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[beta]-galactosidase immobilization onto Poly (Acrylonitrile-Co-Methyl Methacrylate) nanoparticles.


The wide range of commercial applications for polymer micro-and nanoparticles has encouraged much recent research in this field. The polymerization methods available to prepare such monodisperse particles include water-based emulsions, seeded suspension polymerizations, nonaqueous dispersion polymerizations, and precipitation polymerizations. Precipitation polymerizations are unique within this group in that they can lead to monodisperse micro- and nanoparticles free of any added surfactant or stabilizer (1,2).

This technique starts as homogeneous mixtures of monomer, initiator and solvent. During the polymerization, the growing polymer chains phase-separate from the continuous medium by enthalpic precipitation in cases of un-favorable polymer--solvent interactions, or entropic precipitation, in cases where cross linking prevents the polymer and solvent from freely mixing. In good solvents, these polymerizations will often produce turbid macroscopic or microscopic gels, depending largely on the original monomer concentration. In poorer solvents, precipitation polymerization normally produces micro- and nano-meter sized particles (1,2). One of the most important application areas of precipitation polymerization is the synthesis of molecularly imprinted polymers (3,4). Molecular imprinting is a process where a functional monomer and a cross linker are copolymerized in the presence of the imprint molecule that acts as a molecular template. Initially, the functional monomer and the template are connected by a covalent linkage or they are placed nearby through non-covalent interactions. After the polymerization, subsequent removal of the imprint molecule reveals binding sites that are complementary in size and shape to the analyte (5). The high affinity and selectivity of molecularly imprinted polymers and their unique stability enable them to have a wide variety of applications such as separation, sensors and catalysis.

Enzymes have been utilized in a large number of practical applications, particularly in biomedical and biotechnological fields, through immobilization on a variety of supports. Many methods exist for the immobilization of enzymes but usually one of four methods is used:

(i) Physical adsorption,

(ii) Entrapment,

(iii) Copolymerization, and

(iv) Covalent attachment (6-10).

The methods and supports employed for enzyme immobilization are chosen to ensure the highest retention of enzyme activity and its stability and durability. The most important advantage of the former method is the reversibly immobilization of enzyme on the support (11,13). A number of methods for reversible immobilization of enzymes have been reported in the literatures, such as adsorption onto an insoluble material, entrapment in hydro-gels, cross linking with a bi-functional reagent, and covalent linking to an insoluble carrier.

A variety of support materials have been used for immobilization of biocatalyst, and nanoparticles have received increased attention for industrial manufacturing of enzyme-processed products (14). Several kinds of nanoparticles are produced from various polymers with different functional groups. Nanoparticles have wide range of applications in the immobilization of cells and enzymes, bioseparation systems, immunoassays, drug delivery, biosensors and so on.

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 (15,16).

In previous studies, the authors investigate the preparation conditions for the P(AN-co-MMA) nanoparticles (17) and optimized the activation conditions to have finally activated P (AN-co-MMA) nanoparticles ready for enzyme immobilization (18).

In the present study, the immobilization conditions of [beta]-galactosidase onto P(AN-co-MMA) nanoparticles were studied. The obtained biocatalyst was characterized from biochemical points and the stabilities parameters have been evaluated.



Methyl Methacrylate (MMA) was purchased from ACROS (USA), Potassium persulfate ([K.sub.2][S.sub.2][O.sub.8]), Ethylene diamine (EDA) and a-Galactosidase (E.C. were obtained from Siga Chem. Co. (St. Louis, USA), Acrylonitrile (AN) and Glutaraldehyde (GA) was 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.


Preparation of P(AN-co-MMA) nanoparticles

The precipitation polymerization experiments were carried out in a glass test tube. The initiator was dissolved in a cosolvent (1:1) ([H.sub.2]O: ETOH) where the monomers were then added. The initiator Potassium per-sulfate ([K.sub.2][S.sub.2][O.sub.8]) was kept at 0.01 M and the total monomers concentration was at 10 % (V/V) relative to the co-solvent, except otherwise mentioned. The polymerization was induced in a preheated water bath (at 55[degrees]C). The polymer was isolated by centrifuge and washed successively with distilled water to remove any impurities such as residual monomers and initiator. The product was then dried in an oven at 80[degrees]C for 24h. The white powder was obtained as a final product. The mechanism of P(AN-co-MMA) nanoparticles formation is presented in Scheme1.

