Enhanced stability and catalytic activity of immobilized α-amylase on modified Fe3O4 nanoparticles

Chemical Engineering Journal 240 (2014) 426–433

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Enhanced stability and catalytic activity of immobilized a-amylase on modified Fe3O4 nanoparticles Nasrin Sohrabi a,⇑, Nahid Rasouli a, Mehrangiz Torkzadeh b a b

Department of Chemistry, Payame Noor University, PO Box 19395-3697, Tehran, Iran Department of Chemistry, Payame Noor University of Isfahan, Iran

h i g h l i g h t s  a-Amylase enzyme was covalently immobilized on the surface of silica-coated modified magnetite nanoparticles.  Magnetite (Fe3O4) nanoparticles have been considered suitable for immobilization of enzymes.  The EDX spectrum showed that magnetite nanoparticles were successfully coated with silica.  The TEM images showed that the diameter of silica-coated magnetite NPs was about 20 nm.

a r t i c l e

i n f o

Article history: Received 23 May 2013 Received in revised form 7 November 2013 Accepted 23 November 2013 Available online 1 December 2013 Keywords: Magnetite nanoparticles Immobilization a-Amylase Enzyme activity Stability

a b s t r a c t In this work, a-amylase enzyme was covalently immobilized on the surface of silica-coated modified magnetite nanoparticles, for the first time. The synthesis and immobilization process is simple and very fast and consists of the following steps: (1) preparing the magnetic iron oxide nanoparticles using the co-precipitation method, (2) coating NP with silica (SiO2) by sol–gel reaction, (3) preparing the amino-functionalized magnetite NPs by treating silica-coated NPs with 3-aminopropyltriethoxysilane, (4) activating immobilization of a-amylase on the activated amino-functionalized magnetite NPs, and (5) covalently immobilization of a-amylase on the activated-amino-functionalized magnetite NPs. The synthesis steps and characterizations of NPs were examined by FT-IR, XRD, EDX and TEM. The optimum concentration and time for maximum enzyme activity of the immobilized a-amylase are identified to be 150 lg and 4 h, respectively for the hydrolysis of starch. The immobilized a-amylase showed maximal catalytic activity at pH = 6.5 and 45 °C. The kinetic studies shows overall enhancement in the performance of the immobilized enzyme with reference to the free enzyme. Similarly, the thermal stability of the enzyme is found to increase after the immobilization. The Immobilized a-amylase has also been demonstrated to be capable of being reused for six cycles while retaining 85% of the initial activity. By using a magnetically active support, quick separation of amylase from reaction mixture is enabled. The Km values were found as 6.27 and 4.77 mM for free and immobilized enzymes, respectively. The Vmax values for the free and immobilized enzymes were calculated as 2.44 and 11.58 lmol/mg min, respectively. Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction Enzymes have been the subject of intense academic interest for many decades and currently poised to become important industrial catalysts. Under the ambit of green chemistry, biocatalysis is very attractive to produce chemicals, which are also safer. Biocatalysis is slowly but steadily gaining importance in various field of chemical engineering, where chemical synthesis routes are being replaced by enzymatic ones. The major advantage of the enzymatic route is the selectivity with its associated high yield and exclusivity towards the desired product [1]. The main problems of using ⇑ Corresponding author. Tel.: +98 312 5200812; fax: +98 312 5200815. E-mail address: [email protected] (N. Sohrabi). 1385-8947/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cej.2013.11.059

the enzymes are the difficulty of their separation from the solution and their inactivation by organic solvent and extreme pH or temperature. Novel designs with immobilized enzymes and without need of separation are of major concern. This also reduces the loss of enzymes and offer the opportunity to use a continuous reactor with a re-use of the enzyme for many reaction cycles and thus lowering the total production cost of enzyme mediated reactions [2]. Also, the immobilized enzyme may be stabilized against denaturing agents that promote unfolding processes that can destroy the active site [3]. Enhancing both stabilities can be achieved by immobilization and enzyme engineering. Amylases can be regarded as one of the most important and widely used enzymes whose spectrum of application has widen in various branches such as clinical, medicinal and analytical

