Adsorption of urease on PE-MCM-41 and its catalytic effect on hydrolysis of urea

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Colloids and Surfaces B: Biointerfaces 62 (2008) 42–50

Adsorption of urease on PE-MCM-41 and its catalytic effect on hydrolysis of urea Kazi-Zakir Hossain a , Carlos M. Monreal b , Abdelhamid Sayari a,∗ a

b

Department of Chemistry, University of Ottawa, Ottawa K1N 6N5, Canada Environmental Health Team, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa K1A 0C6, Canada Received 4 July 2007; received in revised form 15 September 2007; accepted 16 September 2007 Available online 21 September 2007

Abstract Pore-expanded MCM-41 (PE-MCM-41) silica exhibits a unique combination of high specific surface area (ca. 1000 m2 /g), pore size (up to 25 nm) and pore volume (up to 3.5 cm3 /g). As such, this material is highly suitable for the adsorption of large biomolecules. The current study focused primarily on the application of PE-MCM-41 material as suitable host for urease (nickel-based large metalloenzyme) in controlled hydrolysis of urea. Urease adsorbed on PE-MCM-41, regular MCM-41 and silica gel (SGA) were used as catalysts for urea hydrolysis reaction. Adsorption studies of urease on these materials from aqueous solution at pH 7.2 revealed that the adsorption capacity of PE-MCM-41 (102 mg/g) is significantly higher than that of MCM-41 (56 mg/g) and SGA (21 mg/g). The equilibrium adsorption data were well fitted using the Langmuir–Freundlich model. Furthermore, the kinetic study revealed that the uptake of urease follow the pseudo-first order kinetics. The in vitro urea hydrolysis reaction on pristine urease and different urease-loaded catalysts showed that the rate of hydrolysis reaction is significantly slower on U/PE-MCM-41 compared to that of bulk urease and urease on MCM-41 and SGA. This technique could be an alternative means to the use of urease inhibitors to control the ammonia release from urea fertilizer. © 2007 Elsevier B.V. All rights reserved. Keywords: Urease; Adsorption; Mesoporous silica; Urea; Langmuir–Freundlich isotherm

1. Introduction Urease is a nickel-based metalloenzyme first isolated from seeds of jack bean plant in 1926 [1]. It is found in a variety of bacteria, fungi and plants in nature. It catalyses the hydrolysis of urea, which is the main source of nitrogen used as fertilizer, to form ammonia and carbon dioxide with a rate approximately 1014 times the rate of the un-catalyzed reaction [2,3]. Rapid hydrolysis of urea fertilizer by soil-based bacterial urease, results in unproductive nitrogen evolution and in ammonia volatilization and toxicity, hence alkaline-induced crop damage and subsequent greenhouse gas emission [4]. This phenomenon is quite common in many parts of the world, particularly in agricultural trails in tropical regions where urea fertilizer is used widely because of its low cost, ease in handling and high nitrogen content for fast growth of seasonal crops. Therefore, a urease

∗

Corresponding author. Tel.: +1 613 562 5483; fax: +1 613 562 5170. E-mail address: [email protected] (A. Sayari).

0927-7765/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2007.09.016

inhibitor and/or a means for controlling the rate of the enzymatic hydrolysis of urea can be combined with the urea fertilizer to increase the overall efficiency of nitrogen utilization. The use of urease inhibitors (e.g. phosphoryl and thiophosphoryl amides, quinines, hydroxamic acids) in order to retard the urea hydrolysis has been reported [4]. However, these inhibitors are too expensive and easily decomposed or inactivated to generate any practical benefit [5]. Another possibility to reduce the rate of urea hydrolysis is to immobilize urease within the confined channels of mesoporous silica. Diffusion limitations of the substrate combined with a potential effect of adsorption on the intrinsic activity of the enzyme may bring about significant decrease in the hydrolysis rate. Adsorption-induced immobilization on mesoporous materials has been used for a variety of biologically active species such as amino acids, proteins and enzymes [6–15]. In this process, interactions between the support and the guest molecules are of non-covalent nature, such as hydrogen bonding, electrostatic, van der Waals and hydrophobic or hydrophilic interactions, thus relatively weak. The immo-

