Improving the Stability of Immobilized Penicillin G Acylase via the

Article pubs.acs.org/IECR

Improving the Stability of Immobilized Penicillin G Acylase via the Modification of Supports With Ionic Liquids Huacong Zhou,†,‡ Liangrong Yang,† Wei Li,*,† Qinghui Shou,†,‡ Peng Xu,†,‡ Wensong Li,†,‡ Fuchun Wang,†,‡ Pinhua Yu,†,‡ and Huizhou Liu*,† †

State Key Laboratory of Biochemical Engineering, Key Laboratory of Green Process and Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China ‡ Graduate School of Chinese Academy of Sciences, Beijing, 100039, China S Supporting Information *

ABSTRACT: With the aim of improving the stability of penicillin G acylase (PGA), functional ionic liquids (ILs) were used to modify the surface chemistry of the supports on which they were physically adsorbed. Four kinds of ILsspecifically, 1-methyl3-(triethoxysilylpropyl)-imidazolium salts, with Cl−, BF4−, PF6−, and Tf2N− as the anions (IL-Cl−, BF4−, PF6−, and Tf2N−, respectively)were used to tune the hydrophilic or hydrophobic properties of the ILs. The synthesized ILs were first immobilized on magnetic silica nanoparticles (Fe3O4/SiO2), and the composite material (Fe3O4/SiO2−IL) was then applied to immobilize PGA via physical adsorption. The amount of protein loading, the specific activity, the immobilization yield, and the stability of immobilized PGA were investigated to evaluate the effects of the ILs on the PGA immobilization. The results showed that PGA immobilized on Fe3O4/SiO2−ILs was more stable than that immobilized on Fe3O4/SiO2 with no IL modification. Among the four kinds of ILs tested, the hydrophilic ILs (IL-Cl−, and IL-BF4−) were superior to the hydrophobic ones (IL-PF6−, and IL-Tf2N−) for PGA immobilization. The reusability of the immobilized PGA on Fe3O4/SiO2−IL (BF4−) was evaluated; a high residual activity (70% of the initial activity) was observed after 9 consecutive operation cycles under the experimental conditions. This activity was 1.8 times higher than that of immobilized PGA on naked Fe3O4/SiO2 (40%).

1. INTRODUCTION Penicillin G acylase (EC 3.5.1.11, PGA) is widely used to catalyze the hydrolysis of the amidic bonds of penicillin G and cephalosporin G to produce 6-aminopenicillanic acid (6-APA) and 7-aminodeacetoxycephalosporanic acid (7-ADCA), which are pharmaceutical intermediates in the manufacture of some semisynthetic antibiotics.1 However, native PGA is sensitive to the external environment, and can not be easily recycled, hampering its use in industrial production.2 Hence, the immobilization of PGA was proposed to overcome the existing problems.1,3−5 In past decades, different methods have been developed to immobilize enzymes; these methods included entrapment,6−8 solid carriers,1,9 and carrier-free immobilization techniques.10 Among these, the immobilization of enzymes on solid carriers is the most widely used, due to the rigidity of the carriers; this enables the use of various reactor configurations, and provides a high substrate availability for the enzyme. 11 The two approaches most often applied to control the interactions between the enzyme and the support in this method are covalent attachment and physical adsorption.1 The covalent attachment approach can typically fix enzymes tightly on the support, because of the formation of covalent bonds between the enzyme molecules and the functional groups on the surface of supports; groups such as aldehyde groups,12,13 epoxy groups,14 thiol groups,15 or biopolymers.16,17 However, one of the drawbacks of the covalent attachment approach is that the covalent bonds may be formed at the active sites of the enzymes, thus decreasing the activity of the enzymes.2,18,19 By comparison, physical adsorption is the simplest method of © 2012 American Chemical Society

enzyme immobilization, and has less damaging effects on the enzyme activity.20,21 Various materials have been used to immobilize PGA via physical adsorption, including molecular sieves,2 metal affinity membranes,22 and organic polymers.23 In these studies, the question of how to avoid the leaching of enzymes from the supports became a key point for immobilization. To decrease the leaching of enzymes, different materials were used to modify the surface of supports in attempts to enhance the interaction between the enzyme and supports.2,23,24 Some improvements have been achieved, but the final goal has not yet been reached. For example, only approximately 38% of the initial activity of PGA (ImPGA) immobilized on NH2-modified molecular sieve SBA-15 was maintained after 10 cycles.2 In recent years, the novel green solvents known as ionic liquids (ILs) have drawn increasing attention due to their unique properties.25,26 They are composed solely of ions, and have low melting point, no or very low volatility, thermal stability, wide liquid range, and excellent solvation properties.27−30 Potential applications of ILs have been found in many fields, including in reaction media,31,32 adsorbents for gases,33−37 separation processes,38,39 electrochemistry,40 and catalysts.41,42 It is noteworthy that ILs have been confirmed to be suitable for the reaction media of enzyme catalysis, which is an improvement over traditional organic solvents.43−45 Due to Received: Revised: Accepted: Published: 4582

November 28, 2011 March 4, 2012 March 10, 2012 March 10, 2012 dx.doi.org/10.1021/ie202745c | Ind. Eng. Chem. Res. 2012, 51, 4582−4590

Industrial & Engineering Chemistry Research

Article

the unique properties of ILs, including their tunable hydrophilicity/hydrophobicity, charged cations or anions, and properties similar to ions in solutions, it is possible for enzymes to interact stably with ILs via synergetic interaction forces, thus improving the reusability of the immobilized enzyme.46 In this work, to enhance the stability of PGA on supports, ILs were used to modify the surface of supports. Magnetic silica nanoparticles (Fe3O4/SiO2) composed of an inner magnetic Fe3O4 core and an outer silica shell were chosen as a support because of their special features5 and wide application fields.47−49 Silane group-functionalized ILs with different anions (Cl−, BF4−, PF6−, Tf2N−) were synthesized, and were used to modify the Fe3O4/SiO2 particles. The enzyme loading, immobilized yield, and stability of ImPGA were studied. To evaluate the effects of ILs, the reusability of ImPGA was also investigated.

Scheme 1. Preparation routes for (a) functional ionic liquids, (b) magnetic silica nanoparticles (Fe3O4/SiO2), and (c) immobilization of ionic liquids and PGA adsorption.