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chemistry. Beside their use in starch saccharification they can be used in food, baking, brewing, detergent, textile and paper industries [4–6]. Alpha-amylases (E.C.3.2.1.1) catalyses the hydrolysis of internal a-1,4-glycosidic linkages in starch in low molecular weight products, such as glucose, maltose and maltotriose units [7]. The various types of supports have been previously investigated for the effective immobilization of a-amylase such as functionalized glass beads [8], mesoporous silica [9], amberlite MB 150, chitosan beads [10], gelatin [11], alginate [12], poly(hydroxyethyl methacrylate), copolymers of styrene and hydroxyethyl ethacrylate [13], poly(acrylamide) [14], modified poly(N-isopropylacrylamide) [15], polyaniline [16], porous nitrocellulose [17], silver nanoparticles doped gum acacia–gelatin–silica nanohybrid [18] and tamarined gum–silica nanohybrid [19]. In the Past few decades, materials of nano dimension have been dramatically emerging and have reached an inevitable position in all the disciplines of science and technology. Among these nanomaterials, magnetic nanoparticles are considered unique since they possess excellent and unusual magnetic character. The magnetite (Fe3O4) nanoparticles have been considered suitable for immobilization of enzymes because of their multifunctional characteristics, including small size, superparamagnetism and low toxicity and most importantly, their easy separation from the reaction system. In the magnetic separation techniques, there is no need for expensive liquid chromatography systems, centrifuges, filters or other equipments [20]. Although there have been many significant developments in the synthesis of magnetic nanoparticles, maintaining the stability of these particles for a long time without agglomeration or precipitation is an important issue. A silica shell does not only protect the magnetic cores, but can also prevent the direct contact of the magnetic core with additional agents linked to the silica surface thus avoiding unwanted interactions. A protective shell does not only serve to protect the magnetic nanoparticles against degradation, but can also be used for further functionalization with specific components such as catalytically active species, various drugs, specific binding sites or other functional groups. The easy separation and controlled placement of these functionalized magnetic nanoparticles by means of an external magnetic field enables their application as catalyst supports in immobilized enzyme processes and the construction of magnetically controllable bio-electrocatalytic systems [21–23]. Alpha-amylase was covalently immobilized onto magnetite NPs via glutaraldehyde [24], active dialdehyde groups [25], adepic acid [26] and 3-aminopropyl triethoxysilane [27]. This is the first time that the trichlorotriazine

is used to the immobilization of a-amylase onto magnetite NPs. Also, the kinetic and stability studies are more comprehensive than the previous works mentioned (Table 1). In the present study, a-amylase was covalently immobilized on a new enzyme carrier. For this purpose, inexpensive and renewable Fe3O4 nanoparticles were synthesized by co-precipitation method and then, silica-coated MNPs were prepared through the sol–gel reaction. The silica-coated magnetite nanoparticles were treated with 3-aminopropyl triethoxysilane (APTES) in order to yield the amino-functionalized magnetic nanoparticles. The amino-functionalized magnetic nanoparticles were activated with trichlorotriazine (TCT) and then a-amylase was covalently immobilized on them. The current work aims to develop a magnetically responsive enzyme carrier via a simple and straight forward immobilization which enables better binding of the enzyme to the magnetite substrate as well as in better reuse of the enzyme through its recovery using a magnetic field. The results reveal that silica-coated modified magnetite nanoparticles are promising carries for enzyme immobilization. 2. Experimental 2.1. Materials The materials used in this study include a-amylase (EC 3.2.1.1 from B. subtilis with an activity of 50 U/mg), Tetraethyl orthosilicate (TEOS), 3-aminopropyltriethoxysilane (APTES), Trichlorotriazine (TCT), 3,5-dinitrosalicylic acid (DNS), Maltose, Starch and sodium potassium tartrate (4H2O) were purchased from Sigma–Aldrich. Ferric chloride hexahydrate (FeCl3
6H2O), Ferrous chloride tetrahydrate (FeCl2
4H2O), Coomassie Brilliant Blue G250 and Bovine Serum Albumin (BSA) were purchased from Merck. 2.2. Synthesis of magnetite nanoparticles Although, various methods have been reported for synthesis of magnetite NPs in the literature [28–30], the chemical co-precipitation method, as a simple and fast method was used in this study [31]. According to this method, 1.25 g of FeCl2
4H2O and 3.4 g of FeCl3
6H2O with molar ratio 1:2 were dissolved in 100 mL deionized water. The flow of nitrogen gas removed oxygen and created an inert condition. The solution was heated to 60 °C

