Mesoporous silica particles were prepared by the sol-gel method from different alkoxysilane precursors and used as a host matrix for encapsulation of glucoamylase, an enzyme widely used in fermentative industry. The aim was to investigate the physico-chemical properties of the different silica powders and their effect on the enzyme kinetics. The encapsulated enzymes followed Michaelis-Menten kinetics. The Michaelis constant (KM) and the maximum rate of starch hydrolysis reaction (Vmax) were calculated according to the Michaelis-Menten and Lineweaver-Burke plots. The values of the Michaelis constant (KM) of the encapsulated enzymes were higher than those of the free enzyme. The temperature and pH inﬂ uence on the activity of free and immobilized glucoamylase were also compared. The results of this study show that the enzymes immobilized in organic/inorganic hybrid silica matrixes (obtained by the sol-gel method), allowing the entrapped glucoamylase to retain its biological activity, are suitable for many different applications, (medicinal, clinical, analytical)

A. Chiriac

B. Vlad-Oros

G. Preda

M. Dragomirescu

Z. Dudas

Processing and Application of Ceramics

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63Processing and Application of Ceramics 1 [1–2] (2007) 63–67Entrapment of glucoamylase by sol-gel technique in PhTES/TEOS hybrid matrixesB. Vlad-Oros1*, G. Preda1, Z. Dudas1, M. Dragomirescu2, A. Chiriac11Department of Chemistry, Faculty of Chemistry, Biology, Geography, West University of Timisoara, 300115, Timisoara, Romania,2Faculty of Animal Science and Biotechnologies, Banat University of Agricultural Sciences and Veterinary Medicine, 300645, Timisoara, RomaniaReceived 31 August 2007; received in revised form 16 November 2007; accepted 23 November 2007AbstractMesoporous silica particles were prepared by the sol-gel method from different alkoxysilane precursors and used as a host matrix for encapsulation of glucoamylase, an enzyme widely used in fermentative industry. The aim was to investigate the physico-chemical properties of the different silica powders and their effect on the enzyme kinetics. The encapsulated enzymes followed Michaelis-Menten kinetics. The Michaelis constant (KM) and the maximum rate of starch hydrolysis reaction (Vmax) were calculated according to the Michaelis-Menten and Lineweaver-Burke plots. The values of the Michaelis constant (KM) of the encapsulated enzymes were higher than those of the free enzyme. The temperature and pH infl uence on the activity of free and immobilized glucoamylase were also compared. The results of this study show that the enzymes immobilized in organic/in-organic hybrid silica matrixes (obtained by the sol-gel method), allowing the entrapped glucoamylase to retain its biological activity, are suitable for many different applications, (medicinal, clinical, analytical).Keywords: glucoamylase, phenyltriethoxysilane, sol-gel, kineticsI. IntroductionDue to the combination of unique properties, meso-porous silica matrixes are very promising materials for biotechnological applications. They possess such excel-lent basic features as large and accessible surface area, open porosity, narrow and uniform pore size distribu-tion and very convenient pore size [1].Gels are mostly interesting because an enzyme can be immobilized by building the porous network around it, obtaining effi cient and easy recyclable biocatalysts. The preference for silica is because the corresponding gels can easily be tailored to a large range of porous tex-tures, network structures, surfaces functionalities and processing conditions. The pH, gelation time, shaping, transparency or hydrophobicity can be adapted to a par-ticular enzyme or application [2,3].Biotechnology industries need biocatalysts easy to manipulate and recycle, thus the obtaining of hetero ge-* Corresponding author: tel: +40 256 592 625,fax: +40 256 592 620, e-mail: bvladoros@yahoo.comneous biocatalyst is an important subject for those fi elds. There are different methods of immobilization (adsorp-tion, covalent binding, cross-linking) among them is