2. EXPERIMENTAL SECTION 2.1. Chemicals. Penicillin G acylase was purchased from Zhejiang Haider Biochemical Corp., Hangzhou, China. Penicillin G potassium salt was kindly provided by North China Pharmaceutical Group, Shijiazhuang, China. 6-Aminopenicillanic acid (6-APA) was purchased from Tokyo Chemical Industry Co., Ltd., Japan. p-Dimethylaminobenzaldehyde (PDAB), γ-aminopropyltriethoxysilane (APTES), and tetraethyl orthosilicate (TEOS) were obtained from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. Ferric chloride hexahydrate (FeCl3·6H2O) and ferrous chloride tetrahydrate (FeCl 2 ·4H 2 O) were from Xilong Chemical Co., Ltd., Guangdong, China. Other reagents and solvents were provided by Beijing Chemical Reagents Company, Beijing, China. 2.2. Preparation of IL-Modified Supports. The functional ILs with different anions (Cl−, BF4−, PF6−, and Tf2N−) were synthesized according to the existing literature.50 The ILs are abbreviated here as IL-X−, where X denotes the anions. The Fe3O4 particles and Fe3O4/SiO2 were prepared according to previously described methods.46 The Fe3O4/SiO2 were modified by functional ILs via the hydrolysis of silane groups and the subsequent reaction with −OH on the surface of particles.51 The IL-modified products are denoted as Fe3O4/ SiO2−ILs. All modification routes are illustrated in Scheme 1. To give a comparison with Fe3O4/SiO2−ILs, aminopropylfunctionalized magnetic silica nanoparticles (Fe3O4/SiO2− NH2) were also prepared. The typical procedure was as follows: 3.069 g of γ-aminopropyltriethoxysilane (APTES) was added into 60 mL of the 1:1 mixtures of ethanol/water at a final concentration of 5% (w/t). Then 0.495 g of Fe3O4/SiO2 was dispersed into the above system under ultrasonication for 5 min. Silanization reactions were performed at 60 °C under vigorous stirring for 24 h. The product was separated with a magnet, washed five times (10 mL each) with fresh solvent mixture, and dried at 50 °C in vacuo for 24 h. The carriers were activated with glutaraldehyde before using to immobilize PGA. Glutaraldehyde solution (2.3 mL 50% (v/v)) was added into 5 mL of phosphate buffer (0.1 M, pH 8.0) containing 0.4 g of Fe3O4/SiO2 powder under N2, and the system was kept at 30 °C for 2 h. After this, the powder was separated, washed, and dried in vacuo for 24 h. The obtained products were denoted as Fe3O4/SiO2−CHO. 2.3. Characterization. The size and morphology of the particles were characterized using TEM on a JEM-2010 microscope (Japan). The particle size distributions were calculated using data from 300 particles selected from different

regions of several TEM images. FT-IR spectra were recorded on a Bruker Vecter 22 FTIR spectrometer, with a resolution of 2 cm−1. The amount of ILs or NH2− on the surface of the support was analyzed via C, H, and N elemental analysis performed using a CE-440 element analyzer. Magnetic measurements were performed on a Princeton Applied Research vibrating sample magnetometer model 155 (VSM), at room temperature; and the magnetic moment was measured in the magnetic field range −10 000 to 10 000 Oe. The results presented are the average of at least two independent measurements. 2.4. Immobilization of PGA. The detailed immobilizing process was as follows:52 the commercial PGA solution (23 mg protein/mL) was diluted using 0.1 M pH 8.0 phosphate buffer to a final protein concentration of 1.1−3.5 mg/mL. The pH values of the diluted PGA solutions were adjusted to 9.0 with 0.1 M HCl and NaOH. Carrier (0.02 g) was then added to 5 mL of the PGA solutions, and the mixture was ultrasonicated for 2 min to make sure that the carriers were well suspended. The adsorption process was performed in an incubator at 37 °C and 180 rpm for 5 h. The protein concentration of the PGA solutions was detected before and after immobilization using Bradford’s dye binding assay.53 Into 5 mL of Coomassie Brilliant Blue G-250 solution was added 1 mL of protein sample. The mixture was stirred and then was allowed to react at room temperature for 5 min. The absorbance at 595 nm was recorded. The protein content was calculated according to the standard curve. The PGA loading (Q (mg protein/g carrier)) on carriers was calculated by mass balance. The PGA loading, and the other parameters used to characterize the performance of carriers, including the immobilization yield (IMY (%)) and the specific activity (SA, IU/mg protein), are described as follows, respectively: Q (mg protein/g carrier) = 4583

Q1 − Q 2 m

dx.doi.org/10.1021/ie202745c | Ind. Eng. Chem. Res. 2012, 51, 4582−4590

Industrial & Engineering Chemistry Research IMY(%) =

Article

Q1 − Q 2 × 100% Q1

Specific activity(IU/mg) =

3. RESULTS AND DISCUSSION 3.1. Synthesis of ILs and Preparation of Carriers. The structural and elemental analysis results for the ILs are presented in Table 1. These results showed that the C and H

A Q

Table 1. Structures of Functional Ionic Liquids and Elemental Analysis Resultsa

where Q1 (mg) is the total amount of protein in solution before immobilization, Q2 (mg) is the total amount of protein in the residual solution after immobilization, m [g] is the dry weight of carriers used for immobilization, and A (IU) is the apparent activity of the immobilized PGA. 2.5. Activity Assay of Native and Immobilized PGA. Vials containing 18 mL of 2% (w/t) penicillin G potassium solutions in 0.1 M pH 9.0 phosphate buffer were incubated at 37 °C, and 0.1 mL native PGA solution (23 mg protein/ml) or 0.02 g immobilized enzyme was then added. The system was kept at 37 °C to allow the enzymatic hydrolysis reaction to proceed for exactly 5 min. After this time, 9 mL of ethanol was added, and the vials were shaken vigorously to stop the reaction. The denatured enzyme precipitate and carriers were removed by centrifugation performed at 7000 rpm for 5 min, and the 6-APA content in the supernatant was detected using PDAB method.54 Into the mixture containing 3 mL of 2 M sodium acetate and 1 mL 0.5% PDAB solution, 1 mL sample was added and the system was allowed to react at room temperature for 3 min. The absorbance at 415 nm was recorded and the content of 6-APA was calculated according to the standard curve. One unit of enzyme activity (IU) was defined as the amount of enzyme required to produce 1 μmol 6-APA per min under the assay conditions. The specific activity of enzyme is the amount of enzyme activity units contained per milligram enzyme. The apparent activity of ImPGA is the amount of enzyme activity units contained by the ImPGA on 20 mg of carriers. The reusability of ImPGA was examined by measuring the enzyme activity in consecutive operations. The vials containing 15 mL of 2% (w/t) penicillin G potassium solutions in 0.1 M pH 9.0 phosphate buffer were preheated to 37 °C, and 20 mg of ImPGA on different supports was then added. After reaction at 37 °C for exactly 5 min, the production of 6-APA was analyzed using the same method mentioned above. After one batch, the ImPGA was separated using a magnet, and washed once with 0.1 M pH 9.0 phosphate buffer. The substrate solution was added to start the next operation, under the same reaction conditions. Here, the activity is expressed in relative units (%), where the initial activity of ImPGA was set at 100%. 2.6. Kinetic Studies. To perform pseudo-second-order kinetic assays, a series of vials containing 0.05 g of dry carriers and 12 mL of diluted PGA solutions pH 9.0 (3.0 mg protein/ ml) were incubated at 37 °C and 180 rpm for 15−420 min. At time t, one of the vials was taken out and the amount of protein loading qt was measured. The pseudo-second-order model was expressed in the following linear form:55

