Biomacromolecules 2005, 6, 1027-1030
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Advantages of the Pre-Immobilization of Enzymes on Porous Supports for Their Entrapment in Sol-Gels Lorena Betancor,† Fernando Lo´ pez-Gallego,† Aurelio Hidalgo,† Manuel Fuentes,† Ondrej Podrasky,‡ Gabriela Kuncova,‡ Jose´ M. Guisa´ n,*,† and Roberto Ferna´ ndez-Lafuente*,† Departamento de Biocata´ lisis, Instituto de Cata´ lisis, CSIC, Campus Universidad Auto´ noma, Cantoblanco, 28049 Madrid, Spain, and Department of Biotechnology and Environmental Processes, Institute of Chemical Process Fundamentals, Academy of Science of the Czech Republic, 165 02 Prague 6, Czech Republic Received November 2, 2004; Revised Manuscript Received November 19, 2004
In this work, we have compared the entrapment of free or previously immobilized glucose oxidase using a sol-gel technique. The preimmobilization was carried out on Sepabeads (a porous support) derivatized with glutaraldehyde as the functional group. The prior immobilization of the enzyme permitted to maintain the enzyme activity intact after the formation of the sol-gel. In fact, only 10% of the enzyme activity was lost whereas the soluble enzyme lost 60% of its initial activity. Additionally, enzyme leakage from the sol-gel matrix was avoided, which was relatively high when entrapping the soluble enzyme (39% of the enzyme activity was released after 16 h of incubation in a buffered solution). Moreover, the immobilized enzyme, inside the porous support, cannot be in contact with the sol-gel, and, therefore, it maintained the stability achieved by means of the multipoint covalent attachment on the Sepabeads support. Introduction Enzymes are commonly used for the development of biosensors because of their high specificity. However, considering their moderate stability, in many instances it is necessary to stabilize them, for example, by covalent immobilization, physical adsorption, cross-linking, encapsulation, or entrapment.1 Glucose oxidase (GOX) is one of the most extensively used enzymes in biosensors. This enzyme has been used in the development of devices for the self-monitoring of diabetic patients2,3 the determination of glucose in food and beverages4,5 and in fermentation tanks.6 In this context, new immobilization and signal transduction systems are being continuously developed. Some of these systems are quite sophisticated and expensive, which represent new challenges to achieve a better stability of the biological component of the biosensor.7 The sol-gel encapsulation of enzymes has recently seen important developments motivated by its wide applications in biosensors.8,9 Some of the advantages of this system for the preparation of biosensors are its hydrophilicity, biocompatibility, and easy integration into the sensing and transduction platform.10 Nevertheless, it has been reported that enzyme leakage is one of the main drawbacks of the physical entrapment of a * To whom correspondence should be addressed: Dr. Roberto Ferna´ndez-Lafuente and Dr. Jose´ M. Guisan. Fax: 34 91 585 47 60. Tel.: 34 91 585 48 09. E-mail:
[email protected] (R.F.-L.);
[email protected] (J.M.G.). † CSIC, Campus Universidad Auto ´ noma. ‡ Academy of Science of the Czech Republic.