en-trapment, method that do not affect chemical integri-ty of enzyme. In the last years, inorganic materials are preferred for entrapment because of their advantages in comparison with the organic ones. Among the inorgan-ic materials, the silica matrixes, obtained by the sol-gel method, are used the most. The sol-gel method is a mild technique that makes compatible the synthesis of inor-ganic materials with biomolecules. In silica gels, espe-cially the mesoporous ones, both enzyme and cells were successfully immobilized [4,5]. The simultaneous im-mobilization of enzymes and cells allows one-pot syn-thesis of different products.Amyloglucosidase (glucoamylase, AMG) [α–1,4-D-glucan glucohydrolase (E.C. 3.2.1.3) ], is one of the most important enzyme for all starch based industries (food, textile, brewing, paper and alcohol industries) and also for the pharmaceutical and fi ne chemical in-dustries [6–8].64B. Vlad-Oros et al. / Processing and Application of Ceramics 1 [1–2] (2007) 63–67The aim of this paper was the immobilization of glu-coamylase, as a preliminary step in simultaneous im-mobilization of enzymes and cells, to obtain ethanol from starch. To prepare the hybrid silica matrixes, by the sol-gel method, different alkoxysilane derivatives (RSi (OEt)3 with R = methyl, ethyl, propyl, phenyl etc.) are used as gel precursors. To entrap glucoamylase we chose three matrixes: PhTES / TEOS (about which there are few literature data) and TEOS (for comparison). The physicochemical properties of the matrixes were stud-ied and then the effects of the temperature and pH on the immobilized glucoamylase activities and the kinetic parameters of these biomaterials were also investigated and compared with those of the native enzyme.II. Experimental ProcedureGlucoamylase from Aspergillus niger (300 U/mL) was obtained from Novo. Tetraethoxysilane (TEOS) and phenyltriethoxysilane (PhTES) were purchased from Aldrich (USA) and soluble Zulkowski starch from Merck (Germany). All other chemicals were of analyti-cal grade and were used without further purifi cation.For entrapping enzyme in silica matrixes, we used the sol-gel method of immobilization in one-step [9], TEOS and PhTES as gel precursors, NaF as catalyst, isopropyl alcohol as solvent and polyvinyl alcohol (PVA) as additive.Matrixes were prepared using tetraethoxysilane (TEOS) and a mixture of tetraethoxysilane (TEOS) and phenyltriethoxysilane (PhTES) in different molar ratios (PhTES: TEOS 1: 1 and 2: 1). 1.4 mL mixture of alk-oxides and 0.780 mL buffered enzymatic solution (22.5 U AMG in McIlvaine buffer, pH 4.6) were stirred with 200 μL 4% PVA 22.000 (polyvinyl alcohol 22.000), 100 μL NaF 1 M and 200 μL isopropyl alcohol. In a few minutes the mixture became homogenous and, shortly after, the gelation occurred. The gels were left overnight for aging (4°C), washed and dried (4°C).Specifi c surface areas were calculated from N2 ad-sorption isotherms at 77 K measured on a Quanta-chrome NOVA 2000 instrument using the multi-point Brunauer-Emmett-Teller (BET) method. The total pore volume (Vp) was calculated in the last point of adsorp-tion branch. Pore size distribution curves were calcu-lated from the adsorption (Dp [Ad]) and desorption (Dp [Ds]) branch of the isotherms using the Barrett– Joyner–Halenda (BJH) method.Enzyme assay: 0.5 mL soluble starch (1%), 0.4 mL citrate-phosphate buffer (McIlvaine buffer) pH 4.6 and 0.1 mL enzyme solution or 0.05 g immobilized biocat-alyst were kept for 5 min at 25°C. 1 mL 3,5-dinitrosal-icylic acid (DNS, 1%) was added. The samples were boiled in water 10 min, 10 mL water was added and then absorbance at 540 nm against blank (soluble starch, ci-trate-phosphate buffer, 3,5-dinitrosalicylic acid and dis-tilled water) was measured. One unit of AMG activity was defi ned as the amount of enzyme required to pro-duce 1 μmol of glucose in 5 min under the defi ned as-say condition [10].The effect of the temperature on the activity of na-tive and immobilized enzyme was estimated by mea-suring reducing sugars, released by action of enzyme at various temperatures. The test tubes were