Porcine pancreatic Lipase Immobilized in Amino-Functionalized Short

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Porcine pancreatic Lipase Immobilized in Amino-Functionalized Short Rod-Shaped Mesoporous Silica Prepared Using Poly(ethylene glycol) and Triblock Copolymer as Templates Chunfeng Wang,†,‡ Guowei Zhou,*,†,§ Yunqiang Xu,†,§ and Jing Chen†,§ †

Shandong Provincial Key Laboratory of Fine Chemicals, ‡School of Material Science and Engineering, and §School of Chemistry, Shandong Polytechnic University, Jinan 250353, Shandong, People’s Republic of China ABSTRACT: Mesoporous materials have large ratio surface areas, an average pore diameter, and ordered pore arrangement, showing promising application in the fields of catalysis, adsorption, separation, and functional materials. In this study, short rodshaped mesoporous silica (SRSMS) were prepared using poly(ethylene glycol) (PEG) and P123 as dual templates and functionalized with (3-aminopropyl)triethoxysilane (APTES) by a postsynthesis-grafting method. The synthesis conditions, including PEG chain length and PEG concentration, when the dual templates were added to the reaction system are discussed. The results show that the d100 increased continuously with the increase of PEG concentration and molecular weights (MW). When PEG with different MW was added, the pore size of SRSMS changed a little, while it increased with increasing PEG content. Pore volumes and surface areas of SRSMS decreased, while the two-dimensional hexagonal p6mm mesoscopic structure remained after functionalization. Porcine pancreatic lipase (PPL) was used as a model enzyme for studying the effect of amino-functionalization on loading amount and enzymatic activity. As a result, functionalized SRSMS immobilized PPL showed the better reusability, higher catalytic activity, and thermal stability.

1. INTRODUCTION Ordered mesoporous materials were achieved by supramolecular-templating methods with surfactant as template for framework porosities, in which their structural properties can be finely controlled by experimental conditions.1,2 Among various surfactants, polyethylene glycol (PEG) has been the most frequently used due to its ease of cross-linking into hydrogels and of controlling its molecular weight.35 However, only a limited number of investigations have prepared ordered mesoporous silica using PEG as template. Recently, polymersurfactant complexes have been used as templates to synthesize mesoporous materials because their synergic structure is totally different from that of pure polymers or pure surfactant systems.610 Both surfactant and polymer can prevent direct precipitation and aggregation as stabilizers for crystals. Moreover, they can be advantageous for maintaining the structural stability of the template, which plays an important role in the uniformity and polydispersity of materials.11 For example, association of PEG with the polymer could facilitate the formation of spherical and monolithic micelles.1113 Yang et al.11 prepared mesoporous silica spheres with a high yield and uniform morphology through water-in-oil emulsion with mixtures of PEG and sodium dodecylsulphate (SDS) as templates. Sun et al.12 synthesized monolithic silica materials with bimodal pore structures using a new dual-templating system of PEG and P123, and they introduced a subcritical water oxidation method to remove the organic templates. Although the dual-template method has r 2011 American Chemical Society

been displayed to have many advantages in the synthesis of mesoporous materials, it has not been used to prepare short rodshaped ordered mesoprous silica. Lipases exhibit high selectivity and specificity under very mild and environmentally friendly conditions but are sensitive to denaturation or inactivation by extremes of pH and temperature because it has difficulty maintaining structural stability and biocatalytic efficiency under such extreme conditions. Entrapment of lipase molecules in the mesoporous materials matrix occurs apparently due to the extreme porosity of the mesoporous materials, which has uniform specific areas and pore volumes. Li et al.14,15 applied such materials to immobilize Porcine pancreatic lipase (PPL) and successfully preserved PPL functions under extreme conditions. Zhou et al.16 immobilized lipase from Candida rugosa (CRL) on mesoporous materials and successfully increased CRL applicability. Lipase is an extraordinary enzyme that presents greater affinity to act as the interface of the hydrophobic lipid substrate in a hydrophilic aqueous medium.17 Mesoporous materials with hydrophilic silanol groups would inevitably interfere with substrate accessibility to the immobilized lipase, thus leading to decreased lipase activity. Consequently, hydrophobic aminopropyl groups introduced from further functionalization of mesoporous materials are the Received: July 18, 2011 Revised: September 1, 2011 Published: October 10, 2011 22191

dx.doi.org/10.1021/jp206836v | J. Phys. Chem. C 2011, 115, 22191–22199

The Journal of Physical Chemistry C

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Table 1. Elemental Analysis of SRSMS1 before and after Functionalization elements samples