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bilization techniques, however, could affect their catalytic activity. Urease has received particular attention because of its wellunderstood structural characteristics and catalytic effect on urea hydrolysis process [2,3,16]. Literature reports on urease adsorption by physical immobilization onto solid substrates for the purpose of catalysis and sensing of urea include adsorption onto inorganic support surfaces [17–21], polymeric membranes [22–25], microcapsules [26,27] and sol–gel derived cast films [28,29]. Urease adsorption on activated charcoal for clinical application suffers from particulate release [17], while clay minerals [18] exhibit low adsorption capacity (21.2 mg/g). Urease immobilization on polymeric membranes such as dye attached polyamide membrane [22] by adsorption resulted in an increase of the enzyme thermal stability, while encapsulation in polymeric microshells [26,27] through layer-by-layer technique opened up the possibility of preparing a biocatalytic reactor. Urease adsorbed on sol–gel derived films [28,29] resulted in high thermal and storage stability of the enzyme. It is expected that pore-confined adsorption of the enzyme on PE-MCM-41 could increase its stability while decreasing the permeation of the substrate to the active sites of the enzyme, hence reducing its catalytic activity. However, to our knowledge, the immobilization of urease on mesoporous silica and its potential in controlled hydrolysis of urea has not been explored. Sayari et al. [30,31] reported the preparation of pore-expanded MCM-41 silica (PEMCM-41) possessing high specific surface area (ca. 1000 m2 /g), large pore size (up to 25 nm) and pore volume (up to 3.5 cm3 /g) via post-synthesis hydrothermal treatment of as-synthesized MCM-41 in the presence of N,N-dimethyldecylamine. These materials opened up many opportunities in adsorption and catalytic applications [32–40]. In the present work, we report the adsorption of urease onto the PE-MCM-41 with a specific surface area, pore size and pore volume of ca. 920 m2 /g, 10.4 nm and 2.04 cm3 /g, respectively, at 25 ◦ C from aqueous solutions at different pHs within the range of 5.0–9.0. However, the catalytic effect on urea hydrolysis reaction was investigated using mesoporous silicas loaded with urease at near neutral pH 7.2. The reason of this particular adsorption condition is associated with the fact that most field crops grow well in a soil with pH ranging from 6 to 8 at ambient temperature [41]. For comparison, regular MCM-41 and silica gel adsorbent (SGA) were also used in this study. This study revealed that the amount of urease adsorbed on PE-MCM-41 was higher as compared to MCM41 and SGA. However, the catalytic effect of urease-loaded PE-MCM-41 appeared to slow the urea hydrolysis rate much more efficiently than MCM-41 and SGA. This new technique of controlled-hydrolysis of urea by large pore PE-MCM-41 could be used in agricultural soils to manage the ammonia release process.

bent of 230–400 mesh particle size were purchased from Sigma–Aldrich. MCM-41 and PE-MCM-41 were synthesized using Cab-O-Sil M5 fumed silica (Cabot Co.). Potassium dihydrogen phosphate was obtained from Fluka (Germany). Ammonium standard solution and pH buffer solutions were purchased from Thermo Electron Co. All other chemicals were obtained from Sigma–Aldrich and used as supplied.

2. Experimental

2.4. Adsorption kinetics of urease on mesoporous silica

2.1. Materials

The kinetic study of urease adsorption on mesoporous silica adsorbents was carried out in the batch mode in aqueous solution (pH 7.2) with 2 g/L urease concentration. In each adsorption experiment, 100 mg of the different adsorbents was suspended

Urease (35 units/mg; from jack bean; Mw 480 kDa) was obtained from Fluka. Urea (ultra pure) and silica gel adsor-