Table 1 Comparison of properties of immobilized a-amylase in the present work and other studies [36–42].

a-Amylase

pH opt

T opt. (°C) Km

Free Immobilized on cyclic carbonate bearing hybrid material Free Immobilized on coconut fibre Free Immobilized on zirconia-coated alkylamine glass Free Immobilized on functionalized glass beads Free Immobilized on P(MMA- AA) microspheres via CDI Via SOCl2 Free Immobilized on Na-bentonite Free Immobilized on gum acacia stabilized magnetite nanoparticles Free Immobilized on modified magnetite nanoparticles

6.5 6.5

30 30

15.8 mg mL 9.09 mg mL

4.9 5.7 5.6 5.6–6.2 6.5 5.5 7.5 6.5 6.0 6.2 6.2 7 7

48 48 55 60 30 50 40 55 55 40 60 40 40

11.66 mg mL 1 13.07 mg mL 1 0.80% 0.45% Not available Not available 2.51 g L 1 31.37 g L 1 30.02 g L 1 5.69 g L 23 g L 2.2 ± 0.3 mg mL 2.9 ± 0.1 mg mL

6.5 6.5

45 45

6.22 mM 4.77 mM

Vmax 1

58  10 5.2  10

1

1 1

Activity retained after storage References 3

mg mL 1min 3 mg mL 1min

68.97 U mL 1 65.36 U gm fibre 1 760 lmol min 1 L 1 764 lmol min 1 L 1 Not available Not available 1.67  10 3 g L 1 min 1 1.66  10–3 g L 1 min 1 1.61  10 3 g L 1 min 1 5.86 mol mL 0.94 mol mL 3.4 ± 0.7 lmol mL 1 min 5.3 ± 0.3 lmol mL 1 min 2.44 lmol mg 1 min 1 11.58 lmol mg 1 min 1

1 1

1 1

Lost all in 15 days %70 after 25 days

[1] [1]

Not available Not available Not available 100% after 15 days Lost all in 15 days 80% after 25 days – 24.5% after 30 days 52.5% after 30 days 40% after 12 days 35% after 12 days Not available Not available

[2] [2] [3] [3] [4] [4] [5] [5] [5] [6] [6] [7] [7]

40.61% after 12 days 79.99% after 12 days

This study This study

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when it turned a dark orange color. Then 6 mL of ammonium hydroxide (25%) was added to the solution, and the mixture was stirred for 30 min. The black precipitate that formed was washed three times with de-ionized water and ethanol. 2.3. Preparation of silica coated magnetite nanoparticles The classical Stöber method [29] (base-catalyzed hydrolysis and condensation of TEOS) was used for coating magnetite nanoparticles with a silica shell. The amount of 0.145 g of Fe3O4 was mixed with 40 mL of ethanol. This suspension was dispersed under ultrasonication for 10 min in the presence of a constant N2 flux. Then, 6 mL of water and 3 mL of ammonium hydroxide solution were added to the suspension at room temperature followed by the addition of 0.4 mL of TEOS. Then, the mixture was stirred for 5 h. The obtained silica-coated Fe3O4 nanoparticles were washed several times with ethanol and water and dried under vacuum at room temperature.

were suspended in 0.5 ml phosphate buffer (0.02 M, pH = 6.9) and then this mixture was incubated for 3 min at room temperature with 0.5 ml of the substrate solution after that a-amylase immobilized on magnetic nanoparticles were separated by application of an external magnetic field and then 1 ml of DNS reagent was added to its supernatant. The subsequent procedures were similar to those of free a-amylase. All activity measurement experiments were carried out at least three times. 2.7. Thermal and pH stability The thermal stability of the free and immobilized a-amylase was evaluated by measuring the residual activities of the enzymes after 30 min incubation in phosphate buffer (20 mM, pH = 6.9) and at temperature range of 30–70 °C. The pH stability was evaluated by measuring the residual activity after 30 min incubation of enzyme at specified pH. This parameter was investigated in the pH range of 4–8.

