger [15,16] catalyzes the transglucosylation to the 6OH of the accepting glucose unit and yielded the oligosaccharides
∗ Corresponding author. Tel.: +86 22 2350 0086; fax: +86 22 2350 0086. E-mail address: [email protected] (Z. Jiang). 1369-703X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2009.05.008
with an a-D-(1-6) linkage including isomaltose, panose and isomaltotriose. Compared to free enzyme, immobilized enzyme improved the operational stability and reusability, which could better meet requirement for industrial application. However, as far as we were concerned, immobilized transglucosidase has not yet been utilized for enzymatic production of IMOs. The polymer–inorganic hybrid carrier have found increased application in enzyme immobilization due to its moderate hydrophilicity, controllable transport characteristics, and good physicochemical stability [17,18], which may create a benign microenvironment for enzymes [19]. At present, two kinds of configurations can be found for polymer–inorganic hybrid carriers: the mixed–matrix configuration [20] and the core–shell configuration [21]. Compared with the mixed–matrix configuration, the core–shell configuration could create a more nature-like environment for the immobilized enzyme. Chitosan and alginate are natural cationic or anionic polysaccharides, respectively, which have been successfully utilized in biomacromolecule encapsulation [22–25]. Furthermore, chitosan has been regarded as an efficient structure-directing agent for inorganic minerals [26]. Calcium phosphate, a principal component of hard tissues such as bone and tooth enamel [27,28], is of superior biocompatibility, insolubility and mechanical stability, which have been demonstrated to be suitable for the enzyme immobilization [29]. In this work, alginate–chitosan–calcium phosphate hybrid capsules (Alg–Chi–CaP) were employed to immobilize transglucosidase for efficient conversion of maltose to IMOs. The capsules
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were produced by a one-step method in which the deposition of a semi-permeable alginate–chitosan film around droplets of sodium alginate was coupled with in situ precipitation of calcium phosphate. Ca2+ cross-linked alginate containing transglucosidase was first coated with chitosan and then coated with calcium phosphate to format Alg–Chi–CaP capsules with enhanced mechanical strength and decreased enzyme leakage. The optimum catalytic condition, kinetic parameters, the recycling and storage stability of the immobilized transglucosidase were studied extensively.
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2.4. Immobilization of transglucosidase Sodium alginate [2.0% (w/v)] dissolved in HPO4 2− solution containing 3 mg/ml transglucosidase was prepared. For one batch, 1 ml of the mixture was added dropwise into a gently stirring Ca2+ -containing chitosan solution [1.0% (w/v)] with a syringe. The capsules formed were left immersed in the chitosan solution for 30 min, before being filtered off and rinsed in an excess of distilled water. 2.5. Activity assays of transglucosidase
2. Materials and methods 2.1. Materials Transglucosidase (EC3.2.1.20, from A. niger) was obtained from megazyme. Chitosan (deacetylation degree 75–85%, viscosity 20–200 cps) were obtained from Sigma–Aldrich. Sodium alginate (average molecular weight, 6.27 × 105 ) was obtained from Shanghai Tianlian, China. Maltose was obtained from Guangfu, China. All other reagents used were of analytical grade and were used without further purification.
2.2. Preparation of polymer–inorganic hybrid capsule Sodium alginate and disodium hydrogen phosphate (100 mM) solution were mixed to a final concentration of 2.0% (w/v). Chitosan and calcium chloride solution (100 mM) were mixed to a final concentration of 1.0% (w/v). Alginate–chitosan–calcium phosphate hybrid capsules (Alg–Chi–CaP) capsules were prepared by the dropwise addition of alginate solution into a gently stirring chitosan solution, using a 5 ml syringe with a 0.9 mm diameter needle as shown in Fig. 1. The capsules formed were left in the chitosan solution for 30 min, before being filtered off and rinsed in an excess of distilled water. Alginate–chitosan capsules (Alg–Chi) capsules were prepared following the same procedure, with the exception of alginate mixing in distilled water instead of HPO4 2− ions. All procedures were carried out at room temperature and pH 7.0.
The activities of free and immobilized transglucosidase were determined by the transglycosylation of maltose. The free or immobilized transglucosidase and 5 ml, 100 mg/ml maltose solution were mixed and the system was incubated in a water bath with constant shaking at different temperature for 10 min. The reaction was stopped by adding two times volume of acetonitrile reagent. Incubation was performed in a boiling water bath for 5 min. An enzyme activity unit (U) was defined as the amount of enzyme liberating 1 mg maltose per minute under the assay conditions. Each result was an average of four or five separate experiments. Maltose was quantified using high performance liquid chromatography (HPLC) operating on an analytical column (Tsk-gel Amide80, 5 m, 4.6 mm id × 250 mm). After 10 min reaction, 60 l of the digested maltose solution was diluted with 140 l of acetonitrile. HPLC was used to analyse the samples. For the elution conditions; the mobile phase was 70% acetonitrile and 30% water. Temperature was kept constant at 50 ◦ C, with a flow rate of 0.8 ml/min and an injection volume of 20 l. The detector was a Knauer Differential-Refractometer. In each set of experiment, a standard curve was plotted with maltose solutions of different concentrations. 2.6. Immobilization efficiency The immobilization efficiency of capsules was determined using the following equation. immobilization efficiency (%) = 100 −
2.3. Characterizations of capsules Intact capsules were observed with Zoom Stereo Microscope (Olympus SZ2-ILST). The morphology of capsule surface was observed with scanning electron microscopy (SEM, XL30, PHILIPS, Holand) using an accelerating voltage of 20 kV; before analyzing, the capsule was first freeze-dried and gold coated.
