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Lactose hydrolysis using immobilized a-galactosidase enzyme onto nano-copolymers particles.

Introduction

Enzymes, through immobilization on different kinds of supports, have been applied in variant applications including biomedical and biotechnological ones. Many strategies for enzymes immobilization namely; physical adsorption, entrapment, co-polymerization and covalent attachment, have been used [1-5].

The main advantage for enzyme immobilization is the easy separation of the enzyme from the reaction mixture (substrates and products) and its reusability for tens of time, which reduces the enzyme and the enzymatic products cost tremendously. Beside this splendid advantage, the immobilization process imparts many other advantages to the enzyme such as:

* The ability to stop the reaction rapidly by removing the enzyme from the reaction solution (or vice versa)

* Product is not contaminated with the enzyme

* Easy separation of enzyme from the product (especially useful in food and pharmaceutical industries)

* Enhancement of enzyme stability against pH, temperature, solvents, contaminants, and impurities.

The main disadvantage of immobilization process is the loss of part on enzyme activity during the immobilization step due to many reasons including de-naturation, misorientation and substrate-product diffusion limitation. Diffusion limitation for substrate and product comes in the front of these causes. A generic solution to this problem increases the range of applications for immobilized enzymes. Different strategies for overcoming this problem have been investigated. The advantages and disadvantages of each strategy were summarized and discussed by Mohy Eldin [6]. A simple and new strategy has been recently investigated concerning the immobilization of enzyme over the surface of nonporous carriers. Recently reported work in this area has revealed the great potential for the use of nonporous [7-13], nanofibrous [8-14], and nanoparticles [9, 15-18] materials as a new class of carriers for biocatalysts. The effective enzyme loading on nanomaterials can be considerably high (e.g., it can reach over 10 wt % with particles smaller than 100 nm), and a large surface area per unit mass is also provided to facilitate reaction kinetics. The considerable specific surface area afforded by nanoparticles is not the only size-dependent feature important to the performance of attached enzymes.

Alexey et al. [19] used lysozyme as a model enzyme, and demonstrated that the structure and activity of an enzyme are strongly dependent upon the size of its carrier. Less perturbation of lysozyme's secondary structure is observed when the protein is adsorbed onto smaller nanoparticles under similar attachment conditions. And the structural information strictly correlates with activity recovery for adsorption on the smaller nanoparticles. In short, their pioneering experiment suggested that smaller nanoparticles, because of their greater surface curvature, promote the retention of more native-like protein structure and function as compared to their larger (and hence less curved) particle counterparts.

The mobility of catalyst in solution, is an another crucial factor in determining the activity recovery, and that it also provides an explanation for the higher activities usually observed in enzymes attached to nanoparticles rather than when attached to larger particls [19]. According to Jia et al. work with CT as the model enzyme, they demonstrated that the mobile state of the immobilized enzymes is a key factor in their activity recovery. They believed that "unlike solid materials of large size, nanoparticles dispersed in a solution are mobile and show Brownian motion. In that sense, the enzymes attached to the nanoparticles are not immobilized and are thus different from traditional immobilization. Such solution behavior may point to a transitional region between homogeneous catalysis with free native enzymes and a heterogeneous one with immobilized enzymes. According to the Stokes-Einstein equation, owing to their relatively larger size the mobility and diffusivity of the nanoparticles should be smaller than those of native enzymes" [20, 21]. Both theoretical modeling and experimental measurements early studies demonstrated the mobility-activity relation. It was also believed that the deterioration in the intrinsic activities of tethered enzymes would cause loss of catalyst mobility, in addition to other factors such as conformation changes of protein.

It was found that the reactions catalyzed by enzymes can also affect the motion of nanoparticles [22-24]. This may be referred to the attachment of enzymes to nanoparticles functioned as "nanomotors" [20].

a-Galactosidase from different sources is currently used in the production of lactose free milk products. Hydrolysis of lactose improves product sweetness, makes milk consumption by people who suffer from lactose intolerance possible, and increases product quality and process efficiency in the dairy industry. This hydrolysis reaction could also be applied to the upgrading of cheese whey, a product of cheese processing, disposal of which constitutes a problem [25-26].

