S O g O a b c a A R R A A K B I S A G 1 b a t p m i p n s e c m p c c v 3 f r m 1 h Process Biochemistry 48 (2013) 1054–1058 Contents lists available at SciVerse ScienceDirect Process Biochemistry jo u r n al homep age: www.elsev ier .com/ locate /procbio hort communication ptimization of the immobilization of sweet potato amylase using lutaraldehyde-agarose support. Characterization of the immobilized enzyme lga Luisa Tavanoa,∗, Roberto Fernandez-Lafuenteb, Antonio José Goulart c, Rubens Monti c Department of Nutrition, ICS–Universidade Federal do Triângulo Mineiro, Rua Getulio Guaritá, 159 Uberaba, MG, CEP 38025 360, Brazil Department of Biocatalysis, ICP-CSIC, Campus UAM-CSIC, Cantoblanco, Madrid ZC 28049, Spain Department of Food Nutrition, Faculty of Pharmaceutical Sciences, UNESP–São Paulo State University, Rodovia Araraquara-Jaú, Km1, 14840-000, Araraquara, SP, Brazil r t i c l e i n f o rticle history: eceived 25 March 2013 eceived in revised form 29 April 2013 ccepted 13 May 2013 vailable online 20 May 2013 a b s t r a c t A simplified procedure for the preparation of immobilized beta-amylase using non-purified extract from fresh sweet potato tubers is established in this paper, using differently activated agarose supports. Beta-amylase glutaraldehyde derivative was the preparation with best features, presenting improved temperature and pH stability and activity. The possibility of reusing the amylase was also shown, when this immobilized enzyme was fully active for five cycles of use. However, immobilization decreased eywords: eta-amylase mmobilization weet potato garose enzyme activity to around 15%. This seems to be mainly due to diffusion limitations of the starch inside the pores of the biocatalyst particles. A fifteen-fold increase in the Km was noticed, while the decrease of Vmax was only 30% (10.1 U mg−1 protein and 7.03 U mg−1 protein for free and immobilized preparations, respectively). © 2013 Elsevier Ltd. All rights reserved. lutaraldehyde . Introduction Beta-amylases are enzymes that attack the alpha-1,4-glucan onds from the non-reducing ends of starch, and convert it nd other carbohydrate polymers to maltose units. Maltose, a wo glucose disaccharide, has many applications in food and harmaceutical industries [1–4]. Amylases are widely present in icroorganisms, plants and animals, and have found applications n numerous industries, mainly starch liquefaction [5–7]. Maltose, roduced via beta-amylolysis, confers to cooked roots the sweet- ess characteristic of the traditional sweet potato [8]. Although tarch liquefaction can be accomplished via chemical processes, nzymatic hydrolysis can be performed under mild conditions, and ould avoid the extreme conditions required by the chemical treat- ents. Furthermore, the enzymatic process did not produce water ollution, which is very common in chemical processes [9]. The use of immobilized enzymes in industrial processes, in omparison with the use of soluble enzymes, could reduce pro- ess costs by reducing the quantity of enzyme required, since the ∗ Corresponding author at: Nutrition Department–Science Health Institute, Uni- ersidade Federal do Triangulo Mineiro–Brazil, Rua Getúlio Guaritá, 159–sala 21–CEP 38025 440–Uberaba - Minas Gerais, Brazil. Tel.: +55 34 3318 5839; ax: +55 34 3318 5839. E-mail addresses: tavanool@yahoo.com.br (O.L. Tavano), fl@icp.csic.es (R. Fernandez-Lafuente), antoniogoulart@yahoo.com (A.J. Goulart), ontiru@fcfar.unesp.br (R. Monti). 359-5113/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. ttp://dx.doi.org/10.1016/j.procbio.2013.05.009 immobilized derivative can be recovered at the end of a hydrolysis cycle and reused, as long as the enzyme remains active for sev- eral reaction cycles. Thus, enzyme immobilization and stabilization should be related terms and, in fact, a proper immobilization may improve enzyme stability via multipoint or multisubunit immo- bilization [10,11]. Enzyme immobilization and its application in continuous processes are desirable, as long as an end product of high purity is obtained, as is typical of enzymatic processes [12,13]. Different supports can be used to immobilize an enzyme via differ- ent physical or chemical phenomena, perhaps involving different regions of the protein and yielding different orientations [14]. As