element content (%) ionic liquids IL-Cl− IL-BF4− IL-PF6− IL-Tf2N−

Cexp/Ctheo 48.13 41.56 35.93 27.36

± ± ± ±

0.04/48.37 0.22/41.72 0.07/36.11 0.07/27.50

Hexp/Htheo 8.42 7.30 6.29 4.79

± ± ± ±

0.08/8.37 0.14/7.22 0.08/6.25 0.03/4.76

Nexp/Ntheo 9.39 8.02 7.01 5.27

± ± ± ±

0.09/8.68 0.08/7.49 0.09/6.48 0.03/4.94

a

The subscript exp denotes experimental values, while theo denotes theoretical values. These data were the average values calculated from three repeated measurements. The data after the symbol “±” were the standard deviation of three times’ determined results.

contents of the four kinds of ILs were very close to their theoretical values, while the N contents were slightly higher than the theoretical values, which might have been caused by trace amounts of unreacted N-methylimidazole. No further purification was conducted, because N-methylimidazole cannot be immobilized on carriers, thus has no effect on the properties of carriers. The as-prepared nanoparticles were characterized by FT-IR (Figure 1). The strong band at 580 cm−1 corresponded to Fe−

Figure 1. FT-IR spectra for (1) Fe3O4 nanoparticles, (2) Fe3O4/SiO2, (3) ionic liquids ([C1C(S)Im]Cl−), and (4) Fe3O4/SiO2−ILs ([C1C(S)Im]Cl−).

O vibrations for the naked Fe3O4, and the bands at 3425 and 1632 cm−1 represented the stretching and bending vibrations of Fe−OH, respectively.46 For Fe3O4/SiO2, the weak band at 800 cm−1 was characteristic of Si−O−Fe, which implied that SiO2 was chemically bonded with Fe3O4.56 The band at 1100 cm−1 represented Si−O bonds. For Fe3O4/SiO2−ILs, the weak peaks at 2972, 2930, and 2871 cm−1 were the stretching vibrations of −CH3 and −CH2− in ILs, respectively; those at 1462 cm−1 and 1379 cm−1 corresponded to their bending vibrations.46 These results confirmed that Fe3O4/SiO2 had been successfully modified with ILs through covalent bonds. The size and morphology of the resultant nanoparticles were characterized using TEM (Figure 2). It can be seen that the

t 1 1 = + t 2 qt q k2qe e

where qt and qe are the amounts of PGA absorbed at time t and at equilibrium time (mg/g), respectively, and k2 is the rate constant of the pseudo-second-order adsorption (g/(mg·min)). qe and k2 were calculated from the slope and intercept of plots of t/qt vs t. 4584



Article

Figure 3. VSM for (1) magnetic Fe3O4 nanoparticles, (2) Fe3O4/SiO2, (3) Fe3O4/SiO2−ILs ([C1C(S)Im]Cl−), and (4) ImPGA on Fe3O4/ SiO2−ILs (dry). The inset illustrates the magnetic separation of ImPGA on Fe3O4/SiO2−ILs from the reaction system under the application of an external magnetic field. The time from state (A) to state (B) was less than 1 min.

Tf2N−) showed higher protein loading than that on supports modified using hydrophilic ILs (anions as Cl− and BF4−), but their specific activity was relative low. It is likely that this was related to the hydrophilicity/hydrophobicity properties of the ILs. The hydrophobicity of ILs with the same cations often follows the following order: Tf2N− > PF6− > BF4− > Cl−.57 The hydrophobic IL layers may interact with the hydrophobic parts on the surface of the PGA molecules, and thus increase the PGA loading.58 However, excessive hydrophobicity is adverse to the activity of PGA, because PGA is inherently a hydrophilic protein.59 Therefore, with the increasing hydrophobicity of ILs, the specific activity of ImPGA decreased. The higher specific activity of ImPGA on Fe3O4/SiO2−IL (Cl− and BF4−) among the supports modified using ILs may be attributed to the structure features of the ILs. The cations with short carbon chain (−CH3) and the anions (Cl− and BF4−) made the IL moderately hydrophilic, which facilitated the PGA molecules interacting with the support surface at a stereo confirmation beneficial for catalysis. In Shi’s study,12 the PGA-adsorption performance of NH2−-functionalized Fe3O4/SiO2 for PGA was investigated, and the results indicated that the specific activity of ImPGA was 32.9 IU/mg protein, and the immobilization yield was about 2%. NH2−-functionalized molecular sieves SBA15 were used as supports to physically immobilize PGA in another study;2 the protein loading was 0.5−0.7 mg/20 mg of carriers, and specific activity was 0.5−1.8 IU/mg protein. Larger improvements in protein loading, specific activity, and immobilization yield have therefore been achieved for ImPGA on IL-modified Fe3O4/SiO2 than for other supports. The stability of PGA on different supports was evaluated by rinsing ImPGA with a phosphate buffer solution containing Triton X-100 at a final concentration of 0.5%, then measuring the activity retention (AR). It has been reported that the enzyme molecules immobilized by weak forces or by enzyme− enzyme interactions could be desorbed from carriers by Triton X-100 at a certain concentration.46,60 After the support was modified with ILs, 77.0−95.8% of the initial activity of ImPGA was retained; this was significantly higher than the values for the ImPGA on supports with no IL modification. IL-modified Fe3O4/SiO2 showed some advantages over the previously reported Fe3O4/SiO2−NH2 and SBA-15−NH2, in

Figure 2. TEM images of different magnetic particles, and their size distributions: (A) TEM of naked Fe3O4 nanoparticles, (B) size distribution of Fe3O4, (C) TEM of Fe3O4/SiO2, (D) size distribution of Fe3O4/SiO2, (E) TEM of Fe3O4/SiO2−ILs ([C1C(S)Im]Cl−). The inset in (C) shows the typical core−shell-structure Fe3O4/SiO2, with a scale bar of 20 nm.