N (wt %)

C (wt %)

O (wt %)

Si (wt %)

SRSMS1

0.00

0.00

50.73

49.27

NH-SRSMS1

3.07

10.83

43.46

41.89

Figure 2. SAXRD patterns of (a) SRSMS and (b) NH-SRSMS: (i) SRSMS1, (ii) SRSMS2, and (iii) SRSMS3.

Figure 1. EDS results of (a) SRSMS1 and (b) NH-SRSMS1.

preferred preparation carriers for immobilized lipases. Xu et al.18 immobilized lipase on amino-functionalization SBA-15 and proved that PPL immobilized on functionalized SBA-15 has higher loading amount and catalytic activity as compared to PPL immobilized on unfunctionalized SBA-15. On the basis of the hydrophilic characteristic of SRSMS due to the SiOH hydroxyl groups on the surface, we fabricated aminopropyl-functionalization SRSMS to enhance the loading proportion and activity of the immobilized lipase.

2. EXPERIMENTAL SECTION 2.1. Chemicals. Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (P123, EO20PO70EO20), PPL, (3-aminopropyl)triethoxysilane (APTES, 98%), and triacetin (99%, C9H14O6) were purchased from Aldrich. PEG (MW = 6000, 10 000, 20 000, Sinopharm Chemical Reagent Co., Ltd., chemical purity), tetraethoxysilane (TEOS), and other chemicals were of analytical grade and were all obtained from Tianjin Chemical Agent Co. (China). All chemicals were employed without further purification. 2.2. Synthesis of Short Rod-Shaped Mesoporous Silica. Typical synthesis was performed using PEG with MW of 6000, 10 000, and 20 000 and P123 as the structure-directing agents. Molar ratio of TEOS:P123:PEG:HCl:H2O was 1:0.017:(0;

0.0088; 0.0132; 0.0179; 0.022):5.71:192. First, P123 and PEG were dissolved in a mixture of water and 2 M HCl aqueous solution. The resulting solution was stirred at 308 K until the solution became clear. TEOS was added dropwise into the solution under vigorous stirring. The reaction solution was stirred at 308 K for 20 h, followed by transfer into a Teflon-lined autoclave and heating, and then storing at 373 K for 24 h under static conditions. Finally, the white solid products were collected by filtration, washed with water, air-dried at room temperature, and calcined at 773 K in flowing air for 4 h in a tube furnace for removal of the organic template. Mesoporous silica samples were denoted as xSRSMS(y), where x and y represent the PEG/P123 molar ratio and MW of PEG, respectively. Samples 0.512SRSMS(6000), 0.512SRSMS(10 000), and 0.512SRSMS(20 000) were denoted as SRSMS1, SRSMS2, and SRSMS3, respectively. Samples 0.256SRSMS(6000), 0.768SRSMS(6000), 1.025SRSMS(6000), and 1.281SRSMS(6000) were denoted as SRSMS4, SRSMS5, SRSMS6, and SRSMS7, respectively. 2.3. Postsynthesis-Grafting Functionalization of Short Rod-Shaped Mesoporous Silica. To improve the stability of immobilized PPL, APTES was used in the functionalization process.19 1.0 g of SRSMS was suspended in 30 mL of anhydrous toluene, and 2 mmol of APTES was added per gram of SRSMS. The reaction mixture was refluxed for 2 h, and the resulting amino-functionalized material was collected by filtration, washed properly with toluene for removal of unreacted reagent, and dried under vacuum. Samples obtained were designated as NHSRSMSx (x = 17). 2.4. Synthesis of PPL Immobilized in Mesoporous Supports. Phosphate buffer (pH 7.0) was prepared beforehand. First, 40 mg of the support was immersed into 20 mL of phosphate buffer, 22192

dx.doi.org/10.1021/jp206836v |J. Phys. Chem. C 2011, 115, 22191–22199


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Table 2. Structural Properties of Samples Prepared with Different PEG MW d100 (nm)

a0 (nm)

