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Behaviors of enzyme immobilization onto functional microspheres
International Journal of Biological Macromolecules 37 (2005) 263–267
Behaviors of enzyme immobilization onto functional microspheres Shaogui Wu a,b , Bailing Liu b,∗ , Songjun Li a,b a
Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu 610041, China b Graduate School of Chinese Academy of Sciences, Beijing 100039, China Received 17 May 2005; received in revised form 6 October 2005; accepted 6 December 2005
Abstract Micron-grade monodisperse PMMA microspheres, whose surfaces were modified with functional groups by co-polymerisation using functional monomer, were prepared via dispersion polymerisation. Characterized by their large specific surface area, high adsorption ability, favourable biocompatibility, these monodisperse micron-sized PMMA microspheres were employed as the supporting material in the enzyme immobilization in present work. The influential factors on the activity of immobilized enzyme including pH, temperature, time etc were preliminarily investigated. The results concluded from the experiments indicated that the immobilization procedure could promote the resistance of enzyme against temperature, pH shift and some other tough reaction conditions meanwhile prolong the enzymatic lifetime for storage. © 2005 Elsevier B.V. All rights reserved. Keywords: Enzyme immobilization; Monodisperse microsphere; Dispersion polymerization
1. Introduction The immobilization technique has a wide application in continuous use or re-use of industrial enzymes. It can greatly increase enzymatic activity and stability so as to work at some rude conditions such as extreme pH and temperature, or even in the presence of organic solvents [1]. Another obvious advantage of immobilized enzymes over free enzymes is the facile handling of the reaction, e.g. the almost instant separation of product (which is not contaminated by enzyme) or the easy automatic control of the reactions within a whole production campaign. Therefore, the enzyme immobilization plays an important role in industry currently. Many carrier materials used in enzyme immobilization have been reported such as porous silica gel [2], porous zirconia [3], polymer membrane [4] and so on. Also, the use of polymer microspheres as supports for immobilization of enzymes was frequently reported, such as polypropylene, polystyrene, poly vinyltoluene microspheres, etc. However, most of the microspheres reported were characterized with average diameter greater than 100 m and a wide size distribution, which decreased the immobilizing capacity owing to the low specific surface area.
∗
Corresponding author. Tel.: +86 28 85260436. E-mail address: [email protected] (B. Liu).
0141-8130/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2005.12.007
Monodisperse micron-sized microspheres are characterized by their large specific surface area, great absorption capacity, high surface reactivity and are widely used in analytical chemistry, biochemistry, immune iatrology, standard metrology and many other high-tech fields. As a commonly used material, polymeric microspheres are usually endowed with many novel functions to meet the requirements for various applications. For example, scintillation proximity assay beads (SPA beads, a kind of monodisperse micron-grade functional scintillant beads) has widely been used in the High Throughput Screening (HTS) for drug discovery [5,6]. Some special drug targets (bio-macromolecules) have been immobilized on the surface of the beads, which have been successfully used for screening new drug. Additionally, the monodisperse microngrade functional microspheres are also playing important roles in many other fields such as enzyme linked immunosorbent assay (ELISA), immunofluorescence assay (FIA), etc. In the present work, monodisperse micron-grade functional polymethylmethacrylate (PMMA) particles were employed as the carrier for enzyme immobilization, whose surface had been modified with functional groups. These polymeric microspheres were prepared using dispersion polymerization technique. Enzymes immobilized onto this support can be easily separated from the reaction medium and used in batch and continuous systems. Trypsin as the model enzyme and casein as substrate were employed in the present study. The experiments
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S. Wu et al. / International Journal of Biological Macromolecules 37 (2005) 263–267