[transglucosidase]solution × Vsolution × 100 [transglucosidase]droplet × Vdroplet
(1)
where [transglucosidase]solution and [transglucosidase]droplet are the concentration of transglucosidase in the final solution and in the original liquid droplet, Vsolution and Vdroplet represent the volume of the solution and liquid drop, respectively. The transglucosidase concentration was determined by the enzyme activity.
Fig. 1. Schematic illustration of the enzyme immobilization process.
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2.7. Stirring speed
2.11. Recycling stability and storage stability
An experiment was done to choose the optimum stirring speed. The optimum stirring speed of reaction were determined by immersing transglucosidase-containing capsules into 5 ml, 100 mg/ml of maltose at 50 ◦ C and pH 6.0 for 30 min, and the concentration of transglucosidase changes were monitored at specified time intervals.
The recycling stabilities of the immobilized transglucosidase were evaluated by measuring the enzyme activity in each successive reaction cycle, and were expressed by recycling efficiency. The immobilized transglucosidase were allowed to take effect in maltose solution with pH 6 and 50 ◦ C (optimum). The immobilized transglucosidase was then filtered off, rinsed with distilled water, and then, used in the next reaction cycle. The process was repeated for 15 times. The activity found for each repetition was compared with the initial activity assuming it possessed 100% activity.
2.8. Leakage of transglucosidase from Alg–Chi and Alg–Chi–CaP capsules The leakage characteristics of capsules were determined by immersing capsules into 5 ml of deionized water at room temperature and 600 rpm/min, and the concentration of transglucosidase changes were monitored at specified time intervals. The leakage was calculated using the following equation: leakage (%) =
[transglucosidase]solution × Vsolution × 100 [transglucosidase]droplet × Vdroplet
(2)
2.9. Optimum conditions for enzyme activity The optimum temperature of the free and immobilized transglucosidase was evaluated by adding the enzyme into the maltose solution for 10 min under different temperature condition. Temperatures of 5, 25, 37, 50, 60, and 70 ◦ C were used for the experiment. The activity found at each temperature was compared with the activity at optimum temperature assuming it possessed 100% activity. enzyme activity at the specific temperature × 100 activity (%) = enzyme activity at optimum temperature (3)
The optimum pH of the free and immobilized transglucosidase was evaluated by adding the enzyme into the maltose solution for 10 min. Maltose solutions with pH of 2, 3, 4, 5, 6, 7, 8 and 9 were used. The activity found at each pH was compared with the activity at optimum pH assuming it possessed 100% activity. activity(%) =
enzyme activity at the specific pH × 100 enzyme activity at optimum pH
(4)
2.10. Determination of Km and Vmax values Activities of the free and immobilized transglucosidase were determined by using the classical Michaelis–Menten kinetics. In the graphical evaluation of Michaelis–Menten constants and maximum activities, Lineweaver–Burk plots obtained by plotting experimental values were used. Km 1 1 1 = × . + V Vmax Vmax [S]
(5)
V and [S] are the initial reactive rate and initial substrate concentration, respectively. Vmax is the maximum activity attained at infinite initial substrate concentration and Km is the Michaelis–Menten constant. To determine Vmax and the Km , the activity assay was applied for different maltose concentrations (10, 12.5, 16.7, 25, 50 and 100 mM). Activities of free and immobilized transglucosidase were all determined at the optimum conditions. The catalytic efficiencies of both free and immobilized transglucosidase were calculated accordingly.
recycling efficiency (%) =
enzyme activity on the storing for nth day × 100 initial enzyme activity on the 1st cycle
(6)
Many batches of the free and immobilized transglucosidase were prepared and stored in glass vials at 4 ◦ C to determine the storage efficiency. On the 1st, 2nd, 3rd, 5th, 7th, 10th, 13th, 15th, 17th, 20th, 23rd, 27th and 30th day, 1 ml of each type of the free and immobilized transglucosidase was added to 5 ml of maltose solution at pH 6 and under temperature of 50 ◦ C. storage efficiency (%) =
enzyme activity after storing for nth day × 100 initial enzyme activity
(7)
3. Results and discussion 3.1. Characterization of the capsules As shown in Fig. 3, Alg–Chi–CaP capsules were prepared by the dropwise addition of phosphate-containing sodium alginate solution into calcium-containing chitosan solution. Because of interfacial complexation of the oppositely charged polysaccharide, a thin chitosan film formed around the alginate droplets spontaneously. Counter-diffusion of the oppositely charged ions across the polysaccharide interface results in the in situ precipitation of calcium phosphate. The capsules became much stiffer due to further cross-linking between alginate and Ca2+ ions and the deposition of calcium phosphate [30]. The micrographs of Alg–Chi and Alg–Chi–CaP capsules were shown in Fig. 2a and b, respectively. It could be observed that the Alg–Chi capsules were more jelly-like whereas the Alg–Chi–CaP capsules were noticeably harder. Fig. 2c and d showed that the Alg–Chi–CaP capsule had smooth and intact surface structure, in comparison, the Alg–Chi capsule had a wrinkled surface structure, which was ascribed to the polymer shrunk during the freeze drying process. As shown in Fig. 2e and f, the internal surface of the capsule was Ca2+ -alginate hydrogel network and the external surface of the capsule was relatively smooth calcium phosphate layer. 3.2. Immobilization efficiency The immobilization efficiency could be assessed by measuring the transglucosidase concentration in the solution during the immobilization. The immobilization efficiency was 92.6% for Alg–Chi–CaP capsules, whereas it was 70.5% for Alg–Chi capsules. Due to existence of the external inorganic layer, the leakage of transglucosidase was considerably reduced during the capsules formation process.
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Fig. 2. Optical micrographs of (a) Alg–Chi capsules and (b) Alg–Chi–CaP capsules; SEM image of (c) Alg–Chi capsules, (d) Alg–Chi–CaP capsules, (e) Alg–Chi–CaP capsules interior and (f) Alg–Chi–CaP capsules exterior.