Da (nm)

SBETa (m2 g1)

Va (cm3 g1)

TPb (nm)

SRSMS1

9.27

10.70

6.46

635

0.82

4.24

SRSMS2

9.33

10.77

6.34

660

0.88

4.43

SRSMS3

9.63

11.12

6.32

702

0.92

4.80

NH-SRSMS1

9.53

11.00

6.06

312

0.49

4.94

NH-SRSMS2

9.53

11.00

6.00

304

0.50

5.00

NH-SRSMS3

9.69

11.19

5.91

310

0.50

5.28

SRSMS1-PPL

6.36

384

0.60

NH-SRSMS1-PPL

6.02

220

0.38

samples

a

D, SBET, and V stand for BET pore diameter, surface area, and pore volume, respectively. b Pore wall thickness as TP = a0 D.

and the mixture was sonicated for 10 min until it becomes a visually homogeneous suspension. Thereafter, 40 mg of PPL powder was added and stirred magnetically at 298 K to complete the immobilization of PPL. After 6 h, the supernatant was separated from the solid component by centrifugation for 10 min at 6000 rpm and 277 K. The PPL concentration of the supernatant was measured using UV spectrophotometry at 265 nm and was used for recalculation of the PPL content of the supernatant. A mass balance for lipase solution before and after immobilization was applied to calculate the amount of adsorbed lipase using the Bradford method20 according to formula 1: P¼

Ci Cf V W

ð1Þ

where P is the amount of enzyme bound onto supports (mg g1, mg lipase per gram SRSMS), Ci and Cf are the initial and final enzyme concentrations in the reaction medium (mg mL1), respectively, V is the volume of the reaction medium (mL), and W is the weight of the supports (g). Both SRSMS and NH-SRSMS were used similarly as described above for PPL immobilization. Final samples after enzyme immobilization were designated as SRSMSx-PPL and NH-SRSMSx-PPL (x = 17), respectively. 2.5. Activity Assays. Enzymatic activity of free and immobilized PPL can be measured by titrating with NaOH the acetic acid from triacetin hydrolysis, as mentioned by Kosugi et al.21 In the hydrolysis reaction of triacetin, acetic acid is produced as a byproduct, thus lowering the pH of the hydrolysis mixture. The catalytic activities of free and immobilized PPL were determined by measuring the amount of acetic acid required to maintain a constant pH and the volume of consumed NaOH. 2.5.1. Assay Procedure. Activity assay was performed as reported by Ma et al.22 A triacetin emulsion was prepared by vigorously stirring a mixture of triacetin (1.0 g), deionized water (25 mL), and phosphate buffer (pH 7.0, 12.5 mL) for about 10 min.21 PPL was added once pH was stabilized. The mixture was continuously titrated with 0.02 M NaOH solution for 10 min to maintain a constant pH value. The volume of NaOH solution consumed was recorded, and the activity of PPL was calculated in the standard method. PPL activity unit was denoted as U g1, with one lipase activity unit (1 U) defined as the amount of enzyme required to hydrolyze 1 μmol of triacetin per min at 308 K. The activity of immobilized PPL was assayed by the same method. In addition, blank experiments were performed through the same assay procedure using the same amounts of SRSMS without adding supports. Results showed that the volume of NaOH solution consumed by the blank experiments was eliminated,

Figure 3. (a) N2 adsorptiondesorption isotherms and (b) corresponding BJH pore size distribution curves of samples prepared with different PEG MW.

exhibiting that neither support nor substrate could consume significant amounts of NaOH solution. 2.5.2. Optimum Catalysis Condition of Free and Immobilized PPL. The optimum pH and reaction temperature of free and immobilized PPL were determined as relative activity via the procedure described above. Optimum pH values were compared in different buffers within the pH range of 5.09.0 (0.02 M K2HPO4C6H8O7 buffer solution for pH 5.0, 0.02 M phosphate buffer solution for pH 6.08.0, and 0.02 M Na2CO3NaHCO3 buffer solution for pH 9.0) at 308 K. Thermal inactivation experiments were performed in phosphate buffer (pH 7.0) at the temperatures ranging from 293 to 333 K. Absolute activities of free and immobilized PPL were calculated. 2.5.3. Thermal Stability Study of Free and Immobilized PPL. Measurement of the thermal stability of free and immobilized PPL at high temperature was achieved through an activity test. Immobilized PPL were placed in a cuvette and mixed with phosphate buffer (pH 7.0), followed by capping and incubation at 323 K for the desired time. Free and immobilized PPL were 22193