were designed in various reaction conditions to investigate the effects of immobilization on enzymatic activity and stability. 2. Experimental 2.1. Materials Methyl methacrylate (MMA) from Bodi Chemical Co. (China) was purified under reduced pressure distillation prior to use. Acrolein, functional monomer, was obtained from Kunshan Chemical Co. (China). Polyvinyl pyrrolidone steric stabilizer (PVP) (average molecular weight = 30,000) was obtained from Silong Chemical Co. and used as received. Benzoyl peroxide (BPO), initiator, was purchased from the Chemical Factory of Hubei University (China). Trypsin (high purity grade) was obtained from Shanghai Chemical Factory. Casein was purchased from Chengdu Kelong Chemical Factory. Disodium (A.R.) and citric acid (A.R.) were obtained from Guanghua Chemical Corporation of Shantou. Trichloroacetic acid was supplied by TCA Corporation. Methanol used as solvent was purchased from Chengdu Kelong Chemical Factory (China). All chemicals were used as received directly if not declared specially. Double distilled water was used in the experiment. 2.2. Preparation of microspheres An oven-dried, 100 ml, four-necked round-bottomed flask equipped with a mechanical stirrer, a condenser and a nitrogen inlet and outlet, was charged with methanol (38 g), MMA (4.5 g), and PVP (0.42 g). After bubbling nitrogen (1 h), the temperature was increased to 70 ◦ C. Then BPO-methanol solution was added immediately. The temperature was maintained at 70 ◦ C for 12 h. Then functional monomer acrolein was added dropwise. The mixture was stirred for another 12 h and milk-white latex was obtained. After centrifugation at the speed of 1000 rpm (approximately 223.57 g) for 10 min, the product microspheres were separated from the liquid phase and the supernatant was removed. The microspheres were repeatedly washed with doubly distilled water and centrifuged in 3 successive cycles to get rid of residual solvent. The resulted particles were suspended in phosphate buffer (pH 8) for further use. 2.3. Characterization The morphology of the microspheres (diameter ranging from 6 to 12 m) was observed by optical microscope (SEM better) directly. The size and size distribution were determined using a Malvern Master Sizer/E particle size analyzer (UK). Their values were obtained from the size distribution curve along the x- and y-axis, respectively. The size distribution was termed as uniformity. Obviously the smaller the uniformity was, the narrower the size distribution was. The average diameter was calculated from the diameters and volume percentages of all particles. The concentration of aldehyde groups on the surface of microspheres was determined by conductometric titration [7] (a schematic diagram is shown in Fig. 1). Excessive hydrochlorinate (2%, w/v) hydroxyl amino solution was added to 2 ml
Fig. 1. Schematic diagram of conductometric titration.
microspheres-buffer latex. After the mixture was stirred for 20 min, aqueous NaOH (0.01 M) was titrated drop-wise. The continuous titrated NaOH consumed the H+ and resulted in the decrease of the conductance. A conductivity meter (DDS-307) was employed to record the conductance of the reaction system. The concentration of the aldehyde groups on the surface of microspheres was calculated from the conductivity curve. 2.4. Enzyme immobilization To immobilize enzyme, trypsin (3 ml, 1 mg/ml) solution was added to phosphate buffer (4 ml) containing microspheres. The mixture was slightly stirred at room temperature and incubated for 2 h. Then the microspheres were separated from the liquid phase by centrifugation. The microspheres were washed several times with phosphate buffer (pH 8) to remove the unimmobilized enzymes. The immobilized enzyme (trypsin) was quantified by determined the amount of residual enzyme (unbinding) in supernatants UV-2010 spectrophotometer (Hitachi, Japan) at absorbance 280 nm. Then the enzyme-immobilized microspheres were suspended in phosphate buffer (4 ml, pH 8) and kept in refrigerator at 4 ◦ C for further use. 2.5. Characterization of immobilized enzyme Casein was chosen as the substrate to evaluate the activity of the immobilized trypsin. The enzymatic activity was determined through batch experiments, which were performed in a shaking water bath at 37 ◦ C. Three millilitres 1 mg/ml casein solution was added to 4 ml trypsin-immobilized microspheres latex. The reactions were designed under some controlled conditions (pH, temperature, time and so on) to investigate the effects of immobilization on enzymatic activity. Trichloroacetic acid (TCA) was used to terminate the hydrolyzation since it could inactivate enzyme. Then the reaction mixture was centrifuged to separate the sediments from the bulk phase. The casein hydrolysate, tyrosine in the supernatant was assayed spectrophotometrically. The activity unit was defined as the amount of product generated from the casein hydrolyzation catalyzed by 1 mg/ml trypsin at 37 ◦ C (pH 8.0) for 30 min. The relative activity (% of highest activity/original activity) was used to compare the changes between the free and immobilized enzymes. Three parallel experiments were carried out meanwhile and the average value was calculated as the final result. Km and Vmax are two important characteristic parameters of enzymatic catalytic reaction, the relation of which can be expressed as the famous Michaelis–Menten equation (below).