average particle diameters of Fe3O4 and Fe3O4/SiO2 were approximately 10 and 210 nm, respectively. From Figure 2C, it is evident that typical core−shell-structure Fe3O4/SiO2 was formed. The particles modified with ILs (IL-Cl−) (Figure 2E) showed slightly more aggregation than Fe3O4/SiO2, which might have been due to the interaction with the IL layer surrounding the particles’ surface.46 The magnetic properties of magnetic materials are very important for separation and recovery. To measure the magnetic properties of the obtained materials, vibrating sample magnetometer (VSM) measurements were performed (Figure 3). It can be seen that no hysteresis was present in the magnetization, with both remanence and coercivity being zero; this indicated that the magnetic particles were superparamagnetic. The naked Fe3O4 particles showed the largest saturation magnetization (Ms) of 63.712 emu/g. The Ms values for Fe3O4/SiO2, Fe3O4/SiO2−ILs, and ImPGA (dry) were 27.467, 26.752, and 25.354 emu/g, respectively. It can also be seen that the support could be easily recycled via the use of an external magnetic field. 3.2. Immobilization of PGA. The protein loading, specific activity, immobilization yield, and the stability of the enzyme on the supports are the most important characteristics of immobilized enzymes. These four parameters were investigated for PGA immobilized on different supports (Table 2). The supports detailed in Items 1−5 were prepared in this work; previously reported results for supports modified by other methods are also listed to give a comparison. As shown in Table 2, the ImPGA immobilized on Fe3O4/ SiO2 modified using hydrophobic ILs (anions as PF6− and 4585



Article

Table 2. Properties of Different Magnetic Carriers and Their Performance in PGA Immobilizationa item

supports

1 2 3 4 5 6 7 8

Fe3O4/SiO2 Fe3O4/SiO2−IL (Cl−) Fe3O4/SiO2−IL (BF4−) Fe3O4/SiO2−IL (PF6−) Fe3O4/SiO2−IL (Tf2N−) Fe3O4/SiO2−CHO Fe3O4/SiO2−NH212 SBA-15-NH22

Q (mg/20 mg carriers)

SA (IU/mg protein)

± ± ± ± ± ±

21.5 ± 0.4 17.3 ± 0.6 17.6 ± 0.4 9.8 ± 0.3 8.6 ± 0.3 8.1 ± 0.5 32.9 0.5−1.8

1.8 0.6 0.8 1.2 1.5 1.5

0.3 0.1 0.1 0.2 0.2 0.3

25−35

A (IU/20 mg carriers)

IMY (%)

AR (%)

± ± ± ± ± ±

34.8 ± 1.5 11.1 ± 0.4 15 ± 0.4 23.6 ± 0.8 28.5 ± 1.0 29.9 ± 1.2 2.04

74.3 ± 1.1 77 ± 0.9 78 ± 0.9 94.3 ± 1.1 95.8 ± 1.6 97 ± 2.0

38.7 10.4 14.1 11.8 12.9 12.5

0.4 0.8 0.3 0.7 0.7 0.9

a

These data are average values, calculated from three measurements. Q, protein loading; SA, specific activity; A, apparent activity; IMY, immobilization yield; AR, activity retention after washing with 0.5% Triton X-100. The second data after the symbol “±” in Items 1−6 were the standard deviations calculated from three times’ measurements. The data in Items 6 and 7 are from related literature; a blank means that no relevant results were given in the literature.

maximal activities even at pH 11 (6.5 and 4.8 IU/(20 mg carriers) for ImPGA on Fe3O4/SiO2−IL (BF4−) and Fe3O4/ SiO2−ILs (Tf2N−)). This result could likely be attributed to the stabilization effects of immobilization; PGA may have been immobilized via multipoint interactions with the carriers. It is well-known that the immobilization of enzymes on certain supports can cause significant changes in the catalytic behavior of the enzyme, and the optimal pH of immobilized enzymes shifts to a more acidic or basic pH, depending on the surface properties of carriers, and the interactions between enzymes and carriers.62 3.5. Effect of Temperature on the Activity of ImPGA. Temperature is an important factor that can affect the activity of biocatalysts. The effect of reaction temperature (20−80 °C) on the activity of native and immobilized PGA is illustrated in Figure 5. The activity of both native and immobilized PGA

terms of the protein loading, specific activity, and immobilization yield. The Fe3O4/SiO2 supports without any modification exhibited the same immobilization effects as Fe3O4/SiO2 modified with ILs (IL-Cl− or BF4−), but recycling experiments (detailed later in Section 3.7) showed that ImPGA on Fe3O4/ SiO2 had poor reusability. Accordingly, it can be concluded from Table 2 that hydrophilic IL-modified (IL-Cl− and ILBF4−) Fe3O4/SiO2 is a promising support for the immobilization of PGA via physical adsorption. 3.3. Effect of pH on the Activity of ImPGA. The effect of pH on the activity of the native and immobilized PGA was assayed under pH 4−11 at 37 °C (Figure 4). The activity is

Figure 4. Effect of pH on activity of native PGA and immobilized PGA. At the 100% point, the apparent activity of ImPGA on Fe3O4/ SiO2, Fe3O4/SiO2−IL (BF4−), and Fe3O4/SiO2−ILs (Tf2N−) was 34.2, 10.8, and 8.0 IU/(20 mg carriers), respectively, and the apparent activity of native PGA was 763.4 IU/(mL enzyme solution). The error bar was the standard deviation of three times’ determined results.

Figure 5. Effect of temperature on activities of native PGA and immobilized PGA. At the 100% point, the apparent activity of ImPGA on Fe3O4/SiO2, Fe3O4/SiO2−IL (BF4−), and Fe3O4/SiO2−ILs (Tf2N−) was 32.8, 12.2, and 9.5 IU/(20 mg carriers), respectively, and the apparent activity of native PGA was 759.6 IU/(mL enzyme solution). The error bar was the standard deviation of three times’ determined results.

expressed in relative units [%], where the maximal activity value at a certain temperature is set as 100% and activities at other temperatures are expressed as a ratio with maximal activity value. In general, immobilization should result in a shift in the optimal pH of the enzyme.61 Immobilization of PGA on Fe3O4/SiO2−ILs shifted the optimal pH of PGA from 9 to 7, comparing with native PGA. The native PGA possesses an optimal pH of 9, while it shifted to 8 and 7 for ImPGA on Fe3O4/SiO2 and Fe3O4/SiO2−ILs, respectively (Figure 4). ImPGA on Fe3O4/SiO2−ILs performed well within a broader pH range, and showed a higher resistance to high-pH conditions than native PGA and ImPGA on Fe3O4/SiO2. ImPGA on Fe3O4/SiO2−ILs retained approximately 60% of its

increased with increasing temperature in the low temperature range. At high temperatures, enzyme activity decreased sharply. However, ImPGA on different IL-modified Fe3O4/SiO2 showed different responses to temperature. The optimal temperature for ImPGA on Fe3O4/SiO2−ILs (Tf2N−) was the same as for native PGA and ImPGA on Fe3O4/SiO2 (50 °C), while it shifted to 45 °C for Fe3O4/SiO2−IL (BF4−). The resistance of ImPGA on Fe3O4/SiO2−ILs (Tf2N−) to high temperatures was 4586



