Isolation and Purification of α-Amylase Enzyme Using MIP Technique

Zeinab Niroomandi1, Maryam Otadi2*, Homayun Ahmad Panahi1 and Mahdi Goharrokhi3 1Department of Chemistry, Islamic Azad University, Central Tehran Branch, Tehran, Iran.

2Department of Chemical Engineering, Islamic Azad University, Central Tehran Branch, Tehran, Iran. 3Department of Chemical Engineering, South Tehran Campus, Islamic Azad University, Tehran, Iran.

Abstract. This study aims at investigating the immobilization of α-amylase on molecularly imprinted polymers. Molecular imprinting is proved to be an effective method that creates three-dimensional binding sites in polymers. Immobilized enzymes have various applications. For optimum use of enzymes and for the ease of isolation and purification, they should be immobilized. In the present study, after the parameters that are effective on enzyme activity and the degree of its change in immobilized state are examined, the optimum conditions for enzyme immobilization are determined. In doing so, 35 ºC and pH=5 are obtained as optimum data for immobilization. Furthermore, the synthesized molecularly imprinted polymer was characterized by scanning electron microscopy (SEM), Brunauer-Emmet-Teller (BET), CHN analysis.

Keywords: Molecular Imprinting, α-amylase, enzyme isolation and purification.

1. Introduction Enzymes are proteins that catalyze (i.e., increase the rate of) chemical reaction [4]. Amylases are

enzymes which hydrolyze starch molecules [7] to give diverse products including dextrins and progressively smaller polymers composed of glucose units [1]. These enzymes are of great significance in biotechnology [9, 10] their application range includes food [11], fermentation, textile [12], and paper industries [2, 13]. The impossibility of isolation of enzyme from reaction environment and the difficulty of the purification process are the main problems of using enzymes. In order to solve these problems enzymes are immobilized. There are several methods to immobilize enzymes. In this paper α-amylase enzyme is isolated and purified by synthesizing a polymer with specific features using molecular imprinting method (MIP).

In chemistry, molecularly imprinted polymers (MIP) are prepared using a template molecule and functional monomers that assembled around the template and subsequently get cross linked to each other. The functional monomers, which are self-assembled around the template molecule by interaction between functional groups on both the template and monomers, are polymerized to form an imprinted matrix. Then the template molecule is removed from the matrix under certain conditions, leaving behind a cavity complementary in size and shape to the template. The obtained cavity can work as a selective binding site for a specific template molecule [5,6]

The main application of molecular imprinting is in the realm of chromatography and solid phase extraction to separate, pre-concentrate and detect the low molecular weight compounds [18, 5]. Lei Ye and Klaus Mosbach by molecular imprinting technique synthesized artificial antibodies e.g. specific polymer adsorbents. Molecularly imprinted polymers display favorable binding characteristics such as high affinity and specificity toward target molecules. Compared to biological antibodies, imprinted polymers are more stable and can therefore be used under harsher conditions such as in organic solvents * Corresponding author. Tel.: +989123088841; fax: +982144600071. E-mail address: m_otadi@iauctb.ac.ir

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2011 International Conference on Nanotechnology and Biosensors IPCBEE vol.25(2011) © (2011) IACSIT Press, Singapore

and for prolonged period [14]. Jong-Man Kim, Kwang-duk Ahn and etal design a new polymer catalyst by an imprinting technique with labile covalent interactions. The rate of hydrolysis of diphenyl carbonate in the presence of imprinted polymer IP-DVB-THF was found to be 120 times faster than the background uncatalyzed reaction [15]. Florian Meier and Boris Mizaikoff present a feasible way of preparing a molecularly imprinted polymer for oxybutinin, a well-established remedy for the treatment of urinary incontinence in adults [16]. The obtained characteristics render these particles ideally suitable for the selective preconcentraion of oxybutynin in human urine [17]. The 3 above-mentioned people and also most of the scientists investigating molecular imprinting focused on the applications of that, but the issue of enzyme immobilization by molecularly imprinted polymers, to the best of the author's knowledge, has been rarely examined in the previous studies. So, in this paper we try to investigate the immobilization, isolation and purification of enzyme, which is discussed in the following sections.