S. Wu et al. / International Journal of Biological Macromolecules 37 (2005) 263–267
265
A group of casein solutions of different concentrations (0.25, 0.50, 0.75 and 1.00 mg/ml) were used to determine the values of Km ’s and Vmax ’s of free and immobilized enzymes at controlled condition (37 ◦ C, pH 8.0). The detailed process was as similar as described above. The concentration of tyrosine in the supernatant was regarded as the value of initial reaction rate V0 . Each substrate sample was corresponding to a plot and a diagram of the initial reaction rate V0 plotted against the concentrations of substrate solutions [S] could be obtained. The values of Km and Vmax were calculated from the slopes and intercepts of the fitting curves: Km + [S] Km 1 1 1 = = + V0 Vmax [S] Vmax [S] Vmax 3. Results and discussion 3.1. Characterizations of microspheres Dispersion polymerization is a desired technique to prepare micron-grade monodisperse microspheres, which was also employed to synthesis the carrier PMMA microspheres in our study. The morphology of resulted particles was observed using an optical microscopy directly. As shown in Fig. 2, the PMMA microspheres exhibited a very narrow size distribution. The uniformity coefficient obtained from size analyzer was 0.2003 (shown in Fig. 3). Additionally, the average diameter determined by light scattering was 6.1 m. A rude calculation indicated the specific surface area of the PMMA particles was up to 0.51 m2 /g. The structure of the PMMA microspheres was very stable, both chemically and mechanically, as no change in size or size distribution of them was observed when the PMMA microspheres were stirred fiercely. The size and uniformity of the PMMA microspheres were very moderate for the enzyme immobilization. The concentration of aldehyde group on the surface of PMMA microspheres was determined by conductometric titration. The curve of conductance versus the amount of NaOH consumed was given in Fig. 4. The conductance decreased at the
Fig. 2. Photomicrograph of PMMA microspheres.
Fig. 3. Size-distribution of microspheres.
initial section due to reduction of [H+ ]. H+ was mainly consumed by the NaOH titrated in the ratio 1:1 by molar. Therefore, the concentration of aldehyde group on the surface of microspheres could be obtained by calculating the amount of the NaOH titrated. Experiments result indicated that the concentration of aldehyde groups was 55 mmol/ml for PMMA microspheres latex and 0.572 mmol/g for the dried microspheres. 3.2. Km and Vmax Enzyme can be immobilized tightly on and not easily washed out of supporting materials through physical or chemical adsorptions. It was reported that immobilized bio-molecules still retained high bioactivity after the immobilization [7–10]. The immobilization procedure was usually performed at some controlled conditions that were suitable for enzymatic structural integrity and functionality. A feasible immobilization strategy was employing covalently binding enzyme to functional groups on the surface of microspheres. In present study, the surface of
Fig. 4. Conductometric titration curve.
S. Wu et al. / International Journal of Biological Macromolecules 37 (2005) 263–267
266 Table 1 The apparent Km and Vmax
Km Vmax (U mg−1 enzyme)
Free enzyme
Immobilized enzyme
0.272 40
1.357 25
carrier PMMA microspheres was modified with aldehyde group, which could react with the amino group of enzyme to form corresponding Schiff’s base under mild conditions. Km and Vmax are two important parameters in enzymatic catalytic reaction. A group of parallel experiments were performed and the average values of Km ’s and Vmax ’s of both free and immobilized enzymes were given in Table 1, respectively. Km was usually used to evaluate the ability of enzyme and substrate forming complex, which was a characteristic value irrelevant to the concentrations of substrate and enzyme. Generally, Km presented the required concentration of substrate when the reaction rate reached the half of maximum rate (Vmax ). The more Km was, the weaker binding enzyme and substrate formed. As shown in Table 1, the apparent Km for immobilized enzyme was 1.357, nearly five times of that for free enzyme 0.272. Therefore, it was obvious that the interaction of enzyme and substrate had been weakened after immobilization. Apparently inactivation had occurred due to structural or conformational changes, for example the active sites might have been hidden or damaged during the immobilization process. 3.3. Effects of pH on enzyme activity The effects of pH on enzyme activity were investigated within a pH range from pH 5 to 10 (at 37 ◦ C). As shown in Fig. 5, it was obvious that the optimal pH for both enzymes was near 9 [11]. Immobilized enzyme maintained higher relative activities compared with free enzyme at the pH range. Both enzymes maintained rather high relative activities between pH 7 and 10. As the pH decreased (from pH 5 to 7), the relative activities of both enzymes descended and the trend for free enzyme was more obvious. For example when pH was down to 5, the relative activ-
Fig. 6. Effects of temperature on enzyme activity.