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Table 3. Structural Properties of Samples Prepared with Different P123/PEG Molar Ratio samples

P123/PEG (molar ratio)

PEG (MW)

d100 (nm)

a0 (nm)

samples

d100 (nm)

a0 (nm)

SRSMS4

1/0.256

6000

9.20

10.62

NH-SRSMS4

9.28

10.72

SRSMS5

1/0.768

6000

9.33

10.77

NH-SRSMS5

9.53

11.00

SRSMS6

1/1.025

6000

9.53

11.00

NH-SRSMS6

9.58

11.06

Figure 5. TEM of (a) SRSMS1, (b) SRSMS5, (c) SRSMS6, and (d) NH-SRSMS1. Scale bar = 100 nm. Figure 4. SEM of (a) SRSMS4, (b) SRSMS1, (c) SRSMS6, and (d) NH-SRSMS1. Scale bar = 2.0 μm.

collected by centrifugation, and their relative activities were calculated. 2.5.4. Reusability of Immobilized PPL. After testing for initial activity, immobilized PPL was removed from the reaction medium by centrifugation and reused by repeating (five times) batch experiments at 308 K. Relative activity of the immobilized PPL was calculated according to formula 2:

of 100 kV. A defined amount of samples powder was dispersed in ethanol by sonication, dropped on a copper grid, and dried in air. Fourier transformed infrared (FT-IR) spectra were collected on a Bruker Tensor 27 spectrometer with resolution and scan number of 4 cm1 and 32, respectively. Samples were prepared using the standard KBr disk method and were measured at 400 4000 cm1.

3. RESULTS AND DISCUSSION

ð2Þ

3.1. Effect of Synthesis Conditions and Functionalization on the Structure of SRSMS. 3.1.1. Effect of Postsynthesis-Grafting Functionalization. Mesoporous silica is a pure silica material

where Ra is the relative activity of free or immobilized PPL (%), Aa is the activity of immobilized PPL (U g1), and Am is the maximal activity of immobilized PPL (U g1). 2.6. Characterization. Energy-dispersive spectrometer (EDS) and scanning electron microscopy (SEM) studies were performed using a Quanta200 ESEM FelcoHolland microscope operating at an accelerating voltage of 20 kV. Sample powder was mounted on the surface of a silicon wafer and was sputter-coated with gold for two cycles to avoid charging under the electron beam. Small-angle X-ray powder diffraction (SAXRD) patterns were recorded in a Bruker D8 advance diffractometer with Cu Kα radiation (25 kV, 20 mA) and a step size of 0.02° from 0.5° to 5°. Low-temperature N2 adsorptiondesorption experiments were performed on a Gemini V 2380 system. Surface areas were estimated using the Brunauer EmmettTeller (BET) equation, while pore diameter distribution was calculated from the inflection point of the cumulative pore volume versus diameter curve obtained from the corrected form of the BarrettJoynerHalenda (BJH) method.23 Transmission electron microscopy (TEM) studies were obtained on a JEM-100CX II electron microscope with an acceleration voltage