3.3. Stirring speed When stirring speed was greater than 400 rpm/min, it had trivial influence on reaction rate because the internal diffusion controlled the whole diffusion process as shown in Fig. 3. When the stirring
speed was less than 400 rpm/min, the reaction rate reached the highest, which was due to that the external diffusion constituted the control step for the whole diffusion process. In the following experiment, the stirring speed used was 400 rpm/min unless otherwise noted. 3.4. Leakage of transglucosidase To assess the leakage transglucosidase from the Alg–Chi and Alg–Chi–CaP capsules, both capsules were dip in deionized water at 1000 rpm/min. As seen in Fig. 4, extended time studies indicated that over 80% of the immobilized transglucosidase were leaked from the Alg–Chi capsules to the surrounding solution within a period of 5 h. In contrast, capsules prepared with calcium phosphate retained approximately 65% of the trapped transglucosidase after 7 h, indicating that in situ precipitation of calcium phosphate process was successful in significantly reducing the enzyme leakage from the capsules. 3.5. Thermal and pH stabilities of immobilized transglucosidase
Fig. 3. Effects of stirring speed (50 ◦ C, pH 6.0, reaction time 30 min) on the maltose conversion ratio catalyzed by immobilized transglucosidases.
The activity of free and immobilized transglucosidase was assayed at various temperatures (5–70 ◦ C) as shown in Fig. 5a. The results showed that optimum reaction temperature (50 ◦ C) was not affected by immobilization. The transglucosidase immobilized
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Fig. 4. The leakage of transglucosidase from Alg–Chi capsules and Alg–Chi–CaP capsules.
in Alg–Chi–CaP capsules displayed the broadest temperature profile, indicating that immobilization could render enzyme with a more benign environment which protected enzyme from heatinduced denaturation and allowed the enzyme to become less temperature-depending. At 70 ◦ C, approximately 88% and 66% of the initial activity was lost for the free enzyme and immobilized enzyme, respectively. It is well established that thermal inactivation starts with the unfolding of the protein molecule which is followed by irreversible changes due to aggregation and formation of scrambled structures which takes place
more in soluble form as compared to the immobilized state [31,32]. As shown in Fig. 5(b), the effect of pH on the activity of free and immobilized transglucosidase was studied (from pH 2.0 to 9.0.) and found that both free and immobilized transglucosidase were sensitive to the pH changes. The highest activity for immobilized transglucosidase was achieved at pH 6.0, completely consistent with that of free transglucosidase, which further supported that the conformation of the transglucosidase was well preserved after immobilization. Transglucosidase immobilized in Alg–Chi–CaP capsules retained 77% of its maximum activity at pH 2.0 and 53% at pH 9.0, whereas, free transglucosidase retained only 33% of its maximum activity at pH 2.0 and 29% at pH 9.0, respectively. Such changes are generally analyzed as a result of immobilization, which greatly helped in the stabilization of enzyme at a wider pH range [31,33]. Additionally, it was noticed that enzyme-containing capsules preserved higher activity in acid condition than in alkaline condition. The strong resistance of immobilized transglucosidase against the acidic medium was tentatively explained by the buffering effect of the alginate capsule. According to the previous report, the pKa of alginate ranges between 3.4 and 4.4, which was attributed to the ionization of the unbound carboxyl group (not bound to Ca2+ -ion) [34,35]. In acidic medium, the negatively charged alginate core could attract and consume the H+ ions, assisting in preventing H+ ion from diffusing into and contacting with the enzymes [36]. 3.6. Free and immobilized enzyme activity under optimal conditions Under optimum condition (50 ◦ C and pH 6.0), the enzyme activity of free and immobilized transglucosidase in Alg–Chi capsules and Alg–Chi–CaP capsules was investigated. As shown in Fig. 6, the bioconversion process was monitored by measuring the conversion of maltose with the lapse of time. The reaction rate and the final conversion rate using immobilized transglucosidase were slightly lower than that of free transglucosidase. The equilibrium conversion using free transglucosidase was obtained at 95.31% in 13 h, while that in the case of immobilized transglucosidase immobilized in Alg–Chi capsules was 96.99% in 13.83 h and in Alg–Chi–CaP capsules was 97.4% in 13.98 h. The enzyme activity unit was defined as the amount of transglucosidase needed to convert 1.0 mg of maltose/min at 50 ◦ C, pH 6.0. The specific activity of free transglucosidase was 1.22 U/mg, while specific activity of immobilized transglucosidase in Alg–Chi capsules and in Alg–Chi–CaP capsules was 1.17 and 1.18 U/mg, respectively. Lower specific activity of immobilized transglucosidase might be due to the additional diffusion resistance rather than enzyme denaturation.
Fig. 5. (a) Effects of temperature (pH 6.0, reaction time 10 min) on the activity of free and immobilized transglucosidases. (b) Effects of pH value (50 ◦ C, reaction time 10 min) on the activity of free and immobilized transglucosidases.
Fig. 6. Maltose conversion with reaction time (50 ◦ C, pH 6.0).