ity of free enzyme decreased to less than 40% of its maximum value, while the immobilized enzyme still kept more than 80% of its highest activity. A conclusion was drawn that immobilization procedure could improve the enzymatic resistance against pH shift significantly. 3.4. Effects of temperature on enzyme activity The thermal stability of immobilized enzyme was tested in comparison with free enzyme at given temperatures (29, 37, 45, 53 and 61 ◦ C). A diagram on the relative activities of both enzymes at different incubation temperatures was given in Fig. 6. As shown in Fig. 6, at the low temperature section, the catalytic activities of both enzymes increased with the rise of temperature at first and after a maximum value, decreased at higher temperature. The optimal temperature for free enzyme (with the highest catalytic activity) was near 45 and 54 ◦ C for immobilized enzyme. It was obvious that the optimal temperature of enzyme was enhanced by 9 ◦ C after immobilization. As was evident from the data, the immobilized enzyme possessed a better heat-resistance than free enzyme. This might be explained by that the immobilization procedure could protect the enzymatic configuration from distortion or damage by heat exchange. Therefore, immobilized enzyme could work in tougher environment with less activity loss. 3.5. Effects of time on enzyme activity
Fig. 5. Effects of pH on enzyme activity.
Most enzymes suffered severe losses of their bioactivity during storage period especially for free form whose storage stability was very poor even under low temperatures. As shown in Fig. 7, both the relative activities of free and immobilized enzymes decreased with the time. However, the free enzyme lost its activity more quickly than its immobilized form. On the 6th day, the free enzyme kept 70% of its origin activity while the immobilized form still retained 85%. On the 8th day, the relative activity deceased to 55% for free enzyme but only a little change occurred to immobilized enzyme. This suggested that immobilization procedure could prolong the storage period and consequently increase the times of reusability of enzyme. This
S. Wu et al. / International Journal of Biological Macromolecules 37 (2005) 263–267
267
enzymatic bioactivity. Although immobilized enzyme system had some drawbacks such as the resulting of slight inactivation of enzyme (the increase of Km and the decrease of Vmax after immobilization), the thermal stability, pH stability and storage lifetime of immobilized enzyme have been improved effectively. Meanwhile the immobilization procedure could protect enzyme/protein from structural or conformational variations with the environmental changes. More research work should be carried out to extend the application of immobilization enzyme. References
Fig. 7. Effects of time on enzyme activity (ambient temperature).
was very important for enzyme application on a large scale in industry. 4. Conclusion The monodisperse micron-sized polymeric microspheres are a kind of promising supporting material used for enzyme immobilization. In the present study, PMMA microspheres were prepared and covalent binding strategy was employed for enzyme immobilization to investigate the effects of immobilization on
[1] K.K. Ephraim, M.K. Dieter, J. Mol. Catal. B: Enzym. 10 (2000) 157–176. [2] H.H.P. Yiu, P.A. Wright, N.P. Botting, Micropor. Mesopor. Mater. 44 (2001) 763–768. [3] M. Huckel, H.J. Wirth, M.T.W. Hearn, J. Biochem. Biophys. Meth. 31 (1996) 165–179. [4] Q.T. Nguyen, P.H. Zheng, T.Y. Nguyen, P. Rigal, J. Membr. Sci. 213 (2003) 85–95. [5] N.D. Cook, Drug Discov. Today 1 (1996) 287–294. [6] G.S. Sittampalam, S.D. Kahl, W.P. Janzen, Curr. Opin. Chem. Biol. 1 (1997) 384–392. [7] X.M. Zhang, C.H. Yan, Z.H. Sun, Chin. Chem. Bull. 2 (1989) 27–30. [8] A. Subramanian, S.J. Kennel, P.I. Oden, et al., Enzyme Microb. Technol. 24 (1999) 26–34. [9] K. Rajesh, Kaneto, Curr. Appl. Phys. 5 (2005) 178–183. [10] A. Petri, T. Gambicorti, P. Salvadori, J. Mol. Catal. B: Enzym. 27 (2004) 103–106. [11] L.H. Ding, B.J. Qu, React. Funct. Polym. 49 (2001) 67–76.
