Metallochelate immobilization of urease on to amorphous SiO2

Process Biochemistry 40 (2005) 3045–3049 www.elsevier.com/locate/procbio

Metallochelate immobilization of urease on to amorphous SiO2 Ts. Godjevargova a, M. Velikova b, N. Vasileva c,*, N. Dimova a, D. Damyanov b a

Biotechnology Laboratory, Department of Biotechnology, University ‘‘Prof. Dr. Assen Zlatarov’’, 8010 Bourgas, Bulgaria b Physicochemical Laboratory, Department of Physics Chemical, University ‘‘Prof. Dr. Assen Zlatarov’’, 8010 Bourgas, Bulgaria c Biochemistry Laboratory, Department of Biotechnology and Food Products, University of Rousse ‘‘Angel Kanchev’’ – Technology College, 7200 Razgrad, Bulgaria Received 14 July 2004; received in revised form 10 January 2005; accepted 19 February 2005

Abstract Urease was immobilized onto silica gel and vulcasil activated with Ti4+ and V5+. Higher activity was observed for urease immobilized onto vulcasil compared to that bound to silica gel. The carriers activated with Ti4+ showed activity higher than that of carriers activated with V5+. Activity was affected also by the temperature at which the modified carrier was dried. Lower activities were measured with the carriers heated to 550 8C. The highest activity showed urease metallochelate-bound to vulcasil activated with Ti4+ and dried at 110 8C and the values were close to these of the free enzyme. Thermal stability, pHopt, Topt and kinetic parameters of the immobilized urease were studied. The bound enzyme preserved its activity in wider pH and T intervals and showed higher thermal stability compared to the native one. The highest Vmax of the enzyme reaction was measured for urease metallochelate-bound to vulcasil activated with Ti4+ (dried at 110 8C) and the values were close to these of the free enzyme. The activation energies of the enzymes bound to all types of carriers was ca. 4% smaller than that of the free enzyme. # 2005 Elsevier Ltd. All rights reserved. Keywords: Urease; Metallochelate immobilization; Amorphous SiO2; Activation with Ti4+ and V5+

1. Introduction The metallochelate method for immobilization of enzymes has significant prospects. The process of co-ordination immobilization does not require available reagents and takes place under soft conditions for a comparatively short time which is very important for the labile enzymes. Usually, carriers with a high specific area like nickel, aluminium, silicon, etc., oxides are used [1–3]. The method is based on the ability of the transition metals oxides to produce complex compounds with the hydroxyl groups of the carrier and the enzyme [1,2]. The enzymes immobilized by this method show high activity and good performance stability [3]. Various transition metals have been used: Co(II); Ni(II); Cu(II); Vd(III); Ti(III); Ti(IV). Good results were reported for immobilization using Ti(III) and Ti(IV). Some carriers * Corresponding author. Tel.: +359 84 611 012. E-mail address: [email protected] (N. Vasileva). 1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2005.02.008

showed better results with tri-valent titanium while others— with four-valent titanium [3,4]. The effect of heat treatment of the carriers before enzyme immobilization was not clearly established. Thus, some authors suggested heat treatment at 540 8C, while others recommended 110 8C [5]. In some publications, the influences of pore size and carrier specific area (SiO2) on the quantity of bound enzyme and its activity was studied. It was suggested that the specific area is more important factor than the pore size [6]. This present paper describes the preparation and characterization of new catalysts based on urease immobilized onto specifically activated carriers of amorphous SiO2. The enzyme was selected for the experiments because it is widely used for analyses of heavy metals in water and for the removal of toxic compounds during hemodyalysis [7,8]. This enzyme is also very useful in immobilized form since it can be used multiple times and, therefore, as a part of a continuous technological process [9,10].

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Free and immobilized urease activities were determined spectrophotometrically (Specol 11, Carl Zeiss Jena, Germany) at wave length, l = 460 nm. The method is based on the hydrolysis of urea to ammonium [13]. Activities were measured with 3% urea in 0.06 M phosphate buffer pH 5.8 at 30 8C for 5 min. The amount of ammonium produced was determined spectrophotometrically by measuring the intensity of the coloured compound formed after the addition of Nessler reagent.