2.4. Nanoparticles surface modification

2.8. Kinetic parameters

For the surface modification of the MNPs with 2,4,6-trichloro1,3,5-triazine, an adaption of the Wang and Liu et al. method was used [32]. First, the silica surface was functionalized by 3-aminopropyltriethoxy siliane. The obtained MNPs (5 mg) were treated with APTES (300 lL) in ethanol (500 lL) in order to introduce amino groups to the NP surface. The mixture was reacted at room temperature for 2 h, which was followed by heating at 50 °C for 1.5 h. The amine-functionalized nanoparticles were separated from the cooled mixture by an external magnet. These MNPs were washed successively with ethanol and THF. The obtained MNPs were further reacted with 2,4,6-trichloro-1,3,5-triazine (40 mg) in THF (1000 lL) at room temperature for 3 h. The obtained MNPs were washed with THF, ethanol and de-ionized water. Finally, the triazine functionalized MNPs were dried under vacuum at room temperature.

The kinetic parameters of free and immobilized a-amylase were determined by measuring the initial rates of enzymes with different substrate (starch concentration (10–60 mM)). The Km and Vmax values were calculated from Lineweaver and Burk plot.

2.5. a-Amylase immobilization The triazine functionalized MNPs (10 mg) were dispersed in 800 lL of phosphate buffer (20 mM, pH = 6.9). Then, various volumes of the a-amylase enzyme solution (50–300 lg) were added into the suspension and the mixture was shaken at room temperature for 1–7 h. The immobilized a-amylase was removed by magnetic decantation and washed three times with phosphate buffer. The amount of a-amylase immobilized on MNPs was determined by measuring the initial and final concentration of a-amylase in the immobilization medium using the Bradford method [33]. The immobilization yield was 45% at optimum conditions. 2.6. Enzyme assay According to the Bernfeld method [34], the activities of free and immobilized a-amylase were determined in the presence of 1% (w:v) soluble starch as the substrate in phosphate buffer (0.02 M, pH = 6.9) at room temperature. For free a-amylase, a sample of 0.5 ml was incubated for 3 min at room temperature with 0.5 ml of the substrate solution and the enzymatic reaction was interrupted by the addition of 1 ml of DNS reagent. The mixture was heated for 5 min in boiling water and then cooled at room temperature. After addition of 10 ml of distilled water, the absorbance of the digested products was measured spectrophotometrically at wavelength 540 nm. A blank was prepared in the same manner without free a-amylase. A calibration curve established with maltose, 0.3–4 (lmol/ml) in 1 ml of deionized water. For immobilized a-amylase, the tube containing 10 mg of immobilized a-amylase

2.9. Storage stability and catalyst recycling The storage time of free and immobilized a-amylase was determined by carrying out at different times (1–12 days). The residual activities were calculated as percentage of the initial activity. Also the stability of the immobilized a-amylase was measured by reusing it six times. One milliliter of 1% (w:v) starch in 20 mM phosphate buffer (pH = 6.9) was added to the immobilized enzyme and incubated for 5 min under constant shaking for each cycles. At the end of the reaction, immobilized enzyme was taken and washed with distilled water and then added a substrate solution to start a new cycle. The supernatant was assayed for reducing maltose. 2.10. Characterization The size and morphology of magnetic nanoparticles were determined using transmission electron microscopy (Phillips-CM10 operating at 100 kV with a Cu grid); the FT-IR spectra were recorded by a Fourier transform Infrared Spectroscopy (JASCO FT/ IR-4200, Japan), the X-ray diffraction measurement was recorded on a X-ray diffractometer Bruker, D8ADVANCE [(Germany) using Cu Ka radiation (k = 0.1540 nm)] and the EDX spectra was recorded by Energy-dispersive X-ray spectroscopy (EDX) (Philips XL 30). 3. Results and discussion 3.1. Characterization of nanoparticles The background corrected FT-IR spectra of MNPs are presented in Fig. 1. The absorption band around 590.11 cm 1 in the IR spectra of both Fe3O4 and Fe3O4–SiO2 nanoparticles (Fig. 1(a) and (b)) correspond to FeAO bonds. The peaks at 801.27 cm 1 and 1083.80 cm 1 correspond to symmetric stretching of SiAOH and SiAOASi, respectively and the broad band around 3400.11 cm 1 and 1621.01 cm 1 can be assigned to OAH stretching vibrations. In Fig. 2 spectra of pure magnetite nanoparticles, amino-functionalized magnetite NPs and the nanoparticles activated with TCT (Fig. 2(a)–(c)) are presented. The peaks at 1027.87 cm 1 and 2925.48 cm 1 correspond to

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Fig. 4. X-ray patterns of modified MNPs. Fig. 1. FT-IR of (a) uncoated and (b) silica-coated magnetite nanoparticles.

