α-Amylase immobilization onto dye attached magnetic beads: Optimization and characterization

Journal of Molecular Catalysis B: Enzymatic 78 (2012) 16– 23 Contents lists available at SciVerse ScienceDirect Journal of Molecular Catalysis B: Enzymatic jou rn al hom epa ge: www.elsev ier .com �-Amy ma charac Nalan Tü a Department o b Department o c Department o a r t i c l Article history: Received 17 M Received in re Accepted 17 Ja Available onlin Keywords: �-Amylase Magnetic bead Cibacron Blue Dye-ligand affi Enzyme immobilization crylat f Fe3O a nuc nder olym ation A/CB f mag obiliz lity th 35 days whereas the immobilized enzyme lost about 27% of its activity during the same period. It was also observed that the enzyme could be repeatedly adsorbed and desorbed onto the mPHEMA/CB beads. © 2012 Elsevier B.V. All rights reserved. 1. Introdu Biotechn to convent fields. Over enzyme tec their practic in the field of a wider industry, w of bioproce eral, the im processes s aration, dec while offer tion metho entrapmen a bifunction [3–12]. Am tion to a s ∗ Correspon Eylul Universi fax: +90 232 4 E-mail add 1381-1177/$ – doi:10.1016/j. ction ology is currently considered as a useful alternative ional process technology in industrial and analytical the last few decades, intense research in the area of hnology has provided many approaches that facilitate al applications. The newer technological developments of immobilized biocatalysts can offer the possibility and more economical exploitation of biocatalysts in aste treatment, and medicine and in the development ss monitoring devices like the biosensor [1]. In gen- mobilization of enzymes is advantageous in industrial ince it allows for enzyme re-utilization, facilitates sep- reases production costs and the generation of wastes, ing mild reaction conditions [2]. Enzyme immobiliza- ds basically include adsorption to insoluble materials, t in polymeric matrix, encapsulation, crosslinking with al reagent, or covalent linking to an insoluble carrier ong the physical immobilization methods, adsorp- olid support material is the most general, easiest to ding author at: Department of Chemistry, Faculty of Science, Dokuz ty, 35600 Buca, Izmir, Turkey. Tel.: +90 232 4128702; 534188. ress: [email protected] (N. Tüzmen). perform and oldest protocol. Another important advantage of this method is the possibility of reusing of the enzyme and support material for different purposes because of the reversibility of the method [13]. Support materials used in enzyme immobilization are classified as inorganic supports, synthetic polymers or natural macro- molecules [14]. Polymeric materials make suitable candidates due to their reactive functional groups, good mechanical prop- erties, ease of preparation method and ability to accommodate bio-friendly components for improved biocompatibility [15]. Poly(hydroxyethyl methacrylate) (PHEMA), due to its being a bio- compatible synthetic polymer with adequate mechanical strength for most biomedical and biotechnological applications is regarded as a suitable matrix for immobilization of enzymes [16]. Magnetic separation techniques have currently found many applications in different area of biosciences, especially in laboratory scale [17]. Magnetic separations are relatively rapid and easy, requiring a simple apparatus. The necessities for centrifugation, undesirable dilution of the sample and loss of the carrier during washing often complicate the use of non-magnetic enzyme reactors. Supports with a magnetic core overcome these problems [18–20]. Therefore, enzyme immobilized onto surface of magnetic carriers is preferred to native enzyme for the purpose of using in industrial applications now-a day [21]. Recently, dye-ligand affinity chromatography has been used extensively in laboratory and large scale applications [22–26]. see front matter © 2012 Elsevier B.V. All rights reserved. molcatb.2012.01.017 lase immobilization onto dye attached terization zmena,∗, Tülden Kalburcub, Adil Denizli c f Chemistry, Faculty of Science, Dokuz Eylul University, Izmir, Turkey f Chemistry, Faculty of Science and Arts, Aksaray University, Aksaray, Turkey f Chemistry, Faculty of Science, Hacettepe University, Ankara, Turkey e i n f o ay 2011 vised form 17 January 2012 nuary 2012 e 31 January 2012 s F3GA nity chromatography a b s t r a c t Magnetic poly(2-hydroxyethylmetha ization of HEMA in the presence o immobilized to the mPHEMA beads vi ring and hydroxyl groups of HEMA u diameter) carrying 68.3 �mol CB/g p effects of pH, initial protein concentr mum adsorption capacity of mPHEM amounts of �-amylase per unit mass o 5.0. The optimal pH for free and imm enzyme exhibited better thermostabi / locate /molcatb gnetic beads: Optimization and e) [mPHEMA] beads were prepared by suspension polymer- 4 nano-powder. Cibacron Blue F3GA (CB) was covalently leophilic substitution reaction between chloride of its triazine alkaline