2. Materials and methods 2.1. Materials Two commercial grades were used as carriers for urease immobilization:  Silica gel—commercial Russian silica gel type KSK, grain size 2–6 mm and bulk weight 400–500 kg m 3. The modification was carried out with fraction 1.25–1.6 mm.  Vulcasil—commercial product of Bayer (Germany) with average grain size 20 nm and bulk weight 200 kg m 3.

2.5. Determination of the thermal stability of free and immobilized urease The free and bound enzymes were incubated at temperature of 50 8C for 120 min. During incubation, the specific activity of the enzyme was determined at 30 min intervals by the method described above.

The carriers were modified with vapour of TiCl4 (Ktonos Titangesellschaft, Germany) and VOCl3 (Fluka AG, Switzerland). The urease used for all experiments was purchased from Merck (Germany), its activity was 580 U mg 1 protein.

2.6. Kinetic investigations 2.2. Modification of silica carriers The modification of silica gel and vulcasil carriers was performed by the method of molecular deposition [11]. Vulcasil carrier was treated with vapours of TICl4 and VOCl3, while silica gel was treated with TICl4 vapour only. Part of the activated carriers were dried at 110 8C and the other were heated to 550 8C.

The method of Lineweaver–Burk was used to determine the values of Km and Vmax. For this purpose, a series of measurements of the reaction rate were performed under varied conditions: substrate (urea) concentration from 1  10 2 to 1 mol l 1; reaction time 10 min; optimal pH for both free and immobilized enzyme (5.8) and temperatures of 25, 30 and 35 8C.

2.3. Immobilization of urease

2.7. Determination of the activation energy

A 0.1 g modified carrier was weighed into measuring flask and 1 ml 0.1% solution of urease (prepared in sodiumphosphate buffer) was added. The mixture was thoroughly stirred and stored for 24 h at 4 8C. The carrier with the immobilized enzyme was washed with bidistilled water and 5 ml 0.06 M solution of sodium-phosphate buffer (pH 5.8).

The activation energy of the free enzyme and urease immobilized onto modified carriers was calculated using the Arrhenius equation [14]. The highest reaction rate was determined for several temperatures (from 15 to 40 8C), the plot lg V = f(1/T) was drawn and the values of the activation energy for both free and immobilized urease were calculated.

2.4. Analyses The determination of the amount of enzyme bound to the silica carriers was carried out by the method of Lowry et al. [12] and the absorption was registered by spectrophotometer ‘‘Specol 11’’ (Carl Zeiss Jena, Germany) at wave length l = 750 nm. The amount of bound protein was calibrated using urease solutions and determined by a standard curve (actually, it is a straight line).

3. Results and discussion Two commercial grades of amorphous SiO2 (silica gel and vulcasil) were modified with TiCl4 and VOCl3 and used as carriers for metallochelate immobilization of urease. It was necessary first to desiccate the initial carriers in order to remove physically bound water. The desiccated amorphous

Table 1 Characteristics and chemical composition of modified carriers Carrier

Dehydration temperature (8C)

Amount of OH–groups (number of OH nm 2)

Specific area (m2 g 1)

Pore volume (cm3 g 1)

Initial

Modified

Initial

Modified

Silica gel Vulcasil

200 200

4.8 7.4

230 160

216 147

0.90 0.40

0.70 0.30

Ti4+ (mmol g 1)

V5+ (mmol g 1)

Cl (mmol g 1)

0.88 0.63

– –

1.62 0.81

–

0.45

0.70

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Table 2 Basic characteristics of free and metallochelate immobilized urease onto modified amorphous SiO2 No.

Free and immobilized urease

Drying temperature and heating temperature of activated carrier (8C)

Specific activity (U mg 1)

Amount of protein bound (mg g 1)

1 2 3 4 5 6 7

Free enzyme Enzyme immobilized Enzyme immobilized Enzyme immobilized Enzyme immobilized Enzyme immobilized Enzyme immobilized

– 110 110 110 550 550 550

580 21.32 565.14 2.22 8.3 69.90 1.25

– 10.4 1.2 7.1 8.1 0.9 6.5

0.05

0.1

onto onto onto onto onto onto

silica gel activated with Ti4+ vulcasil activated with Ti4+ vulcasil activated with V5+ silica gel activated with Ti4+ vulcasil activated with Ti4+ vulcasil activated with V5+