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In this study, P(AN-co-MMA) nanoparticles were synthesized from the monomers Acrylonitrile and Methyl Methacrylate (Scheme 1) and later on used for covalent immobilization of enzyme. To achieve this goal, reaction between the available ester group on P(AN-co-MMA) nanoparticles surface and the amine groups of diamine was first performed. The introduced amine groups were further activated using symmetric coupling agent, glutaraldehyde (GA), which finally covalently binding with enzyme (Scheme 2).

Materials and Methods

Methyl Methacrylate (MMA) was purchased fromACROS (USA), Potassium Persulfate ([K.sub.2][S.sub.2][O.sub.8]), Ethylene Diamine (EDA) and [alpha]-Galactosidase from Aspergillus oryzae (E.C.3.2.1.23) were obtained from Siga Chem. Co. (St. Louis, USA), Acrylonitrile (AN) and Glutaraldehyde (GA) were obtained from Fluka (packed in Switzerland) and Lactose was obtained from El-Nasr Pharmaceutical Co for Chemicals. (Egypt). All other chemicals used were of analytical reagent grade. Buffer solutions were prepared with distilled water.

Nanoparticles Preparation

The P(AN-co-MMA) copolymers were prepared by precipitation polymerization of Acrylonitrile (AN) and Methyl Methacrylate (MMA) using Potassium Persulfate ([K.sub.2][S.sub.2][O.sub.8]) as initiator. To the reactor, monomers were mixed with the co-solvent from distilled water and Ethanol (1:1), and then followed by initiator injection. The polymerization was carried out at 55[degrees]C for 4 h. The formed copolymer was isolated by centrifugation at 14000 rpm and washed successively with the co-solvent to remove any residual monomers and initiator. The copolymer was then dried in an oven at 60[degrees]C for 24 h where white powder was obtained.

P(AN-co-MMA) nanoparticles surface modification

The surface of P(AN-co-MMA) nanoparticles was treated with a large excess of an aqueous solution of ethylene diamine where 1g P(AN-co-MMA) nanoparticles was mixed with 20 mL solution of 0.025% EdA, in distilled water, and kept in water bath maintained at 80[degrees]C for 1h.

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After completion the reaction, the nanospheres were centrifuged and washed with distilled water to remove unreacted ethylene diamine, then dried [27].

Enzyme immobilization

P(AN-co-MMA) modified nanoparticles, 1g, were activated using 20 mL of 1% glutaraldehyde solution (pH 8.0) at 40[degrees]C for 60 minutes. Then, the P(AN-co-MMA) activated nanoparticles were centrifuged and washed with distilled water to remove unreacted glutaraldehyde and transferred to enzyme Phosphate-Citrate buffer solution of pH 4.4, 20 mL of 0.005g/mL of [alpha]-Galactosidase, and stirred at room temperature, for one hour, then for 16 hours at 4[degrees]C to complete the immobilization process.

Determination of immobilized enzyme activity

The P(AN-co-MMA) copolymers were prepared by precipitation polymerization of Acrylonitrile (AN) and Methyl Methacrylate (MMA) using Potassium Persulfate ([K.sub.2][S.sub.2][O.sub.8]) as initiator. To the reactor, monomers were mixed with the co-solvent from distilled water and Ethanol (1:1), and then followed by initiator injection. The polymerization was carried out at 55[degrees]C for 4 h. The formed copolymer was isolated by centrifugation at 14000 rpm and washed successively with the co-solvent to remove any residual monomers and initiator. The copolymer was then dried in an oven at 60[degrees]C for 24 h where white powder was obtained.

P(AN-co-MMA) nanoparticles surface modification

The surface of P(AN-co-MMA) nanoparticles was treated with a large excess of an aqueous solution of ethylene diamine where 1g P(AN-co-MMA) nanoparticles was mixed with 20 mL solution of 0.025% EDA, in distilled water, and kept in water bath maintained at 80[degrees]C for 1h. After completion the reaction, the nanospheres were centrifuged and washed with distilled water to remove unreacted ethylene diamine, then dried [27].