conditions. The mPHEMA/CB beads (100–140 �m in er were used in �-amylase adsorption studies to assess the , temperature and ionic strength on enzyme activity. Maxi- beads was found to be 401 ± 11 mg/g carrier. The adsorbed netic beads reached a plateau value at about 1.0 mg/mL at pH ed �-amylase was 7.0 and 8.0, respectively. The immobilized an the free one. The free enzyme lost all of its activity within N. Tüzmen et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 16– 23 17 Dye-ligands are commercially available, inexpensive, and can eas- ily be immobilized, especially on matrices bearing hydroxyl groups. Although dyes are all synthetic in nature, they are still classified as affinity ligands because they interact with the active sites of many prote tors, or bind known as r Most of the anthraquin (often a mo the dye liga of electrosta F3GA (CB) is and reversi Starch is �-amylases ing enzyme are of cons d-glucanglu �-1,4 glyco �-Amylase and other detergent a istry [30]. B the reactio bilized �-a been many ent organic on immobil limited. In this s investigated �-amylase. ization. CB w molecules. C bilized to m chloride of molecules u The prepar transform i scope (SEM �-amylase varying diff tration, ion and reusabi of the enzym 2. Materia 2.1. Chemic Aspergill MO, USA) acid, CB we Hydroxyeth Louis, MO, U gen atmosp Darmstadt, sisobutyron re-crystalliz hydrolyzed Aldrich Che obtained fr purity and tered by M experiments unless otherwise stated. Before use, the laboratory glassware was rinsed with water and dried in a dust-free environ- ment. epar HEM aque ntly a twe mole s des meri The with ) wa A/H n wa tical eriza he re per re w most A b aced ere east emp wit pica ams o s so m) i tion iliza , an ntil azi was s for his t pho arac spe btain dzu, with t form dete appr drica as m nic s on a of th cord on th rium ent of C ins mimicking the structure of the substrates, cofac- ing agents for those proteins. A number of textile dyes, eactive dyes, have been used for protein purification. se reactive dyes consist of a chromophore (azo dyes, one, or phathalocyanine), linked to a reactive group no- or dichlorotriazine ring). The interaction between nd and biomolecules can be by complex combination tic, hydrophobic, and hydrogen bonding. Cibacron Blue an anthraquinone textile dye that interacts specifically bly with biomolecules [27]. hydrolyzed to glucose, maltose and dextrin by �- and and other related enzyme [28]. Among starch hydrolyz- s that are produced on industrial scale, �-amylases iderable commercial interest [29]. �-Amylase (1,4 �- canohydrolase, EC 3.2.1.1) catalyzes the hydrolysis of sidic linkages in starch and other related carbohydrates. is applied widely in food industry, beer production, drink manufactures, textile desizing, paper industry, pplications and analysis in medicinal and clinical chem- ut once used, �-amylase cannot retrieve easily from n systems. So, it is very valuable to employ immo- mylase to supercede free �-amylase [31]. There have reports about immobilization of �-amylase on differ- and inorganic supports [32,33,5,34–37], but reports ization of �-amylase to magnetic polymeric beads are tudy, PHEMA based magnetic beads carrying CB were as a novel magnetic carrier for the immobilization of mPHEMA beads were prepared by suspension polymer- as used as the dye-ligand for specific binding of protein B, an anthraquinone textile dye, was covalently immo- PHEMA beads via a nucleophilic reaction between the its triazine ring and the hydroxyl groups of the HEMA nder alkaline conditions with the elimination of HCl. ed mPHEMA/CB beads were characterized by Fourier nfrared (FTIR) spectroscopy, scanning electron micro- ), swelling ratio analysis and elemental analysis. Then, adsorption onto the magnetic beads was optimized by erent parameters such as pH, initial �-amylase concen- ic strength, and temperature. Desorption of �-amylase lity of these magnetic beads and the kinetic parameters e were also tested. ls and methods als us oryzae �-amylase obtained from Sigma (St Louis, was used in this study. Starch, 3,5-dinitrosalicylic re all obtained from Sigma (St. Louis, MO, USA). 2- ylmethacrylate (HEMA) was purchased from Sigma (St. SA) and purified by vacuum distillation under a nitro- here. Ethylene glycol dimethacrylate (EGDMA, Merck, Germany) was used as the crosslinking agent. Azobi- itrile (AIBN, Sigma) was selected as initiator. AIBN was ed from methanol. Poly(vinyl alcohol) (PVA), 99–100% with Mn 100,000 g/mol (Fluka), was obtained from mical Co. (USA). Magnetite nano-powder (Fe3O4) was om Sigma. All other chemicals were of the highest used without further purification. Ultra-pure water fil- illipore S.A.S 67120 Molsheim, France was used for all 2.2. Pr mP in an covale tion be HEMA NaCl a copoly below: of PVA (0.04 g (EGDM solutio magne polym bath. T The tem peratu a ther mPHEM and pl vents w for at l room t sieving A ty Ten gr aqueou (400 rp