Annals of West University of Timisoara

Annals of West University of Timisoara Series Chemistry 15 (2) (2006) 221-226 IMMOBILIZATION OF BACTERIAL ALPHA-AMYLASE IN/ON INORGANIC SUPPORTS Beatrice Vlad-Oros a, Monica Dragomirescu b, Ioana Stan a, Gabriela Preda a, J. Halasz c, A. Chiriac a a West University of Timişoara, Faculty of Chemistry, Biology, Geography, Department of Chemistry, Pestalozzi, 16, Timişoara, 300115, ROMANIA b Banat University of Agricultural Sciences and Veterinary Medicine, Faculty of Animal Science and Biotechnologies, Calea Aradului, 119, Timişoara, 300645, ROMANIA c University of Szeged, Department of Applied and Environmental Chemistry, Rerrich Sq. 1, H-6720 Szeged, HUNGARY Received: Modified: Accepted: SUMMARY Amylases are among the most important hydrolytic enzymes for all starch based industries. In this light, the encapsulation of microbial amylases by sol-gel method has drawn an increasing interest. The aim of this paper was to find the optimal sol gel process for the entrapment of α-amylase. Three different methods to immobilize α-amylase were used: entrapment, by sol-gel method, adsorption and entrapment/deposition. The enzyme was entrapped in silica matrices by sol-gel method, at different water/teos ratio and different enzyme loading, in the presence of stabilizing agent. The activities of the immobilized α-amylase were assayed and compared with that of the native enzyme. Keywords: α-amylase, tetraethoxysilane, sol-gel, entrapment, adsorption, entrapment/deposition INTRODUCTION Bioorganically doped sol-gel materials have drawn great interest because of the various potential applications of such materials in optical and electrochemical sensors, biosensors, diagnostic devices, catalysts and even bioartificial organs [1, 2]. Since the 90 s, when Avnir and co-workers first reported direct immobilization of 221

V LAD-OROS B., D RAGOMIRESCU M. ET AL. alkaline phosphatase in silica matrix by sol-gel technique [3], the encapsulation of biomolecules in sol-gel materials has been the topic for a great number of scientific papers. Enzymes, proteins, antibodies, antigens, viruses, bacteria, and even whole cells have been immobilized in various glass matrices in different forms (fibers, thin films, monoliths or granules). Bioactivity together with the flexibility to shape materials makes these hybrid materials attractive for application ranging from medicine to industry or environmental technology. The entrapped enzymes retain their functionality but have higher thermal, storage and ph stability in comparison with the native ones. The main advantage of using sol-gel materials for entrapment of enzymes is that they permit stabilization of tertiary structure of protein molecules, because of their tight gel structure. The protective action of the sol-gel cage also prevents leaching and allows the penetration of the substrate molecules. However, the activity of an entrapped enzyme is often hindered by internal diffusion, and sometimes, by reduced accessibility in non-templated microporous sol-gel matrices, with pore diameters typically < 15 Å, even if the synthesis is optimized to preserve the labile biomolecules [4]. Amylases, enzymes which hydrolyze starch molecules to form dextrins and smaller polymers composed of glucose units, are significantly important in the today biotechnology with applications in a variety of industry fields (food, fermentation, textile and papers industries). α-amylase is a metalloenzyme, which contains at least one Ca 2+ ion and has a molecular weight of 50-60 kda (microbial α-amylase). The ubiquitous occurrence, the divers application and the low cost of α-amylase make it a good model for investigation [5]. The aim of this paper was to find the optimal sol gel process for the entrapment of α-amylase. Three different methods to immobilize α-amylase were used: entrapment, by sol-gel method, adsorption and entrapment/deposition. The enzyme was entrapped in silica matrices by sol-gel method, at different water/teos ratio and different enzyme loading, in the presence of stabilizing agent. The activities of the immobilized α-amylase were assayed and compared with that of the native enzyme. MATERIALS AND METHODS Bacterial α-amylase (E.C. 3.2.1.1), tetraetoxysilane (TEOS) and polyethylene glycol (PEG-400) were obtained from Fluka. Soluble potatoes starch was purchased from Bender & Hobein. Celite C 22 was from Carlo Erba. Folin-Ciocâteus phenol reagent was obtained from Merck. All other chemicals were of analytical grade and were used without further purification. The immobilization of enzyme was performed in three different ways: 222

