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SBA-15 functionalized by epoxy groups for immobilization of penicillin G acylase
Recent Progress in Mesostructured Mesostructured Materials D. Zhao, S. Qiu, Y. Tang and C. Yu (Editors) © 2007 Elsevier B.V. All rights reserved.
877 877
SBA-15 functionalized by epoxy groups for immobilization of penicillin G acylase Yongjun Lti, Qiaoling Zhao, Yanglong Guo*, Yanqin Wang, Yun Guo and Guanzhong Lu Lab for Advanced Materials, Research Institute of Industrial Catalysis, East China University of Science and Technology, Shanghai 20023 7, P. R. China
1. Introduction The discovery of the large pore mesoporous materials, such as SBA-15 [1], broadens their application in the biotechnological fields, and more molecular sieves of biological interest have really attracted much attention [2]. The studies show that the surface functionalization of mesoporous silica by organic groups can enhance the interaction between the enzyme and the support and increases its operation stability [3]. The organic groups functionalized SBA15 have been used to immobilized penicillin G acylase (PGA) [4,5]. Epoxy groups can react with amino groups (NH2) of the enzyme under mild conditions without pretreatment and cross linkage. The organic copolymer supports with epoxy groups have been developed to immobilize PGA successfully, and appreciative apparent activity and operation stability were achieved [6,7]. However, mesoporous silica functionalized by epoxy groups has not yet been reported. In this paper, SBA-15 functionalized by epoxy groups was prepared, characterized and used as the support for the immobilization of PGA. Effect of the amount of epoxy groups on the apparent activity and operation stability of immobilized PGA (IMP) were also investigated. 2. Experimental Section SBA-15 was synthesized according to Zhao et al. [1]. y-glycidoxypropyltrimethoxylsilane (GPTMS) was used as the functionalization agent. The functionalization of SBA-15 was carried out by a post-synthesis route [3]. Changing the amount of GPTMS in the synthesis solution from 0.5 g, 1 g and 1.5 g, the functionalized samples of SBA-15(0.5), SBA-15(1) and SBA-15(1.5)
878
were prepared. The immobilization of PGA (804 IU/ml for the free enzyme) and assay of IMP apparent activity were the same as Reference [8]. After testing the apparent activity of IMP, IMP was separated by centrifugation, and used repeatedly to assay its apparent activity. 3. Results and Discussion The powder XRD pattern of SBA-15 (Figure 1) agrees well with that reported by Zhao et al. [1], which confirms the successful synthesis of SBA-15. The XRD pattern of SBA-15(0.5) functionalized by GPTMS is similar to that of SBA-15, which indicates that the framework of SBA-15 is not destroyed after the functionalization. Compared with the FT-IR spectrum of GPTMS, the absorption peaks at 2927, 2854, 1461 and 1398 cm"1 in the IR spectrum of SBA-15(0.5) (Figure 2) are attributed to the presence of glycidoxypropyl groups on the functionalized SBA-15. The characteristic absorption peak of epoxy groups should be located at ~910 cm"1, but it is too weak to be seen clearly. The nitrogen adsorption-desorption isotherms of SBA-15 and functionalized SBA-15(0.5) are shown in Figure 3, which belong to the isotherms of type IV. The isotherm of SBA-15(0.5) still retains the characteristic step of the isotherm of SBA-15, in which the capillary condensation step shifts slightly to lower relative pressures. This indicates that the pore size decreases after being functionalized, which can be also confirmed by the pore size distribution curve shown in Figure 4. For the functionalization SBA-15, a significant reduction in the amount of nitrogen adsorbed implies decreasing of its pore volume. 100 100
Absorbance
Intensity
GPTMS
110 110 200
•
V
SBA-15 SBA-15(0.5)
V
x/~^
/
^
ft
910
SBA-15
__
SBA-15(0.5) 1
22
33
4 o
Theta ( 2 Theta ()
Fig. 1 XRD patterns of SBA-15 and the functionalized SBA-15(0.5)
5
4000 4000 3500 3500 3000 3000 2500 2500 2000 2000 1500 15001000 1000 500 500 -1 ) Wavenumber (cm )
Fig. 2 FT-IR spectra of GPTMS, SBA-15 and the functionalized SBA-15(0.5)
The pore size distribution curves of the samples are shown in Figure 4. No obvious difference is observed between two pore size distribution curves, that is, SBA-15(0.5) is structurally similar to SBA-15. However, the pore diameter of SBA-15 is about 1.2 nm larger than that of SBA-15(0.5), which indicates that
879
GPTMS has been grafted on the pore surface of SBA-15 and the surface functionalization results in a decrease in the pore size. SBA-15 12 3
25 20 15
SBA-15 10 5
Pore Volume (cm /g)
Quantity Adsorbed (mmol/g)
14 30
SBA-15(0.5)
10
SBA-15(0.5) 8 6 4 2 0
0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.0
0
5
100 100 Relative activity(%)
Weight (%)
Fig. 5 TGA profiles of SBA-15 and the functionalized SBA-15 samples
15 15
20
25
30 30
35
Fig. 4 Pore size distribution curves of SBA-15 and SBA-15(0.5)
Fig. 3 Nitrogen adsorption-desorption isotherms of SBA-15 and SBA-15(0.5) 100 98 A 96 94 92 90 B 88 C 86 84 D 82 100 100 200 300 400 500 600 700 800 o Tempreture ((°C) C)
10 10
Pore Diameter (nm)
Relative Pressure (P/Po)