2.2. Synthesis of MCM-41 and PE-MCM-41 The pore-expanded mesoporous silica host was prepared via a two-step procedure published earlier [30,31]. The first step was to prepare an MCM-41 mesostructure at a relatively low temperature, e.g. 80 ◦ C. This was achieved using Cab-O-Sil M5 fumed silica as the silica source, cetyltrimethylammonium bromide (CTAB) as the surfactant template and a 25% solution of tetramethylammonium hydroxide in water (TMAOH) for pH adjustment. TMAOH (57.72 g) was added in a Teflon-lined autoclave containing 556 g of distilled water with stirring. CTAB (82 g) was added to the solution and stirred until a homogeneous mixture was formed. Fumed CabO-Sil silica (32 g) was slowly added to the mixture, stirred for 30 min beyond complete dissolution. The molar gel composition was SiO2 :TMAOH:CTAB:H2 O = 1.0:0.32:0.45:67. The gel was heated in an autoclave at 80 ◦ C for 48 h. The pore expansion procedure was carried out by post-synthesis hydrothermal treatment of as-synthesized MCM-41 in the presence of N,Ndimethyldecylamine (DMDA). The as-synthesized MCM-41 (38.5 g) was added to an emulsion of DMDA (48.2 g) in distilled water (578 mL) under stirring for 30 min. The mixture was then heated in an autoclave at 120 ◦ C for 72 h. Both MCM-41 and PE-MCM-41 were collected by filtration, dried at room temperature and prepared for urease immobilization by calcining at 550 ◦ C in air for 5 h. 2.3. Characterization To determine the physical characteristics of each material, nitrogen adsorption–desorption isotherms were measured at 77 K on a Coulter Omnisorp 100 gas analyzer. The pure silica materials were degassed at 250 ◦ C under high vacuum (10−5 Torr) for 2 h prior to the nitrogen adsorption measurements, while the urease-immobilized samples were degassed at 50 ◦ C for 6 h. The specific surface area was determined from the linear part of the BET plot (P/P0 = 0.05–0.15). The average pore size was taken as the peak of the pore size distributions as calculated from the adsorption branch using the KJS (Kruk–Jaroniec–Sayari) method [42]. The total pore volume was determined as the volume of liquid nitrogen adsorbed at P/P0 of 0.995.

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in 10 mL of the urease solution. The suspensions were continuously shaken in a bath with a speed of 180 St/min at 25 ◦ C for different time intervals (30 min to 48 h) and then centrifuged. The urease concentration in the supernatants was measured by UV–vis spectroscopy at 276 nm (Cary 300, Varian). Calibration measurements were performed separately before each experiment, with urease solutions of different concentrations within the range of interest.

ammonium ion specific electrode (Orion 93-18). Calibration measurements were carried out separately before each set of experiments with ammonium standard solutions of different concentrations.

2.5. Adsorption isotherms and urease-loaded mesoporous silicas

Nitrogen adsorption–desorption isotherms for MCM-41, PEMCM-41 and SGA are shown in Fig. 1. The isotherm for MCM-41 is of type IV according to the IUPAC classification and exhibits a H1 hysteresis loop, characteristic of periodic mesoporous materials [43,44]. Similarly, PE-MCM-41 exhibits a type IV isotherm with a broad H1 hysteresis loop occurring at higher relative pressure, which is consistent with pore enlargement. Uniformly sized mesopores are evident from the narrow and sharp capillary condensation step in the isotherms for both MCM-41 and PE-MCM-41. This is consistent with the narrow pore size distribution shown in Fig. 1(inset). Although the isotherm for SGA looks like type IV, the mesopores are devoid of uniformity with very broad pore size distribution as evident in Fig. 1(inset). The textural properties of the different mesoporous adsorbents are summarized in Table 1. The specific surface areas of MCM-41 and PE-MCM-41 are 1078 and 920 m2 g−1 , respectively, which are more than double compared to the specific surface area of SGA (443 m2 g−1 ). On the other hand, the pore size of PE-MCM-41 is 10.4 nm, which is higher than the pore sizes of MCM-41 (3.8 nm) and SGA (6.5 nm).

A series of urease solutions with concentrations ranging from 0.25 to 10 g/L was prepared by dissolving different amounts of the enzyme in de-ionized water. Adsorption isotherms of urease on various mesoporous adsorbents were achieved by equilibrating the adsorbents with aqueous solutions of urease with pH 7.2. In each adsorption experiment, 100 mg of the different adsorbents was suspended in 10 mL of the desired urease solution. The suspensions were continuously shaken in a shaking bath with a speed of 180 St/min at 25 ◦ C until an assumed equilibrium was reached, typically after 24 h and then centrifuged. The urease concentration in the supernatants was measured by UV–vis spectroscopy as mentioned above. The urease containing silica materials were recovered from the reaction mixtures by filtration using a 0.22 ␮m membrane filter (cellulose membrane, Millipore Co.) and washed with de-ionized water. The materials were dried at room temperature for 24 h and finally in a vacuum oven at 50 ◦ C for 12 h. Urease adsorbed samples of MCM-41, PE-MCM-41 and SGA silicas were denoted as U/MCM-41, U/PE-MCM-41 and U/SGA, respectively. The influence of pH on the adsorption of urease onto the mesoporous silica adsorbents was examined as described above, with an enzyme concentration of 10 g/L in the pH range 5.0–9.0 using 25 mM phosphate buffer. Thermogravimetric analysis of native urease and ureaseloaded samples were performed using the thermogravimetric analyzer (Q 500, TA Instrument) coupled with a mass spectrometer (Pfeiffer Thermostar) for determining the urease loading and its stability through the weight loss of the material upon calcination in the temperature range of 25–800 ◦ C, in flowing N2 .