consisting of silica units with SiOH hydroxyl groups on the surface.24,25 EDS images of SRSMS1 and NH-SRSMS1 are shown in Figure 1a and b. In Figure 1a, no signals of C and N were discovered. As expected, two adjacent signals of nitrogen (N) and carbon (C) were observed in Figure 1b; these signals can be attributed to the amino and propyl portions of APTES that have been successfully introduced into SRSMS1. Elemental data of SRSMS1 and NH-SRSMS1 are summarized in Table 1. N content obviously increases from 0 (SRSMS1) to around 3% (NH-SRSMS1), and C content increased from 0 (SRSMS1) to 10% (NH-SRSMS1) after functionalization. Results indicate that APTES had been successfully grafted on the surface of SRSMS1. 3.1.2. Effect of PEG with Different MW. Figure 2a shows SAXRD patterns of SRSMS samples synthesized with PEG of different MW. Three well-resolved peaks that could be indexed as (100), (110), and (200) diffractions were seen, meaning that all samples were mesoporous materials with 2D mesostructure (p6mm).26,27 SAXRD data from three different samples showed that these diffraction peaks shift from high to low angle with increasing PEG MW. Interplanar spacing d100 values of SRSMS1, SRSMS2, and SRSMS3 were 9.27, 9.33, and 9.63 nm, respectively,

Ra ¼

Aa 100 Am

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Table 4. Loading Amounts and Activities of Immobilized PPL samples

P (mg g1)a

activity (U g1)b

SRSMS1-PPL NH-SRSMS1-PPL

270 ( 4.00 410 ( 5.20

238 ( 7.50 443 ( 15.50

PPL

338

a

PPL immobilized in phosphate buffer (pH 7.0) for incubation time of 6 h. b The activities were measured at the optimal catalysis conditions (pH 7.0, 308 K for PPL; pH 7.0, 308 K for SRSMS1-PPL; and pH 8.0, 318 K for NH-SRSMS1-PPL).

Figure 6. SAXRD patterns of (a) SRSMS and (b) NH-SRSMS: (i) SRSMS4, (ii) SRSMS1, (iii) SRSMS5, and (iv) SRSMS6.

with cell parameter a0 correspondingly increasing continuously to 10.70, 10.77, and 11.12 nm (Table 2), respectively (a0 = 2d100/ 30.5). SRSMS1 showed more intense and narrower peaks than SRSMS2 and SRSMS3, indicative of a relatively highly structured ordering. SAXRD patterns of amino-functionalized samples are shown in Figure 2b. All patterns were similar to that observed in unfunctionalized SRSMS, indicating that the hexagonal structure was unchanged after functionalization. As compared to unfunctionalized SRSMS, functionalized SRSMS showed more intense and narrower peaks, with enlarged d100 and a0 (Table 2). N2 adsorptiondesorption isotherms of samples before and after functionalization at 76 K are shown in Figure 3a. All samples exhibited typical type IV isotherms with clear hysteresis loops of H1 type associated with capillary condensation at high relative pressure, which is typical of hexagonal cylindrical channel mesoporous materials.28 This is further evidence that the structure of well-ordered hexagonal arrangements remains unchanged after functionalization. Pore size distribution curves computed from the adsorption isotherms of the samples are shown in Figure 3b, while textural parameters are listed in Table 2. Pore sizes of all unfunctionalized samples were about 6.4 nm, indicating that the MW of PEG exerts little influence on the pore size of SRSMS. Similar results were also reported by Jiang et al.1 The thickness of SRSMS walls was calculated to be 4.24 (SRSMS1), 4.43 (SRSMS2), and 4.80 nm (SRSMS3), respectively. This demonstrates that wall thickness increased continuously with higher PEG MW. However, the surface area and pore volume of the SRSMS2 and SRSMS3 were much higher than those of SRSMS1 (Table 2). The pore size distribution curve of SRSMS1 showed a narrower range than did SRSMS2 and SRSMS3,