Standard deviation

SiO2 was then treated with TiCl4 and VOCl3 to obtain surface complexes [15]. This technique is known as the method of molecular deposition [11]. The bound complexes obtained were then hydrolyzed and subjected to thermal treatment [15,16]. The structure characteristics and the amounts of active groups in the carriers used are presented in Table 1. It can be seen that the amount of hydroxyl groups per unit surface of the initial vulcasil was higher than that of the initial silica gel. It should be noted also that the higher content of hydroxyl groups did not lead to co-ordination bonding of higher amount of Ti4+. This is probably due to the complex mechanism of the surface reactions taking place. With the carrier of modified vulcasil, not all OH– groups interact with TiCl4 [16]. Other important characteristics of the carriers are their specific area and pore volume. The data in Table 1 clearly show that silica gel had a higher specific area and pore volume than vulcasil. Total desiccation of the carrier activated with Ti4+ and 5+ V is important for the stabilization and constitution of the bound complexes which, in turn, is necessary for the metallochelate immobilization. Therefore, part of the activated complexes was dried at 110 8C and the other heated to 550 8C. Thus, the total number of activated carriers studied was 6 and the different carrier types are presented in Table 2. The amount of protein bound to all types of carriers was found to be from 1 to 11 mg g 1 carrier (Table 2). The amount of bound protein was higher for silica gel activated with Ti4+ ions than that observed for vulcasil due to the higher content of Ti4+, specific area and pore volume. It should be noted here that the higher amount of protein lead to lower enzyme activity. This is probably due to local aggregations of protein and more difficult diffusion of the substrate to the active centers of the enzyme molecules bound on surface area and in pores. It should also be noted that activity was affected by the temperature at which the carrier was dried. The results obtained were lower with carrier activated by heating to 550 8C. This could be expected since the degree of chelation decreased due to the decrease of both surface hydrophilicity and the number of active centers able to chelate the enzyme.

The results presented in Table 2 showed that the activity of urease immobilized onto vulcasil modified with Ti4+ and dried at 110 8C was close to that of the free enzyme. In this case, the highest value of the bound enzyme specific activity was measured (565.14 U mg 1 enzyme). Further, pH optimum, T optimum and temperature stability of urease immobilized on all types of carriers were studied (Figs. 1–3) excluding the carriers activated with V5+ because of their very low activity. pHopt (Fig. 1) and Topt (Fig. 2) of bound urease were the same as these of free urease (pHopt = 5.8 and Topt = 30 8C). Temperature and pH curves for urease immobilized by the metallochelate method were wider than those registered for

Fig. 1. Effect of pH on the activity of urease metallochelate-immobilized onto carriers of amorphous SiO2 activated with Ti4+: (a) silica gel; (b) vulcasil. Free enzyme (1); enzyme immobilized onto activated carrier dried at 110 8C (2); enzyme immobilized onto activated carrier heated to 550 8C (3). The activities were measured with 3% urea in 0.06 M phosphate buffer at 30 8C for 5 min.

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Fig. 2. Effect of temperature on the activity of urease metallochelateimmobilized onto carriers of amorphous SiO2 activated with Ti4+: (a) silica gel; (b) vulcasil. Free enzyme (1); enzyme immobilized onto activated carrier dried at 110 8C (2); enzyme immobilized onto activated carrier heated to 550 8C (3). The activities were measured with 3% urea in 0.06 M phosphate buffer, at pH 5.8, for 5 min.

the free enzyme. This suggests that, due to the co-ordination bonding between the urease and carrier, the enzyme was active over a wider temperature and pH interval. This effect was much more pronounced with vulcasil carrier (Figs. 1b and 2b) and especially for urease bound to vulcasil modified with Ti4+ and heated to 110 8C. Thermal stability is an important characteristic for every enzyme. As can be seen from Fig. 3, free urease was fully deactivated after 90 min incubation at 50 8C. In all cases studied, immobilized urease at the same temperature lost about 60% of its activity after 90 min and about 80% after 120 min. Preservation of activity was best pronounced for