3. Results and discussion 3.1. Adsorbents

3.2. Adsorption kinetics Fig. 2 shows the time-resolved uptake of urease over PEMCM-41, MCM-41 and SGA with initial concentration of 2 g/L.

2.6. Catalytic activity in urea hydrolysis reaction A 5 mM aqueous solution of urea was used to study the catalytic activity of native urease (90 ␮g/mL) and ureaseloaded mesoporous silicas (e.g. U/MCM-41, U/PE-MCM-41 and U/SGA). In each hydrolysis experiment, a determined amount of urease-containing silica catalyst (equivalent to the amount of native urease) was suspended in 50 mL of urea solution at room temperature under gentle stirring and covered to avoid evaporation. A controlled experiment using pristine PEMCM-41 was performed by using 50 mg of PE-MCM-41 under otherwise the same conditions. The conversion of urea to ammonia was monitored in situ over time using a Benchtop Multimeter (Orion 5-Star, Thermo Electron Co. USA) equipped with an

Fig. 1. Nitrogen adsorption–desorption isotherms for various adsorbents used in this study (Inset: KJS pore-size distributions).

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Table 1 Textural parameters of various adsorbent and their urease-loaded derivatives from aqueous solution with pH 7.2 at 25 ◦ C Materials

Initial concentration (g/L)

Loading (mg/g)

SBET (m2 /g)

KJS pore size (nm)

Vp (cm3 /g)

PE-MCM-41 PE-MCM-41a MCM-41 SGA U/PE-MCM-41 U/MCM-41 U/SGA

– – – – 10 10 10

– – – – 102 56 21

920 420 1078 443 278 491 377

10.4 3.5 3.8 6.5 2.1 1.6 6.4

2.14 0.27 1.08 0.71 0.17 0.21 0.63

a

Treated in aqueous media for 24 h at 25 ◦ C.

It is obvious from the figure that the urease uptake by PE-MCM41 at the beginning was relatively fast compared to the other adsorbents. PE-MCM-41 with large pore may facilitate the mass transfer inside the channels. However, the adsorption equilibrium is attained for all three adsorbents within 24 h of shaking. The amount of urease adsorbed, qt at any intermediate time, t was calculated from the mass balance equation as follows: qt =

(C0 − Ct )V m

(1)

where C0 is the initial concentration of solution (g/L), Ct the concentration of solution at time t (g/L), V the volume of solution (L) and m is the mass of adsorbent (g). The adsorption process can be described by either pseudo-first order or pseudo-second order kinetics [45]. For pseudo-first order process, the Lagergren rate equation is the one generally used, ln(qe − qt ) = ln qe − k1 t

(2)

where qe is the equilibrium adsorption amount, qt the adsorption amount at time t and k1 is the pseudo-first order rate constant. The

pseudo-second order process can be expressed as the equation, 1 t t = + qt k2 qe2 qe

(3)

where qe is the equilibrium adsorption amount, qt the adsorption amount at time t and k2 is the pseudo-second order rate constant. The plot of ln (qe − qt ) versus t based on pseudo-first order kinetics and the plot of t/qt versus t-based pseudo-second order kinetics are presented in Fig. 3a and b, respectively. The kinetic parameters for adsorption of urease on different mesoporous adsorptions were calculated from Eqs. (2) and (3) and compiled in Table 2. It can be seen from Fig. 3a and b that a linear relation is established during the whole adsorption process with the coefficient (R2 ) higher than 0.99. However, the qe values calculated from pseudo-first order kinetics are closer to the experimental results (Table 2). Therefore, it is inferred that the urease adsorption on mesoporous silica follows the pseudo-first order kinetics. Among the adsorbents used, the order of urease uptake rate (k1 ) was as follows: PE-MCM-41 > MCM-41∼SGA. The adsorption of proteins (e.g. lysozyme, ␤-lactoglobulin and hemoglobin) from aqueous solution on silica powder was also found to follow a pseudo-first order kinetics [46]. 3.3. Effect of pH on urease adsorption

Fig. 2. Time resolved uptakes of urease over PE-MCM-41, MCM-41 and SGA adsorbents.