centra immob filtered times u sodium beads sample after t spectro 2.3. Ch FTIR were o Shima mixed a pelle To beads, a cylin (Hd) w and io shaken height was re based Equilib Elem degree ation of mPHEMA/CB beads A beads were produced by suspension polymerization ous medium. CB, an anthraquinone textile dye, was ttached to the mPHEMA beads via a nucleophilic reac- en chloride of its triazine ring and hydroxyl groups of cules under alkaline conditions with the elimination of cribed in our previous article [38]. A typical suspension zation procedure of mPHEMA beads was summarized as dispersion medium was prepared by dissolving 200 mg in 50 mL of distilled water. The desired amount of AIBN s dissolved within the monomer phase 8.0/4.0/12.0 mL EMA/toluene) with 0.75 g magnetite particles. Then, this s transferred into the dispersion medium placed in a ly stirred (at a constant stirring rate of 600 rpm) glass tion reactor (100 mL) which was in a thermostatic water actor was flushed by bubbling nitrogen and then sealed. ature of the reactor was kept at 65 ◦C for 4 h. The tem- as then raised to 90 ◦C for 2 h and kept constant by ated water bath during the polymerization reaction. eads were separated from the polymerization medium in a Soxhlet extraction apparatus. The porogenic sol- extracted out of the beads with acetone under reflux 12 h. The magnetic beads were dried under vacuum at erature and then the dried beads were fractionated by h standard test sieves. l CB immobilization procedure was performed as below: f mPHEMA beads in 100 mL of the Cibacron Blue F3GA lution containing 4.0 g NaOH were magnetically stirred n a sealed reactor at of 80 ◦C for 4 h. The initial con- of the CB in the medium was 1.0 mg/mL. After the dye tion progress, the CB immobilized magnetic beads were d washed with distilled water and methanol several it ran clear. The mPHEMA/CB beads were stored in 0.02% de at 4 ◦C. The leakage of the CB from the magnetic followed by treating them with fresh human plasma 24 h at room temperature. CB concentration released reatment was measured in the liquid phase by UV- tometer (Schimadzu, Model 1601) at 630 nm. terization of mPHEMA/CB beads ctra of the CB, mPHEMA beads and mPHEMA/CB beads ed by using a FTIR spectrophotometer (FTIR 8000 Series, Japan). The dried beads (about 0.1 g) was thoroughly KBr (0.1 g, IR Grade, Merck, Germany) and pressed into , and the spectrum was then recorded. rmine the equilibrium water uptake of mPHEMA/CB oximately 3.0 g of dried polymer sample were put into l tube. The height of the bed formed by the dried beads easured. Then, 50 mL of buffer solution at a certain pH trength was added into the tube. The sealed tube was rotator at 30 rpm for 24 h. At the end of this period, the e bed formed by swollen the mPHEMA/CB beads (Hs) ed. The equilibrium water uptake ratio was calculated e following expression: water uptake ratio = Hs Hd × 100 (1) al analysis of the mPHEMA/CB beads to evaluate the B incorporated into the mPHEMA beads were subjected 18 N. Tüzmen et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 16– 23 to elemental analysis using a Leco Elemental Analyzer (Model CHNS-932). The degree of magnetism of the mPHEMA beads was measured in a magnetic field by using a vibrating sample magnetometer (Princeton magnetite with an ele Varian). The surf by scanning dried in air a of the dried Electronen ter coated f the desired beads. 2.4. ˛-Amy The ads amylase so period for Initial and UV-spectro adsorbed � calculated b Q = (C0 − C m Here, Q i the beads (m the initial so certain peri of the solut �-Amyla over a rang concentrati NaCl). The �-a three replic were used t Confidence in order to 2.5. Adsorp In order nism of the adsorption adsorption muir and Fr The Lang that the ads assuming m of identical ration on th given by th Qeq = Qmax 1 + b where Qmax equilibrium (mg/L) and Freundli to describe as multipla sites are occupied first and that the binding strength decreases with the increasing degree of site occupation. It may be represented as follows: Kf C 1/ eq Ceq, equil rptio ents esorp orpt NaCl 10 m A/C m an pho ted eads m. tion oun mou mag ter s re th ility as r tivity acti acco libe salic solu or 3 –po mL ter and with . In with duci ed as ted t aine ction ment ty of amo ol m ions. c act M g enz Applied Research, Model 150A, USA). The presence of particles in the polymeric structure was investigated ctron spin resonance (ESR) spectrophotometer (EL 9, ace morphology of the magnetic beads was investigated electron microscopy (SEM). The samples were initially t 25 ◦C for seven days before being analyzed. A fragment bead was mounted on a SEM sample mount (Rastern Microscopy, Leitz-AMR-1000, Germany) and was sput- or 2 min. The surface of the sample was then scanned at magnification to study the morphology of mPHEMA/CB lase adsorption studies orption experiments were conducted with 5 mL �- lution in a batch system for 60 min, the