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Enzyme immobilization

P(AN-co-MMA) modified nanoparticles, 1g, were activated using 20 mL of 1% glutaraldehyde solution (pH 8.0) at 40[degrees]C for 60 minutes. Then, the P(AN-co-MMA) activated nanoparticles were centrifuged and washed with distilled water to remove unreacted glutaraldehyde and transferred to enzyme Phosphate-Citrate buffer solution of pH 4.4, 20 mL of 0.005g/mL of a-Galactosidase, and stirred at room temperature, for one hour, then for 16 hours at 4[degrees]C to complete the immobilization process.

The catalytic activity of the immobilized enzyme was determined by mixing 1 g of catalytic P(AN-co-MMA) nanoparticles with 50 mL of 100 mM lactose in phosphatecitrate solution of pH 4.4 at 40[degrees]C with stirring, 250 rpm, for 30 minutes. Samples, 0.1 mL each, were withdrawn every 5 minutes to assess the produced glucose using glucose Kit. The enzymatic activity was determined by the angular coefficient of the liner plot of the glucose production as a function of time [28]. The retention of activity percentage (RAP) is the ratio of the immobilized enzyme's activity to the activity of the same amount of free enzyme and given as:

Retention of Activity Percentage = (Activity of immobilized enzyme/Activity of free enzyme) x 100

RAP provides information on the role of substrate diffusion in the reaction. A value of RAP =100 is obtained under conditions of complete diffusion, i.e., in case of homogenous reaction with the free enzyme.

Thermal Gravimetric Analysis

The thermal degradation behaviors of the P(AN-co-MMA) nanoparticles, P(MMA) and P(AN) was studied using Thermo Gravimetric Analyzer (Shimadzu TGA- 50, Japan); instrument in the temperature range from 20[degrees]C to 400[degrees]C under nitrogen at a flow rate of 20 mL/min and at a heating rate of 10[degrees]C/min.

Morphological Characterization

The surface morphology of P(AN-co-MMA) nanoparticles was observed using Scanning Electron Microscope (Joel Jsm 6360LA, Japan) at an accelerated voltage of 20 kV. Before observation, samples were mounted on metal grids with double sided adhesive tape and coated with gold in vacuo.

FT-IR Spectroscopic Analysis

The chemical structure of the P (AN-co-MMA), P (MMA) and P(AN) nanoparticles was analyzed by FT-IR spectra. Samples were mixed with KBr to make pellets. FT-IR spectra in the absorbance mode were recorded using FT-IR spectrometer (Shimadzu FTIR- 8400 S, Japan), connected to a PC, and analysis the data by IR Solution software, Version 1.21. The spectra (128 scans at 2 [cm.sup.-1] resolution) were collected with the frequency range of4000-400 [cm.sup.-1]. The FTIR spectra were Fourier-deconvoluted with a resolution enhancement factor of 1.5 and a bandwidth of 15 [cm.sup.-1].

Particle Size Analysis

Particle size of the P(AN-co-MMA) nanoparticles was analyzed using Submicron Particle SizeAnalyzer (Beckman Coulter--USA) using photon correlation spectroscopy (PCS), which determines particle size by measuring the rate of fluctuations in laser light intensity scattered by particles as they diffuse through a fluid operating at a 25 mW laser. A constant amount of particles was dispersed in distilled water, viscosity 1.002 and refractive index 1.33, to prepare a concentration of 1% at temperature 20[degrees]C and was kept under agitation at 150 rpm for 30 minutes.

Results and Discussion

P(AN-co-MMA) nanoparticles were synthesized from the monomers Acrylonitrile and Methyl Methacrylate in ethanol/water medium using ([K.sub.2][S.sub.2][O.sub.8]) as initiator. The ester groups of the P(AN-co-MMA) nanoparticles were converted into amino groups in the presence of ethylene diamine during the modification process [29]. In the following, the modification followed by activation processes of P(AN-co-MMA) nanoparticles has been studied. The changes in P(AN-co-MMA) nanoparticles characters have been followed using different characterization techniques such as FT-IR, SEM and TGA. Different factors affecting the modification and activation processes have been monitored and their impact on the catalytic and retained activity of the immobilized [alpha]-Galactosidase has been discussed.