Article

different supports follow the order Fe3O4/SiO2 < Fe3O4/SiO2− IL (Cl−) < Fe3O4/SiO2−IL (Tf2N−), which meant that hydrophilic and hydrophobic ILs had different effects on the adsorption rate between the PGA and the supports. The results suggested that the hydrophobic ILs might have accelerated the adsorption process of PGA onto the supports. The predicted values qe(theo) for the maximal loading of PGA on Fe3O4/SiO2−IL (Cl−) were very close to the experimental values qe(exp). The differences between the two values for Fe3O4/SiO2 and Fe3O4/SiO2−IL (Tf2N−) may have been caused by experimental errors. 3.7. Reusability of ImPGA. To evaluate the effects of the ILs on the stability of ImPGA on the supports, the reusability of ImPGA on Fe3O4/SiO2−IL (BF4−) was investigated. The reusability of ImPGA on Fe3O4/SiO2 without IL modification is also presented, to give a comparison. The results are shown in Figure 7. ImPGA on Fe3O4/SiO2−IL (BF4−) maintained 70%

better than that of the native enzyme and ImPGA on Fe3O4/ SiO2. This result was different from Shi’s report,12 in which the optimal temperature for both native and immobilized PGA was found to be 45 °C. A possible explanation for this is that different IL layers on the surface had different effects on the conformation of PGA, leading to different enzyme behaviors. Meanwhile, the hydrophobicity of ILs may have played an important role in changing thermo-sensitivity of ImPGA. 3.6. Kinetic Assay of PGA Adsorption on Carriers. To evaluate the effect of the IL layers on the interaction between the enzyme and the supports, the kinetics of PGA immobilization were analyzed using the pseudo-second-order model proposed by Ho and McKay.63 This model is commonly used to describe the kinetics of enzyme adsorption onto support surfaces.64,65 As described in Section 2.6, the plot would give a linear relationship if the pseudo-second-order kinetics are applicable. In PGA immobilization, the k2 value in the kinetic formula represents the absorption rate constant for PGA absorbed onto the carriers. From the qe value, the amount of PGA absorbed at an equilibrium time qe can be predicted theoretically. The results of the kinetic assay are illustrated in Figure 6. The parameters, qe, k2, and R2 were obtained using linear

Figure 7. Reusability of PGA immobilized on Fe3O4/SiO2 and Fe3O4/ SiO2−IL (BF4−). In the first use, the apparent activity of ImPGA PGA on Fe3O4/SiO2 and Fe3O4/SiO2−IL (BF4−) was 39.5 and 14.1 IU/(20 mg carriers), respectively. The error bar was the standard deviation of three times’ determined results.

of its initial activity after 9 consecutive operations under the experimental conditions; this remaining activity was 1.8 times higher than that shown by ImPGA on Fe3O4/SiO2, for which only 40% of the activity was retained. Shah et al.2 immobilized PGA via physical adsorption, using NH2-modified molecular sieves, and studied the reusability of ImPGA. It was found that approximately 43% of the initial activity remained after 9 operations. The residual activity was found to be 77% in Shi’s work,12 where PGA was immobilized using CHO-functionalized silica microspheres, via covalent attachment. In conclusion, the reusability of ImPGA on Fe3O4/SiO2−IL (BF4−) was better than that on NH2-functionalized supports, and was close to the levels shown by covalently immobilized PGA. These results confirmed that IL-BF4− enhanced the stability of PGA on supports, and that it may have potential applications in protein immobilization.

Figure 6. Pseudo-second-order adsorption kinetic plots of PGA onto magnetic carriers with different IL modification. The error bar was the standard deviation of four times’ determined results.

Table 3. Pseudo-Second-Order Reaction Kinetic Constants for PGA Adsorption on Different Supports support Fe3O4/SiO2 Fe3O4/SiO2−IL (Cl−) Fe3O4/SiO2−IL (Tf2N−)

qe(theo)a (mg/g)

k2

qe(exp)b (mg/g)

R2

333.3 212.8

1.75 × 10−4 3.22 × 10−4

295.6 206.4

0.9979 0.9994

285.7

9.42 × 10−3

314.9

0.9988

a

qe(theo) is the PGA loading, calculated using the results of the kinetic assay. bqe(exp) is the PGA loading taken from experimental results.

4. CONCLUSIONS Functional ionic liquids were used to modify the surface of Fe3O4/SiO2, with the aim of improving the stability of PGA physically adsorbed on supports. The main findings can be summarized as follows: 1. The hydrophilic and hydrophobic ionic liquids had different effects on the protein loading and specific activity of ImGA in the immobilizing process. The

regression, and are reported in Table 3. The results showed that the adsorption followed the Ho and McKay equation, as R2 was close to 1. The kinetics of PGA adsorption on the three kinds of magnetic carriers fit the pseudo-second-order model, suggesting that the adsorption mechanism depended on not only the properties of the adsorbate, but also the surface structures of the adsorbent.66 From Table 3, the k2 values of 4587



■

Article

hydrophobic ionic liquids (IL-PF6− and Tf2N−) increased the amount of protein loading on the support, while the hydrophilic ionic liquids (IL-Cl− and BF4−) were in favor of maintaining the specific activity after immobilization. In terms of the most important parameters for immobilized enzymesi.e., the protein loading, specific activity, immobilization yield, and the stability of the enzyme on the surface of the supportsthe hydrophilic ILs (IL-Cl− and IL-BF 4−) were superior to the hydrophobic ones (IL-PF 6 −and Tf2 N −) for PGA immobilization. 2. Ionic liquids, especially hydrophobic ionic liquids, were able to speed up the adsorption of PGA onto the surface of Fe3O4/SiO2, comparing with support with no ionic liquid modification. 3. The reusability of ImPGA was improved after the modification of Fe3O4/SiO2 with IL-BF4−. The ImPGA on Fe3O4/SiO2−IL(BF4−) maintained 70% of its initial activity (9.9 IU/(20 mg carrier)) after 9 consecutive operations under the experimental conditions; this residual activity was 1.8 times higher than that on Fe3O4/SiO2 (40%) with no ionic liquid modification. This good performance of ImPGA on Fe3O4/SiO2− IL(BF4−) might have been due to the suitable hydrophilicity of IL-BF4−, and thus produced moderate interaction between the enzyme molecules and the support surface. The improved reusability makes the Fe3O4/SiO2−IL(BF4−) ideal for potential applications in enzyme immobilization.