Behaviors of enzyme immobilization onto functional microspheres Shaogui Wu a,b , Bailing Liu b,∗ , Songjun Li a,b a
Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu 610041, China b Graduate School of Chinese Academy of Sciences, Beijing 100039, China Received 17 May 2005; received in revised form 6 October 2005; accepted 6 December 2005
Abstract Micron-grade monodisperse PMMA microspheres, whose surfaces were modified with functional groups by co-polymerisation using functional monomer, were prepared via dispersion polymerisation. Characterized by their large specific surface area, high adsorption ability, favourable biocompatibility, these monodisperse micron-sized PMMA microspheres were employed as the supporting material in the enzyme immobilization in present work. The influential factors on the activity of immobilized enzyme including pH, temperature, time etc were preliminarily investigated. The results concluded from the experiments indicated that the immobilization procedure could promote the resistance of enzyme against temperature, pH shift and some other tough reaction conditions meanwhile prolong the enzymatic lifetime for storage. © 2005 Elsevier B.V. All rights reserved. Keywords: Enzyme immobilization; Monodisperse microsphere; Dispersion polymerization
1. Introduction The immobilization technique has a wide application in continuous use or re-use of industrial enzymes. It can greatly increase enzymatic activity and stability so as to work at some rude conditions such as extreme pH and temperature, or even in the presence of organic solvents [1]. Another obvious advantage of immobilized enzymes over free enzymes is the facile handling of the reaction, e.g. the almost instant separation of product (which is not contaminated by enzyme) or the easy automatic control of the reactions within a whole production campaign. Therefore, the enzyme immobilization plays an important role in industry currently. Many carrier materials used in enzyme immobilization have been reported such as porous silica gel [2], porous zirconia [3], polymer membrane [4] and so on. Also, the use of polymer microspheres as supports for immobilization of enzymes was frequently reported, such as polypropylene, polystyrene, poly vinyltoluene microspheres, etc. However, most of the microspheres reported were characterized with average diameter greater than 100 m and a wide size distribution, which decreased the immobilizing capacity owing to the low specific surface area.
∗
Corresponding author. Tel.: +86 28 85260436. E-mail address: [email protected] (B. Liu).