2. Material and Methods:

2.1. Material α- amylase from B. subtilis, Cellulase TC from Trichoderma, Protease from Aspergilus saitoi were

purchased from Sigma. Dinitrosalicylic acid color reagent, soluble starches were purchased from Merck. All other solvents and reagents were laboratory reagents grade or better.

2.2. Methods Polymer synthesis: Emulsion method was used for polymerization. Poly vinyl alcol was solved in

solved in distilled water in 90 oC. Enzyme was separately solved in phosphate buffer 0.01 M, PH= 7; then it was added to the first solution. CuCl2 and 10 ml of AGE/IDA couple was added too. After that, the cross linker, EGDMA (ethylene glycol di meta acrilate), was mixed with toluene and added to the solution. Finally 10 min reflux with N2; then, AIBN was added followed by 6 h reflux with N2 in 70 oC and 3h reflux in 90 oC. The polymer obtained was filtrated and washed with distilled water. Polymer was ground and sieved, after that it was washed. The washing steps ware 24 h with 100 cc phosphate buffer solution, 24 h with 100 cc methanol, twice with 50 cc nitric acid 0.5 M and finally the polymer was thoroughly washed with distilled water until it turned neutral. The precipitate was removed by filtration and dried.

α-Amylase AssayMethod: That of Bernfeld (1955) wherein the rate at which maltose is released from starch is measured by its ability to reduce 3, 5-dinitrosalicylic acid [8]. One unit releases one micromole of maltose per min at 25°C and pH 4.8 under the specified conditions.

/3 min

micromoles maltose releasedUnits mgutes mg enzyme in reaction mixture

=×

The standard curve of ∆A540 was plotted according to maltose milligrams. Then, for the enzyme the amount of maltose released and activity were resulted from standard diagram.

2.3. Determination of Optimum Conditions For Isolation of Enzyme pH: In this step, prepare 0.01 M solutions of free and immobilized enzyme with different pH 4, 5, 6,

7 were prepared; then the activity was assayed. Time: 1 cc of the first stoke of the enzyme (50 ppm), along with 0.01 gr of polymer was stirred in

vortex at different times. It was then Centrifuged and filtrated; A229 Enzyme was measured in the filtrate by uv-vis. The different times at which the solution was stirred were 1, 5, 10, 30, 60, 90, 120 minutes respectively.

2.4. Comparison of the Enzyme Activity of Free and Immobilized Enzyme in Optimum Conditions

pH: first, 0.01 M buffers with PH= 4, 5, 6, 7 were prepared. The amount of absorption of the enzyme on the polymer was measured using equation (2-1) in optimum pH of the enzyme activity (pH= 5).

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∆A540 = A540nm test – A540nm blank Eq. (2-1) Then, the capacity of the polymer was calculated using the same equation:

Polymer capacity= (C0 – Ce) v/w Eq. (2-2) In this equation v was the volume of solution, and w was the weight of the residue.Finally, the assay

process took place. Temperature: the method is based on Bernfeld assay, but in this step the solutions were incubated

in 20, 30, 35, 40, 45 oC in addition to 25 oC. Enzyme Concentrations: solutions of the free enzyme with different concentrations (0.05, .01, 0.15,

0.2, 0.25 M) and solutions of the immobilized enzyme with the same concentrations were prepared; then they were assayed using Bernfeld method.

2.5. Examining the Special Function of Polymer The method was based on 2-2-4 section. A mixture of three enzymes (including α-amylase, protease

and cellulose) was prepared. Then the activity of free and immobilized enzymes in λmax of each enzyme was assayed.