The effect of pH on the adsorption of urease on different mesoporous adsorbents is shown in Fig. 4. It is apparent from the Figure that all three silica adsorbents exhibit similar trends, i.e., the amount of adsorbed urease decreased slowly and linearly as the pH increased from 5.0 to 9.0. The highest adsorption was observed at pH 5, which is close to the isoelectric point (pI) of urease [18]. Near the pI of urease, the net charge of the enzyme is zero and the columbic repulsive force between the urease molecules is minimal. These phenomena enhance secondary interactions such as hydrogen bonding, van der Waals and hydrophobic–hydrophilic interactions leading to increased urease adsorption [7,22]. The net negative charges on silica and urease surfaces gradually develop as pH increases which may result in diminishing enzyme adsorption capacity. Nonetheless, as can be observed from Fig. 4, the amount of adsorbed urease remained relatively high even on negatively charged silica surface at higher pH.

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Fig. 3. Kinetic plots for the adsorption of urease over PE-MCM-41, MCM-41 and SGA adsorbents: (a) pseudo-first order and (b) pseudo-second order model. Table 2 Kinetic parameters for the first- and second-order urease adsorption Adsorbents

qe experimental (mg/g)

First order k1

PE-MCM-41 MCM-41 SGA

78.0 41.9 16.9

(×102 h−1 )

19.46 14.69 15.24

Second order qe calculated (mg/g)

R2

k2 (×102 g mg−1 h−1 )

qe calculated (mg/g)

R2

76.8 40.8 12.9

0.99 0.99 0.99

0.33 0.43 2.06

85.5 48.3 18.3

0.99 0.99 0.99

3.4. Urease adsorption isotherms To evaluate the adsorption capacity of the adsorbents, we carried out adsorption experiments using urease solutions with 0.25–10 g/L initial concentration. The equilibrium sorption data obtained was analyzed based on the commonly used Langmuir and Langmuir–Freundlich models [47]. The Langmuir isotherms model is described by the equation, qe =

KL qm Ce 1 + K L Ce

(4)

where qe (mg/g) is the equilibrium adsorption amount, qm (mg/g) the maximum sorption capacity, Ce the equilibrium concentration of solute (mg/L) and KL is the Langmuir constant. A typical Langmuir–Freundlich model is described by the equation, qe =

Fig. 4. Effect of pH on the adsorption of urease over PE-MCM-41, MCM-41 and SGA adsorbents (initial concentration is 10 mg/mL).

KLF qm Cen 1 + KLF Cen

(5)

where qe (mg/g) is the equilibrium adsorption amount, qm (mg/g) the maximum sorption capacity, KLF the Langmuir–Freundlich constant and n is the intensity of the constant. Fig. 5 shows the urease adsorption isotherms using Eqs. (4) and (5) and the associated parameters are shown in Table 3.

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Fig. 5. Adsorption isotherms of urease over PE-MCM-41, MCM-41 and SGA. Solid lines represent the data fitted by Langmuir–Freundlich (L–F) and dotted lines for Langmuir (L) model.

As can be seen from Fig. 5, the isotherms are well represented by both Langmuir and Langmuir–Freundlich models. However, comparing the qmax values obtained by the models with the experimental qmax values and also the goodness of fitting, the adsorption data appears to be following Langmuir–Freundlich model more closely compared to the Langmuir model. The adsorption capacity of the different adsorbents shows the following order: PE-MCM-41 > MCM-41 > SGA, although MCM-41 has higher surface area as compared to the other adsorbents. A reasonable explanation is that the pore sizes of MCM-41 (3.8 nm) and SGA (6.5 nm) are smaller than the dimension of urease (7.5 nm × 8.0 nm × 8.0 nm) and not accessible to bulky urease molecules. In the case of PE-MCM-41, the presence of uniform large pore system (10.4 nm) rather facilitates the diffusion of urease molecules inside the mesopores. Moreover, the mesopore volume of PE-MCM-41 (2.04 cm3 /g) is also higher than the MCM-41 and SGA (Table 1) and paves the way for more accessible adsorption sites. From our results we can conclude that urease entrapped inside the relatively large pore system of PE-MCM-41, consistent with the adsorption capacity of PE-MCM-41 compared to MCM-41 and SGA. The maxi-