P(AN-co-MMA) nanoparticles surface modification

P(AN-co-MMA) nanoparticles were aminated by treating with a large excess of Ethylene diamine aqueous solution. Thus, 1g P(AN-co-MMA) nanoparticles was mixed with EDA (0.025%) 20 mL solution in distilled water and kept in water bath maintained at 80[degrees]C for 1h. After completion the reaction, the nanoparticles were filtered and washed with distilled water to remove unreacted ethylene diamine, then dried in an air dryer (19).

Enzyme immobilization

P(AN-co-MMA) modified nanoparticles (1g) was activated using 20 mL of glutaraldehyde (1%) of pH 8.0 at 40[degrees]C for 60 minutes. After completion of the activation process, the P (AN-co-MMA) activated nanoparticles were filtered and washed with distilled water to remove unreacted glutaraldehyde. The activated P (AN-co-MMA) nanoparticles were then transferred to 20 mL of enzyme Phosphate- citrate buffer solution (pH 4.4) containing 0.005g/mL of [beta]-galactosidase and stirred at room temperature for one hour then for 16 hours at 4[degrees]C to complete the immobilization process. The mechanism of P (ANco-MMA) nanoparticles modification, activation and immobilization with enzyme is presented in scheme 2.

Determination of the immobilized enzyme activity

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 phosphate-citrate solution (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 (20).

Results and Discussion


After found the best experimental conditions for functionalization and activation process, optimization of the immobilization process represents the last step in this section. Factor affecting this process namely; the enzyme concentration, the immobilization time and the immobilization pH have been studied.

Effect of the enzyme concentration

In order to maximize the amount and the activity of the immobilized [beta]-galactosidase onto the surface of the P(AN-co-MMA) nanoparticles, the initial enzyme concentration was changed between 0.001 g/mL and 0.02 g/mL in the medium. As seen in Figure 1, increase the enzyme concentration up to 0.01 g/mL leads to increase the activity of the immobilized enzyme almost exponentially. This increase in the observed activity is associated with increase of the immobilized enzyme molecules to the fixed aldehyde groups on the surface of the nanoparticles. This leads consequently to increase the density of the immobilized enzyme molecules on the particles' surface which in turn leading to rise of "protein-protein" interaction and a consequence reduction of catalytic activity. This explanation is confirmed by the obtained behavior of the enzyme' retained activity (21).

Effect of the immobilization time

In order to effectively facilitate the covalent coupling and prevent [beta]-galactosidase deactivation for longer reaction time, it is important to choose the optimum reaction time. Figure 2 illustrates the catalytic activity of the immobilized [beta]-galactosidase prepared at different immobilization time.

From the figure it is clear that the catalytic activity increased with prolonged the immobilization time and the maximum value was obtained under immobilization allowed to proceeding for 5h. However, the catalytic activity neglectably decreased in case of prolonged the immobilization time up to 16 hours. It could be concluded that five hours seem to be the optimum time necessary for the immobilization process to reach maximum activity taking into consideration that the immobilization process taking on the particles' surface mainly. Further, the retention of activity shows the same manner which increased with prolonged reaction time and the obtained the highest retained activity after 5hours immobilization time.

Effect of the Immobilization pH

Figure 3 shows the effect of performing the immobilization process under different pH on the catalytic and the retained activity of immobilized [beta]-galactosidase. The maximum catalytic activity was gained at about pH 5.2. The rate of inactivation of a-Galactosidase, like other protein denaturations, was in most cases greatly dependent on the pH of the solution. The protonation and deprotonation of the charged functional groups were dependent up on the pH of the solution. The behavior of the catalytic activity and retention of activity is very similar. They showed approximately the same behavior when the immobilization's pH increase. Such behavior may explained according to the compromise between the denaturation effect of almost neutral medium on the enzyme molecules, which is favored for the deprotonation of amine groups on the surface of enzyme molecules and increase the rate of its coupling with aldehyde groups onto the particles' surface.