equilibrium the adsorption of �-amylase at room temperature. final protein concentrations were determined by photometry (Schimadzu 1601, Japan). The amount of -amylase per unit mass of the mPHEMA/CB beads was y using the following expression: )V (2) s the amount of �-amylase adsorbed onto unit mass of g/g); C0 and C are the concentrations of �-amylase in lution and in the aqueous phase after treatment for the od of the time, respectively (mg/mL); V is the volume ion (mL), and m is the mass of the beads used (g). se adsorption on the mPHEMA/CB beads was studied e of pHs (4.0–9.0), temperatures (15–45 ◦C), �-amylase ons (0.25–2.0 mg/mL) and ionic strengths (0–0.1 M mylase adsorption experiments were performed in ates. For each set of data, standard statistical methods o determine the mean values and standard deviations. intervals of 95% were calculated for each set of samples determine the margin of error. tion isotherms to obtain information about the properties and mecha- sorption process, the experimental results of �-amylase onto the mPHEMA/CB beads were represented by isotherms and fitted with two model equations (Lang- eundlich). muir isotherm model [39] is based on the assumption orption process takes place on a homogeneous surface, onolayer adsorption onto a surface with a finite number sites, so a monolayer of adsorbate is formed at satu- e adsorbent surface. The Langmuir isotherm model is e expression: bCeq Ceq (3) is the maximum adsorption capacity (mg/g), Ceq is the concentration of �-amylase at the equilibrium time b the Langmuir constant (L/mg). ch [40] presented a fairly satisfactory empirical model non-ideal adsorption on heterogeneous surfaces as well yer adsorption. It is assumed that the stronger binding Qeq = where at the to adso repres 2.6. D Des 1.0 M mode, mPHEM 200 rp spectro calcula netic b mediu Desorp = Am A The and wa and we reusab cycle w 2.7. Ac The mined groups dinitro starch ature f sodium in 100 ing wa added sured 540 nm pared Non-re and us conver ity obt the fra attach quanti as the 1.0 �m condit Specifi = m n (4) represents the equilibrium concentration of �-amylase ibrium time (mg/L), n, the Freundlich constant related n intensity (dimensionless), Kf, the Freundlich constant the relative adsorption capacity (mg/g). tion and reusability of the mPHEMA/CB beads ion experiments of �-amylase were performed using solution. In the desorption experiment at a batch L desorption solution was added �-amylase adsorbed B beads and stirred at room temperature for 2 h at d the final concentration was measured with a UV- tometer (Schimadzu 1601, Japan). Desorption ratio was from the amount of �-amylase adsorbed on the mag- and the final protein concentration in the desorption ratio (%) t of ˛ − amylase desorbed nt of ˛-amylase adsorbed × 100 (5) netic beads were washed with 50 mM NaOH solution everal times and reequilibrated in the adsorption buffer en reused for enzyme adsorption. In order to show the of the mPHEMA/CB beads, the adsorption–desorption epeated five times using the same group of beads. assays of free and immobilized ˛-amylase vities of free and immobilized �-amylase were deter- rding to the method of Bernfeld wherein the reducing rated from starch are measured by the reduction of 3,5- ylic acid [41]. 0.1 mL of enzyme solution and 0.1 mL of tion (1%) were mixed and incubated at room temper- min and then 0.2 mL of the assay mixture containing tassium tartarate (30 g), NaOH (1.6 g) and DNSA (1.0 g) distilled water. This solution was incubated in boil- for 5 min. After cooling, distilled water (2.0 mL) was the intensity of the red color developed was mea- a UV-spectrophotometer (Schimadzu 1601, Japan) at each set of experiments, a calibration curve was pre- maltose solutions (0.1 mL) of various concentrations. ng ends of starch were determined as described above, blanks in the activity measurements. The results were o relative activities (percentage of the maximum activ- d in that series). The residual activity was defined as of total hydrolytic activity recovered after covalent on the mPHEMA/CB beads compared with the same free enzyme. One unit of �-amylase activity is defined unt of enzyme, which produces reducing ends equal to altose in 1 min at 25 ◦C and pH 6.9 under the specified ivity (Units/mg) icromoles maltose liberated yme in reaction mixture × 3 min (6) N. Tüzmen et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 16– 23 19 Table 1 Some properties of the mPHEMA beads. Bead size 100–140 �m Specific surface area 56 m2/g Average por Swelling rat Cibacron Blu Density Volume frac Volume frac Magnetic fie 2.8. Immob The effic was calcula Immobiliza = Specifi Spec 2.9. Steady The acti (5.0–9.0) an temperatur effect of ion ing the NaC concentrati range. Mich Lineweaver determinat 2.10. Storag The acti in phospha every day a the enzyme phate buffe 3. Results 3.1. Propert Some pr vious article The scan mPHEMA b of the non-m