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mum adsorption capacity of urease adsorbed on PE-MCM-41 was found to be ca. 102 mg g−1 which is almost twice as high as the adsorption capacity of MCM-41 (56 mg/g) and five times higher than the capacity of SGA (21 mg/g). Nitrogen adsorption–desorption isotherms for the ureaseloaded adsorbents (10 g/L initial concentration) were measured. The isotherms were of type I (Fig. is not included) and the specific surface area, pore diameter and pore volume of all samples were reduced substantially. The results are shown in Table 1. The specific surface area, pore diameter and pore volume of PE-MCM-41 were 920 m2 g−1 , 10.4 nm and 2.04 cm3 g−1 , respectively. These parameters of PE-MCM-41 decreased on urease loading. For example, the specific surface area of U/PEMCM-41 was reduced from 920 to 278 m2 g−1 which represents ca. 70% reduction, while the specific pore volume was reduced from 2.04 to 0.17 cm3 g−1 corresponding to 91% decrease. This large reduction of the specific surface area and pore volume were primarily attributed to the adsorption of urease molecule in the mesopore channels of PE-MCM-41. It is interesting to note that the actual volume of urease adsorbed (102 mg/g) in the mesopore channels of PE-MCM-41 is only 0.89 cm3 /g (assuming that the net volume of one urease molecule is 6.89 × 10−18 cm3 ), which is 43.8% of the total free volume of the PE-MCM-41. This large difference in pore volume can be rationalized by the fact that the additional shrinkage of PE-MCM-41 silica (e.g. specific pore volume was reduced from 2.04 to 0.27 cm3 g−1 ) resulted after treating the adsorbent with water as shown in Table 1. 3.5. Stability of the adsorbed urease Fig. 6a–c shows the TG-DTG plots of PE-MCM-41, native urease and U/ PE-MCM-41. Fig. 6a indicates that the rate of weight loss was maximum at 94 ◦ C (Ia ) due to the removal of the physically adsorbed water. Another small peak at 321 ◦ C (IIa ) is assigned to the loss of water formed from the surface hydroxyl group. The total weight loss up to 800 ◦ C was only ca. 10.5%. Pristine urease shows four prominent degradation peaks at 152 ◦ C (Iu ), 204 ◦ C (IIu ), 238 ◦ C (IIIu ) and 347 ◦ C (IVu ) provided in Fig. 6b. This degradation pattern of the urease changes substantially after adsorption of urease on PE-MCM41 as shown in Fig. 6c. For example, the weight loss peak at 152 ◦ C (Iu ) totally disappeared; the 204 ◦ C (IIu ) and 238 ◦ C (IIIu ) events moved to higher temperature (236 ◦ C (IIu ), 347 ◦ C (IIIu ), respectively). Moreover, the weight loss peak intensity in the U/PE-MCM-41 material decreased significantly compared to the pristine uresae (e.g. 347 ◦ C (IIIu )) in Fig. 6c. From these observations, it can be concluded that the decomposition of the

Table 3 Adsorption parameters for urease adsorption onto PE-MCM-41, MCM-41 and SGA adsorbents Adsorbents

PE-MCM-41 MCM-41 SGA

Langmuir (n = 1)

Langmuir–Freundlich

qmax experimental (mg/g)

qmax calculated (mg/g)

KL

102 56 21

108.6 57.8 23.4

2.0 2.1 1.7

(×103

L g−1 )

R2

qmax calculated (mg/g)

KLF (×103 L g−1 )

n

R2

0.98 0.99 0.99

102.9 57.6 26.0

0.5 1.9 8.5

1.26 1.02 0.71

0.99 0.99 0.99

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Fig. 6. Decomposition profiles for (a) PE-MCM-41, (b) Pristine urease and (c) U/PE-MCM-41.