biocatalyst using the sol-gel technique.11-13 This problem has led in some instances to the design of complicated protocols for the preparation of matrixes having a pore size adequate to allow the flow of substrates and products but small enough to prevent the elution of the entrapped biocomponent.11,14,15 Although in some instances an improvement in enzyme stability has been described by sol-gel entrapment16 during sol-gel formation, some enzymes may be inactivated by the trapping conditions or by negative interactions with the solgel components, which may promote a reduction of enzyme stability.15,17 Considering all these facts, the entrapment of enzymes that have been previously immobilized in porous supports may present several advantages. First, the large size of the support particle could prevent leakage of the biocatalyst even when sol-gel materials with large pores are used. Second, the immobilization inside the pores of the support could reduce any negative interaction between the enzyme and the solgel matrix. And third, specific immobilization protocols may be used to greatly improve the stability of the immobilized enzyme via multipoint covalent attachment.18-20 Experimental Section All experiments were performed at least in triplicate and the results are presented as their mean value. The experimental error was never over 5%. Materials. GOX from Aspergillus niger, 2,2′-azino-bis(3-ethylbenzathiazoline-6-sulfonic acid) (ABTS), horseradish peroxidase, glutaraldehyde, and tetramethyl orthosilicate
10.1021/bm0493077 CCC: $30.25 © 2005 American Chemical Society Published on Web 01/08/2005
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(TMOS) were purchased from Sigma (St. Luis, MO). Glucose was from Panreac (Barcelona, Spain). The aminated Sepabeads (EC-EA2, average particle size 20 µm) were kindly prepared and donated by Resindion SRL (Mitsubishi Chemical Corp.). All other reagents were of analytical grade. Activation of the matrix with glutaraldehyde was performed as previously described in ref 21. Activity Assay of Soluble GOX. GOX activity was determined spectrophotometrically by an increase in absorbance at 414 nm resulting from the oxidation of ABTS through a peroxidase-coupled system.22 The reaction mixture consisted of 1 mL of 100 mM sodium phosphate buffer at pH 6.0, 0.5 mL of 1 M glucose, 0.1 mL of a 10 mg/mL ABTS solution prepared in distilled water, and 0.1 mL of a 2 mg/mL peroxidase solution in 100 mM sodium phosphate buffer at pH 6.0. One enzymatic unit causes the oxidation of 1 µm of ABTS/ min at 25 °C and pH 6.0 under the specified conditions. GOX Immobilization on Sepabeads Supports Activated with Glutaraldehyde. A total of 10 g of Sepabeads ECEA2 activated with glutaraldehyde (glutaraldehyde-Sepabeads) was incubated with 30 mL of an enzymatic solution containing different GOX concentrations (from 2 to 100 mg/g of support) in 25 mM sodium phosphate buffer at pH 7.0. Periodically, samples of supernatant and suspension were withdrawn and their activities were assayed as described above. The suspension was left for 16 h at 25 °C under gentle shaking. After that, the immobilized preparation was filtered and washed with 5 volumes of 25 mM sodium phosphate buffer at pH 7.0. Entrapment of Different GOX Preparations in SolGel Derived Matrixes. The entrapment of GOX in silica gels was carried out by preparing thin layers of immobilized enzyme on 2.5 × 3 cm glass slides. Preparation of the Glass Slides. The glass slides were washed successively with toluene, acetone, and ethanol, and afterward, they were incubated in 1 M NaOH overnight. After that, they were washed thoroughly with double distilled water and sonicated for 20 min. The slides were dried at 120 °C for 2 h. Entrapment. A stock solution of pre-polymerized TMOS was prepared by acid catalysis. A total of 4.1 g of TMOS and 2 mL of ultrapure water were mixed and incubated for 5 min at -20 °C. This biphasic solution was vigorously shaken while 0.5 mL of 0.1 M HCl was added. The mixture was left for 5 min at -20 °C and then stored at 4 °C. After 24 h, 0.5 mL of pre-polymerized TMOS was mixed with 0.5 mL of a 50 mM solution of NaOH and vigorously agitated while 0.5 mL of free GOX solution or immobilized GOX suspension containing 0.2 UI/mL was added. The mixture was poured over the glass slides previously treated as mentioned above. The polymerization was completed within the following 2 min after enzyme addition. The amount of enzyme on each glass slide was calculated by weighing the slides before and after pouring the gels. Activity Assay of GOX Entrapped in Sol-Gel Derived Matrixes. GOX activity was measured in the sol-gel by immersion of the glass slides in 10-mL spectrophotometer cuvettes. The reaction mixture was prepared with 6 mL of
Betancor et al.
Figure 1. Glutaraldehyde-Sepabeads loading capacity with GOX. The experiment was carried out offering different amounts of enzyme to 1 g of support at pH 7 and 25 °C, showing the immobilization yield in each case.