has been previously described, reversible immobilization and cova- lent immobilization may be more or less adequate depending on the requirements [15]. The ionic exchange of a very stable enzyme on an anion exchanger (e.g., aminated supports) may provide a way of reusing the enzyme several cycles, and recover the support and reuse it again after inactivation [15]. However, if stabilization is pursued, an intense multipoint covalent attachment between the enzyme and the support should be the objective of the immobiliza- tion [10]. This strategy can produce a very high stabilization of the enzyme by reducing the mobility of the enzyme structure, although may be difficult to optimize. The choice of an appropriate support and suitable immobiliza- tion conditions can favor reaching high stabilization factors and good activity values of immobilized amylases, simplifying the use of this enzyme in different industrial applications. Glyoxyl [16] and glutaraldehyde [17] activated supports have been described as ade- dx.doi.org/10.1016/j.procbio.2013.05.009 http://www.sciencedirect.com/science/journal/13595113 http://www.elsevier.com/locate/procbio mailto:tavanool@yahoo.com.br mailto:rfl@icp.csic.es mailto:antoniogoulart@yahoo.com mailto:montiru@fcfar.unesp.br dx.doi.org/10.1016/j.procbio.2013.05.009 iochem q G L b b a t e a f a l e S f p l a s l i t l t 2 2 t E p 2 l 2 r w l o A u s 2 s 2 c d A w m 2 w t w d s s a g O.L. Tavano et al. / Process B uate to produce stabilization via multipoint covalent attachment. lyoxyl-agarose support immobilizes the enzyme by the richest in ys residues areas [18,19], and glutaraldehyde-agarose can immo- ilize by the most reactive amino group (likely the terminal one) or y the most negatively charged areas, or even by hydrophobic inter- ctions depending on the conditions of immobilization [17,20]. In amylase immobilization, one problem to be considered is he large size of the substrate, the starch [6,21]. In these cases, nzyme activity may decrease by different causes. As it occurs for ny enzyme, the activity may decrease if the enzyme areas relevant or its activity become distorted [22]. But in this case, the enzyme ctivity will also be hindered by two diffusion problems. First, the arge size of the substrate may produce diffusion limitations to the ntry of this large molecule to the pores of the biocatalyst [15]. econd, if the active center is oriented towards the support sur- ace, it will be unavailable for the substrate [14]. Moreover, sweet otato beta amylase presents an additional problem for immobi- ization. Crystallographic studies demonstrate that the enzyme is tetramers [23,24]. This means that immobilization of all enzyme ubunits will be convenient to prevent subunits dissociation, stabi- ized also each monomer via multipoint covalent attachment, and n any case, reducing the risks of final product contamination by he enzyme [11]. In this study, amylase extracted from sweet potato was immobi- ized on agarose beads activated following different protocols and he best derivative was further characterized. . Material and methods .1. Materials Agarose 6B beads were from Amersham Biosciences. Soluble starch potato, dini- rosalicylic acid, sodium periodate, sodium bohohydride were from Sigma-Aldrich. thylendiamine was from Fluka, and glutaraldehyde 25% was from Vetec. The sweet otatoes used were obtained at the local market. .2. Methods Experiments were performed by triplicate and the values are the mean of at east 3 independent experiments. Standard deviations were always under 10%. .2.1. Amylase extraction The sweet potatoes used were obtained at the local market. The fresh tuberous oots were washed, peeled and diced. Fresh sweet potato tuber just after collection ere used, to avoid high alpha amylase contamination, considering that alpha amy- ase tended to increase during storage [8,25]. For the beta-amylase extraction, 50 g f the potato pieces were added to 100 mL of cold distilled water and pulverized. fter centrifugation (7000 × g/20 min/4 ◦C) the supernatant (amylase extract) was sed for all assays. The enzyme extract presented about 4.7 mg protein mL−1 and a pecific activity of 17.05 U mg−1 protein. No further purification methods were used. .2.2. Protein determination Protein concentration was determined by