IMMOBILIZATION OF BACTERIAL ALPHA-AMYLASE IN/ON INORGANIC SUPPORTS 1. A silica sol was obtained from TEOS, ethanol and water (1.25:1.25:1, v/v), in acid catalysis (HCl 1N). Then the sol was mixed with ethanol and water (1.33:1.33:1, v/v), 5 drops NH 3 12% and 1.25 ml buffered enzymatic solution, containing 25 mg α-amylase. The gelation occurred in a few minutes. The gels were left overnight for aging, washed with acetone and distilled water and dried. 2. The gelation was also performed in the presence of 1 g inorganic support, celite (entrapment/deposition). 3. α-amylase was immobilized, by physical adsorption, on inorganic supports (celite and xerogel obtained by sol-gel technique but in the absence of the enzyme): 25 mg α-amylase in 1.8 ml distilled water and 0.5 g support were stirred for 3 h at 25 C, washed with water and acetone, filtered and dried. The activity of α amylase was spectrophotometrically assayed by using soluble potatoes starch as substrate (Hitachi 1100 spectrophotometer). Residual starch concentration assay (I 2 /I - ): 0.5 ml soluble starch (0.4%), 0.4 ml phosphate buffer (0.05 M, ph 5.2) and 0.01 g immobilized biocatalyst were kept for 5 min. at 25ºC. 5 ml solution I 2 /I - M/1000 and 15 ml distilled water were added. The samples were filtered. The absorbance was measured at 595 nm against distilled water. One unit of α-amylase activity was defined as the amount of enzyme required to hydrolyze 1 mg starch in 5 min at 25 C when 2 mg starch was present at the start of the reaction. Protein concentration was determined by the Lowry method [6]: 0.2 ml enzyme solution, 5 ml cupper alkaline reagent and 0.5 ml Folin-Ciocalteus phenol-reagent were kept for 30 min. at 25 C. The samples were filtered and assayed at 660 nm against water. A bovine serum albumin calibration curve was used. The surface area measurements were performed by gas adsorption in the Quantachrome instruments, NOVA 2000 Series, high speed gas sorption analyzer version 7.02. The samples were previously degassed to below 50 mm Hg at room temperature and analyses were performed at 77 K, using liquid nitrogen. The equilibration interval was 5 s. Specific surface areas were determined by the BET method in the 0.05 0.3 relative pressure and pore diameter distributions were calculated by BJH model on adsorption branch. RESULTS AND DISCUSSION The molar ratio of water to precursor influences the structural evolution of sol-gel materials because of the role of water in the hydrolysis and condensation processes [7]. 2 moles of water are produced in the condensation step per mole Si(OH) 4, and 4 moles of water are consumed in the hydrolysis step for a net stoichiometric consumption of 2 moles water per mole TEOS in the overall reaction. Theoretically, a water:teos ratio, R, of 2 is sufficient for complete hydrolysis and condensation as water is formed in the 223

V LAD-OROS B., D RAGOMIRESCU M. ET AL. condensation process, but generally the reactions do not go to completion under these conditions because of the formation of intermediate species [8]. In this study, R values of 6, 9, 12, 18, 24 and 30 were used. The best results were obtained for a ratio of 24. Activity slightly increased with increasing R value up to 24 and then decreased at higher R values. 18 16 I 2 /I - Activity (U/g) 14 12 10 8 6 4 2 0 6 9 12 18 24 30 Figure 1. Influence of H 2O/TEOS ratio on immobilized α-amylase activity r TEOS/H 2 O To optimize the immobilization parameters the enzyme loading was also assayed. The enzyme activity increases with enzyme loading until a maximum, then decreases, probably because of internal diffusional limitations and crowding effect on the enzyme molecules. Despite very high enzyme loading the immobilization yields remained relatively small (5-15%). The best results were obtained for 75 mg α-amylase/5ml sol. I2/I - Activity (U/g) 224 20 18 16 14 12 10 8 6 4 2 0 25 50 75 100 mg α-amylase Figure 2. Influence of enzyme loading on the immobilization efficiency Generally additives such as polyethylene glycol, polyvinyl alcohol, bovine serum albumin, gelatin are used to preserve enzyme activity. To evaluate the effect of PEG concentration, the entrapment of α-amylase was performed according to method 1 in the presence of PEG as additive. Instead of water we added PEG-400 4% solution in an