100 mM sodium phosphate buffer at pH 6.0, 2.5 mL of 1 M glucose, 0.5 mL of a 10 mg/mL ABTS solution prepared in distilled water, and 0.5 mL of a 2 mg/mL peroxidase solution in 100 mM sodium phosphate buffer at pH 6.0. One enzymatic unit causes the oxidation of 1 µm of ABTS/ min at 25 °C and pH 6.0 under the specified conditions. Thermal Inactivation of Different GOX Preparations. Samples of the entrapped and nonentrapped preparations containing 0.03 IU/mL were incubated at 50 °C in 25 mM sodium phosphate buffer of pH 7.0, and, periodically, samples were withdrawn and their remaining activities were assayed as described above. In the case of the entrapped preparations, the glass slides were incubated in thermostated buffer in Petri dishes. Determination of Eluted Activity. The glass slides containing the entrapped enzyme were placed in a Petri dish containing 50 mL of 25 mM sodium phosphate buffer at pH 7.0 and 4 °C and submitted to the turbulence generated by a 2-cm magnetic stirrer at 1000 rpm. Periodically, samples of the supernatant were withdrawn and assayed for activity as previously described. Results Immobilization of the Enzyme on GlutaraldehydeSepabeads. The full immobilization of GOX on glutaraldehyde-Sepabeads occurred within 1 h of incubation. A 100% immobilization yield as well as a very high expressed activity (more than 90%) of the derivative was obtained. Figure 1 shows the studies to optimize the loading capacity of the support; even 80 mg of protein/g of support could be immobilized, without any negative effect on the expressed activity. This permitted a high volumetric activity. Figure 2 shows the thermal inactivation courses of the free enzyme and the immobilized enzyme. The immobilized enzyme was much more stable than the soluble enzyme; this immobilized preparation was used for further studies. Entrapment of Different GOX Preparations on the Sol-Gel. Both the immobilized and the soluble GOX were entrapped in sol-gels derived from TMOS. Table 1 shows the results of the expressed activity in each case. The recovered activity in the case of the Sepabeads derivative is much higher than that of the soluble enzyme after the gel polymerization. In fact, only 10% of the enzyme activity was lost using the previously immobilized preparation, while the soluble enzyme lost around 53% of its initial activity.
Enzymes on Porous Supports for Sol-Gels Entrapment
Figure 2. Thermal stability of soluble and Sepabeads-immobilized GOX. Diamonds, glutaraldehyde-Sepabeads-GOX derivative; squares, soluble GOX. The Sepabeads-immobilized GOX suspension and soluble GOX solution contained 0.03 IU/mL and were incubated at 45 °C in 25 mM sodium phosphate buffer at pH 7.0. More details are described in Methods. Table 1. Analysis of the Expressed and Eluted Activities of Different Entrapped Preparations entrapped preparation
expressed activitya (%)
eluted activityb (%)
retained activityc (%)
soluble GOX Sepabeads-GOX
47 92
39 0
28 92
a Relative to the initial entrapped activity. b Activity measured in the buffer solution after the leakage experiment (see Methods). c Expressed activity after the leakage experiment.
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Figure 4. Thermal stability of different GOX preparations. Diamonds, soluble GOX; squares, soluble GOX entrapped in the sol-gel matrix; triangles, Sepabeads-GOX suspension; circles, Sepabeads-GOX entrapped in the sol-gel matrix. In all cases 0.03 IU/mL were incubated in 25 mM sodium phosphate buffer at pH 7.0 at 50 °C. More details are described in Methods.
suggests that the sol-gel matrix may generate some unfavorable interactions with the enzyme.15,17 However, the previously immobilized enzyme keeps its activity almost unaltered after the entrapment in sol-gel matrixes. Figure 4 shows that the final stabilization of the immobilized enzyme was much higher than that of the entrapped soluble enzyme. Discussion
Figure 3. Thermal stability of soluble GOX and GOX entrapped in the sol-gel derived matrix. The experiment was carried out incubating 0.03 IU/mL at 50 °C in 25 mM sodium phosphate buffer at pH 7.0. Diamonds, soluble GOX; squares, soluble GOX entrapped in the solgel matrix. Further details are described in Methods.