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3.7. Kinetic studies The Michaelis–Menten kinetics of free and immobilized transglucosidase was studied and the corresponding Michaelis constant (Km ) and maximum reaction rate (Vmax) were calculated from Lineweaver–Burk plots. The value of the maximum reaction rate (Vmax ) and the Michaelis constant (Km ) evaluated from Lineweaver–Burk plots were shown. Both the Km and the Vmax values were changed by the immobilization processes. The Vmax value of the transglucosidase in Alg–Chi capsules (1.025 U/mg) and Alg–Chi–CaP capsules (1.165 U/mg) was found to be lower than that of free transglucosidase (1.239 U/mg). The Km value of the transglucosidase in Alg–Chi (16.015 mM) and Alg–Chi–CaP (22.37 mM) capsules were found to be higher than that of the free transglucosidase (9.094 mM). The decreased Vmax value of the immobilized transglucosidase could be due to the additional diffusion limitation to the substrate (maltose) and the product (IMOs) caused by the carrier, which induced the low accessibility of the substrate to the active sites of the transglucosidase and consequently resulted in a lower possibility of enzyme–substrate complex formation. The Km value was known as the affinity of the enzymes toward substrates and the lower values of Km meant the higher affinity between enzymes and substrates. [37] The increase in Km after immobilization indicated a weaker binding between the maltose molecules and the immobilized transglucosidase. 3.8. Recycling stability and storage stability Enzymes were very sensitive to environmental conditions and might lose their activities quite easily. Thus, it was meaningful to characterize their recycling stability and storage stability for industrial application. To determine the recycling stability of the immobilized enzyme, the activity of transglucosidase in capsules was measured sequentially 15 times at the optimum condition (50 ◦ C and pH 6.0). After one cycle, capsules containing enzyme were removed from the reaction medium and washed twice with distilled water. As shown in Fig. 7, after the 7th reaction cycles, the transglucosidase in Alg–Chi capsule lost half of its initial activity, whereas in the Alg–Chi–CaP capsule, after the 15th cycles, it still retained 65% of its initial activity. The difference in recycling stability was due to the leakage of transglucosidase during the multiple soaking, separation, and washing processes employed in the reaction cycles. Whereas, for the transglucosidase immobilized in Alg–Chi–CaP capsule, transglucosidase leakage was prevented during recycling process and the recycling stability was increased substantially. The pore size of the inorganic layer should meet two requirements: it must be big enough for the substrates and products to pass through freely but small enough to effectively prevent the enzyme from leaking. Since BET analysis showed the average pore diameter of the Alg–Chi–CaP capsule was slightly bigger than 3 nm [38], it could be concluded that Alg–Chi–CaP capsule was effective as it allowed for the substrates and products (0.6 nm) to pass through, and meanwhile prevented transglucosidase which was larger than 3 nm from leaking from capsules. In addition, the free and immobilized enzymes were stored without any buffer at 4 ◦ C and their activities were tested for 30 days. The storage stability of free and immobilized transglucosidase was shown. Taking the initial activity level to be 100%, the relative activity of the free transglucosidase, transglucosidase immobilized in Alg–Chi and Alg–Chi–CaP capsules was decreased to 38%, 78%, and 85% after 30 days, respectively. The little differences in the storage stability of the Alg–Chi and Alg–Chi–CaP capsule indicated that the calcium phosphate played trivial role in improving storage stability. It was reasonably believed that the immobilized transglucosidase would exhibit a distinct advantage over free enzyme in
Fig. 7. (a) Recycling stability of immobilized transglucosidases in Alg–Chi capsules and Alg–Chi–CaP capsules. (b) Storage stability of free and immobilized transglucosidases.
long-time storage, owing to crowded microenvironment created by the biomimetic alginate capsule, which closely imitated the effects of crowding and confinement in a living cell. Alginate and transglucosidase (pI = 5.1) both bear net negative charges in a neutral pH environment. Therefore, the conformational transition of transglucosidase from a folded to an unfolded state was substantially inhibited by the electrostatic repulsion between transglucosidase and alginate molecules. Additionally, the biocompatible alginate helped the enzymes avoid the unfavorable attack possibly arising from the outside storage environment effectively. 4. Conclusions A facile method for preparing alginate–chitosan–calcium phosphate hybrid capsules (Alg–Chi–CaP) as efficient transglucosidase immobilization carrier was proposed. The biocompatible alginate-core accommodated the suitable microenvironment for transglucosidase, the outer calcium phosphate shell of capsule ensured the facile accessibility of immobilized transglucosidase for substrates and prevent transglucosidase from leaking out effectively. Owing to the synergy effect of hydrophilic polymers and mechanically stable calcium phosphate, the immobilized transglucosidase displayed higher thermal stability and pH stability than that in the free form and retained more than 60% initial activity after 15 repeated cycles. Additionally, compared to free transglucosidase, the immobilized transglucosidase exhibited improved storage stability. The facile immobilization process and the enhanced stability set an encouraging example for converting natural compounds into high value-added functional products.
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Immobilized transglucosidase in biomimetic polymer–inorganic hybrid capsules for efficient conversion of maltose to isomaltooligosaccharides Lei Zhang, Yanjun Jiang, Zhongyi Jiang ∗ , Xiaohui Sun, Jiafu Shi, Wei Cheng, Qianyun Sun Key Laboratory for Green Chemical Technology of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, 92 Weijin Road, Nankai District, Tianjin 300072, PR China
a r t i c l e
i n f o
Article history: Received 21 January 2009 Received in revised form 6 May 2009 Accepted 8 May 2009
Keywords: Enzyme Immobilized Biocatalyst preparation Kinetics Biomimetic hybrid capsule Isomaltooligosaccharide
a b s t r a c t Isomaltooligosaccharides (IMOs) are relatively new functional food ingredients which have great potential to improve the quality of many foods due to their low calories, no cariogenicity and safety for diabetics. To convert maltose to IMOs efficiently, ␣-transglucosidase was immobilized in a kind of alginate–chitosan–calcium phosphate hybrid capsules (Alg–Chi–CaP), which were prepared through a facile bio-inspired mineralization process. The surface morphology of Alg–Chi–CaP capsule and alginate–chitosan capsule (Alg–Chi) was characterized by scanning electron microscopy (SEM). Due to the presence of inorganic shell, immobilization efficiency of transglucosidase in Alg–Chi–CaP capsules was higher than that in Alg–Chi capsules. The optimal temperature (60 ◦ C) and pH (6.0) value for enzymatic conversion catalyzed by transglucosidase immobilized Alg–Chi–CaP capsules were identical to those catalyzed by free transglucosidase. As compared to the free enzyme, transglucosidase in Alg–Chi–CaP capsules exhibited significantly higher recycling stability and storage stability in a broader temperature and pH range. © 2009 Elsevier B.V. All rights reserved.