Bradford method [26], using bovine erum albumin (BSA) as standard. .2.3. Enzymatic activity Enzyme activity was determined using starch as substrate (1% w/v in 100 mM itrate-phosphate buffer at pH 6.0). Starch hydrolysis (50 ◦C) was monitored by etermination of reducing sugar using dinitrosalicylic acid method at 540 nm [27]. standard curve was prepared with maltose. One beta-amylase activity unit (U) as defined as the amount of enzyme capable of producing 1 �Mol of maltose per inute under assay conditions. .2.4. Supports preparation Agarose 6B was from Amersham Biosciences. Glyoxyl-agarose was prepared ith the maximal activation degree, as previously described [28]. The consump- ion of periodate (directly related to the aldehyde residue generated in the support) as checked after oxidation process, adding 0.2 mL of the supernatant of the oxi- izing suspension to a mixture of 1.5 mL of 10% (w/v) KI and 1.5 mL of saturated odium bicarbonate. The absorbance was read at 419 nm, considering the initial odium periodate solution as 100% (0% aldehyde production). Monoaminoethyl-N- minoethyl (MANAE)-agarose, was prepared as described elsewhere [29] and the lutaraldehyde-agarose support was prepared as previously described [17]. istry 48 (2013) 1054–1058 1055 2.2.5. Enzyme Immobilization The different immobilization supports were suspended in an enzyme solu- tion (10 g agarose beads:100 mL enzyme solutions in immobilization buffers). The immobilization buffers differ for each immobilization protocol: 100 mM sodium bicarbonate buffer pH 10.2 when immobilization on a glyoxyl support was per- formed [18]; 5 mM sodium phosphate buffer pH 7.0 for immobilization on MANAE support; or 200 mM sodium phosphate buffer pH 7.0 for immobilization on glu- taraldehyde support [17,20]. The immobilization suspensions were gently stirred at room temperature. Different enzyme concentrations were tested. For all immo- bilization experiments, samples of the suspensions and the supernatants were periodically withdrawn and enzyme activity of the samples was determined as described above. A reference enzyme suspension identical to each immobilization suspension was prepared, using inert agarose, as a reference. The biocatalysts prepared using glyoxyl agarose were reduced by adding sodium borohydride to reach 1 mg mL−1 [16] and maintained under agitation for 30 min. When using glutaraldehyde supports, the immobilized enzyme was recovered by filtration and resuspended in 10 volumes of 100 mM bicarbonate buffer at pH 10.2 containing 1 mg mL−1 sodium borohydride for 30 min under mild stirring. This treat- ment reduces the remaining aldehyde groups and the imino bonds. The resulting derivatives were washed with abundant distilled water and, before use, with activity buffer. 2.2.6. Effect of temperature and pH on the free enzyme and derivatives Enzyme activity was determined as described above, using different tempera- tures and different pH values (citrate-phosphate buffer for pH 3.0–8.0 range and glycine –NaOH buffer for pH 9.0 and 10.0). 2.2.7. Thermal and pH stability Aliquots of the immobilized and free enzyme samples were incubated at differ- ent pH values for 30 min, at 25 ◦C, and the remaining activity was assayed at pH 6.0, for pH stability determination. When thermal stability was assayed, aliquots of the immobilized and free enzyme were incubated at pH 6.0, at 60 ◦C. Periodically, sam- ples of these suspensions or solutions were withdrawn, placed in ice baths for 30 s and the remaining activities assayed. For both studies the initial activity is regarded as 100%, and residual activity was expressed as a percentage of initial activity. 2.2.8. Km and Vmax determination In order to calculate the Km and Vmax values, starch was used in increasing concentrations, as described by Chang and Juang [30] (using 1.0–16.0 mg mL−1), prepared in pH 6.0, to free enzyme, and at pH 6.5 for the enzyme immobilized form, at 50 ◦C. Lineweaver-Burk plots were used to determine the kinetic parameters. 2.2.9. Reuse assay The immobilized enzyme was used as described in 2.2.3 but for several cycles. At the end of each cycle, the derivative was washed with distilled water and activity buffer and a new substrate solution was added to start a new round of reaction. The initial and remaining activities were assayed at pH 6.0, 50 ◦C (initial activity at these conditions is regarded as 100%). 