However, t containing tals (diame On the othe spherical sh FTIR spe evaluated i summarize ment of CB 3.2. Optimi 3.2.1. Effect The amo showed a m Table 2 Some fundamental IR frequencies in (cm−1) of CB, mPHEMA and mPHEMA/CB beads. Vibration type CB mPHEMA mPHEMA-CB beads 3455 3446 1739 with a shoulder at 1650 1730, intensive 1727 1565, sharp band C 1617 1615 1610 1226 1022 1085 500–700 890 890 lic O H 3466 3446 tic CH2 2987 2987 tic CH3 2955 2955 out of plain 1263, 1158 C 1158 gher pH values (Fig. 1). Specific interactions (hydrophobic trostatic or hydrogen bonding) between �-amylase and dye les ral g ) and bet nform re) a nity s acid I, th ary, e io ctro ason pH ctro . Effect adso cent twee affin for t ed t 5) (F mPH tion ents Und 00 50 00 e size 814 nm io 62.1% e F3GA content 68.3 �mol/g 1.1 g/mL tion of polymer 94.5% tion of magnetite particles 5.5% Fe3O4 ld resonance 2250 G ilization efficiency iency of immobilization onto the mPHEMA/CB beads ted using following formula [40]: tion (%) c activity of immobilized enzyme ific activity of soluble enzyme × 100 (7) state kinetics vity assays were carried out over a range of pH values d temperatures (15–45 ◦C) to determine the pH and e profiles for the free and immobilized enzyme. The ic strength on �-amylase activity was studied by vary- l concentration from 0 to 0.1 M. The effect of substrate on was tested in the 0.25–2.5 g/L starch concentration aelis constant (Km) and Vmax were determined using –Burk plot. All parameters were the mean of triplicate ions from three independent preparations. e stability vities of free and immobilized �-amylase (0.5 mg/mL) te buffer (50 mM, pH 7.0) were periodically measured s mentioned above. At the end of activity assay period, -immobilized magnetic beads were washed with phos- r (0.05 M, pH 7.0) at least three times. and discussion ies of mPHEMA/CB beads operties of the mPHEMA beads were given in our pre- [42] and summarized in Table 1. ning electron pictures of non-magnetic PHEMA and eads were given in our previous article [42]. Surface agnetic PHEMA beads has contained no macro-pores. he surface of the mPHEMA beads has rough surface macro-pores due to the abrasion of magnetite crys- ter 20 N. Tüzmen et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 16– 23 0 50 100 150 200 250 300 350 400 450 0 Q ( m g /g m ic ro b e a d ) mPHEMA mPHEMA-Cibacron Blue Fig. 2. Effect amylase (aque 2.5 4.5 6.5 8.5 10.5 120 S p e s if ic A c ti v it y ( IU ) Fig. 3. Immob 25 ◦C). magnetic be maximum tion efficie [43,1]. It sho adsorbed n for �-amyla may have r mechanism ing caused and by grou molecules ( The acti adsorption amylase/g c sites on the 3.2.3. Adsor In this w undlich mo model. The Table 3 Langmuir and Langmuir Qmax: 666.7 b: 1.7 R2: 0.8113 0 50 100 150 200 250 0 Q ( m g /g m ic ro b e a d ) ffect o ration lecu s ads ngmu ing th rmol dicat was ngm otein s. Freu , res ads es p 2.2521.751.51.2510.750.50.25 Initial Amylase Concentration (mg/mL) of the initial enzyme concentration on the amount of adsorbed �- ous solution in 100 mM acetate at pH 5.0; T: 25 ◦C). Fig. 4. E concent for mo well a the La involv of inte fore in beads the La and pr proces The and 1.8 ity and indicat 440360280200 Q (mg/g microbead) ilization efficiency (aqueous solution in 100 mM acetate at pH 5.0; T: ads was found as 401 ± 11 mg �-amylase/g carrier and immobilization obtained was 69.9%. This immobiliza- ncy was as high as given results in previous reports uld also be noted that negligible amounts of �-amylase on-specifically on the plain mPHEMA (1.3 ± 0.2 mg/g se). This increase in the �-amylase coupling capacity esulted from cooperative effect of different interaction s such as hydrophobic, electrostatic or hydrogen bond- by the acidic groups and aromatic structures on the CB ps on the side chains of amino acids on the �-amylase Fig. 3). vity of �-amylase was increased by increasing the capacity (Q) and then reached a plateau above 400 mg �- arrier because of the saturation of the active adsorption magnetic beads. ption isotherms ork, the correlation obtained from the fitting of the Fre- del (Table 3) was better than the fit using the Langmuir Freundlich model is an empirical model, which allows Freundlich isotherm constants for �-amylase adsorption. Freundlich Kf: 418.9 n: 1.8 R2: 0.9342 418.9 sugge medium on 3.2.4. Effect To deter tion, adsorp NaCl. As sh the mPHEM the NaCl c increase in the dye mo interaction immobilize because it solution ca number of t would decr of the �-am It is also su the reducti 3.2.5. Effect In order amylase ad range of te the adsorp 76.73% from was observ der Waals a known that in hydropho over 35 ◦C m surface or s 0.120.10.080.060.040.02 Ionic Strength (M NaCl) f the ionic strength on the amount of adsorbed �-amylase (�-amylase : 0.5 mg/mL; aqueous solution in 100 mM acetate at pH 5.0; T: 25 ◦C). lar interactions between the surface and adsorbate as orbate–adsorbate interactions