1. Introduction In recent years, considerable R&D efforts have been devoted to oligosaccharide engineering with the focus on exploring the function of oligosaccharides for mammalian metabolic process [1–3]. Oligosaccharides are relatively new functional food ingredients which have great potential to improve the quality of many foods. Various oligosaccharides [4], including isomaltooligosaccharides (IMOs) [5], fructooligosaccharide [6] and soybean oligosaccharides [7], have been widely used for food or feed additives [8] and scaffold [9,10] due to their potential advantages such as bifidusstimulating activity [11], low calorific value [12] and low cariogenic properties [13] etc. IMOs, comparing with other oligosaccharides, have received peculiar attention since they are very stable in acid solution, relatively low in price and have extensive sources [4]. Commercially available IMO is defined as saccharides that have 40% ␣-(1-6) glucosidic linkages among the total linkages. IMOs with the degrees of polymerization (DP) ranging from 2 to 6 are produced from corn starch by serial reactions of starch with ␣-amylase and -amylase and transglucosidase [14]. ␣-transglucosidase from Aspergillus niger [15,16] catalyzes the transglucosylation to the 6OH of the accepting glucose unit and yielded the oligosaccharides
∗ Corresponding author. Tel.: +86 22 2350 0086; fax: +86 22 2350 0086. E-mail address: [email protected] (Z. Jiang). 1369-703X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2009.05.008
with an a-D-(1-6) linkage including isomaltose, panose and isomaltotriose. Compared to free enzyme, immobilized enzyme improved the operational stability and reusability, which could better meet requirement for industrial application. However, as far as we were concerned, immobilized transglucosidase has not yet been utilized for enzymatic production of IMOs. The polymer–inorganic hybrid carrier have found increased application in enzyme immobilization due to its moderate hydrophilicity, controllable transport characteristics, and good physicochemical stability [17,18], which may create a benign microenvironment for enzymes [19]. At present, two kinds of configurations can be found for polymer–inorganic hybrid carriers: the mixed–matrix configuration [20] and the core–shell configuration [21]. Compared with the mixed–matrix configuration, the core–shell configuration could create a more nature-like environment for the immobilized enzyme. Chitosan and alginate are natural cationic or anionic polysaccharides, respectively, which have been successfully utilized in biomacromolecule encapsulation [22–25]. Furthermore, chitosan has been regarded as an efficient structure-directing agent for inorganic minerals [26]. Calcium phosphate, a principal component of hard tissues such as bone and tooth enamel [27,28], is of superior biocompatibility, insolubility and mechanical stability, which have been demonstrated to be suitable for the enzyme immobilization [29]. In this work, alginate–chitosan–calcium phosphate hybrid capsules (Alg–Chi–CaP) were employed to immobilize transglucosidase for efficient conversion of maltose to IMOs. The capsules
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were produced by a one-step method in which the deposition of a semi-permeable alginate–chitosan film around droplets of sodium alginate was coupled with in situ precipitation of calcium phosphate. Ca2+ cross-linked alginate containing transglucosidase was first coated with chitosan and then coated with calcium phosphate to format Alg–Chi–CaP capsules with enhanced mechanical strength and decreased enzyme leakage. The optimum catalytic condition, kinetic parameters, the recycling and storage stability of the immobilized transglucosidase were studied extensively.
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2.4. Immobilization of transglucosidase Sodium alginate [2.0% (w/v)] dissolved in HPO4 2− solution containing 3 mg/ml transglucosidase was prepared. For one batch, 1 ml of the mixture was added dropwise into a gently stirring Ca2+ -containing chitosan solution [1.0% (w/v)] with a syringe. The capsules formed were left immersed in the chitosan solution for 30 min, before being filtered off and rinsed in an excess of distilled water. 2.5. Activity assays of transglucosidase
2. Materials and methods 2.1. Materials Transglucosidase (EC3.2.1.20, from A. niger) was obtained from megazyme. Chitosan (deacetylation degree 75–85%, viscosity 20–200 cps) were obtained from Sigma–Aldrich. Sodium alginate (average molecular weight, 6.27 × 105 ) was obtained from Shanghai Tianlian, China. Maltose was obtained from Guangfu, China. All other reagents used were of analytical grade and were used without further purification.
2.2. Preparation of polymer–inorganic hybrid capsule Sodium alginate and disodium hydrogen phosphate (100 mM) solution were mixed to a final concentration of 2.0% (w/v). Chitosan and calcium chloride solution (100 mM) were mixed to a final concentration of 1.0% (w/v). Alginate–chitosan–calcium phosphate hybrid capsules (Alg–Chi–CaP) capsules were prepared by the dropwise addition of alginate solution into a gently stirring chitosan solution, using a 5 ml syringe with a 0.9 mm diameter needle as shown in Fig. 1. The capsules formed were left in the chitosan solution for 30 min, before being filtered off and rinsed in an excess of distilled water. Alginate–chitosan capsules (Alg–Chi) capsules were prepared following the same procedure, with the exception of alginate mixing in distilled water instead of HPO4 2− ions. All procedures were carried out at room temperature and pH 7.0.