Biochemical Characterization

Effect of Substrate's Temperature

The effect of variation the substrate's temperature on the free and immobilized [beta]-galactosidase activities were investigated, by using lactose as substrates, and the obtained data are shown in Figure 4. The optimum temperature of the free [beta]-galactosidase appeared at 50[degrees]C where the optimum temperature of the immobilized [beta]-galactosidase was shifted to higher temperature; 60[degrees]C. Inspection the temperature's profile of the immobilized [beta]-galactosidase shows that the immobilization process induced thermal stability at temperature above 50[degrees]C, while the relative activity of the free [beta]-galactosidase starts to decline. The increase in the optimum temperature may be caused by changing the physical and the chemical properties of the enzyme. This could be explained by creation of conformational limitations on the enzyme movements as a result of formation of covalent bonds between the enzyme and the support. In general, the effect of changes in temperature on the rates of enzyme-catalyzed reactions does not provide much information on the mechanism of biocatalysts. However, these effects can be important in indicating structural changes in enzyme (22).

Arrhenius plots (Figure 5) in the temperature range from 25[degrees]C to optimum appear linear and activation energies were found to be 7.47 and 5.54 kcal [mole.sup.-1] for the free and the immobilized forms. The activation energy of an enzyme reaction may or may not change as a consequence of the immobilization process. For example, the activation energies of immobilized glucoamylase and [beta]-galactosidase were almost the same as their free counterparts. On the other hand, the activation energy of invertase covalently immobilized onto pHEMA membrane increased in comparison to that of the free enzyme (23).

Effect of Substrate's pH

The effect of variation the substrate's pH on the activity of the free and the immobilized forms for lactose hydrolysis was examined in the pH range from 2.0 to 7.0 at 40[degrees]C and the results are presented in Figure 6. The change in optimum pH depends on the charge of the enzyme and/or of the water insoluble matrix. This change is useful in understanding the structure-function relationship of enzyme and to compare the activity of free and immobilized enzyme as a function of pH.

The pH value for optimum activity for the free a-Galactosidase was found to be at 4.4. On the other hand, the optimal pH for the immobilized [beta]-galactosidase was extended between pH 4.4 and 5.2. The shift to neutral region for the immobilized enzyme can be due to the basic nature of the amino functionalized surface of the P(AN-co-MMA) nanoparticles, the amino groups on the nanoparticles surface can prevent the uniform distribution of hydrogen ions between the surface and the bulk solution. Furthermore, the pH profiles of the immobilized [beta]-galactosidase display an improved stability on both sides of the optimum pH value, in comparison to that of the free form, which means that the immobilization method preserved the enzyme activity in a wider pH range. These results could probably be attributed to the stabilization of a-Galactosidase molecules by forming multipoint attachments on the glutaraldehyde activated P(AN-co-MMA) nanoparticles.




Kinetic Studies of immobilization

Kinetic parameters for the activity of the free and the immobilized [beta]-galactosidase, [K.sub.m] and [V.sub.max], were assayed at substrate concentration from 20 to 250 mM. [V.sub.max] defines the highest possible velocity when all the enzyme is saturated with substrate, therefore, this parameter reflects the intrinsic characteristics of the immobilized enzyme, but may be affected by diffusion constrains. [K.sub.m] is defined as the substrate concentration that gives a reaction velocity of 1/2 [V.sub.max]. This parameter reflects the effective characteristics of the enzyme and depends upon both partition and diffusion effects.