Modification process

Surface modification of P(AN-co-MMA) nanoparticles was carried out using ethylene diamine (EDA) to be functionalized with terminal primary amine groups. Factors namely; ethylene diamine concentration, reaction time, reaction temperature and P(AN-co-MMA) amount have been studied. The success of any immobilization process is governed basically by keeping almost, if not all, of the enzyme activity after completion of the immobilization process. This factor knows as retention of activity percentage (RAP). The effect of different factors affecting the catalytic and retained activity of the P(AN-co-MMA) nanoparticles is studied and discussed in the fallowing.

Effect of EDA Concentration

The effect of variation EDA concentration on the catalytic activity was investigated (Figure 1). Contentious decrease of the activity has been observed with increase of EDA concentration from 0.025% to 0.5%, where the activity has been decreased by about 30%. Further slight activity decrement, about 6%, has been observed with increasing EDA concentration up to 1%. The determining effect of EDA concentration in the range of 0.025 to 0.5% implies the importance of the selection of optimum concentration of EDA. Similar behavior was noticed by other authors for the same enzyme and other different enzymes covalently immobilized onto glutaraldehyde activated matrices [30-33]. They explain this behavior based on the presumption that "immobilization of the enzyme may occur directly by covalent attachment or first ironically exchange the enzyme molecules and later covalently binding. This may depends on the of support's activation degree. At very low activation degree, the ionic exchange step will be neglect able and the activation step will be the domain one. At higher activation degree, a mixture of adsorption followed by covalent binding will be existence. In this way, the orientation of the enzyme molecules will be affected first and consequently the enzyme activity and retained activity".

This explanation is reinforced by the fact that fixed number of enzyme molecules has been immobilized since no trace of enzyme activity was detected in the immobilization medium after completion of the immobilization process. In addition, formation of Schiff's base results from reaction between aldehyde groups of GA and primary amine groups on the surface of nanoparticles from one side and enzyme molecules from other side induces ionic changes in the microenvironment around the immobilized enzyme. Possible hydrophobic interactions between the immobilized enzyme molecules and the hydrophobic moieties of P(AN-co-MMA) structure could be contributed also. The individual or synergetic effect of the mentioned factors could give us a reasonable explanation of the presented behavior in Figure 1. The effect of variation the EDA concentration on the retained catalytic activity of immobilized enzyme molecules was shown in Figure 1. A similar behavior to that of the catalytic activity was observed. The retained catalytic activity was decreased by 36% with increases the concentration of EDA up to 1%. This was expected since the amount of immobilized enzyme is fixed. The observed sensitivity of immobilized enzyme' retained activity indicates the importance of optimizing the number of attachment point between the enzyme molecule and the matrix. Different behavior of the same enzyme has been observed by El-Aassar et al [34] when immobilized on nanofibers form of the same matrix using PEI instead of EDA in the modification process. This difference could be referred to the nature of PEI, as a polymer, hosting the enzyme molecules away from the fibers surface and offering different "micro-environment".

Effect of Reaction Temperature with EDA

Figure 2 show the effect of variation reaction temperature with EDA on the catalytic and retained activity of immobilized enzyme. From the figure it is clear that increases the temperature, within studied range (30[degrees]C-90[degrees]C), has a liner negative effect on the catalytic activity through two stages. In the first stage, 30[degrees]C-65[degrees]C, very small activity decline was observed. In the second stage, 70[degrees]C-90[degrees]C, very fast decline of activity was observed. The retention of activity showed the same behavior. Two explanations could give an interpretation of the obtained results. The first one focused on increase the possibility of consuming the EDA molecule in cross-linking of PMMA ester groups [35]. Hence, reduced the available aldehyde groups of GA which binding covalently with enzyme molecules. As a result, the amount of immobilized enzyme reduced and hence the catalytic activity. Since the amount of immobilized enzyme was found constant under different reaction temperatures, the above mentioned explanation was excluded. Under these circumstances, increasing the reaction temperature could lead to increase the density of attached amine groups to the nanoparticles surface which causes, consequently, to bind the enzyme molecules with multi covalent attachment bonds on the nanocopolymer particles surface. This raises the possibilities of binding the enzyme active site and hence "freezing" of the conformational structures of enzyme molecules which make their active centers not free accessible to the substrate.