The activities of free and immobilized transglucosidase were determined by the transglycosylation of maltose. The free or immobilized transglucosidase and 5 ml, 100 mg/ml maltose solution were mixed and the system was incubated in a water bath with constant shaking at different temperature for 10 min. The reaction was stopped by adding two times volume of acetonitrile reagent. Incubation was performed in a boiling water bath for 5 min. An enzyme activity unit (U) was defined as the amount of enzyme liberating 1 mg maltose per minute under the assay conditions. Each result was an average of four or five separate experiments. Maltose was quantified using high performance liquid chromatography (HPLC) operating on an analytical column (Tsk-gel Amide80, 5 m, 4.6 mm id × 250 mm). After 10 min reaction, 60 l of the digested maltose solution was diluted with 140 l of acetonitrile. HPLC was used to analyse the samples. For the elution conditions; the mobile phase was 70% acetonitrile and 30% water. Temperature was kept constant at 50 ◦ C, with a flow rate of 0.8 ml/min and an injection volume of 20 l. The detector was a Knauer Differential-Refractometer. In each set of experiment, a standard curve was plotted with maltose solutions of different concentrations. 2.6. Immobilization efficiency The immobilization efficiency of capsules was determined using the following equation. immobilization efficiency (%) = 100 −
2.3. Characterizations of capsules Intact capsules were observed with Zoom Stereo Microscope (Olympus SZ2-ILST). The morphology of capsule surface was observed with scanning electron microscopy (SEM, XL30, PHILIPS, Holand) using an accelerating voltage of 20 kV; before analyzing, the capsule was first freeze-dried and gold coated.
[transglucosidase]solution × Vsolution × 100 [transglucosidase]droplet × Vdroplet
(1)
where [transglucosidase]solution and [transglucosidase]droplet are the concentration of transglucosidase in the final solution and in the original liquid droplet, Vsolution and Vdroplet represent the volume of the solution and liquid drop, respectively. The transglucosidase concentration was determined by the enzyme activity.
Fig. 1. Schematic illustration of the enzyme immobilization process.
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2.7. Stirring speed
2.11. Recycling stability and storage stability
An experiment was done to choose the optimum stirring speed. The optimum stirring speed of reaction were determined by immersing transglucosidase-containing capsules into 5 ml, 100 mg/ml of maltose at 50 ◦ C and pH 6.0 for 30 min, and the concentration of transglucosidase changes were monitored at specified time intervals.
The recycling stabilities of the immobilized transglucosidase were evaluated by measuring the enzyme activity in each successive reaction cycle, and were expressed by recycling efficiency. The immobilized transglucosidase were allowed to take effect in maltose solution with pH 6 and 50 ◦ C (optimum). The immobilized transglucosidase was then filtered off, rinsed with distilled water, and then, used in the next reaction cycle. The process was repeated for 15 times. The activity found for each repetition was compared with the initial activity assuming it possessed 100% activity.
2.8. Leakage of transglucosidase from Alg–Chi and Alg–Chi–CaP capsules The leakage characteristics of capsules were determined by immersing capsules into 5 ml of deionized water at room temperature and 600 rpm/min, and the concentration of transglucosidase changes were monitored at specified time intervals. The leakage was calculated using the following equation: leakage (%) =
[transglucosidase]solution × Vsolution × 100 [transglucosidase]droplet × Vdroplet
(2)
2.9. Optimum conditions for enzyme activity The optimum temperature of the free and immobilized transglucosidase was evaluated by adding the enzyme into the maltose solution for 10 min under different temperature condition. Temperatures of 5, 25, 37, 50, 60, and 70 ◦ C were used for the experiment. The activity found at each temperature was compared with the activity at optimum temperature assuming it possessed 100% activity. enzyme activity at the specific temperature × 100 activity (%) = enzyme activity at optimum temperature (3)
The optimum pH of the free and immobilized transglucosidase was evaluated by adding the enzyme into the maltose solution for 10 min. Maltose solutions with pH of 2, 3, 4, 5, 6, 7, 8 and 9 were used. The activity found at each pH was compared with the activity at optimum pH assuming it possessed 100% activity. activity(%) =
enzyme activity at the specific pH × 100 enzyme activity at optimum pH
(4)
2.10. Determination of Km and Vmax values Activities of the free and immobilized transglucosidase were determined by using the classical Michaelis–Menten kinetics. In the graphical evaluation of Michaelis–Menten constants and maximum activities, Lineweaver–Burk plots obtained by plotting experimental values were used. Km 1 1 1 = × . + V Vmax Vmax [S]
(5)
V and [S] are the initial reactive rate and initial substrate concentration, respectively. Vmax is the maximum activity attained at infinite initial substrate concentration and Km is the Michaelis–Menten constant. To determine Vmax and the Km , the activity assay was applied for different maltose concentrations (10, 12.5, 16.7, 25, 50 and 100 mM). Activities of free and immobilized transglucosidase were all determined at the optimum conditions. The catalytic efficiencies of both free and immobilized transglucosidase were calculated accordingly.