3. Results

3.1. The Result of Polymer Synthesize Figure 1 shows a SEM of polymer (MIP and NIP). Polymer was continuously formed. Polymer

crystals were in the form of cotton and string. The sizes of the particles were so vast that they were observed in the range of 5-500 μm. Porous with the size of 100-200 nm were observed on the surface of polymer. Like that of porous, the size of the holes did not exceed nm.

The result of CHN and BET analyses were as follow: Table 1: The Result of CHN analysis

Analysis name Element name Sample 1 Sample 2

CH

N

C

H

N

59.35%

7.135%

0.29%

60.36%

7.12%

0.17%

Table 2: The result of BET analysis

Surface area (N2/ gr) 545.2

Hole volume (cc/gr) 0.75 Hole diameter (nm) 1.42

3.2. The Result of the Comparision of Optimization Parameters of Immobilization the Enzyme on Polymer

pH: Figure 2 shows that enzyme in pH= 5 have the maximum activity. The enzyme activity decreased in PH= 6, 7 but in PH= 8 the enzyme showed minimum activity. Decrease of enzyme activity can be due to the formation of a bad ion state for enzyme or substrate or both of them, inhibition of enzyme, or some of these factors.

Time: figure 3 indicates that enzyme activity increases until it reaches 90 min, but the activity decreases as it approaches to 120 min. At the end of the reaction, there was no substrate in the solution, and that’s why enzyme activity decreased as the time increased.

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3.3. The Result of the Comparison of Different Parameters in Free and Immobilized Enzyme

pH: pH affected the enzyme activity; and each enzyme shows the maximum activity in specific pH. So, for the purpose of optimum use of enzyme, it is necessary to determine its optimal pH. Therefore, in order to present more tangible results the effect of different pH on the immobilized enzyme activity was investigated.

The method was fully described in previous sections. The absorption of free and immobilized enzyme in A540 was determined and ∆A540 was calculated by eq (2-1). Now the amount of maltose released was determined using standard curve and the enzyme activity in different pH was obtained by eq (2-1). Results are shown in figure 4.

Temperatures: as it was indicated in figure 5 both of free and immobilized enzyme showed maximum activity in 35 oC. Also with immobilization of enzyme the activity was decrease.

Enzyme Concentrations: the figure 6 shows that the more enzymes is concentrated, the more its activity increases.

3.4. The Result of the Examination of the Special Function of Polymer As it was shown in table 3, the amount of polymer absorption for protease and cellulose is minute;

that is, only amylase had selectivity to polymer, and the binding sites of polymer were fairly exclusive to amylase.

Table 3: The amount of absorption of enzyme on the polymer

Enzyme Adsorption of polymer Absorption of free enzyme Absorption of immobilized enzyme

α-amylase 0.41573 0.45286 0.249524

protease 0.13878 0.13928 0.13902

cellulase 0.09772 0.09792 0.09652

Diagrams and Figures

Fig. 1: SEM of polymer: (a) MIP; (b) NIP

00.00050.001

0.00150.002

0.00250.003

0.00350.004

0.00450.005

0 1 2 3 4 5 6 7 8 9

pH

ads

Fig. 2: pH optimization of immobilized enzyme

0102030405060708090

100

0 20 40 60 80 100 120 140

time (min)

%ad

s.

Fig. 3: Time optimization of immobilized enzyme

103

0

0.2

0.4

0.6

0.8

1

0 1 2 3 4 5 6 7 8

pH.

ads.

Fig. 4: Comparison of activity of free and immobilized enzyme in different pH (▼ free enzyme, ■ immobilized enzyme)

0

0.2

0.4

0.6

0.8

1

1.2

0 10 20 30 40 50

temprature

ads.

Fig. 5: Comparison of activity of free and immobilized enzyme in different temperatures (▼ free enzyme, ■

immobilized enzyme)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 0.05 0.1 0.15 0.2 0.25 0.3

enzmy conc.

ads.

-\

Fig. 6: Comparison of activity of the free and immobilized enzyme in different concentrations (▼ free enzyme, ■

immobilized enzyme)

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