Enzyme Leakage from the Sol-Gel. Eluted GOX activity was not detected from the sol-gel when using the immobilized glutaraldehyde-Sepabeads-GOX, even after exposure to turbulence for some hours (Table 1). This could be expected because the average pore diameter of the silica matrix was 78 Å (determined by mercury intrusion porosimeter)23 and the particle size of the Sepabeads was almost 10-fold higher. However, the sol-gel matrixes prepared with soluble GOX released 39% of their initial activity to the supernatant after being incubated during 16 h at 4 °C in a buffered solution (see Methods). Therefore, the final activity retained after the leakage assays was considerably much higher for the pre-immobilized preparation (92% of the initial activity) than for the soluble enzyme (only 28% of the initial activity). Thermal Stability of Sol-Gel Entrapped Samples. Figure 3 shows that the trapping of the soluble enzyme in the sol-gel resulted in a decrease of enzyme stability. This
Sol-gel trapping is an enzyme immobilization technique used in biosensor development where the enzyme should be in near contact with an electrode or other transducer. However, this technique may have some problems: leakage of the protein and negative matrix-enzyme interactions. On the other hand, immobilization of enzymes on pre-existing porous supports may give good stabilizations and protect the enzyme inside the pores from any negative interaction, but they can hardly be used in biosensor design. Here, we propose to consider both immobilization technologies not as parallel solutions to the enzyme immobilization but as complementary tools. Thus, in this work, we have shown some of the advantages that the previous immobilization of an enzyme on porous supports may have for its entrapment in sol-gel matrixes. The use of a porous support, such as Sepabeads, prevented the negative interactions of the silica matrix with the enzyme, which caused a high recovery of enzyme activity compared to the encapsulation of the soluble enzyme. Additionally, the lack of protein-matrix interactions when using porous supports may keep the enzyme properties unaltered after the trapping of the immobilized enzyme in the sol-gel, independently of the matrix properties. Finally, the loss of activity by enzyme leakage, very important when using the soluble enzyme, was fully prevented by this protocol. Moreover, it is possible to greatly stabilize the enzyme by using a suitable immobilization protocol and to keep this stabilization after the enzyme entrapment, independently of the trapping matrixes used. Hence, the pre-immobilization of the enzyme on a porous support may be a first step in the preparation of a sol-gel, taking benefits from both techniques: the enzyme stabilization possibility by multipoint covalent attachment on pre-existing supports and the use of sol-gel matrixes for the preparation of biosensors. We can
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assume that these advantages may be extrapolated to any other encapsulation method. Acknowledgment. This work has been partially founded by the European Project EU: GRD2001-C40477 and the Spanish CICYT (BIO2000-0747-C05-02 and BIO20012259). The authors thank Resindion SRL for material delivery and financial support. We gratefully recognize the postdoctoral fellowships for A.H. (Gobierno Vasco) as well as the predoctoral fellowship for F.L.-G. from Spanish MCYT. References and Notes (1) Malhotra, B.; Singhal, R.; Chaubey, A.; Sharma, S.; Kumar, A. Recent trends in biosensors. Curr. Appl. Phys. 2005, 5, 92-97. (2) Shichiri, M.; Asakawa, N.; Yamasaki, Y.; Kawamori, R.; Abe, H. Telemetry glucose monitoring device with needle-type glucose sensor: a useful tool for blood glucose monitoring in diabetic individuals. Diabetes Care 1986, 9, 298-301. (3) Jaffari, S. A.; Turner, A. P. Recent advances in amperometric glucose biosensors for in vivo monitoring. Physiological Measurement 1995, 16, 1-15. (4) Tothill, I.; Newman, J.; White, S. F.; Turner, A. Monitoring of the glucose concentration during microbial fermentation using a novel mass-producible biosensor suitable for on-line use. Enzyme Microb. Technol. 1997, 20, 590-596. (5) Yang, M.; Yang, Y.; Liu, B.; Shen, G.; Yu, R. Amperometric glucose biosensor based on chitosan with improved selectivity and stability. Sens. Actuators, B 2004, 101, 269-276. (6) Brooks, S.; Ashby, R.; Turner, A P. F.; Calder, M. R.; Clarke, D. J. Development of an On-line Glucose Sensor for Fermentation Monitoring. Biosensors 1987-1988, 3, 45-56. (7) Malhotra, B.; Chaubey, A. Biosensors for clinical diagnostics industry. Sens. Actuators, B 2003, 91, 117-120. (8) Pierre, A. C. The Sol-Gel Encapsulation of Enzymes. Biocatal. Biotransform. 2004, 22, 145-170. (9) Kuncova, G.; Szilva, J.; Hetflejs, J.; Sabata, S. Catalysis in Organic Solvents with Lipase Immobilized by Sol-Gel Technique. J. Sol.Gel Sci. Technol. 2003, 26, 1183-1187. (10) Singh, A. K.; Gupta, A.; Mulchandani, A.; Chen, W.; Bhatia, R. B.; Schoeniger, J.; Ashley, C. S.; Brinker, C. J. Encapsulation of enzymes and cells in sol-gel matrices for biosensor applications. Proc. SPIEInt. Soc. Opt. Eng. 1999, 3858-3864.