3. Results and discussion 3.1. Selection of the immobilization support Derivatives were prepared using high activated supports with the objective of achieving an intense multipoint covalent attach- ment or an intense adsorption [10,15]. Through titration of the remaining sodium periodate, an activation degree of 106 micromoles of glyoxyl groups mL−1 packed agarose beads was determined. Therefore, we can assume that each enzyme molecule has many reactive groups under its surface after immobilization. If the enzyme interacts with the supports through many points, enzyme stability by their three-dimensional structure “rigidifica- tion” may be obtained [10,31]. The beta-amylase immobilization was performed by using glyoxyl, MANAE and glutaraldehyde-agarose supports under the conditions described in methods. Different protein:support ratios were tested for each support (data not shown). The expressed activity gradually increased whilst 100% immobilization was main- tained; with the highest possible loads being obtained using MANAE and glutaraldehyde supports (Table 1). Using the glyoxyl- agarose support, it was not possible to achieve high percentages of immobilization, even using the lowest protein loading. This low loading using glyoxyl supports could be due to the lack of a region bearing many exposed Lys residues able to react with the enzyme 1056 O.L. Tavano et al. / Process Biochemistry 48 (2013) 1054–1058 Table 1 Immobilization parameters of �-amylase from sweet potato on differently activated agarose supports. Support Initial protein charge (�g g−1 support) Immobilization (%)a Activity Expectedb (U g−1 derivative) Derivative Activity (U g−1 derivative) Activity Recoveredc Glyoxyl 40 47.2% 0.35 0.0734 20.6% MANAE 154.8 100% 2.6 0.1934 7.3% Glutaraldehyde 163.9 100% 2.8 0.4643 16.6% a (Initial protein concentration − final protein concentration in supernatant solution/initial protein concentration) × 100. b −1 ein. ( t p t e a l c i p w o g a t a 3 g t i o b v 3 c s t t m i e b i e t e [ c a e l [ T K g the optimum pH, from 5.0 to 5.6. Very interestingly, the immobi- lized amylase maintained high activity in all the range of pH values studied, even in the most extreme ones (pH 3.0, and 10.0), while the free enzyme dramatically decreased its activity by just moving A 0 10 20 30 40 50 60 70 80 90 100 2 3 4 5 6 7 8 9 10 11 pH Ac tiv ity (% ) B 30 40 50 60 70 80 90 100 Re si du al a ct iv ity (% ) Considering specific activity of enzyme extract equivalent to 17.05 U mg prot c (Total activity in derivative/Total initial activity) × 100. glyoxyl groups are able only to fix protein to the support if mul- ipoint covalent attachment is established [18]), or due to steric roblems for the reaction between an area rich in Lys of the pro- ein and the support due to the presence of sugar chains, as this nzyme is glycosylated [32]. Although the three supports were ble to immobilize the enzyme, the expressed activities were very ow, especially when the MANAE support was used (Table 1). This ould be due to enzyme desorption during washings if the enzyme mmobilization was quite weak. However, the activity of the sus- ension before washing was as low as the activity recovered after ashings, suggesting that a real inactivation of the enzyme had ccurred. The glutaraldehyde-agarose derivative was the support that ives the highest immobilization yields and expressed activity mong the three assayed ones (Table 1). Based on these observa- ions, the glutaraldehyde immobilized derivative has been used for ll further studies. .2. Kinetic parameters of beta-amylase immobilized on lutaraldehyde-agarose beads The moderate activity recovered even in the case of the glu- araldehyde support may be due to the three reasons explained n introduction: enzyme distortions, external diffusion limitations r steric problems to the enzyme-substrate interaction generated y the support surface. Immobilization procedure raised the Km alue by about 15 times, while decreasing the Vmax by only a 0% (Table 2). The apparent increase in Km and the relative good onservation of Vmax for the glutaraldehyde derivative (Table 2) uggested that the main problem is just diffusion problems for he entry of the