in solution. In contrast ir model is based on a reversible adsorption process e exchange of adsorbed and free proteins in the absence ecular interactions. The results of the modeling there- ed that the adsorption of �-amylase to the mPHEMA/CB more complicated than the ideal process described by uir model and that both protein–protein interactions –surface interactions were important in the adsorption ndlich constants, Kf and n were calculated to be 418.9 pectively. Kf and n are indicators of adsorption capac- orption intensity [44]. A value of n > 1 for the CB ligand ositive co-operativity in bonding while a Kf value of sts easy adsorption of �-amylase from the adsorption to the mPHEMA/CB beads. of ionic strength mine the effect of ionic strength on �-amylase adsorp- tion studies were carried out in the range of 0.0–0.1 M own in Fig. 4, the amount of �-amylase adsorbed onto A/CB beads decreased significantly (53.4%, p < 0.05) as oncentration was increased from zero to 0.1 M. The NaCl concentration could promote the adsorption of lecules onto the polymer surface through hydrophobic s. Moreover, the hydrophobic interactions between the d dye molecules themselves would also become strong, has been observed that the addition of salt to a dye used the stacking of the free dye molecules. Thus, the he immobilized dye molecules accessible to �-amylase ease as the ionic strength increased, and the adsorption ylase to the immobilized dye would become difficult. ggested that an increase in NaCl concentration result in on of electrostatic interactions [45]. of temperature to determine the effect of temperature on the �- sorption, adsorption studies were performed over a mperatures from 15 ◦C to 45 ◦C. As seen in Fig. 5, tion capacity of the magnetic beads increased about 15 ◦C to 35 ◦C and maximum �-amylase adsorption ed at 35 ◦C. This increase may be due to increasing van ttraction forces, which increase with temperature. It is increasing the temperature enhanced protein retention bic interaction. The decrease observed at temperatures ay be due to the conformational changes on enzyme tructural deformation of the matrix. N. Tüzmen et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 16– 23 21 0 50 100 150 200 250 5040302010 Q ( m g /g m ic ro b e a d ) Temperature (ºC) Fig. 5. Effect of temperature on the amount of adsorbed �-amylase (�-amylase concentration: 0.5 mg/mL; aqueous solution in 100 mM acetate at pH 5.0). 3.3. Desorption and reusability of the mPHEMA/CB beads Desorption studies were performed for 2 h in a batch system with 1 M NaCl and more than 90% of the adsorbed �-amylase was desorbed from the mPHEMA/CB beads. It should be noted that there was no CB r In order adsorption– same beads tion capaci that the mP immobiliza capacity. 3.4. Steady Environm in free or in at various p to determin The beh immediate an altered Depending matrix and the immed thus cause 100 150 200 250 0 Q ( m g /g m ic ro b e a d ) Fig. 6. Reusab aqueous soluti was repeated 0 20 40 60 80 100 10987654 R e ta in e d A c ti v it y % pH Free Amylase Immobilized Amylase Fig. 7. Effect of the medium pH on the activity of free and immobilized �-amylase (�-amylase concentration: 0.5 mg/mL; T: 25 ◦C). The pH effect on the activity of free and immobilized �-amylase was studied at various pHs (pH 5.0–9.0) at 25 ◦C and the results are presented in Fig. 7. The optimal pH for free and immobilized �-amylase was 7.0 and 8.0, respectively. The pH profile of the immobilized �-amylase was much broader than that of the free enzyme, indicating that this method preserves the enzyme activ- ity in a wi ide r A/C ilize d im re of y [11 lase 47] a �-am owar lite [ form nviro tem ilize ◦C (p lase ed at e (Fi ected n op activ ak a 0 elease in this process. to show the reusability of the mPHEMA/CB beads, the desorption cycle was repeated five times using the . There was no remarkable reduction in the adsorp- ty of the magnetic beads (Fig. 6). This result showed HEMA/CB beads could be repeatedly used in enzyme tion without detectable losses in its initial adsorption state kinetics ental parameters affect the activity of enzymes either immobilized forms. Therefore, studies were carried out H, temperature and substrate concentrations in order e the optimum conditions. avior of an enzyme molecule may be modified by its microenvironment. An enzyme in solution can have pH optimum upon immobilization on a solid matrix. upon the surface and residual charges on the solid the nature of the enzyme bound, the pH value in iate vicinity of the enzyme molecule may change and a shift in the pH optimum of the enzyme activity [46]. basic s mPHEM immob free an structu activit �-amy beads [ hand, units t Amber the con local e The immob 15–45 �-amy obtain amylas as exp up to a the de to a pe 10 654321 Reuse Time ility of the mPHEMA/CB beads (�-amylase concentration: 0.5 mg/mL; on in 100 mM acetate at pH 5.0; T: 25 ◦C; Adsorption–desorption cycle 5 times). 