Effect of Reaction Time with EDA

Figure 3 illustrated the effect of variation reaction time with EDA. The obtained results revealed that both the activity and retained activity increased linearly with increase the reaction time till reaching its maximum value at 50 minutes. Further increase of the reaction time up to 60 minutes did not lead to a significant change in the catalytic activity. The creation of increasingly number of free amine groups on particles surface increased the chance of formation of multi covalent bonds between the particles surface and the enzyme molecules which leads to fix the enzyme molecules in the better conformational structure. This results are in accordance with previous published ones by El-Aassar et at [34].

Effect of P(AN-co-MMA) nanoparticles amount

Figure 4 show the correlation between the activity and the retained activity, of covalently immobilized a-galactosidase, and the variation of P(AN-co-MMA) nanoparticles amount. Two trends were noticed from inspection of the figure. The first is a linear activity increment with an increase of PVC amount in the range from 0.25 to 1.0 g. The second trend of the curve started from 1 to 2.5 g in which the activity increment rate was found to be lower and tends to level off. Two observations have to be taken into consideration to explain the obtained results. The first is the increase in the number of induced amine groups with the surface area of the particles. The second is the constant amount of immobilized enzyme. This leads to reduce the "density" of enzyme molecules immobilized on the particles' surface and prevention of the protein-protein interaction. Similar results have been obtained previousely by the authors [33].

Activation process

Induced primary amine groups on the particles' surface, from the previous step, were activated using glutaraldehyde to facilitate the covalent binding of a-galactosidase molecules via Schiff base formation. Accordingly, the control of aldehyde groups content through studying the factors affecting the activation step namely; glutaraldehyde concentration, reaction time, reaction temperature and reaction pH is an important issue to understand and optimize the the activity and retained activity of immobilized enzyme.

Effect of Glutaraldehyde Concentration

Glutaraldehyde has two roles. The first is to facilitate the covalent binding of a-galactosidase and the second as a spacer to increase the distance between the enzymes molecules and the particles' surface [36]. Figure 5 show the effect of variation glutaraldehyde concentration on the activity and retained activity of immobilized a-galactosidase. Inspection the figure show two phases. The first phase is sharp and linear increase of the activity with increase glutaraldehyde concentration up to 1%. The second phase is lower rate increase of the activity with further increase of GA concentration to 6%. The same behavior has been observed by the author [33 & 34]. Two explanations could be given for the observed relative decrease of the activity increment rate in the second phase of the curve. The first is the incorporation of the GA molecules in crosslinking free amine end groups of EDA rather than coupling the enzyme molecules to the particles surface. This observation has been found previously by the authors in immobilizing Penicillin G Acylase enzyme to aminated Nylone membranes [37]. The fact that amount of immobilized enzyme molecules changed with variation of GA concentration excluded the previous explanation.

The second explanation is increasing the GA concentration increases the possibilities of binding the enzyme molecules with a higher number of covalent bonds in which fixed the enzyme three dimensional structures and reduced of its active center availability for substrates. Simultaneously, the protein density on the particles surface increased and causes "protein-protein" interaction which consequently leads to reduce the activity of immobilized enzyme [38]. The leveling off all activity and retained activity at GA concentration above 6% is expected since all the enzyme molecules have been immobilized and kept almost all of their catalytic activity; 95% retention of activity.

Effect of Reaction Temperature with GA

Figure 6 show the effect of variation the reaction temperature with GA on the catalytic activity and retained activity of immobilized enzyme. From illustrated data it is clear that the activity and retained activity increased with activation temperature up to 40[degrees]C. Further increase in the glutaraldehyde reaction temperature (up to 80[degrees]C) has insignificant effect. Increase the reaction temperature with GA may be leads to formation of poly GA with longer chains.