substrate inside the biocatalyst particle. In fact, he milling (just by magnetic stirring) of the catalyst particle per- itted to increase the observed activity. The negative effects of mmobilization on both kinetic constants decreased the catalytic fficiency of beta-amylase (Vmax/Km values showed in Table 2) y a 20 fold factor. Chang and Juang [30] also observed a decrease n apparent affinity between beta-amylase and starch when the nzyme was immobilized on chitosan-clay composite, although he Km increase was only about 2.5 times. The Km for the free nzyme was very near to our study (2.4 mg mL−1). In Roy and Edge’s 33] study, when the enzyme was immobilized on polystyrene ation exchange resin equilibrated with Al3+ íons, its Km increased bout 6 times. Differences on the particles size, pores diameter and nzyme loading may drastically influence the effect of diffusional imitations on the expressed activity of immobilized enzymes 15,22]. able 2 inetic parameters for starch hydrolysis with free and immobilized �-amylase- lutaraldehyde biocatalyst. Enzyme preparation Km (mg mL−1) Vmax (U mg−1 prot) Vmax/Km Soluble 2.17 10.1 4.7 Glutaraldehyde-agarose 34.47 7.03 0.20 3.3. Characterization of the beta amylase immobilized on glutaraladehyde agarose beads The goal of these experiments was not to find the optimal conditions for the different enzyme preparations, but to show a first comparison between the immobilized and the free enzyme. Fig. 1A shows the effect of pH on activity of free and immobilized beta-amylase. Both preparations have better results when citrate- phosphate buffer was used, but the free enzyme presented optimal pH value at pH 6.0, while the immobilized amylase had a maximum activity at pH 6.5. Roy and Edge [33] also observed an increase in 0 10 20 3 4 5 6 7 8 9 10 pH of i ncubation Fig. 1. Effect of pH on free (open symbol) and immobilized (closed symbol) beta- amylase activity (A) and stability (B). The effect of pH on the enzyme activity (A) was measured at 50 ◦C. The effect of pH on enzyme stability (B) was measured as detailed in Methods. The enzyme preparations were incubated at different pH values for 30 min at room temperature (25 ◦C), and the remaining activity was assayed in pH 6.0, at 50 ◦C (initial activity is regarded as 100%). For all measurements were used citrate-phosphate buffer (pH 3.0–8.0) and Glicin-NaOH buffer (pH 9.0–10.0). O.L. Tavano et al. / Process Biochemistry 48 (2013) 1054–1058 1057 A 0 10 20 30 40 50 60 70 80 90 100 20 25 30 35 40 45 50 55 60 65 70 75 80 temperature (°C) A ct iv ity ( % ) B 0 10 20 30 40 50 60 70 80 90 100 0 3 6 9 12 15 18 21 24 Time (h) R es id ua l a ct iv ity (% ) Fig. 2. Effect of temperature on activity (A) of free (open symbol) and immobi- l o a o t t o e s t e 1 a [ O i a i e h s u t t i i w n e o o l a 0 20 40 60 80 100 120 A ct iv ity (% ) 1° use 2° use 3°use 4°use 5°use reaction cycles Fig. 3. Re-use of beta-amylase glutaraldehyde-agarose derivative for several reac- tion cycles. The initial and remaining activities were assayed at pH 6.0, 50 ◦C (initial ized (closed symbol) beta-amylase from sweet potato. Thermal inactivation courses f free and immobilized enzyme (B) were studied at 60 ◦C and pH 6.0. Remaining ctivity was assayed at 50 ◦C (The initial activity is regarded as 100%). ne pH unit far from the optimal pH value. In fact, at pHs higher han 8.0, the immobilized enzyme showed greater activity than he free form (Fig. 1A). This could be related to a higher stability f the enzyme. In fact, Fig. 1B shows that immobilization greatly nhanced the stability of the enzyme at the highest and lowest tudied pH values. The free enzyme can not withstand the incuba- ion at pH < 4.0 or pH > 7.5 for 30 min. In contrast, the immobilized nzyme presented a retained activity over 70% even at pH 3.0 or 0.0. Thus, the improved activity of the immobilized enzyme at lkaline and acid pH value can be associated to this higher stability 22], perhaps produced by prevention of enzyme dissociation [11]. ther authors have also identified an increased performance of the mmobilized beta-amylase in extreme ranges of pH. For example, ctivity of beta-amylase was improved at pH 4.0 and pH > 7.0 when mmobilized on Chitopearl BCW 3505 beads [34]. The optimum temperature for activity was also increased after