50 75 10 R e ta in e d A c ti v it y % Fig. 8. Effect (�-amylase co 5.0). der pH range. There was a shift of 1.0 U towards the esulting from immobilization of �-amylase onto the B beads. This shift towards increased basicity for the d �-amylase is natural as the microenvironment of the mobilized enzyme is quite different. The charge and the support material has significant effects on enzyme ]. A shift towards acidic region has been observed when was immobilized on poly(methylacrylate-acrylic acid) nd on zirconium dynamic membrane [48]. On the other ylase purified from mung bean showed a shift of 1.4 ds the basic side upon immobilization on Chitosan and 1]. The shift towards basic region can be explained by ational changes on the enzyme and the pH change in nment of the bead. perature dependence of the activities of free and d �-amylase was studied over a temperature range of H 5.0,) and temperature profiles of free and immobilized are shown in Fig. 8. The maximum catalytic activity was 25 ◦C for free �-amylase and 35 ◦C for immobilized �- g. 8). The behavior of the activity with temperature was : increased temperature caused an increased activity, timum reaction temperature and then overlapped with ation of the enzyme. As a result, the activity increased nd then decreased. In general, the effect of changes in 50403020 Temperature(ºC) Free Amylase Immobilized Amylase of temperature on the activity of free and immobilized �-amylase ncentration: 0.5 mg/mL; aqueous solution in 100 mM acetate at pH 22 N. Tüzmen et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 16– 23 Table 4 Storage stabilities of different carriers. Loss of activity for free �-amylase Loss of activity for immobilized �-amylase Reference All activity within 10 days 30% for poly(EGDMA-VIM)-Cu2+–�-amylase All activity within 20 days 25% for carbodiimide (CDI)–�-amylase, 37.5 (EDA)–�-amylase, 5% for hexamethylene di within 30 days All activity within 20 days 47.5% for SOCl2–�-amylase and 75.5% for CD All activity within 15 days 30% for cyclic carbonate functional hybrid m All activity within 15 days 20% for functionalized glass beads–�-amyla All activity within 35 days 27%for mPHEMA-CB–�-amylase within 35 d temperature on the rate of enzyme catalyzed reactions does not provide much information on the mechanism of catalysis. However, these effects can be important indicators of structural changes in enzymes [49]. As seen in Fig. 5, the immobilized enzyme exhibited better thermo-stability than the free one. It was demonstrated by the authors that the thermal stability of enzymes might be drasti- cally increased if they are attached to a complementary surface of a relatively rigid support in a multipoint [50]. The dependence of the activity on ionic strength was esti- mated both free and immobilized enzymes at 25 ◦C and optimal pHs (Fig. 9) with increa of structura between io salt ions. Catalytic evaluated b as substrat 0.780 ± 0.02 the immobi of the enzy size the hig results wer strate was n for the free and 5.98 U/ amylase wa increase m enzyme sur 3.5. Storage Enzyme tal conditio to characte industrial u 60 80 100 0 R e ta in e d A c ti v it y % Fig. 9. Effect o (�-amylase co 5.0; T: 25 ◦C). 0 20 40 60 80 100 0 n e d A c ti v it y % Fig. mobilized �-amylase preparations were stored in a phos- uffer (50 mM, pH 7.0) at 4 ◦C and the activity measurements arried out for a period of 5 weeks. The free enzyme lost all ctivity within 35 days, whereas the immobilized enzyme out 27% of its activity during the same period (Fig. 10). This activity is natural in enzyme activity, and immobilization antly prevented this phenomenon. These results are also in ent with the literature [10,11,47,51,52] (Table 4). Improved e stability by immobilization has also been reported by vari- er groups [53,54]. clusion ecent years magnetic beads have been widely used in the atography for fast and effective separation and purification olecules. The most significant advantage of using mag- eads in chromatographic applications is the easy removal m from the reaction media in the presence of magnetic dditionally, magnetic supports can easily be stabilized in idized reactors that are externally exposed to magnetic hich enables continuous separation of biomolecules like es and proteins. Furthermore, the usage of these kinds of als can seriously reduce the investment and production . Activities of free and immobilized enzymes decreased sing ionic strength. This observation may be the result l orientations of enzymes depends on the influence nic groups of amino acids on the �-amylase surface and properties of the free and immobilized enzymes were y using Lineweaver–Burk plot with soluble starch e. Michaelis constant (Km) values were found to be mg/mL for the free enzyme and 0.785 ± 0.04 mg/mL for lized �-amylase. The Km value is known as the affinity mes to substrates and the lower values of Km empha- her