30.01°, 35.81°, 43.38°, 57.41°, and 62.98° (Fig. 4) show good consistency with the reported data and can be indexed to pure phase of Fe3O4 structure. The average crystallite size of the Fe3O4 nanoparticles calculated from Scherrer’s equation was 13 nm. The EDX spectrum of silica-coated nanoparticles is shown in Fig. 5. The atomic weight ratio of O:Si:Fe was 36.31:29.47:34.22, which indicates the magnetite nanoparticles are successfully coated by silica. Fig. 6(a) shows the TEM image of Fe3O4–SiO2. The mean diameter determined from the TEM image is about 20 nm [35]. Fig. 6(b) shows TEM image of modified MNPs. The average size of nanoparticles in Fig 6(b) (30 nm) is larger than that of (20 nm) Fig 6(a). The obtained results revealed that the size of silica-coated nanoparticles increased, because two layers of APTES and TCT were added to the surface of silica-coated nanoparticles. 3.2. a-Amylase immobilization parameters Fig. 2. FT-IR of (a) pure MNPs (b) MNPs–APTES (c) MNPs–APTES–TCT.

symmetric stretching of SiAOASi and CAH respectively and the broad band around 3434.61 cm 1 and 1629.55 cm 1 can be assigned to ANH2. The peak at 1613.55 cm 1 correspond to aromatic C@N peak at 1000–1600 cm 1 region, indicating the presence of TCT. The FT-IR spectra confirmed the binding of a-amylase onto magnetic nanoparticles. The binding of a-amylase to modified MNPs was confirmed by FT-IR analysis. Fig. 3 shows the FT-IR spectra of the modified MNPs with and without immobilization a-amylase. The characteristic bands of proteins are at 1656 cm 1 and 1531 cm 1. The peaks at 1657 cm 1and 1535.12 cm 1 after immobilization of a-amylase on the magnetite NPs correspond to peptide binding. It shows that a-amylase effectively present in the samples confirming binding of a-amylase to modified MNPs. The powder X-ray diffraction (XRD) patterns were used to identify the crystalline structure of MNPs. The XRD peaks with 2h at

Fig. 3. FT-IR spectra of the modified nanoparticles without (a) and with (b) bound a-amylase. The spectrum (c) is the spectrum of pure a-amylase.

3.2.1. Time of immobilization The amount of immobilized a-amylase and the relative activity of a-amylase versus reaction time are shown in Figs. 7 and 8. The results indicated that by increasing the reaction time from 1 to 4 h, the amount of immobilized a-amylase increased and remained constant after about 5 h. It seems that most of TCT groups on the surface of magnetic nanoparticles were blocked by amino-group of a-amylase after this time. The obtained results also revealed that the relative activity of a-amylase was increased with reaction time up to 4 h and then, it decreased (Fig. 8). The obtained results can be related to the saturation of TCT groups on NPs by a-amylase molecules at 4 h and reduction of enzyme activity after this time due to denaturation. Therefore, the optimal time was 4 h. 3.2.2. Concentration of a-amylase The effect of different amounts of the a-amylase (50–300 lg) added on amount of immobilized a-amylase and relative activity

Fig. 5. EDX spectrum of silica-coated magnetite nanoparticles.

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Fig. 6. TEM images of (a) silica-coated magnetite (b) modified MNPs.

Fig. 7. Effect of reaction time on the amount of immobilized a-amylase: immobilization condition: the initial amount of a-amylase: 150 lg, 10 mg of NPs in phosphate buffer (20 mM, pH = 6.9, t = 25 °C).

Fig. 9. Effect of the initial amount of a-amylase on the amount of immobilized aamylase: Immobilization condition: t = 4 h, 5 mg of NPs in phosphate buffer (20 mM, pH = 6.9, t = 25 °C).

Fig. 8. The relative activity of immobilized a-amylase enzyme versus the reaction time.

Fig. 10. The effect different amounts of the a-amylase added on relative activity of the immobilized a-amylase.

of the immobilized a-amylase are shown in Figs. 9 and 10. The amount of the a-amylase loading increased greatly with the initial a-amylase concentration and the relative activity reached a maximum value at an initial a-amylase amount of 150 lg. It is considered that the higher a-amylase loading makes the a-amylase form an intermolecular steric hindrance, which restrains the diffusion of the substrate and product. Therefore, the relative activity decreased slowly above 150 lg of a-amylase. It is reasonable to conclude that the binding sites on the surface of the magnetic microspheres are limited and the enzyme molecules need enough space for catalyzing the reaction of the substrate.