recycling efficiency (%) =
enzyme activity on the storing for nth day × 100 initial enzyme activity on the 1st cycle
(6)
Many batches of the free and immobilized transglucosidase were prepared and stored in glass vials at 4 ◦ C to determine the storage efficiency. On the 1st, 2nd, 3rd, 5th, 7th, 10th, 13th, 15th, 17th, 20th, 23rd, 27th and 30th day, 1 ml of each type of the free and immobilized transglucosidase was added to 5 ml of maltose solution at pH 6 and under temperature of 50 ◦ C. storage efficiency (%) =
enzyme activity after storing for nth day × 100 initial enzyme activity
(7)
3. Results and discussion 3.1. Characterization of the capsules As shown in Fig. 3, Alg–Chi–CaP capsules were prepared by the dropwise addition of phosphate-containing sodium alginate solution into calcium-containing chitosan solution. Because of interfacial complexation of the oppositely charged polysaccharide, a thin chitosan film formed around the alginate droplets spontaneously. Counter-diffusion of the oppositely charged ions across the polysaccharide interface results in the in situ precipitation of calcium phosphate. The capsules became much stiffer due to further cross-linking between alginate and Ca2+ ions and the deposition of calcium phosphate [30]. The micrographs of Alg–Chi and Alg–Chi–CaP capsules were shown in Fig. 2a and b, respectively. It could be observed that the Alg–Chi capsules were more jelly-like whereas the Alg–Chi–CaP capsules were noticeably harder. Fig. 2c and d showed that the Alg–Chi–CaP capsule had smooth and intact surface structure, in comparison, the Alg–Chi capsule had a wrinkled surface structure, which was ascribed to the polymer shrunk during the freeze drying process. As shown in Fig. 2e and f, the internal surface of the capsule was Ca2+ -alginate hydrogel network and the external surface of the capsule was relatively smooth calcium phosphate layer. 3.2. Immobilization efficiency The immobilization efficiency could be assessed by measuring the transglucosidase concentration in the solution during the immobilization. The immobilization efficiency was 92.6% for Alg–Chi–CaP capsules, whereas it was 70.5% for Alg–Chi capsules. Due to existence of the external inorganic layer, the leakage of transglucosidase was considerably reduced during the capsules formation process.
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Fig. 2. Optical micrographs of (a) Alg–Chi capsules and (b) Alg–Chi–CaP capsules; SEM image of (c) Alg–Chi capsules, (d) Alg–Chi–CaP capsules, (e) Alg–Chi–CaP capsules interior and (f) Alg–Chi–CaP capsules exterior.
3.3. Stirring speed When stirring speed was greater than 400 rpm/min, it had trivial influence on reaction rate because the internal diffusion controlled the whole diffusion process as shown in Fig. 3. When the stirring
speed was less than 400 rpm/min, the reaction rate reached the highest, which was due to that the external diffusion constituted the control step for the whole diffusion process. In the following experiment, the stirring speed used was 400 rpm/min unless otherwise noted. 3.4. Leakage of transglucosidase To assess the leakage transglucosidase from the Alg–Chi and Alg–Chi–CaP capsules, both capsules were dip in deionized water at 1000 rpm/min. As seen in Fig. 4, extended time studies indicated that over 80% of the immobilized transglucosidase were leaked from the Alg–Chi capsules to the surrounding solution within a period of 5 h. In contrast, capsules prepared with calcium phosphate retained approximately 65% of the trapped transglucosidase after 7 h, indicating that in situ precipitation of calcium phosphate process was successful in significantly reducing the enzyme leakage from the capsules. 3.5. Thermal and pH stabilities of immobilized transglucosidase
Fig. 3. Effects of stirring speed (50 ◦ C, pH 6.0, reaction time 30 min) on the maltose conversion ratio catalyzed by immobilized transglucosidases.
The activity of free and immobilized transglucosidase was assayed at various temperatures (5–70 ◦ C) as shown in Fig. 5a. The results showed that optimum reaction temperature (50 ◦ C) was not affected by immobilization. The transglucosidase immobilized
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Fig. 4. The leakage of transglucosidase from Alg–Chi capsules and Alg–Chi–CaP capsules.
in Alg–Chi–CaP capsules displayed the broadest temperature profile, indicating that immobilization could render enzyme with a more benign environment which protected enzyme from heatinduced denaturation and allowed the enzyme to become less temperature-depending. At 70 ◦ C, approximately 88% and 66% of the initial activity was lost for the free enzyme and immobilized enzyme, respectively. It is well established that thermal inactivation starts with the unfolding of the protein molecule which is followed by irreversible changes due to aggregation and formation of scrambled structures which takes place
more in soluble form as compared to the immobilized state [31,32]. As shown in Fig. 5(b), the effect of pH on the activity of free and immobilized transglucosidase was studied (from pH 2.0 to 9.0.) and found that both free and immobilized transglucosidase were sensitive to the pH changes. The highest activity for immobilized transglucosidase was achieved at pH 6.0, completely consistent with that of free transglucosidase, which further supported that the conformation of the transglucosidase was well preserved after immobilization. Transglucosidase immobilized in Alg–Chi–CaP capsules retained 77% of its maximum activity at pH 2.0 and 53% at pH 9.0, whereas, free transglucosidase retained only 33% of its maximum activity at pH 2.0 and 29% at pH 9.0, respectively. Such changes are generally analyzed as a result of immobilization, which greatly helped in the stabilization of enzyme at a wider pH range [31,33]. Additionally, it was noticed that enzyme-containing capsules preserved higher activity in acid condition than in alkaline condition. The strong resistance of immobilized transglucosidase against the acidic medium was tentatively explained by the buffering effect of the alginate capsule. According to the previous report, the pKa of alginate ranges between 3.4 and 4.4, which was attributed to the ionization of the unbound carboxyl group (not bound to Ca2+ -ion) [34,35]. In acidic medium, the negatively charged alginate core could attract and consume the H+ ions, assisting in preventing H+ ion from diffusing into and contacting with the enzymes [36]. 3.6. Free and immobilized enzyme activity under optimal conditions Under optimum condition (50 ◦ C and pH 6.0), the enzyme activity of free and immobilized transglucosidase in Alg–Chi capsules and Alg–Chi–CaP capsules was investigated. As shown in Fig. 6, the bioconversion process was monitored by measuring the conversion of maltose with the lapse of time. The reaction rate and the final conversion rate using immobilized transglucosidase were slightly lower than that of free transglucosidase. The equilibrium conversion using free transglucosidase was obtained at 95.31% in 13 h, while that in the case of immobilized transglucosidase immobilized in Alg–Chi capsules was 96.99% in 13.83 h and in Alg–Chi–CaP capsules was 97.4% in 13.98 h. The enzyme activity unit was defined as the amount of transglucosidase needed to convert 1.0 mg of maltose/min at 50 ◦ C, pH 6.0. The specific activity of free transglucosidase was 1.22 U/mg, while specific activity of immobilized transglucosidase in Alg–Chi capsules and in Alg–Chi–CaP capsules was 1.17 and 1.18 U/mg, respectively. Lower specific activity of immobilized transglucosidase might be due to the additional diffusion resistance rather than enzyme denaturation.