0141-8130/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2005.12.007
Monodisperse micron-sized microspheres are characterized by their large specific surface area, great absorption capacity, high surface reactivity and are widely used in analytical chemistry, biochemistry, immune iatrology, standard metrology and many other high-tech fields. As a commonly used material, polymeric microspheres are usually endowed with many novel functions to meet the requirements for various applications. For example, scintillation proximity assay beads (SPA beads, a kind of monodisperse micron-grade functional scintillant beads) has widely been used in the High Throughput Screening (HTS) for drug discovery [5,6]. Some special drug targets (bio-macromolecules) have been immobilized on the surface of the beads, which have been successfully used for screening new drug. Additionally, the monodisperse microngrade functional microspheres are also playing important roles in many other fields such as enzyme linked immunosorbent assay (ELISA), immunofluorescence assay (FIA), etc. In the present work, monodisperse micron-grade functional polymethylmethacrylate (PMMA) particles were employed as the carrier for enzyme immobilization, whose surface had been modified with functional groups. These polymeric microspheres were prepared using dispersion polymerization technique. Enzymes immobilized onto this support can be easily separated from the reaction medium and used in batch and continuous systems. Trypsin as the model enzyme and casein as substrate were employed in the present study. The experiments
264
S. Wu et al. / International Journal of Biological Macromolecules 37 (2005) 263–267
were designed in various reaction conditions to investigate the effects of immobilization on enzymatic activity and stability. 2. Experimental 2.1. Materials Methyl methacrylate (MMA) from Bodi Chemical Co. (China) was purified under reduced pressure distillation prior to use. Acrolein, functional monomer, was obtained from Kunshan Chemical Co. (China). Polyvinyl pyrrolidone steric stabilizer (PVP) (average molecular weight = 30,000) was obtained from Silong Chemical Co. and used as received. Benzoyl peroxide (BPO), initiator, was purchased from the Chemical Factory of Hubei University (China). Trypsin (high purity grade) was obtained from Shanghai Chemical Factory. Casein was purchased from Chengdu Kelong Chemical Factory. Disodium (A.R.) and citric acid (A.R.) were obtained from Guanghua Chemical Corporation of Shantou. Trichloroacetic acid was supplied by TCA Corporation. Methanol used as solvent was purchased from Chengdu Kelong Chemical Factory (China). All chemicals were used as received directly if not declared specially. Double distilled water was used in the experiment. 2.2. Preparation of microspheres An oven-dried, 100 ml, four-necked round-bottomed flask equipped with a mechanical stirrer, a condenser and a nitrogen inlet and outlet, was charged with methanol (38 g), MMA (4.5 g), and PVP (0.42 g). After bubbling nitrogen (1 h), the temperature was increased to 70 ◦ C. Then BPO-methanol solution was added immediately. The temperature was maintained at 70 ◦ C for 12 h. Then functional monomer acrolein was added dropwise. The mixture was stirred for another 12 h and milk-white latex was obtained. After centrifugation at the speed of 1000 rpm (approximately 223.57 g) for 10 min, the product microspheres were separated from the liquid phase and the supernatant was removed. The microspheres were repeatedly washed with doubly distilled water and centrifuged in 3 successive cycles to get rid of residual solvent. The resulted particles were suspended in phosphate buffer (pH 8) for further use. 2.3. Characterization The morphology of the microspheres (diameter ranging from 6 to 12 m) was observed by optical microscope (SEM better) directly. The size and size distribution were determined using a Malvern Master Sizer/E particle size analyzer (UK). Their values were obtained from the size distribution curve along the x- and y-axis, respectively. The size distribution was termed as uniformity. Obviously the smaller the uniformity was, the narrower the size distribution was. The average diameter was calculated from the diameters and volume percentages of all particles. The concentration of aldehyde groups on the surface of microspheres was determined by conductometric titration [7] (a schematic diagram is shown in Fig. 1). Excessive hydrochlorinate (2%, w/v) hydroxyl amino solution was added to 2 ml
Fig. 1. Schematic diagram of conductometric titration.
microspheres-buffer latex. After the mixture was stirred for 20 min, aqueous NaOH (0.01 M) was titrated drop-wise. The continuous titrated NaOH consumed the H+ and resulted in the decrease of the conductance. A conductivity meter (DDS-307) was employed to record the conductance of the reaction system. The concentration of the aldehyde groups on the surface of microspheres was calculated from the conductivity curve. 2.4. Enzyme immobilization To immobilize enzyme, trypsin (3 ml, 1 mg/ml) solution was added to phosphate buffer (4 ml) containing microspheres. The mixture was slightly stirred at room temperature and incubated for 2 h. Then the microspheres were separated from the liquid phase by centrifugation. The microspheres were washed several times with phosphate buffer (pH 8) to remove the unimmobilized enzymes. The immobilized enzyme (trypsin) was quantified by determined the amount of residual enzyme (unbinding) in supernatants UV-2010 spectrophotometer (Hitachi, Japan) at absorbance 280 nm. Then the enzyme-immobilized microspheres were suspended in phosphate buffer (4 ml, pH 8) and kept in refrigerator at 4 ◦ C for further use. 2.5. Characterization of immobilized enzyme Casein was chosen as the substrate to evaluate the activity of the immobilized trypsin. The enzymatic activity was determined through batch experiments, which were performed in a shaking water bath at 37 ◦ C. Three millilitres 1 mg/ml casein solution was added to 4 ml trypsin-immobilized microspheres latex. The reactions were designed under some controlled conditions (pH, temperature, time and so on) to investigate the effects of immobilization on enzymatic activity. Trichloroacetic acid (TCA) was used to terminate the hydrolyzation since it could inactivate enzyme. Then the reaction mixture was centrifuged to separate the sediments from the bulk phase. The casein hydrolysate, tyrosine in the supernatant was assayed spectrophotometrically. The activity unit was defined as the amount of product generated from the casein hydrolyzation catalyzed by 1 mg/ml trypsin at 37 ◦ C (pH 8.0) for 30 min. The relative activity (% of highest activity/original activity) was used to compare the changes between the free and immobilized enzymes. Three parallel experiments were carried out meanwhile and the average value was calculated as the final result. Km and Vmax are two important characteristic parameters of enzymatic catalytic reaction, the relation of which can be expressed as the famous Michaelis–Menten equation (below).