stored in a water bath at specifi c temperature (25, 30, 37, 45, 60, 70, 80 and 90°C).The effect of the pH on the activity of native and en-trapped enzyme was investigated by reducing sugars as-say, but in the presence of citric acid 0.1 M – Na2HPO4 0.2 M buffer ranged from pH 2.6 to 8 (McIlvaine buf-fer, [11,12]) at room temperature.a) b)Figure 1. Adsorption/desorption isotherm (a) and pore size distribution curves (b) of TEOS matrix containing glucoamylase65B. Vlad-Oros et al. / Processing and Application of Ceramics 1 [1–2] (2007) 63–67Figure 2. The effect of the pH on activity of immobilized and native glucoamylaseDetermination of kinetics parameters of native and immobilized enzyme: KM and Vmax were determined by measuring initial rates of Zulkowsky starch hydrolysis. Kinetics studies were conducted in citrate – phosphate buffer 0.15 M, pH 4.6, at 37°C in a 50 mL stirred jack-eted batch reactor. The starch concentrations were 1.5– 6.25 mg/mL. The reaction was started by addition of the enzyme (4 mL native enzyme and 40 mg immobilized enzyme) and 1 mL samples were collected every 2 min-utes, during the fi rst 20 minutes of the reaction. The re-ducing sugars assay was used for analyze the samples [13].III. Results and DiscussionThe aim of this work was to investigate the physico-chemical and biochemical properties of the glucoamy-lase immobilized in PhTES / TEOS and TEOS matrix-es.Physicochemical characterizationThe specifi c surface area and pore size distribution were determined by N2 adsorption and desorption meth-od at 77 K. The gels demonstrate the characteristics of mesoporous materials as it can be seen in Fig. 1 – iso-therm type IV with H3 hysteresis, according to IUPAC [14,15]. All the gels have presented the same type of isotherm. From their isotherms, the textural properties of the gels were determined as summarized in Table 1. The gels present mesoporous volumes and the introduc-tion of the organic groups into the gels results in a slight Table 1. Textural properties of glucoamylase – entrapped gelsMatrixDp (Ads)[Å]Dp (Des)[Å]SBET[m2/g]Vp[cm3/g]TEOS 33.01–231.36 38.39–256.77 153.1 0.686PhTES : TEOS 1 : 1 33.19–232.16 22.92–255.57 256.2 0.679PhTES : TEOS 2 : 1 28.65–229.35 33.03–253.04 214.7 0.595decrease in the pore volume. Pores diameters are quite similar, so probably other factors infl uenced the enzy-matic activities (increased hydrophobicity, modifi ed microenvironment).Biochemical characterizationIn biotechnological applications, the main parame-ters of the immobilized biocatalysts are pH and temper-ature, inhibitors and activators, substrate concentration. This work presents the immobilization infl uence on the temperature and pH profi le and also on the kinetic pa-rameters of the glucoamylase.The infl uence of the pH on the activity of the native and immobilized glucoamylases was investigated in the pH range 2.6–8, at room temperature. The optimum pH of the native enzyme was 4.6. After the immobiliza-tion, the optimum pH of the entrapped enzymes varies very little. Thus glucoamylase immobilized in matrix-es based on TEOS and PhTES : TEOS 2 : 1 has an op-timum pH of 5 and for the enzyme immobilized on ma-trix based on PhTES : TEOS 1 : 1 the optimum pH was 4 (Fig. 2).Figure 3. Temperature effect on activity of immobilized and native glucoamylaseThe effect of the temperature on the activities of the native and immobilized glucoamylase in the range 20– 90°C, was also studied. The optimal temperature for the native glucoamylase was 60°C and remains the same for the enzyme immobilized in matrix obtained from TEOS. In the case of the matrixes based on PhTES: TEOS, the optimal temperature for the entrapped en-66B. Vlad-Oros et al. / Processing and Application of Ceramics 1 [1–2] (2007) 63–67zyme was 37°C, decreased by 23°C in comparison to native glucoamylase (Fig. 3). Therefore, kinetic param-eters were determined at this temperature and not in standard conditions.Figure 4. Initial rates (v) vs. substrate concentration ([S]) plots of native and immobilized glucoamylaseA typical profi le for Zulkowsky starch hydrolysis using soluble