(3) Norouzian, D.; Hosseinzadeh, A.; Inanlou, D.; Moazami, N. Various Techniques Used to Immobilize Naringinase Produced by Penicillium Decombens PTCC 5248. World J. Microbiol. Biotechnol. 1999, 15, 501. (4) Kotha, A.; Selvaraj, L.; Rajan, C. R.; Ponrathnam, S.; Kumar, K. K.; Ambekar, G. R.; Shewale, J. G. Adsorption and Expression of Penicillin G Acylase Immobilized onto Methacrylate Polymers Generated with Varying Pore Generating Solvent Volume. Appl. Biochem. Biotechnol. 1991, 30, 297. (5) Xiao, Q. G.; Tao, X.; Chen, J. F. Silica Nanotubes Based on Needle-Like Calcium Carbonate: Fabrication and Immobilization for Glucose Oxidase. Ind. Eng. Chem. Res. 2007, 46, 459. (6) He, J.; Li, X. F.; Evans, D. G.; Duan, X.; Li, C. Y. A New Support for the Immobilization of Penicillin Acylase. J. Mol. Catal., B 2000, 11, 45. (7) Wilson, L.; Illanes, A.; Pessela, B. C. C.; Abian, O.; FernándezLafuente, R.; Guisán, J. M. Encapsulation of Crosslinked Penicillin G Acylase Aggregates in Lentikats: Evaluation of a Novel Biocatalyst in Organic Media. Biotechnol. Bioeng. 2004, 86, 558. (8) Bernardino, S.; Estrela, N.; Ochoa-Mendes, V.; Fernandes, P.; Fonseca, L. P. Optimization in the Immobilization of Penicillin G Acylase by Entrapment in Xerogel Particles with Magnetic Properties. J. Sol-Gel Sci. Technol. 2011, 58, 545. (9) Mahmood, I.; Guo, C.; Xia, H. S.; Ma, J. H.; Jiang, Y. Y.; Liu, H. Z. Lipase Immobilization on Oleic Acid-Pluronic (L-64) Block Copolymer Coated Magnetic Nanoparticles, for Hydrolysis at the Oil/Water Interface. Ind. Eng. Chem. Res. 2008, 47, 6379. (10) Cao, L. Q.; Rantwijk, F.v.; Sheldon, R. A. Cross-Linked Enzyme Aggregates: A Simple and Effective Method for the Immobilization of Penicillin Acylase. Org. Lett. 2000, 2, 1361. (11) Brady, D.; Jordaan, J. Advances in Enzyme Immobilisation. Biotechnol. Lett. 2009, 31, 1639. (12) Shi, B. F.; Wang, Y. Q.; Ren, J. W.; Liu, X. H.; Zhang, Y.; Guo, Y. L.; Guo, Y.; Lu, G. Z. Superparamagnetic AminopropylFunctionalized Silica Core-Shell Microspheres as Magnetically Separable Carriers for Immobilization of Penicillin G Acylase. J. Mol. Catal., B 2010, 63, 50. (13) Danial, E. N.; Elnashar, M. M. M.; Awad, G. E. A. Immobilized Inulinase on Grafted Alginate Beads Prepared by the One-Step and the Two-Steps Methods. Ind. Eng. Chem. Res. 2010, 49, 3120. (14) Wang, W.; Deng, L.; Peng, Z. H.; Xiao, X. Study of the Epoxydized Magnetic Hydroxyl Particles as a Carrier for Immobilizing Penicillin G Acylase. Enzyme Microb. Technol. 2007, 40, 255. (15) Grazú, V.; Abian, O.; Mateo, C.; Batista-Viera, F.; FernándezLafuente, R.; Guisán, J. M. Stabilization of Enzymes by Multipoint Immobilization of Thiolated Proteins on New Epoxy-Thiol Supports. Biotechnol. Bioeng. 2005, 90, 597. (16) Elnashar, M. M. M.; Danial, E. N.; Awad, G. E. A. Novel Carrier of Grated Alginate for Covalent Immobilization of Inulinase. Ind. Eng. Chem. Res. 2009, 48, 9781. (17) Elnashar, M. M. M.; Yassin, M. A.; Kahil, T. Novel Thermally and Mechanically Stable Hydrogel for Enzyme Immobilization of Penicillin G Acylase Via Covalent Technique. J. Appl. Polym. Sci. 2008, 109, 4105. (18) Hartmann, M.; Jung, D. Biocatalysis with Enzymes Immobilized on Mesoporous Hosts: The Status Quo and Future Trends. J. Mater. Chem. 2010, 20, 844. (19) Hirsh, S. L.; Bilek, M. M. M.; Nosworthy, N. J.; Kondyurin, A.; dos Remedios, C. G.; McKenzie, D. R. A Comparison of Covalent Immobilization and Physical Adsorption of a Cellulase Enzyme Mixture. Langmuir 2010, 26, 14380. (20) Sousa, H. A.; Rodrignes, C.; Klein, E.; Afonso, C. A. M.; Crespo, J. G. Immobilisation of Pig Liver Esterase in Hollow Fibre Membranes. Enzyme Microb. Technol. 2001, 29, 625. (21) Li, W.; Zhang, J. L.; Zhang, C. X.; Feng, X. Y.; Han, B. X.; Yang, G. Y. Synthesis of α-Chymotrypsin/Polymer Composites by a Reverse Micelle/Gas Antisolvent Method. Colloids Surf., B 2007, 59, 11. (22) Ke, Y.-M.; Chen, C.-I.; Kao, P.-M.; Chen, H.-B.; Huang, H.-C.; Yao, C.-J.; Liu, Y.-C. Preparation of the Immobilized Metal Affinity

ASSOCIATED CONTENT

S Supporting Information *

Detailed experimental procedures of preparation of supports and other relating contents. This information is available free of charge via the Internet at http://pubs.acs.org/.