This leads to two opposite effects on the activity. The first is reducing the free terminal aldehyde end groups available for enzyme immobilization and consequently the amount of immobilized enzyme. The second is kept the attached enzyme molecules fare from the particles surface which consequently leads to reduce the occurrence of protein-protein interaction and, at the same time, increases the chances of substrate to reach the enzyme molecules. The synergetic effect of both two determined the obtained behavior.

Similar behavior was observed by El-Aassar [34] but with higher effect of the temperature on the activity in the range from 30 to 50oC where the maximum activity was observed. The current results showed advantage in this direction which the maximum activity was obtained at 40[degrees]C.

Effect of Reaction Time with GA

Variation of reaction time with GA clearly affected the catalytic activity and retained activity of immobilized enzyme (Figure 7). The behavior of the catalytic activity and retained activity is very similar, they showed approximately the same behavior when the reaction time increase. Linear increment has been observed with increasing reaction time up to 30 minutes after which, exponential increment obtained with reaction time increase up to 60 minutes which the highest values was obtained and finally, leveling off was observed with increasing reaction time up to 90 minutes. It can be ascribed to the reason that at higher reaction time with GA, higher amount of a-Galactosidase was introduced onto the surface of the P (AN-co-MMA) nanoparticles. The retained activity of immobilized enzyme on nanoparticles or nanofibers of P (AN-co-MMA) in this work and El-Aassar work [34] showed higher values than enzyme immobilized on aminated PVC particles [33].

Effect of GA Solution' pH

Figure 8 show the effect of variation the pH of GA solution on the catalytic activity and retained activity of immobilized enzyme. From illustrated data it is clear that the activity almost linearly increased with pH up to its highest value at 11.0. This behavior may be referred to the de-protonation effect of alkaline medium on the amine groups which increase its reactivity to react with end aldehyde groups of GA. This consequently leads to eliminate the negative effect of first adsorption step with un-activated protonated amino groups and consequently leads to immobilize the enzyme in best conformational structure and gains better activity [39]. Furthermore, linear increase of the retained activity of immobilized a-galactosidase has been observed with glutaraldehyde pH increase up to pH 11.0. The same trend has been observed by Mohy Eldin et al [33, 40] with immobilization of PGA onto amino functionalized nylon particles. At high reaction' pH, the possibility of glutaraldehyde polymerization is raised. According to the published results by Roberto Fern'andez-Lafuente et al [41], using lower concentrations or shorter reaction times, an average of more than one glutaraldehyde molecule and less than two glutaraldehyde molecules were incorporated. The use of more drastic conditions (e.g., pH over 8, higher glutaraldehyde concentrations) yielded an uncontrolled reaction that generated the polymerization of glutaraldehyde in solution. These results are in agreement with those previously reported by Monsan [42]. Polymerized glutraraldehyde at higher pH (10) plays double functions. The first is the regular binding one. The other one is "spacer" which put the enzyme molecules away from the support surface and makes them more available for the substrates. This normally leads to have more activity and higher retained activity for the immobilized enzyme. El-Aassar show different behavior with nanofibers [34] where the influence of pH was clear with increasing pH up to 6, after that increasing the pH up to 12 has no significant effect. In comparison with the obtained results, nanoparticles proved to have higher retained activity of immobilized enzyme, 75%, compared to 45% of enzyme immobilized onto nanofibers by El-Aassar [34]. This difference of behavior may be referred to the nature of PIE which composed of primary, secondary and tertiary amines.

P(AN-co-MMA) nanoparticles characterization

Proves of surface functionalization process have been obtained through performing FT-IR and TGA analysis of the P(AN-co-MMA) nanoparticles. In addition, morphology of P(AN-co-MMA) nanoparticles has been monitored through SEM micrographs, and the particle size of P(AN-co-MMA) nanoparticles has been measured. The obtained results are discussed in the following.