indicating higher ordering of the sample, which is consistent with SAXRD results. As compared to SRSMS1, NH-SRSMS1 exhibited a marked decrease in BET surface area, pore volume, and average pore size, with increased wall thickness (Table 2). Findings demonstrate that the amino groups by APTES were anchored on the surface of SRSMS1. 3.1.3. Effect of PEG Concentration. To investigate the effect of PEG concentration on structure, we fabricated SRSMS samples using various PEG concentrations. Synthesis conditions are listed in Table 3. SEM images of the representative SRSMS samples synthesized with different P123/PEG molar ratios are shown in Figure 4. All samples exhibited short rod-shaped morphology, with SRSMS1 samples (Figure 4b) exhibiting better morphology and polydispersity. When the P123/PEG molar ratio was low (e.g., 0.256), the silica skeleton was stacked densely (Figure 4a), and high PEG content led to the formation of broken irregular particles (Figure 4c). SEM image of NH-SRSMS1 (Figure 4d) revealed inerratic short rod-shaped macrostructure, indicating that there was no change in silica morphology after modification with APTES. Figure 5 shows representative TEM images of calcined samples prepared with different P123/PEG molar ratios. Materials clearly showed well-ordered parallel straight mesochannel arraying along the long axis. Pore size increased with increasing PEG concentration, which may be attributed to the size of micelles formed by P123 and PEG during synthesis. Low PEG concentrations lead to small micelles and small pore size, whereas polymers conglomerate to form large micelles at high PEG concentration to form large mesopores.29 Figure 5d demonstrates that the ordered mesostructure of NH-SRSMS1 is preserved after modification. Average pore diameters of SRSMS1 and NH-SRSMS1 estimated from TEM images were uniform nanosize of 67 nm, which were confirmed by nitrogen adsorptiondesorption. SAXRD patterns of SRSMS synthesized with different P123/ PEG molar ratios are shown in Figure 6a. SRSMS4, SRSMS1, SRSMS5, and SRSMS6 displayed three diffraction peaks, indicating that all samples were mesoporous materials. SRSMS1 exhibited more intense and narrower diffraction peaks than did SRSMS4, SRSMS5, and SRSMS6, demonstrating that it has a relatively highly structured ordering. The positions of the three reflections of SRSMS shifted from high to low angle with increasing PEG content, with corresponding continuous increases in d100 and a0 (Table 3). SAXRD patterns of NHSRSMS4, NH-SRSMS1, NH-SRSMS5, and NH-SRSMS6 are shown in Figure 6b. They exhibited patterns similar to that of unfunctionalized samples, indicating that the hexagonal structure is maintained after functionalization. Values of d100 and a0 (Table 3) were increased after functionalization. 3.2. Immobilization of PPL onto Mesoporous Supports. Pore size of SRSMS1 and NH-SRSMS1 was determined to be 22195



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Scheme 1. Schematic Representation for PPL Immobilization onto SRSMS and Functionalized SRSMS Prepared Using PEG and P123 as Templates

Figure 7. FT-IR spectra of SRSMS1, NH-SRSMS1, SRSMS1-PPL, and NH-SRSMS1-PPL.

67 nm, which is large enough to accommodate PPL molecules (∼4 nm). Table 4 reveals that loading proportion increased from 270 (SRSMS1-PPL) to 410 mg g1 (NH-SRSMS1-PPL) after functionalization. Similar observations have been reported for the adsorption of PPL onto SBA-15.18 SRSMS1-PPL activity was 238 U g1 at optimum catalysis conditions, whereas the value for NH-SRSMS1-PPL almost doubled to 443 U g1. Activity of SRSMS1-PPL was found to be less than that of free PPL, which may be attributed to the substrate’s diffusional limitation into the porous material and a lower degree of dispersion into the mesoporous matrix.30 NH-SRSMS1-PPL activity was higher than that of free PPL, suggesting that amino group modification could play an important role in enhancing the adaptability of immobilized PPL. A possible mechanism for PPL immobilization onto SRSMS and functionalized SRSMS is illustrated in Scheme 1. Amino groups on the NH-SRSMS surface compete for intergroup forces between NH2; PPL would be stronger than the weak intermolecular forces (van der Waals interactions and hydrogen bonding) between OH on the SRSMS surface and PPL, so that PPL will become tightly anchored on NH-SRSMS1 support. 3.2.1. Effect of Immobilized PPL on SRSMS Structure before and after Functionalization. As is shown in Figure 7, the presence of an intense absorption band at 1083 cm1 corresponds to asymmetric SiO stretching vibration, while bands at 798 cm1 are attributed to symmetric SiO stretching vibration. The band at 958 cm1

Figure 8. (a) N2 adsorptiondesorption isotherms and (b) corresponding BJH pore size distribution curves of SRSMS1, NH-SRSMS1, SRSMS1-PPL, and NH-SRSMS1-PPL.