nzyme immobilization (Fig. 2A). The free enzyme presented the ighest activity at 50 ◦C; however, the immobilized enzyme pre- ented maximum activity at 60 ◦C, maintaining a high activity even p to 80 ◦C (at this temperature the free enzyme was almost inac- ive). Fig. 2B shows that the enzyme was actually more stable than he free enzyme at 60 ◦C, and that may be the explanation for this mproved features found in the immobilized enzyme. After 12 h of ncubation, the immobilized derivative retained about 60% activity, hilst the free form retained just 30% activity (Fig. 2B). The time eeded to reach 50% of residual activity was about 3 h for the free nzyme and about 19 h for immobilized preparation. Rigidification f the enzyme structure via multipoint attachment and reduction f dissociation problems via multi-subunit immobilization are the ikeliest explanations, for these good results. The application of this biocatalyst in industry requires the oper- tional stability of immobilized enzymes to be high enough. Fig. 3 activity is regarded as 100%). At the end of each cycle, the derivative was washed with distilled water and activity buffer and a new substrate solution was added to start a new round of reaction. shows that the activity of this immobilized enzyme was fully retained for five cycles of use. This suggested that the enzyme did not suffer dissociation during use, as enzyme is submitted to several washings/dilution and the activity did not decrease. 4. Conclusions It has been established a simplified procedure for the prepa- ration of immobilized beta-amylase using non-purified extract from fresh sweet potato tubers. Although the procedure was very straightforward, an enzymatic derivative preparation with good catalytic properties was obtained. Beta-amylase from sweet potato is a promising element for study, in both its free or immobilized form because of its easy-to-find and cheap source, and its stabil- ity and immobilization possibility. Beta-amylase glutaraldehyde derivative has shown to be very interesting and offer advantages over their free form, very likely to the multipoint immobilization of the enzyme. It is possible to produce very stable derivatives, with both improved temperature and pH stability. The only drawback of the immobilization is the decrease in enzyme activity, that seems to be mainly due to diffusion limitations of the starch inside the pores of the enzyme, that could be modulated by using supports with higher pore diameter (e.g., agarose 4%) or by the use of lower par- ticle size (this point may promote some difficulty to the industrial management of the biocatalyst) [22]. Acknowledgements We would like to thank CNPq and PADC-FCFAr for financial support. We gratefully recognize the support from the Spanish Gov- ernment, grant CTQ2009-07568. The help and comments from Dr. Ángel Berenguer (Instituto de Materiales, Universidad de Alicante) are kindly acknowledged. References [1] Reddy NS, Nimmangadda A, Sambasiva RKRS. An overview of the microbial �-amylase family. African J Biotechnol 2003;2:645–8. [2] Schramm M, Loyter A. Purification of alpha-amylase by precipitation of amylase-glycogen complex. Method Enzymol 1996;9:533–7. [3] Guzmán-Maldonado H, Paredes-López O. Amylolytic enzymes and products derived from starch: A review. Crit Rev Food Nutr 1995;35:373–403. [4] Coutinho PM, Reilly PJ. Glucoamylase strutuctural, functional and evolutionary relationships. Proteins 1997;29:334–47. [5] Pandey A, Nigam P, Soccol CR, Socool VT, Singh D, Mohan R. Advances in micro- bial amylases. 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amylase using glutaraldehyde-agarose support. Characterization of the i... 1 Introduction 2 Material and methods 2.1 Materials 2.2 Methods 2.2.1 Amylase extraction 2.2.2 Protein determination 2.2.3 Enzymatic activity 2.2.4 Supports preparation 2.2.5 Enzyme Immobilization 2.2.6 Effect of temperature and pH on the free enzyme and derivatives 2.2.7 Thermal and pH stability 2.2.8 Km and Vmax determination 2.2.9 Reuse assay 3 Results and discussion 3.1 Selection of the immobilization support 3.2 Kinetic parameters of beta-amylase immobilized on glutaraldehyde-agarose beads 3.3 Characterization of the beta amylase immobilized on glutaraladehyde agarose beads 4 Conclusions Acknowledgements References