■

AUTHOR INFORMATION

Corresponding Author

*Tel.: +86-10-82544911. Fax: +86-10-62554264. E-mail: wli@ cnu.edu.cn (W.L.), [email protected] (H.Z.L.). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported financially by National Natural Science Foundation of China (21136009 and 21106162), the Innovative Research Group Science Fund (20221603), the National Technology Research and Development Program of China (2009CB219904), and State Key Laboratory of Chemical Engineering (SKL-ChE-11A04). We thank Hongshuai Gao, Qingfen Liu, and Zhentao An (Institute of Process Engineering, Chinese Academy of Sciences, Beijing, China) for their help in experiments, and are grateful to Professor Xin Hou (College of Life Sciences, Inner Mongolia University, Huhhot, China) for his help with language.

■

REFERENCES

(1) Kallenberg, A. I.; Rantwijk, F.v.; Sheldon, R. A. Immobilization of Penicillin G Acylase: The Key to Optimum Performance. Adv. Synth. Catal. 2005, 347, 905. (2) Shah, P.; Sridevi, N.; Prabhune, A.; Ramaswamy, V. Structural Features of Penicillin Acylase Adsorption on APTES Functionalized SBA-15. Microporous Mesoporous Mater. 2008, 116, 157. 4588



Article

Membrane with High Amount of Metal Ions and Protein Adsorption Efficiencies. Process Biochem. 2010, 45, 500. (23) Gao, B. J.; Wang, X. P.; Shen, Y. L. Studies on Characters of Immobilizing Penicillin G Acylase on a Novel Composite Support Pei/ Sio2. Biochem. Eng. J. 2006, 28, 140. (24) Kang, Y.; He, J.; Guo, X. D.; Guo, X.; Song, Z. H. Influence of Pore Diameters on the Immobilization of Lipase in SBA-15. Ind. Eng. Chem. Res. 2007, 46, 4474. (25) Myakonkaya, O.; Eastoe, J.; Mutch, K. J.; Grillo, I. PolymerInduced Recovery of Nanoparticles from Microemulsions. Phys. Chem. Chem. Phys. 2011, 13, 3059. (26) Hu, S. Q.; Zhang, Z. F.; Zhou, Y. X.; Han, B. X.; Fan, H. L.; Li, W. J.; Song, J. L.; Xie, Y. Conversion of Fructose to 5Hydroxymethylfurfural Using Ionic Liquids Prepared from Renewable Materials. Green Chem. 2008, 10, 1280. (27) Seddon, K. R.; Stark, A.; Torres, M.-J. Influence of Chloride, Water, and Organic Solvents on the Physical Properties of Ionic Liquids. Pure Appl. Chem. 2000, 72, 2275. (28) Amarasekara, A. S.; Wiredu, B. Degradation of Cellulose in Dilute Aqueous Solutions of Acidic Ionic Liquid 1-(1-Propylsulfonic)3-Methylimidazolium Chloride, and P-Toluenesulfonic Acid at Moderate Temperatures and Pressures. Ind. Eng. Chem. Res. 2011, 50, 12276. (29) Amarasekara, A. S.; Owereh, O. S. Hydrolysis and Decomposition of Cellulose in Bronsted Acidic Ionic Liquids under Mild Conditions. Ind. Eng. Chem. Res. 2009, 48, 10152. (30) Liu, J. H.; Cheng, S. Q.; Zhang, J. L.; Feng, X. Y.; Fu, X. G.; Han, B. X. Reverse Micelles in Carbon Dioxide with Ionic-Liquid Domains. Angew. Chem., Int. Ed. 2007, 46, 3313. (31) Xia, M.; Lu, Y.o. A Novel Neutral Ionic Liquid-Catalyzed Solvent-Free Synthesis of 2,4,5-Trisubstituted Imidazoles under Microwave Irradiation. J. Mol. Catal., A 2007, 265, 205. (32) Li, Z. H.; Liu, Z. M.; Zhang, J. L.; Han, B. X.; Du, J. M.; Gao, Y.n.; Jiang, T. Synthesis of Single-Crystal Gold Nanosheets of Large Size in Ionic Liquids. J. Phys. Chem. B 2005, 109, 14445. (33) Baltus, R. E.; Culbertson, B. H.; Dai, S.; Luo, H.; DePaoli, D. W. Low-Pressure Solubility of Carbon Dioxide in Room-Temperature Ionic Liquids Measured with a Quartz Crystal Microbalance. J. Phys. Chem. B 2004, 108, 721. (34) Zhang, S. J.; Chen, Y. H.; Li, F. W.; Lu, X. M.; Dai, W. B.; Mori, R. Fixation and Conversion of CO2 Using Ionic Liquids. Catal. Today 2006, 115, 61. (35) Harper, N. D.; Nizio, K. D.; Hendsbee, A. D.; Masuda, J. D.; Robertson, K. N.; Murphy, L. J.; Johnson, M. B.; Pye, C. C.; Clyburne, J. A. C. Survey of Carbon Dioxide Capture in Phosphonium-Based Ionic Liquids and End-Capped Polyethylene Glycol Using DETA (DETA = Diethylenetriamine) as a Model Absorbent. Ind. Eng. Chem. Res. 2011, 50, 2822. (36) Moganty, S. S.; Baltus, R. E. Diffusivity of Carbon Dioxide in Room-Temperature Ionic Liquids. Ind. Eng. Chem. Res. 2010, 49, 9370. (37) Moganty, S. S.; Baltus, R. E. Regular Solution Theory for Low Pressure Carbon Dioxide Solubility in Room Temperature Ionic Liquids: Ionic Liquid Solubility Parameter from Activation Energy of Viscosity. Ind. Eng. Chem. Res. 2010, 49, 5846. (38) Dai, S.; Ju, Y. H.; Barnes, C. E. Solvent Extraction of Strontium Nitrate by a Crown Ether Using Room-Temperature Ionic Liquids. J. Chem. Soc., Dalton Trans. 1999, 8, 1201. (39) Belluomini, G. J.; Pendergast, J. G.; Domke, C. H.; Ussing, B. R. Performance of Several Ionic Liquids for the Separation of 1-Octene from N-Octane. Ind. Eng. Chem. Res. 2009, 48, 11168. (40) Sherren, C. N.; Mu, C.; Webb, M. I.; McKenzie, I.; McCollum, B. M.; Brodovitch, J.-C.; Percival, P. W.; Storr, T.; Seddon, K. R.; Clyburne, J. A. C.; Walsby, C. J. Merging the Chemistry of ElectronRich Olefins with Imidazolium Ionic Liquids: Radicals and HydrogenAtom Adducts. Chem. Sci. 2011, 2, 2173. (41) Cole, A. C.; Jensen, J. L.; Ntai, I.; Tran, K. L. T.; Weaver, K. J.; Forbes, D. C.; Davis, J. H. Jr. Novel Brønsted Acidic Ionic Liquids and Their Use as Dual Solvent-Catalysts. J. Am. Chem. Soc. 2002, 124, 5962.