IMMOBILIZATION OF BACTERIAL ALPHA-AMYLASE IN/ON INORGANIC SUPPORTS appropriate volume so the product would contain different amounts of additive. The results showed that the addition of 2 ml PEG-400 4% solution slightly improved the entrapped enzyme activity. 8 I2/I - Activity (U/g) 7.8 7.6 7.4 7.2 7 Figure 3. Influence of PEG on immobilized α-amylase 6.8 0 1 2 3 ml PEG α-amylase was entrapped using a combined method, attempting to improve the performance of the immobilized enzyme by exploiting the positive attributes of each technique. We immobilized α-amylase by sol-gel technique - method 1 and then we added different inorganic supports celite and silica. For comparison α-amylase was immobilized on silica and celite by physical bonding, according to method 3. The effectiveness of these three immobilization techniques (entrapment, entrapment/deposition and adsorption) was compared (Figure 4). Though the adsorption gives higher activities, because of the weak bonds involved desorption of the enzyme is one of the main disadvantages of this method. Therefore the method of choice should be the one who combine the positive characteristics of different immobilization methods for obtaining improved entrapped enzyme. I 2 /I - Activity (U/g) 70 60 50 40 30 20 10 0 1 - silica/celite (Entrapment/deposition) 2 - celite (Physical bonding) 3 - silica (Entrapment) 4 - silica (Physical bonding) 1 2 3 4 Figure 4. The influence of the immobilization method We investigated the main physical properties of the catalysts, obtained by sol-gel method. Brunauer-Emmett-Teller (BET) surface area and pore parameters were determined 225

V LAD-OROS B., D RAGOMIRESCU M. ET AL. by N 2 adsorption-desorption isotherms and are summerized in the Table I. The quite low activities obtained suggest that the method should be fine-tuned in order to obtain mesopore networks which are much more favorable for the enzymatic activity, provide enough freedom for the conformational transitions and reduce diffusional hindrances. Table I. The catalyst surface parameters Immobilized enzyme Total pore volume [cm 3 /g] BET area [m 2 /g] Pore max. radius [Å] α-amylase 0.375 324.7 83.27 CONCLUSION These preliminary results suggest a simple route to immobilize enzymes in sol-gel materials which can be readily used in various fields of industry. The immobilization conditions (e.g. H 2 O/TEOS ratio, enzyme loading, additives, etc.) should be optimized as these parameters have a significant role to play in the enhancement of enzymatic activities. Sol-gel encapsulation is a versatile method of immobilization that offers several advantages compared to the other techniques. Progress has still to be made in order to improve the performance of the immobilized enzymes. REFERENCES 1. Gill I., Ballesteros A., "Bioencapsulation within synthetic polymers (Part 1): sol-gel encapsulated biologicals", Trends Biotechnol., 18 (2000) 282-296 2. Livage J., Coradin T., Roux C., Encapsulation of biomolecules in silica gels, J. Phys.:Condens. Matter, 13 (2001) R673-R691 3. Braun S., Rappoport S., Zusman R., Avnir D., Ottolenghi M., Biochemically active sol-gel glasses: The trapping of enzymes., Matter. Lett., 10 (1990) 1 4. Badjic J., Kostic N.M., Effects of encapsulation in sol-gel silica glass on esterase activity, conformational stability, and unfolding of bovine carbonic anhydrase II. Chem. Mater., 11 (1999) 3671 3679 5. Gupta R., Gigras P., Mohapatra H., Goswami V.K., Chauhan B. "Microbial α-amylases: A biotechnological perspective", Process Biochem., 38 (2003) 1599-1616 6. Lowry O.H., Rosebrough N.I., Farr A.L., Randall R.J, Protein measurement with the Folin- Phenol reagents. J. Biol. Chem., 193 (1951) 265 275 7. McDonagh C., Sheridan F., Butler T., MacCraith B.D., Characterization of sol-gel-derived silica films, J. Non-Crystalline Solids, 194 (1996) 72-77 8. Brinker J., Scherer G.W., Sol-Gel Science: The Physics and Chemistry of Sol-Gel Processing, Academic Press, Boston, 1990 226