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265
A group of casein solutions of different concentrations (0.25, 0.50, 0.75 and 1.00 mg/ml) were used to determine the values of Km ’s and Vmax ’s of free and immobilized enzymes at controlled condition (37 ◦ C, pH 8.0). The detailed process was as similar as described above. The concentration of tyrosine in the supernatant was regarded as the value of initial reaction rate V0 . Each substrate sample was corresponding to a plot and a diagram of the initial reaction rate V0 plotted against the concentrations of substrate solutions [S] could be obtained. The values of Km and Vmax were calculated from the slopes and intercepts of the fitting curves: Km + [S] Km 1 1 1 = = + V0 Vmax [S] Vmax [S] Vmax 3. Results and discussion 3.1. Characterizations of microspheres Dispersion polymerization is a desired technique to prepare micron-grade monodisperse microspheres, which was also employed to synthesis the carrier PMMA microspheres in our study. The morphology of resulted particles was observed using an optical microscopy directly. As shown in Fig. 2, the PMMA microspheres exhibited a very narrow size distribution. The uniformity coefficient obtained from size analyzer was 0.2003 (shown in Fig. 3). Additionally, the average diameter determined by light scattering was 6.1 m. A rude calculation indicated the specific surface area of the PMMA particles was up to 0.51 m2 /g. The structure of the PMMA microspheres was very stable, both chemically and mechanically, as no change in size or size distribution of them was observed when the PMMA microspheres were stirred fiercely. The size and uniformity of the PMMA microspheres were very moderate for the enzyme immobilization. The concentration of aldehyde group on the surface of PMMA microspheres was determined by conductometric titration. The curve of conductance versus the amount of NaOH consumed was given in Fig. 4. The conductance decreased at the
Fig. 2. Photomicrograph of PMMA microspheres.
Fig. 3. Size-distribution of microspheres.
initial section due to reduction of [H+ ]. H+ was mainly consumed by the NaOH titrated in the ratio 1:1 by molar. Therefore, the concentration of aldehyde group on the surface of microspheres could be obtained by calculating the amount of the NaOH titrated. Experiments result indicated that the concentration of aldehyde groups was 55 mmol/ml for PMMA microspheres latex and 0.572 mmol/g for the dried microspheres. 3.2. Km and Vmax Enzyme can be immobilized tightly on and not easily washed out of supporting materials through physical or chemical adsorptions. It was reported that immobilized bio-molecules still retained high bioactivity after the immobilization [7–10]. The immobilization procedure was usually performed at some controlled conditions that were suitable for enzymatic structural integrity and functionality. A feasible immobilization strategy was employing covalently binding enzyme to functional groups on the surface of microspheres. In present study, the surface of
Fig. 4. Conductometric titration curve.