3.2.3. Thermal and pH stability The effect of pH on the free and immobilized a-amylase was investigated in the range of pH = 4.0–8.0 and results were presented in Fig. 11. As shown the pH of maximum activity for both free and immobilized enzyme was found to be 6.5. The results show that the immobilized a-amylase is more stable than the free a-amylase. The activity of the free and immobilized a-amylase was assayed at various temperatures (30–70 °C). As it can be seen in Fig. 12, the maximum activities were observed at 45 °C for both free and immobilized enzymes. The relative activity loss was calculated as approximately 51.45% for the immobilized enzyme and

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Fig. 11. pH stability of the immobilized and free a-amylase.

Fig. 12. Thermal stability of the immobilized and free a-amylase.

75.65% for the free one. The immobilized enzyme had a higher activity than the free one. Thus, the immobilized enzyme was much more stable than the free enzyme. The comparison of properties of immobilized a-amylase in the present work and other studies were added in Table 1.

3.2.4. Storage stability and catalyst recycling Fig. 13 illustrates the storage stabilities of the free and immobilized enzymes were stored in 0.02 M phosphate buffer (pH = 6.9) at 4 °C and their activities were tested for 12 days. The immobilized enzyme retained 79.99% of its activity after 12 days but the free

Fig. 13. Storage stability of the immobilized and free a-amylase.

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indicate that the affinity of the a-amylase to its substrate was increased by immobilization. Since magnetic nanoparticles were so small and nonporous, it could be imagined that the a-amylase molecule might be expanded over the particle surface with a better orientation leading to higher affinity to substrate and more available active sites. The Vmax values for the free and immobilized enzymes were calculated as 2.44 and 11.58 lmol/mg min, respectively. The improvement of Vmax may also be due to more efficient conformation of the immobilized a-amylase with respect to the free a-amylase (Table 1). 4. Conclusions

Fig. 14. Catalyst recycling of the immobilized a-amylase.

The present study aims to investigate a new method for the covalent immobilization of a-amylase on the surface of silicacoated magnetite nanoparticles. The functional group on the MNPs for covalent attachment of a-amylase molecules was TCT. The TEM images indicated that the diameter of silica-coated magnetite NPs was about 20 nm and the diameter of modified magnetite NPs was about 30 nm. The XRD pattern proved that the immobilization process did not result in the phase change of Fe3O4. The FT-IR spectrum was used for confirming the immobilization of a-amylase onto NPs. The EDX spectrum revealed that the magnetite nanoparticles were successfully coated with silica. The optimized concentration and reaction time for the immobilization step were determined 150 lg and 4 h respectively. The results demonstrated the substantial improvement of thermal, pH and storage stability of the a-amylase due to immobilization. After six consecutive operations, the immobilized a-amylase could still retain 85% of its initial activity. Moreover, the kinetic studies confirm the Michaelis–Menten behavior and suggests overall enhancement in the performance of the immobilized enzyme with reference to the free enzyme. Acknowledgement The financial support of Research Council of Payame Noor University of Isfahan is gratefully acknowledged. References

Fig. 15. (a) Lineweaver Burk plot for immobilized a-amylase (b) for free a-amylase.

enzyme retained 40.61% of its activity after 12 days. The obtained results showed that through immobilization, the enzyme gained more stable character than the free one. Fig. 14 shows the residual activity as a function number of cycles. The results showed that the immobilized a-amylase remained 85.22% of its initial activity after six cycles. 3.2.5. Kinetic parameters Fig. 15(a) and (b) represents Lineweaver Burk plot for the immobilized a-amylase and free a-amylase. The catalytic properties of the free and immobilized enzymes were evaluated by using soluble starch (10–60 mM) as a substrate. The Michaelis–Menten constant Km and the maximum activity Vmax of the free and immobilized enzymes were estimated at pH = 6.9 and 25 °C. The Km values were found as 6.27 mM and 4.77 mM for free and immobilized enzymes, orderly. The Km value is known as the affinity of the enzymes to substrates and the lower values of Km emphasize the higher affinity between enzymes and substrates. The results

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