urease adsorbed in PE-MCM-41 is suppressed by the protection of the adsorbent resulting in increased thermal stability. The stability of adsorbed urease in liquid reaction media was examined by enzyme leaching experiment. We have examined a urease leaching test in urea hydrolysis reaction (data is not included), and it was found that no urease leaching occurred. From the foregoing discussion, it is clear that the uresae stability increased upon adsorption onto the PE-MCM-41. 3.6. Catalytic effect on urea hydrolysis reaction It is well known that urea hydrolysis into ammonia and carbon dioxide is catalyzed by the enzyme urease [2,3]. Fig. 7 shows the

hydrolysis of urea by U/MCM-41, U/PE-MCM-41 and U/SGA in comparison to the native urease and pristine PE-MCM-41. The urease-free PE-MCM-41 exhibited negligible conversion of urea to ammonia (0.03%) over 36 h, while pure urease converted 100% of the urea within 60 min (Fig. 7, inset). The catalytic effect of U/MCM-41 and U/SGA exhibit comparable behaviors as they achieve complete urea conversion within ca. 30 h. However, in the presence of U/PE-MCM-41, urea hydrolysis showed a much slower release of ammonia (e.g. 32% conversion at 36 h, Fig. 7 and 89% of urea total conversion after 21 days of reaction time, Fig. 8). These results reveal that the pure silica support material is unable to catalyze the hydrolysis of urea, whereas the free enzyme urease is a too powerful catalyst

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Fig. 7. Hydrolysis of urea in aqueous solution to ammonia by U/PE-MCM-41, U/MCM-41, U/SGA and native urease (inset: urea hydrolysis to ammonia in the presence of native urease).

leading to a fast reaction that would prevent the conservation of urea-derived nitrogen in soil. Urease immobilized on solid substrate could be useful for slow decomposition of urea in this purpose since the immobilization of the enzyme on solid substrate reduces its catalytic activity [27]. Among the catalysts

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used in this study, the order of urea hydrolysis reaction rate was as follows: U  U/MCM-41∼U/SGA  U/PE-MCM-41. A reasonable explanation of this trend is based on two assumptions: (i) adsorbed urease is inherently less active than pristine urease, and (ii) urease located inside the pore channels is at least apparently, less active then urease adsorbed on the external surface. As mentioned earlier, it is believed that in U/MCM-41 and U/SGA the adsorbed urease is located primarily on the external surface. Thus, though it remained high, their catalytic activity is more than one order of magnitude lower than unsupported urease. On the contrary, in U/PE-MCM-41 urease resides mostly inside the pore channels and shows approximately another order of magnitude decrease in catalytic activity. The lower activity of U/PE-MCM-41 may be due to either inaccessibility of urease because of confinement, or slow diffusion of urea inside the now hardly porous material, or a combination thereof. This means of controlling the rate of the enzymatic hydrolysis of urea using PEMCM-41 could be combined with the urea fertilizer to increase the overall efficiency of nitrogen utilization. PE-MCM-41 could be added to agricultural trails few days before the addition of urea fertilizer. Soil based urease may adsorb inside the large pore PE-MCM-41 and limits its activity substantially to hydrolyze the fertilizer as we have shown. Thus, this would allow the fertilizer nitrogen to be released over a longer period of time during the growing season of field crops. 4. Conclusion This study mainly focused on the adsorption of urease on PE-MCM-41, regular MCM-41 and SGA with different textural properties at ambient conditions (25 ◦ C and neutral pH). The adsorption kinetics revealed that the amount of urease adsorbed follows the trend: PE-MCM-41 > MCM-41 > SGA. Furthermore, the adsorption rate constant values indicated that the adsorption is faster on PE-MCM-41 compared than on MCM-41 and SGA. This trend of urease adsorption on PEMCM-41 is due to enhanced mass transfer through the large pores of PE-MCM-41. The maximum urease loading at pH 7.2 was 102 mg/g of PE-MCM-41. Enhanced stability of the urease after adsorption was observed. The urease-loaded catalysts were used for in vitro urea hydrolysis reaction. Urease adsorption within the pore system of PE-MCM-41 appears to significantly reduce the rate of urea hydrolysis. Therefore PE-MCM-41 could be used in agricultural soil to reduce the rate of urea hydrolysis process, providing a useful model for the control of nitrogen release in soils, which may lead to improved management of nitrogen fertilizer for crops nutrition as well as to the reduction of ammonia gas emission from urea fertilizer. Acknowledgments

Fig. 8. Catalytic performance of U/PE-MCM-41: slow conversion of urea to ammonia by U/PE-MCM-41.

A.S. is the Government of Canada Research Chair in Catalysis by Nanostructured Materials (2001–2008). We thank the Technology and Innovation Program of Natural Resources Canada for financial support. Thanks to Harlick, Tsyganok and T.V.M. Rao for fruitful discussions.

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