S. Wu et al. / International Journal of Biological Macromolecules 37 (2005) 263–267
266 Table 1 The apparent Km and Vmax
Km Vmax (U mg−1 enzyme)
Free enzyme
Immobilized enzyme
0.272 40
1.357 25
carrier PMMA microspheres was modified with aldehyde group, which could react with the amino group of enzyme to form corresponding Schiff’s base under mild conditions. Km and Vmax are two important parameters in enzymatic catalytic reaction. A group of parallel experiments were performed and the average values of Km ’s and Vmax ’s of both free and immobilized enzymes were given in Table 1, respectively. Km was usually used to evaluate the ability of enzyme and substrate forming complex, which was a characteristic value irrelevant to the concentrations of substrate and enzyme. Generally, Km presented the required concentration of substrate when the reaction rate reached the half of maximum rate (Vmax ). The more Km was, the weaker binding enzyme and substrate formed. As shown in Table 1, the apparent Km for immobilized enzyme was 1.357, nearly five times of that for free enzyme 0.272. Therefore, it was obvious that the interaction of enzyme and substrate had been weakened after immobilization. Apparently inactivation had occurred due to structural or conformational changes, for example the active sites might have been hidden or damaged during the immobilization process. 3.3. Effects of pH on enzyme activity The effects of pH on enzyme activity were investigated within a pH range from pH 5 to 10 (at 37 ◦ C). As shown in Fig. 5, it was obvious that the optimal pH for both enzymes was near 9 [11]. Immobilized enzyme maintained higher relative activities compared with free enzyme at the pH range. Both enzymes maintained rather high relative activities between pH 7 and 10. As the pH decreased (from pH 5 to 7), the relative activities of both enzymes descended and the trend for free enzyme was more obvious. For example when pH was down to 5, the relative activ-
Fig. 6. Effects of temperature on enzyme activity.
ity of free enzyme decreased to less than 40% of its maximum value, while the immobilized enzyme still kept more than 80% of its highest activity. A conclusion was drawn that immobilization procedure could improve the enzymatic resistance against pH shift significantly. 3.4. Effects of temperature on enzyme activity The thermal stability of immobilized enzyme was tested in comparison with free enzyme at given temperatures (29, 37, 45, 53 and 61 ◦ C). A diagram on the relative activities of both enzymes at different incubation temperatures was given in Fig. 6. As shown in Fig. 6, at the low temperature section, the catalytic activities of both enzymes increased with the rise of temperature at first and after a maximum value, decreased at higher temperature. The optimal temperature for free enzyme (with the highest catalytic activity) was near 45 and 54 ◦ C for immobilized enzyme. It was obvious that the optimal temperature of enzyme was enhanced by 9 ◦ C after immobilization. As was evident from the data, the immobilized enzyme possessed a better heat-resistance than free enzyme. This might be explained by that the immobilization procedure could protect the enzymatic configuration from distortion or damage by heat exchange. Therefore, immobilized enzyme could work in tougher environment with less activity loss. 3.5. Effects of time on enzyme activity
Fig. 5. Effects of pH on enzyme activity.
Most enzymes suffered severe losses of their bioactivity during storage period especially for free form whose storage stability was very poor even under low temperatures. As shown in Fig. 7, both the relative activities of free and immobilized enzymes decreased with the time. However, the free enzyme lost its activity more quickly than its immobilized form. On the 6th day, the free enzyme kept 70% of its origin activity while the immobilized form still retained 85%. On the 8th day, the relative activity deceased to 55% for free enzyme but only a little change occurred to immobilized enzyme. This suggested that immobilization procedure could prolong the storage period and consequently increase the times of reusability of enzyme. This
S. Wu et al. / International Journal of Biological Macromolecules 37 (2005) 263–267
267
enzymatic bioactivity. Although immobilized enzyme system had some drawbacks such as the resulting of slight inactivation of enzyme (the increase of Km and the decrease of Vmax after immobilization), the thermal stability, pH stability and storage lifetime of immobilized enzyme have been improved effectively. Meanwhile the immobilization procedure could protect enzyme/protein from structural or conformational variations with the environmental changes. More research work should be carried out to extend the application of immobilization enzyme. References
Fig. 7. Effects of time on enzyme activity (ambient temperature).
was very important for enzyme application on a large scale in industry. 4. Conclusion The monodisperse micron-sized polymeric microspheres are a kind of promising supporting material used for enzyme immobilization. In the present study, PMMA microspheres were prepared and covalent binding strategy was employed for enzyme immobilization to investigate the effects of immobilization on
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