and immobilized glucoamylase is shown in Fig. 4. Table 2 summarizes the kinetic parameters obtained by fi tting the experimental data to the Line-weaver-Burke linearization. The Michaelis constant values, for the immobilized glucoamylase, are higher than the KM of the native one, indicating a lower affi ni-ty to the substrate. These results could indicate the pres-ence of partitioning and diffusional effects in the pores of the sol-gel derived matrix or that glucoamylase suf-fers a conformational change, which affects the active site upon its entrapment into the matrixes (KM increas-es). In the case of the PhTES : TEOS matrixes, the Vmax is increased compared to the maximum velocity of the native enzyme. The immobilization in the TEOS ma-trix may also lead to a partial protein inactivation or se-questration (Vmax decreases). However, the Vmax/KM ratio shows that the best catalytic effi ciency was determined for the native enzyme. Among the immobilized ones, the Vmax/KM ratio indicates that glucoamylase entrapped in the most hydrophobic matrix provides the best ef-fectiveness when compared with the native counterpart. Thus, for entrapped enzymes, the best catalytic effi cien-cy was obtained in the case of PhTES : TEOS 2 : 1.Table 2. Kinetic parameters for the native and immobilized α-amylase, estimated by fi tting the experimental data to the Lineweaver-Burke linearizationSampleVmax KM Vmax100/KMNative enzyme 0.24  1.24 19.35TEOS 0.09  1.12  8.03PhTES : TEOS 1 : 1 0.66 10.71  6.16PhTES : TEOS 2 : 1 0.33  2.44 13.52IV. ConclusionsThe studied matrixes present mesoporous volumes, which allow a lower capillary pressure on the gel net-work and a faster internal diffusion of the substrate than the microporous sol-gel materials. However, the intro-duction of the organic groups into the gels determines a slight decrease in the pore volume.The slight variation in the optimum pH and tempera-ture proves that the sol-gel immobilization of glucoam-ylase in TEOS and PhTES : TEOS matrixes permits their use in the same pH and temperature conditions as the native counterpart.The encapsulated glucoamylase follows the Michae-lis-Menten kinetics. The Vmax/KM ratio, that shows the catalytic effi ciency, indicates PhTES : TEOS 2 : 1 as the most favorable matrix for glucoamylase immobiliza-tion, among those studied.Our results suggest that the sol-gel entrapment in TEOS and PhTES : TEOS matrixes is biocompatible al-lowing the entrapped glucoamylase to retain its biolog-ical activity.Acknowledgements: This work was supported by CEEX project, contract number 38/2005, subprogram 9 MATNANTECH.References1. D. Fattakhova-Rohlfi ng, M. Wark, J. Rathousky, “Elec-trode layers for electrochemical applications based on functionalized mesoporous silica fi lms”, Sensors and Actuators B: Chemical, 126 [1] (2007) 78–812. A.C. Pierre, “The Sol-Gel Encapsulation of Enzymes”, Biocatal. Biotransform., 22 [3] (2004) 145–1703. T. Ogoshi, Y. Chujo, “Organic-inorganic polymer hy-brids prepared by the sol-gel method”, Composite In-terfaces, 11 [8–9] (2005) 539–5664. Y. Wei, J. Xu, Q. Feng, H. Dong, M. 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Simpelkamp, “Effi cient im-mobilization of lipases by entrapment in hydrophobic sol-gel materials”, Biotechnol. Bioeng., 49 [5] (1996) 527–53410. ***BDH Enzyme Catalog, Poole, 1981.11. T.C. McIlvaine, “A buffer solution for colorimetric comparison”, J. Biol. Chem., 49 (1921) 183–186.12. D.D. Perrin, B. Dempsey, Buffers for pH and Metal Ion Control, Chapman and Hall, London, 1983.13. G. Preda, M. Dragomirescu, F. Peter, Biocatalizatori enzimatici. Obtinere, caracterizare, aplicatii, Mirton, Timisoara, 2003.14. IUPAC Recommendations, Pure Appl. Chem. 57 (1994) 1739–1758.15. F. Rosquerol, J. Rosquerol, K. Sing, Adsorption by Powder & Porous Solids, Academic Press, San Diego, 1999. 1728.

Entrapment of glucoamylase by sol-gel technique in PhTES/TEOS hybrid matrixes

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Entrapment of glucoamylase by sol-gel technique in PhTES/TEOS hybrid matrixes

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