associated with bending of SiOH bonds can be observed in the SRSMS1 spectra. No CH2 group absorption peaks were observed in the FT-IR spectrum of SRSMS1, indicating that the PEG and P123 templates have been removed after heat treatment at 773 K. The absorption bands at 3430 and 1640 cm1 demonstrate the presence of water molecules. After the functionalization process, the presence of absorption bands at 30002900 cm1 can be attributed to stretching vibration of the CH2 group from APTES. In additional, the absorption bands at 1536 cm1, which can be assigned to twisting vibration of NH, can sufficiently prove that APTES has been grafted onto the SRSMS surface.31 The spectra of supports with 22196



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Figure 9. Effect of different supports on activity of immobilized PPL. Figure 11. Activity of free and immobilized PPL as a function of incubation temperature.

Figure 10. Activity of free and immobilized PPL as a function of medium pH.

immobilized PPL exhibit a band at 1676 cm1, which can be assigned to the deformation of the CdO (from CONH).32 These results confirm that PPL has been successfully immobilized on the supports. Figure 8 shows the N2 adsorptiondesorption isotherms and the corresponding very narrow pore size distribution curves computed from the adsorption isotherms of SRSMS1, NHSRSMS1, SRSMS1-PPL, and NH-SRSMS1-PPL. Isotherms of all samples were type IV isotherms with clear hysteresis loops of H1 type associated with capillary condensation at high relative pressure, indicating that the structure of well-ordered hexagonal arrangements remained unchanged after PPL loading. After PPL immobilization, we discovered that average pore sizes decreased only slightly from 6.46 (SRSMS1) to 6.41 nm (SRSMS1-PPL) and from 6.06 (NH-SRSMS1) to 6.02 nm (NH-SRSMS1-PPL), respectively. Similar results have been reported for the adsorption of CRL onto SBA-15.16 The BET surface areas and pore volumes of SRSMS1 significantly decreased from 635 to 384 m2 g1 and from 0.82 to 0.60 cm3 g1 after PPL immobilization, while that of NH-SRSMS1 decreased from 312 to 220 m2 g1 and from 0.49 to 0.38 cm3 g1. This finding further confirms that most of the PPL were immobilized inside the pore channels. 3.2.2. Effect of Different Supports on Enzymatic Activity of Immobilized PPL. Figure 9a shows the activity of PPL immobilized on SRSMS synthesized with different P123/PEG molar ratios at 308 K. PPL immobilized on SRSMS4 prepared with the lowest P123/PEG molar ratio (0.256) exhibited an activity of 140 U g1, whereas PPL immobilized on SRSMS1 obtained with a P123/PEG molar ratio of 0.512 showed the highest activity at 238 U g1. When PEG concentration was further increased, the resulting SRSMS-immobilized PPL displayed decreased hydrolysis

activity of 202, 177, and 102 U g1, respectively. Activity of PPL immobilized on SRSMS synthesized with different MW is shown in Figure 9b. Activity of SRSMS1-PPL, SRSMS2-PPL, and SRSMS3-PPL suggests that hydrolysis activity decreased continuously with increasing PEG MW. Findings clearly demonstrate that SRSMS1 carrier provides superior support. 3.2.3. Effect of pH on Enzymatic Activity of Free and Immobilized PPL. Figure 10 illustrates the effect of medium pH on the activity of free and immobilized PPL at 308 K. Optimal catalysis pH of free PPL and SRSMS1-PPL was 7.0, while that of NH-SRSMS1-PPL was 8.0. Free PPL activity fluctuated within a narrow pH range.33 SRSMS1-PPL and NH-SRSMS1-PPL maintained high activity within a pH range of 7.09.0. The reason for this is not completely clear, but a possible explanation is that the abundant silanol groups on SRSMS1 surface and aminoporpyl moieties on NH-SRSMS1 surface caused the partitioning of protons between the bulk phase and PPL microenvironment, thus causing a shift in the optimal pH value.15 We also found that the highest hydrolysis activity of SRSMS1-PPL was 238 U g1, whereas the value almost doubled for that of NH-SRSMS1-PPL at 410 U g1. Activity improvement could be due to the induction of structure screen function by the supports. Findings clearly prove that functionalization-grafted hydrophobic groups on SRSMS1 improve the activity of immobilized PPL. 3.2.4. Effect of Reaction Temperature on Enzymatic Activity of Free and Immobilized PPL. The effect of temperature profile on free and immobilized PPL catalytic activity performance was studied in the range 293333 K (Figure 11). For free PPL and SRSMS1-PPL, the optimal reaction temperature was 308 K, while that for NH-SRSMS1-PPL was 318 K. As compared to free PPL, SRSMS1-PPL and NH-SRSMS1-PPL exhibited excellent adaptability in a wider temperature range.34 Thus, immobilized PPL has better heat resistance than free PPL within the range of 293333 K. Similar observations have been described for PPL adsorption onto SBA-15.18 This could be because pores of supports can protect the enzyme from injury due to direct exposure.14 We also found that NH-SRSMS1-PPL exhibited the highest activity at 410 U g1, while SRSMS1-PPL had activity of 238 U g1 only. This could be attributed to the stronger interaction between the PPL molecule and the NH-SRSMS1 support, resulting in prevention of PPL heat denaturation and enhancement of the adaptability of immobilized PPL.35 3.2.5. Thermal Stability of Free and Immobilized PPL. Figure 12 shows the variation in hydrolysis activity of free and immobilized PPL. The two kinds of immobilized PPL maintained 22197