The kinetic parameters [K.sub.m] and [V.sub.max] of the free and immobilized enzymes were determined by using lactose as substrate. The activities of free and immobilized [beta]-galactosidase for various concentrations of the substrate were plotted in the form of Lineweaver-Burk plots, as shown in Figure 7, and [K.sub.m] and [V.sub.max] values were calculated from the intercepts on x- and y-axes, respectively. [K.sub.m] values were estimated at 54.5 and 117.3 mM for the free and the immobilized [beta]-galactosidase, respectively. The apparent [K.sub.m] value of the immobilized [beta]-galactosidase was two times higher than that of the free enzyme. The [V.sub.max] value of the immobilized enzyme increased about 3.5-fold, compared to the free enzyme.

When a biocatalyst is immobilized, kinetic parameters [K.sub.m] and [V.sub.max] undergo variations with respect to the corresponding parameters of the free form, revealing an affinity change for the substrate. These variations are attributed to several factors such as protein conformational changes induced by the support, steric hindrances and substrates diffusion effects. These factors may operate simultaneously or separately, alternating the microenvironment around the bound enzyme. In the construction of enzyme reactors and biosensors, it is very important to know the variations in the apparent kinetic parameters that appear as a result of immobilization. The increase in [V.sub.max] value as a result of immobilization should be related with the increase in K value, since an increase in the [K.sub.m] value leads a decrease in the affinity of the enzyme for its substrate. This increase in the [K.sub.m] values was either due to the conformational changes of the enzyme resulting in a lower possibility of forming a substrate enzyme complex, or to the lower accessibility of the substrate to the active sites of the immobilized enzyme caused by the increased diffusion limitation.

Thermal stability

Thermal stability of immobilized enzymes is one of the most important criteria of their applications. In general, activity of immobilized enzymes, especially in a covalently bound system, was more resistant than that of the soluble form against heat. Consequently, thermal stability experiment was conducted with the free and the immobilized a-Galactosidase. As was evident from Figure 8, the free a-Galactosidase lost about 87.2 % of its initial activity while the immobilized a-Galactosidase lost about 66.7% of its initial activity after a 300 min heat treatment at 60[degrees]C. These results suggest that the thermal stability of immobilized [beta]-galactosidase becomes significantly higher than that of the free enzyme at high temperature. This is may be referred to the acquired protection of covalently immobilized enzyme from conformational changes causing effect of the environment. Similar results have been previously reported for various covalently immobilized enzymes (23,24).




pH stability

The reduction in activity was observed during 300 minutes incubation at two pH values namely; 4.2 and 7. The pH stability of the immobilized [beta]-galactosidase compared to its free counterpart has recognized especially at pH 4.2, white the decrease at pH 7 is almost identical with the free enzyme; Figure 9. The observed stability of the immobilized form at pH 4.2 may be explained by the partition effect of the formed Schiff base between the enzyme molecules and the particles' surface. This is in agreement with cited results demonstrated that the resistance of [beta]-galactosidase to pH was strengthened and its pH stability is better than that of free [beta]-galactosidase (25).

Effect of the Stirring Rate

The effect of variation the stirring rate on the activity of the free and the immobilized [beta]-galactosidase has been explored and the results are presented in figure 10. It is clear that the activity of the free and the immobilized [beta]-galactosidase increased with increase the stirring rate up to 300 rpm with higher rate for the immobilized one. Further increase of the stirring rate has a neglect effect on the activity of the immobilized enzyme while a lower rate increase of activity the free enzyme was observed at 600 rpm. The activity increment may be due to reduction of the substrate' external diffusion limitation effect especially in case of the immobilized form where the immobilized enzyme can be more easily contacted by the substrates with the increase of the stirring rate (26). One can assume that if the enzyme had enough time, stirring rate and of course enough flexibility, it could tumble on a smooth surface and become activated for interaction with the substrate.