(42) Zhao, G. Y.; Jiang, T.; Gao, H. X.; Han, B. X.; Huang, J.; Sun, D. H. Mannich Reaction Using Acidic Ionic Liquids as Catalysts and Solvents. Green Chem. 2004, 6, 75. (43) Noritomi, H.; Suzuki, K.; Kikuta, M.; Kato, S. Catalytic Activity of α-Chymotrypsin in Enzymatic Peptide Synthesis in Ionic Liquids. Biochem. Eng. J. 2009, 47, 27. (44) Hernández-Fernández, F. J.; Ríos, A.P.d.l.; Tomás-Alonso, F.; Gómez, D.; Víllora, G. Stability of Hydrolase Enzymes in Ionic Liquids. Can. J. Chem. Eng. 2009, 87, 910. (45) Lau, R. M.; Rantwijk, F.v.; Seddon, K. R.; Sheldon, R. A. LipaseCatalyzed Reactions in Ionic Liquids. Org. Lett. 2000, 2, 4189. (46) Jiang, Y. Y.; Guo, C.; Xia, H. S.; Mahmood, I.; Liu, C. Z.; Liu, H. Z. Magnetic Nanoparticles Supported Ionic Liquids for Lipase Immobilization Enzyme Activity in Catalyzing Esterification. J. Mol. Catal., B 2009, 58, 103. (47) Liu, G.; Guan, Y. P.; Ge, Y.; Xie, L. Preparation of Monodisperse Magnetic Polystyrene Microspheres and Its Surface Chemical Modification. J. Appl. Polym. Sci. 2011, 120, 3278. (48) Wang, Q.; Guan, Y. P.; Ren, X. F.; Cha, G. J.; Yang, M. Z. Rapid Extraction of Low Concentration Heavy Metal Ions by Magnetic Fluids in High Gradient Magnetic Separator. Sep. Purif. Technol. 2011, 82, 185. (49) Elnashar, M. M. M. Review Article: Immobilized Molecules Using Biomaterials and Nanobiotechnology. J. Biomater. Nanobiotechnol. 2010, 1, 61. (50) Valkenberg, M. H.; deCastro, C.; Holderich, W. F. Immobilisation of Chloroaluminate Ionic Liquids on Silica Materials. Top. Catal. 2001, 14, 139. (51) Mehnert, C. P.; Cook, R. A.; Dispenziere, N. C.; Afework, M. Supported Ionic Liquid Catalysis-A New Concept for Homogeneous Hydroformylation Catalysis. J. Am. Chem. Soc. 2002, 124, 12932. (52) Shi, B. F.; Wang, Y. S.; Guo, Y. L.; Wang, Y. Q.; Wang, Y.; Guo, Y.; Zhang, Z. G.; Liu, X. H.; Lu, G. Z. Aminopropyl-Functionalized Silicas Synthesized by W/O Microemulsion for Immobilization of Penicillin G Acylase. Catal. Today 2009, 148, 184. (53) Bradford, M. M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 1976, 72, 248. (54) Shewale, J. G.; Kumar, K. K.; Ambekar, G. R. Evaluation of Determination of 6-Aminopenicillanic Acid by P-Dimethlaminobenzaldehyde. Biotechnol. Tech. 1987, 1, 69. (55) Crini, G. Kinetic and Equilibrium Studies on the Removal of Cationic Dyes from Aqueous Solution by Adsorption onto a Cyclodextrin Polymer. Dyes Pigm. 2008, 77, 415. (56) Haddad, P. S.; Duarte, E. L.; Baptista, M. S.; Goya, G. F.; Leite, C. A. P.; Itri, R. Synthesis and Characterization of Silica-Coated Magnetic Nanoparticles. Prog. Colloid Polym. Sci. 2004, 128, 232. (57) Huddleston, J. G.; Visser, A. E.; Reichert, W. M.; Willauer, H. D.; Broker, G. A.; Rogers, R. D. Characterization and Comparison of Hydrophilic and Hydrophobic Room Temperature Ionic Liquids Incorporating the Imidazolium Cation. Green Chem. 2001, 3, 156. (58) Ríos, A.P.d.l.; Hernández-Fernández, F. J.; Rubio, M.; Gómez, D.; Víllora, G. Stabilization of Native Penicillin G Acylase by Ionic Liquids. J. Chem. Technol. Biotechnol. 2007, 82, 190. (59) Skrob, F.; Becka, S.; Plhackova, K.; Fotopulosova, V.; Kyslik, P. Novel Penicillin G Acylase from Achromobacter sp. CCM 4824. Enzyme Microb. Technol. 2003, 32, 738. (60) Palomo, J. M.; Orziz, C.; Fernandez-Lorente, G.; Fuentes, M.; Guisan, J. M.; Fernandez-Lafuente, R. Lipase-Lipase Interactions as a New Tool to Immobilize and Modulate the Lipase Properties. Enzyme Microb. Technol. 2005, 36, 447. (61) Reshmi, R.; Sanjay, G.; Sugunan, S. Enhanced Activity and Stability of α-Amylase Immobilized on Alumina. Catal. Commun. 2006, 7, 460. (62) Arslan, A.; Kiralp, S.; Toppare, L.; Bozkurt, A. Novel Conducting Polymer Electrolyte Biosensor Based on Poly (1-Vinyl Imidazole) and Poly (Acrylic Acid) Networks. Langmuir 2006, 22, 2912. 4589



Article

(63) Ho, Y. S.; McKay, G.; Wase, D. A. J.; Forster, C. F. Study of the Sorption of Divalent Metal Ions on to Peat. Adsorpt. Sci. Technol. 2000, 18, 639. (64) Daoud, F. B. O.; Kaddour, S.; Sadoun, T. Adsorption of Cellulase Aspergillus niger on a Commercial Activated Carbon: Kinetics and Equilibrium Studies. Colloids Surf., B 2010, 75, 93. (65) Bayramolu, G.; Yilmaz, M.; Arica, M. Y. Affinity Dye-Ligand Poly (Hydroxyethyl Methacrylate)/Chitosan Composite Membrane for Adsorption Lysozyme and Kinetic Properties. Biochem. Eng. J. 2003, 13, 35. (66) Iram, M.; Guo, C.; Guan, Y. P.; Ishfaq, A.; Liu, H. Z. Adsorption and Magnetic Removal of Neutral Red Dye from Aqueous Solution Using Fe3O4 Hollow Nanospheres. J. Hazard. Mater. 2010, 181, 1039.

4590


Improving the Stability of Immobilized Penicillin G Acylase via the

Recommend Documents