Fig. 3. Thermal stability at 50 8C of urease metallochelate-immobilized onto carriers of amorphous SiO2 activated with Ti4+ for 120 min: (a) silica gel; (b) vulcasil. Free enzyme (1); enzyme immobilized onto activated carrier dried at 110 8C (2); enzyme immobilized onto activated carrier heated to 550 8C (3). The activities were measured with 3% urea in 0.06 M phosphate buffer, at pH 5.8.

the enzyme metallochelate-immobilized on the vulcasil carrier dried at 110 8C and activated with Ti4+ ((loosing about 60% of its initial activity after 120 min)—Fig. 3b, curve 2). The effect of substrate concentration on enzyme reaction rate for urease immobilized on all types of carriers, except these activated with V5+, was studied. Experiments were carried out at three different temperatures: 25, 30 and 35 8C. Using the plots 1/V = f(1/S), Michaelis constant (Km) and maximal reaction rate (Vmax) were determined for each type of immobilized enzyme and values are presented in Table 3.

Table 3 Kinetic parameters of the free and metallochelate immobilized urease onto modified amorphous SiO2 No.

1 2 3 4 5

Free and immobilized urease

Free enzyme Enzyme immobilized Enzyme immobilized Enzyme immobilized Enzyme immobilized Standard deviation

onto onto onto onto

silica gel activated with Ti4+ vulcasil activated with Ti4+ silica gel activated with Ti4+ vulcasil activated with Ti4+

Drying temperature and heating temperature of activated carrier (8C) – 110 110 550 550

Vmax  106 (mol min 1 mg 1)

Km  103 (mol l 1)

25 8C

30 8C

35 8C

25 8C

30 8C

35 8C

3.8 0.071 2.5 0.042 0.28

4.3 0.083 3.12 0.055 0.40

4.2 0.078 2.98 0.052 0.35

18 23.7 27.6 26.6 28

17 22.2 26 25 27

17.4 22.4 27 25.6 27.5

0.005

0.2

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Table 4 Activation energy of the free and metallochelate immobilized urease onto modified amorphous SiO2 No.

Free and immobilized urease

Drying temperature and heating temperature of activated carrier (8C)

Activation energy (cal mol 1)

1 2 3 4 5

Free enzyme Enzyme immobilized Enzyme immobilized Enzyme immobilized Enzyme immobilized

– 110 110 550 550

5085 4950 5055 4900 5030

onto onto onto onto

silica gel activated with Ti4+ vulcasil activated with Ti4+ silica gel activated with Ti4+ vulcasil activated with Ti4+

Standard deviation

A comparison of the results obtained revealed that the enzyme reaction rate observed with urease bound to vulcasil was higher than that with silica gel carrier. From all the immobilized systems studied, the highest Vmax was measured with urease bound to vulcasil activated with Ti4+ and dried at 110 8C. As was expected, the reaction with free urease had the highest Vmax. Further, the results presented in Table 3 showed that the temperature at which the enzyme reaction was carried out exerted a significant effect on the values of Vmax. The latter was highest at 30 8C (temperature optimum of the enzyme) and lowest at 25 8C. The Michaelis constant Km was determined for all types of enzymes (Table 3). Once again a dependence on carrier type was observed. Thus, the Km for urease bound to vulcasil showed slightly higher values then these for silica gel carrier. The difference among Km values for the different carriers was comparatively low and it may be concluded that the Km for all types of enzymes was close to that of the free urease. Therefore, the method used for immobilization provides good preservation of the affinity between substrate and enzyme. Schmidt-Steffen and Straube [13], Chen and Chiu [17] and Pozniak et al. [18] reported that Km for urease covalently bonded to the polymer membrane increased by an order compared to Km for free urease. It can be concluded from the results obtained for Km that the metallochelate immobilization provides good preservation of the affinity between enzyme and substrate due to the weaker bonds between the enzyme and the carrier compared the bonds obtained by chemical immobilization. The effect of the temperature (15–40 8E) on the enzyme reaction rate was also studied. The plot lg V = f(1/T) was drawn and the activation energy of the enzyme reaction was determined. The results are presented in Table 4. It should be noted that the activation energy was higher for enzymes showing higher Vmax. The activation energy of the bound enzymes was by ca. 4% smaller than that of the free enzyme. This means that metallochelate immobilization is a very good method for binding enzymes to carriers.

0.5

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