Storage stability

Enzymes are very delicate biocatalysts and lose their activity even during storage. Therefore, storage stability is a factor which should be examined. In this study, the free and the immobilized [beta]-galactosidase were stored in Phosphate/Citrate buffer (pH 4.4) at 4[degrees]C; Figure 11. From the figure it is clear that the activity of both forms has decreased linearly with time. the immobilized a-Galactosidase, 48% decrease in activity was detected during 6-week storage period while the activity lose of the free a-Galactosidase was about 65%. These results revealed that the immobilized a-Galactosidase exhibits higher storage stability than that of the free form. The possible generated multipoint covalent attachments between enzyme and glutaraldehyde activated P(AN-co-MMA) nanoparticles should also convey a higher conformational stability to the immobilized enzyme. The relative severe decrease in activity of the free [beta]-galactosidase might be due to its susceptible autolysis during the storage time. It should note that the immobilization of [beta]-galactosidase could reduce the autolysis effect.

Operational Stability

The successful integration of enzymes into analytical devices requires not only high storage stabilities but also high operational stabilities. Operational stability or Reuse stability for the immobilized enzyme is very important in economics, and an increased stability can make the immobilized enzyme more advantageous than its free counterpart. To investigate the reuse stability, the immobilized enzyme was washed with Phosphate/ Citrate buffer (pH 4.4) after each catalysis run and reintroduced into a fresh lactose solution for another hydrolysis at 40[degrees]C. Figure 12 shows the effect of repeated use on the activity of the immobilized enzyme. After 10 reuses (50 minutes), the residual activities of the immobilized enzymes were 48 % of the intial activity. Normally, the inactivation of the enzyme caused by the denaturation of the protein and the leakage of the protein from the support upon use, however, in our case the second reason could be excluded due to the covalent immobilization. From our previous experience with immobilized [beta]-galactosidase onto aminated PVC particles (27), incomplete recovery of the immobilized enzyme after each cycle could be the main reason of activity decline. To confirm our proposal, the weight of the catalytic particles was determined. It was found that 25% of the catalytic particles has been lost after ten reuse cycles. The loss of the catalytic particles with the lowest size and the highest surface area, which probably carry most of the immobilized enzyme amount, may be the main cause of activity loss.





One of the most important aims of enzyme technology is to enhance the conformational stability of the enzyme. The extent of stabilization depends on the enzyme structure, the immobilization methods, and the support's type. In this study, the attention is focused on optimization of the immobilization conditions of [beta]-galactosidase on the poly (acrylonitrile-comethyl methacrylate) nanoparticles; P(AN-co-MMA).

Biochemical characterization of the immobilized enzyme show shift of both the optimum temperature and the optimum pH to higher values compared with its free counterpart. On the other hand, the study of the kinetic parameters show that the apparent constant [K.sub.m] of the immobilized [beta]-galactosidase was about two times than that of the free one while the apparent V value of the immobilized [beta]-galactosidase was 3.5 times larger than that of the free one.

Furthermore, the immobilization process induced thermal, pH and storage stability for the immobilized enzyme. Finally, a high operational stability, obtained with the immobilized [beta]-galactosidase, indicates that the immobilized [beta]-galactosidase could successfully be used in the hydrolysis process of lactose either in milk de-lactose or in whey treatment.

The observation that the activity of the immobilized enzyme becomes less sensitive to reaction conditions than that of the free counterpart recommended the applications of the poly (acrylonitrile-co-methyl methacrylate) nanoparticles to enzymes immobilization.


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M.S. Mohy Eldin (a) *, M.R. EL-Aassar (a), A.A. EL-Zatahry (a), M.M.B. EL-Sabbah (b)

(a) Group of Biotechnology Applications, Polymer Materials Research Department, Advanced Technology and New Material Research Institute, Scientific Research and Technological Applications City, New Borg El-Arab City 21934, Alexandria, Egypt

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

Received 25 January 2015; Accepted 22 May 2015; Published online 13 June 2015

(#) Corresponding author: Prof. M. S. Mohy Eldin, E-mail:
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Title Annotation:Original Article
Author:Eldin, M.S. Mohy; Aassar, M.R. EL-; Zatahry, A.A. EL-; Sabbah, M.M.B. EL-
Publication:Trends in Biomaterials and Artificial Organs
Article Type:Report
Geographic Code:1USA
Date:Jul 1, 2015
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