affinity between enzymes and substrates [50]. The e shown that the affinity of the �-amylase to its sub- ot significantly changed by immobilization. Vmax values and immobilized enzymes were calculated as 4.09 U/mg mg, respectively. The Vmax value of the immobilized �- s higher than that of the free enzyme (p < 0.05). This ight be attributed to conformational changes on the face. stability s are very sensitive biocatalysts against environmen- ns and may lose their activities quite easily [51]. Thus, rize storage stability of an enzyme for preparative or ses is meaningful. To determine storage stability, free Free Amylase R e ta i and im phate b were c of its a lost ab loss of signific agreem storag ous oth 4. Con In r chrom of biom netic b of the field. A the flu area, w enzym materi 0.120.10.080.060.040.02 Ionic Strength (M NaCl) Immobilized Amylase f the ionic strength on the activity of free and immobilized �-amylase ncentration: 0.5 mg/mL; aqueous solution in 100 mM acetate at pH costs. Biom to biologic easiness of novel magn the immob tion from a pH 5.0. Sev gation of ne within 10 days [10] % for ethylene diamine amine (HMDA)–�-amylase [11] I–�-amylase within 30 days [47] atrix–�-amylase within 25 days [51] se within 25 days [52] ays This study 403530252015105 Days Immobilized Amylase Free Amylase 10. Storage stabilities of free and immobilized �-amylase. imetic dye ligands have several advantages compared ligands in the aspects of high adsorption capacity, rigid, immobilization and economy. Therefore, we used a etic affinity matrix incorporated CB as dye ligand for ilization of �-amylase. Maximum �-amylase adsorp- queous solution was determined as 401 ± 11 mg/g at eral publications have appeared describing the investi- w supports for �-amylase immobilization, for example, N. Tüzmen et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 16– 23 23 �-amylase covalently immobilized onto poly(methylmethacrylate- acrylic acid) beads. The amount of enzyme immobilized on 1.0 g of support was about 4.0 mg [47]. The amount of enzyme immobilized per gram of cellulose-coated magnetite (CCM) nanoparticles attains saturation value, i.e., 18.2 mg amylase/g CCM nanoparticles [21]. Turunc et al. demonstrated that the amount of adsorbed �-amylase was found as 34.4 mg/g of cyclic carbonate and methacrylate func- tional multicomponent inorganic matrix (CTM) [51]. Rozie et al. showed that the amount of adsorbed �-amylase was found as 185 mg/g of crosslinked patato starch [55]. In another study, 375 mg �-amylase was adsorbed onto the 1.0 gram of grafted Na-alginate beads [56]. The highest adsorption observed with the present novel dye attached mPHEMA beads appears to be quite promising. Also, these dye-attached magnetic beads can be used repeatedly for �-amylase immobilization. The reversibility of the binding is another asset which enables users to recharge the support with fresh enzyme upon substantial loss of adsorbed enzyme activity, leading to systems with easy rejuvenation properties. The immo- bilized �-amylase preparation retains much of its activity over a wider pH range than the free form does. Immobilized enzyme also showed a higher level of activity over a wider temperature range than the free enzyme did. When compared to the soluble enzyme under similar conditions, the higher storage stability of the immobi- lized �-amy advantages Acknowled We than References [1] P. Tripath 69. [2] R.A. Sheld [3] I. Chibata [4] G. Palmie Enzyme M [5] L. Cong, R [6] J.M. More Sanchez, [7] W. Tische [8] H. Tumtu [9] L. Giorno [10] A. Kara, B 61. [11] N. Hasırc [12] P. Vidinha 121 (2006 [13] M.E. C¸ orm (2010) 15 [14] P. Ye, Z.K. Xu, A.F. Che, J. Wu, F. Seta, Biomaterials 26 (2005) 6394. [15] V. Rebros, M. Rosenberg, Z. Mlichova, L. Kristofıkova, Food Chem. 102 (2007) 784. [16] A. Denizli, G. Ozkan, M.Y. Arica, J. Appl. Polym. Sci. 78 (2000) 81. [17] B. Xue, Y. Sun, J. Chromatogr. A 921 (2001) 109. [18] I. Safarik, M. Safarikova, F. Weyda, E.M. Szablewska, A.S. Waniewska, J. 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Anal. ) 563. α-Amylase immobilization onto dye attached magnetic beads: Optimization and characterization 1 Introduction 2 Materials and methods 2.1 Chemicals 2.2 Preparation of mPHEMA/CB beads 2.3 Characterization of mPHEMA/CB beads 2.4 α-Amylase adsorption studies 2.5 Adsorption isotherms 2.6 Desorption and reusability of the mPHEMA/CB beads 2.7 Activity assays of free and immobilized α-amylase 2.8 Immobilization efficiency 2.9 Steady state kinetics 2.10 Storage stability 3 Results and discussion 3.1 Properties of mPHEMA/CB beads 3.2 Optimization of α-amylase adsorption 3.2.1 Effect of pH 3.2.2 Effect of the initial α-amylase concentration 3.2.3 Adsorption isotherms 3.2.4 Effect of ionic strength 3.2.5 Effect of temperature 3.3 Desorption and reusability of the mPHEMA/CB beads 3.4 Steady state kinetics 3.5 Storage stability 4 Conclusion Acknowledgment References