Figure 12. Activity of free and immobilized PPL as a function of incubation time at 323 K.

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and MW of PEG. Properties of PPL immobilized into different supports and into functionalized NH-SRSMS were investigated. PPL immobilized on SRSMS1 support showed higher activity as compared to SRSMS2-PPL, SRSMS3-PPL, SRSMS4-PPL, SRSMS5-PPL, SRSMS6-PPL, and SRSMS7-PPL. In addition, NH-SRSMS1-PPL exhibited greater loading amount and higher activity than SRSMS1-PPL. The optimal catalytic pH values of SRSMS1-PPL and NH-SRSMS1-PPL were pH 7.0 and 8.0, respectively. Apparent optimal temperature of NH-SRSMS1PPL was 318 K, which was 10 K higher than that of free PPL. NHSRSMS1-PPL exhibited excellent thermal stability as compared to SRSMS1-PPL. The relative activity of SRSMS1-PPL was maintained at levels exceeding 50% of its original activity, with a numerical value of 57% for NH-SRSMS1-PPL after five reuses.

’ AUTHOR INFORMATION Corresponding Author

*Phone: +86-531-89631696. E-mail: [email protected].

’ ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Grant No. 20976100), the Natural Science Foundation of Shandong Province (Grant No. ZR2010BM013), and the Scientific and Technological Development Plan of Shandong Province (Grant No. 2010GGX10306). ’ REFERENCES Figure 13. Relative activity of immobilized PPL as a function of reuse numbers.

over 100 U g1 of hydrolysis activity for 90 min, while free PPL preserved only 40 U g1 for 80 min at 323 K. In addition, the activity of NH-SRSMS1-PPL remained comparatively stable at over 102 U g1 after incubation for 150 min as compared to SRSMS1-PPL (89 U g1). This difference indicates that aminofunctionalization improves the thermal stability of immobilized PPL. This can be attributed to stronger intermolecular forces between abundant NH2 on the NH-SRSMS1 support surface and PPL as compared to that between OH on the SRSMS1 support and PPL,36,37 thus preventing thermal denaturation of NH-SRSMS1-PPL and improving the thermal stability of NHSRSMS1-PPL. These findings demonstrate the usefulness of this type of surface modification. 3.2.6. Capability for Repeated Use of Immobilized PPL. Variation in relative activity of immobilized PPL after five cycles at 308 K is shown in Figure 13. NH-SRSMS1-PPL has more than 57% of hydrolysis activity after the fifth reuse, while SRSMS1PPL preserved only 50% activity. These results further confirm that NH-SRSMS1-PPL has better stability than SRSMS1-PPL, which can be due to the strong interaction for immobilization because of a suitable “counter-functional group” on the surface of the NH-SRSMS1 support.36

4. CONCLUSIONS SRSMS materials were successfully synthesized using PEG and P123 as templates and functionalized with APTES by postsynthesis-grafting method. Morphology and pore size distribution of SRSMS could be adjusted by controlling the concentration

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