FT-IR Analysis

Figure 9 presents the FTIR spectra of polymers, (PAN) and (PMMA), and their copolymer, P(AN-co-MMA). PAN is characteristic of the adsorption peaks at 1629 [cm.sup."1] and 2246 [cm.sup."1], which correspond to the bonds C=C and Ca"N respectively [43-44]. PMMA is characteristic of the adsorption peaks at 1631 [cm.sup."1] and 1732[cm.sup."1], which correspond to the bonds C=C and C=O respectively [45-46].

By comparing the FTIR spectrum of the copolymer with that of individual polymers, It can be found that the P(AN-co-MMA) keeps the absorptions bands at 1730[cm.sup."1] and 2243 [cm.sup."1] for C=O and Ca"N, respectively. C=C loses the absorption at 1629 or 1631[cm.sup."1] for indicating that the copolymer maintains the main characteristics of the monomers.

TGA Analysis

The thermal stability of the P(AN-co-MMA) nanoparticles was analyzed under N2 atmosphere at temperature ranged from R.T to 600[degrees]C at a heating rate of 10[degrees]C [min.sup."1]. Compared with the TGA curves of PMMA and PAN show their thermal stability up to 265[degrees]C and 269[degrees]C, respectively, the result show the decomposition of the copolymer at temperature higher than 306[degrees]C (Figure 10). Therefore, the copolymer nanoparticles gained thermal stability. Apparently, the copolymerization between MMA and AN can improve the thermal stability of the polymer with single monomer.

SEM Analysis

Figure 11 show SEM images of PAN, PMMA, and P(ANco-MMA). From the figure, it is clear the changes in the surface morphology of copolymer nanoparticles and formation spherical particles.

Particle size Analysis

Since the immobilization process occurs on the surface of P(AN-co-MMA) nanoparticles, so the surface area is a determined factor. Particle size distribution affecting directly the surface area, increasing the particle size distribution range of particles automatically leads to increase standard deviation error. Data obtained show that how much wide is the distribution of P(AN-co-MMA) nanoparticles, from 10 to 110 nm, which explain the variation of the obtained catalytic activity of immobilized enzyme prepared under the same conditions.

Conclusion

[alpha]-galactosidase enzyme has been successfully immobilized through covalent bond on P(AN-co-MMA) nanoparticles with high catalytic activity reached to 12500 units/kg and 96% retained catalytic activity.

P(AN-co-MMA) nanoparticles were first successfully modified with EDA and as a result its surface has been functionalized with amine groups, and then activated using glutaraldehyde as a coupling agent. Minimum EDA concentration and minimum reaction temperature with EDA gave the best results of catalytic activity of immobilized enzyme. The optimum conditions within studied ranges are 0.025% EDA and 30[degrees]C respectively. On the other hand, the optimum conditions of activation process using glutaraldehyde within studied range are GA 6%, pH= 11.0, 90 minutes reaction time at 65[degrees]C.

Proves for the modification and activation steps have been extracted from FT-IR and TGA analysis, and surface morphology changes have been monitored through SEM micrographs.

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M.S. Mohy Eldin (a)*, M.R. Elaassar (a), A.A. El.Zatahrya (b), M.B. El-Sabbah (c)

(a) Polymer materials research Department, Institute of Advanced Technology and New Material, Mubarak City for Scientific Research and Technology Applications, New Borg El-Arab City 21934, Alexandria, Egypt

(b) Department of Chemistry, Faculty of Science, King Saud Univesity, Riadh, KSA

(c) Department of Chemistry, Faculty of Science, Al-Azhar University, Cairo, Egypt

* Corresponding author: Prof. M.S. Mohy Eldin, m.mohyeldin@mucsat.sci.eg

Received 8 July 2014; Accepted 5 December 2014; Available online 10 December 2014
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Title Annotation:Original Articled
Author:Eldin, M.S. Mohy; Elaassar, M.R.; El.Zatahrya, A.A.; Sabbah, M.B., El-
Publication:Trends in Biomaterials and Artificial Organs
Article Type:Report
Date:Oct 1, 2014
Words:6196
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