Fig. 5. (a) Effects of temperature (pH 6.0, reaction time 10 min) on the activity of free and immobilized transglucosidases. (b) Effects of pH value (50 ◦ C, reaction time 10 min) on the activity of free and immobilized transglucosidases.
Fig. 6. Maltose conversion with reaction time (50 ◦ C, pH 6.0).
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3.7. Kinetic studies The Michaelis–Menten kinetics of free and immobilized transglucosidase was studied and the corresponding Michaelis constant (Km ) and maximum reaction rate (Vmax) were calculated from Lineweaver–Burk plots. The value of the maximum reaction rate (Vmax ) and the Michaelis constant (Km ) evaluated from Lineweaver–Burk plots were shown. Both the Km and the Vmax values were changed by the immobilization processes. The Vmax value of the transglucosidase in Alg–Chi capsules (1.025 U/mg) and Alg–Chi–CaP capsules (1.165 U/mg) was found to be lower than that of free transglucosidase (1.239 U/mg). The Km value of the transglucosidase in Alg–Chi (16.015 mM) and Alg–Chi–CaP (22.37 mM) capsules were found to be higher than that of the free transglucosidase (9.094 mM). The decreased Vmax value of the immobilized transglucosidase could be due to the additional diffusion limitation to the substrate (maltose) and the product (IMOs) caused by the carrier, which induced the low accessibility of the substrate to the active sites of the transglucosidase and consequently resulted in a lower possibility of enzyme–substrate complex formation. The Km value was known as the affinity of the enzymes toward substrates and the lower values of Km meant the higher affinity between enzymes and substrates. [37] The increase in Km after immobilization indicated a weaker binding between the maltose molecules and the immobilized transglucosidase. 3.8. Recycling stability and storage stability Enzymes were very sensitive to environmental conditions and might lose their activities quite easily. Thus, it was meaningful to characterize their recycling stability and storage stability for industrial application. To determine the recycling stability of the immobilized enzyme, the activity of transglucosidase in capsules was measured sequentially 15 times at the optimum condition (50 ◦ C and pH 6.0). After one cycle, capsules containing enzyme were removed from the reaction medium and washed twice with distilled water. As shown in Fig. 7, after the 7th reaction cycles, the transglucosidase in Alg–Chi capsule lost half of its initial activity, whereas in the Alg–Chi–CaP capsule, after the 15th cycles, it still retained 65% of its initial activity. The difference in recycling stability was due to the leakage of transglucosidase during the multiple soaking, separation, and washing processes employed in the reaction cycles. Whereas, for the transglucosidase immobilized in Alg–Chi–CaP capsule, transglucosidase leakage was prevented during recycling process and the recycling stability was increased substantially. The pore size of the inorganic layer should meet two requirements: it must be big enough for the substrates and products to pass through freely but small enough to effectively prevent the enzyme from leaking. Since BET analysis showed the average pore diameter of the Alg–Chi–CaP capsule was slightly bigger than 3 nm [38], it could be concluded that Alg–Chi–CaP capsule was effective as it allowed for the substrates and products (0.6 nm) to pass through, and meanwhile prevented transglucosidase which was larger than 3 nm from leaking from capsules. In addition, the free and immobilized enzymes were stored without any buffer at 4 ◦ C and their activities were tested for 30 days. The storage stability of free and immobilized transglucosidase was shown. Taking the initial activity level to be 100%, the relative activity of the free transglucosidase, transglucosidase immobilized in Alg–Chi and Alg–Chi–CaP capsules was decreased to 38%, 78%, and 85% after 30 days, respectively. The little differences in the storage stability of the Alg–Chi and Alg–Chi–CaP capsule indicated that the calcium phosphate played trivial role in improving storage stability. It was reasonably believed that the immobilized transglucosidase would exhibit a distinct advantage over free enzyme in
Fig. 7. (a) Recycling stability of immobilized transglucosidases in Alg–Chi capsules and Alg–Chi–CaP capsules. (b) Storage stability of free and immobilized transglucosidases.
long-time storage, owing to crowded microenvironment created by the biomimetic alginate capsule, which closely imitated the effects of crowding and confinement in a living cell. Alginate and transglucosidase (pI = 5.1) both bear net negative charges in a neutral pH environment. Therefore, the conformational transition of transglucosidase from a folded to an unfolded state was substantially inhibited by the electrostatic repulsion between transglucosidase and alginate molecules. Additionally, the biocompatible alginate helped the enzymes avoid the unfavorable attack possibly arising from the outside storage environment effectively. 4. Conclusions A facile method for preparing alginate–chitosan–calcium phosphate hybrid capsules (Alg–Chi–CaP) as efficient transglucosidase immobilization carrier was proposed. The biocompatible alginate-core accommodated the suitable microenvironment for transglucosidase, the outer calcium phosphate shell of capsule ensured the facile accessibility of immobilized transglucosidase for substrates and prevent transglucosidase from leaking out effectively. Owing to the synergy effect of hydrophilic polymers and mechanically stable calcium phosphate, the immobilized transglucosidase displayed higher thermal stability and pH stability than that in the free form and retained more than 60% initial activity after 15 repeated cycles. Additionally, compared to free transglucosidase, the immobilized transglucosidase exhibited improved storage stability. The facile immobilization process and the enhanced stability set an encouraging example for converting natural compounds into high value-added functional products.
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