Betancor et al. (11) Przybyt, M.; Bialkowska, B. Enzyme electrodes constructed on the basis of oxygen electrode with oxidases immobilised by sol-gel technique. Mater. Sci. 2002, 20, 63-79. (12) Blandino, A.; Macias, M.; Cantero, D. Glucose oxidase release from calcium alginate gel capsules. Enzyme Microb. Technol. 2000, 27, 319-324. (13) Veronese, F. M.; Mammucari, C.; Schiavon, F.; Schiavon, O.; Lora, S.; Secundo, F.; Chilin, A.; Guiotto, A. Pegylated enzyme entrapped in poly(vinyl alcohol) hydrogel for biocatalytic application II. Farmaco 2001, 56, 541-547. (14) Blandino, A.; Macı´as, M.; Cantero, D. Immobilization of glucose oxidase within calcium alginate gel capsules. Process Biochem. 2001, 36, 601-606. (15) Jin, W.; Brennan, J. D. Properties and Applications of Proteins Encapsulated within Sol-Gel Derived Materials. Anal. Chim. Acta 2002, 461, 1-36. (16) Zhou, H. X.; Dill, K. A. Stabilization of proteins in confined spaces. Biochemistry 2001, 40, 11289-11293. (17) Lin, J.; Brown, C. W. Sol-gel glass as a matrix for chemical and biochemical sensing. Trends Anal. Chem. 1997, 16, 200-211. (18) Blanco, R. M.; Guisa´n, J. M. Stabilization of enzymes by multipoint covalent attachment to agarose aldehyde gels. Borohydride reduction of trypsin-agarose derivatives. Enzyme Microb. Technol. 1989, 11, 361-363. (19) Guisa´n, J. M. Aldehyde agarose gels as activated supports for immobilization-stabilization of enzymes. Enzyme Microb. Technol. 1988, 10, 375-382. (20) Ichikawa, S.; Takano, K.; Kuroiwa, T.; Hiruta, O.; Sato, S.; Mukataka, S. Immobilization and stabilization of chitosanase by multipoint attachment to agar gel support. J. Biosci. Bioeng. 2002, 93, 201206. (21) Guisan, J. M.; Rodriguez, V.; Rosell, C. M.; Soler, G.; Bastida, A.; Ferna´ndez-Lafuente, R.; Garcı´a-Junceda, E. In Methods in Biotechnology, 1: Immobilization of enzymes and cells; Bickerstaff, G., Ed.; Humana Press: Totowa, NJ, 1997; Vol. 1, p 261. (22) Bateman, R. C., Jr.; Evans, J. A. Using the Glucose Oxidase/ Peroxidase System in Enzyme Kinetics. J. Chem. Educ. 1995, 72, A240. (23) Kassama, L. S.; Ngadi, M. O. Pore structure characterization of deepfat-fried chicken meat. J. Food Eng. 2005, 66, 369-375.
BM0493077