PGA/SB A-15(0.5) PGA/SBA-15(0.5)
90 80 70
PGA/SB A- 1 5 PGA/SBA-15
60 50 40 30
0
11 22 33 4 4 5 5 6 6 7 7 8 8 Recycle time time
10 11 11 9 10
Fig. 6 Operation stability of PGA immobilized on SBA-15 and SBA-15(0.5)
The TGA profiles of SBA-15(A) and SBA-15(0.5)(B), SBA-15(1.5)(C), SBA-15(1)(D) are shown in Figure 5. The results show that SBA-15 has a slight weight loss, mainly due to surface dehydration or dehydroxylation. However, the functionalized SBA-15 samples have a remarkable weight loss at 300-500 °C, which can be attributed to the decomposition of the incorporated glycidoxypropyl groups. With an increase in the GPTMS concentration in the synthesis solution, the weight loss of the functionalized SBA-15 increases slightly. SBA-15(1) has the largest weight loss. It is possible that too much GPTMS molecules blocked their entrance into the inner channel. The results above show that glycidoxypropyl groups are chemically grafted on the surface of SBA-15. The results in Table 1 show that, the apparent activity of PGA immobilized on SBA-15(0.5) is higher than that on SBA-15. When the amount of glycidoxypropyl groups grafted on SBA-15 is higher, its pore size diminishes obviously to block more enzymes and reactants entering into the channel,
880
therefore the apparent activity of PGA immobilized on SBA-15(1) and SBA15( 1.5) are lower than that of PGA/SBA-15. The operation stability (Figure 6) of PGA immobilized on SBA-15(0.5) is better than that on SBA-15. After being recycled for 10 times, PGA immobilized on SBA-15(0.5) preserved 88% of the initial apparent activity, and that on SBA-15 only preserved 65%. It indicates that PGA has been immobilized on the functionalized SBA-15 by covalent bond and the combination between PGA and the functionalized SBA-15 is more stable than that between PGA and SBA-15 by hydrogen bond only. Table 1 Apparent activity of IMP and BJH adsorption pore diameter of functionalized SBA-15 Supports Apparent activity (IU/g dry support) BJH adsorption pore diameter(nm)
SBA-15 1343 8.2
SBA-15(0.5) 1408 7.3
SBA-15(1) 1316 6.9
SBA-15(1.5) 1191 6.7
4. Conclusion SBA-15 was successfully functionalized by glycidoxypropyl groups without destroy its framework, and the presence of glycidoxypropyl groups on the surface of SBA-15 leads to a decrease in the pore size. PGA immobilized on the functionalized SBA-15 by covalent bond is more stable than that on SBA-15, which improves obviously the apparent activity and operation stability of PGA/functionalized SBA-15. This project was supported financially by National Basic Research Program of China (No. 2004CB719500), Shanghai Rising-Star Program (No. 04QMX1431) and Program for Outstanding Young Teacher of Shanghai Universities (No. 04YQHB050). 5. References [1] D. Y. Zhao, J. L. Feng, Q. S. Huo, N. Melosh, G. H. Fredrickson, B. F. Chmelka and G. D. Stucky, Science, 279 (1998) 548. [2] H. H. P. Yiu and P. A. Wright, J. Mater. Chem., 15 (2005) 3690. [3] J. F. Kennedy and A. Wiseman (Ed.). Handbook of Enzyme Biotechnology, Ellis Horwood, London, (1995), 235. [4] A. S. M. Chong and X. S. Zhao, Catal. Today, 93-95 (2004) 293. [5] A. S. M. Chong and X. S. Zhao, Appl. Surf. Sci., 237 (2004) 398. [6] P. Xue, G. Z. Lu, Y. L. Guo, Y Guo and Y. S. Wang, Chem. J. Chinese U., 25 (2004) 361. [7] J. Torres-Bacete, M. Arroyo, R. Torres-Guzman, I. de la Mata, M. P. Castillon and C. Acebal, Biotechnol. Lett., 22 (2000) 1011. [8] P. Xue, G. Z. Lu, Y. L. Guo, Y. S. Wang and Y Guo, J. Mol. Catal. B, 30 (2004) 75.
877 877
SBA-15 functionalized by epoxy groups for immobilization of penicillin G acylase Yongjun Lti, Qiaoling Zhao, Yanglong Guo*, Yanqin Wang, Yun Guo and Guanzhong Lu Lab for Advanced Materials, Research Institute of Industrial Catalysis, East China University of Science and Technology, Shanghai 20023 7, P. R. China
1. Introduction The discovery of the large pore mesoporous materials, such as SBA-15 [1], broadens their application in the biotechnological fields, and more molecular sieves of biological interest have really attracted much attention [2]. The studies show that the surface functionalization of mesoporous silica by organic groups can enhance the interaction between the enzyme and the support and increases its operation stability [3]. The organic groups functionalized SBA15 have been used to immobilized penicillin G acylase (PGA) [4,5]. Epoxy groups can react with amino groups (NH2) of the enzyme under mild conditions without pretreatment and cross linkage. The organic copolymer supports with epoxy groups have been developed to immobilize PGA successfully, and appreciative apparent activity and operation stability were achieved [6,7]. However, mesoporous silica functionalized by epoxy groups has not yet been reported. In this paper, SBA-15 functionalized by epoxy groups was prepared, characterized and used as the support for the immobilization of PGA. Effect of the amount of epoxy groups on the apparent activity and operation stability of immobilized PGA (IMP) were also investigated. 2. Experimental Section SBA-15 was synthesized according to Zhao et al. [1]. y-glycidoxypropyltrimethoxylsilane (GPTMS) was used as the functionalization agent. The functionalization of SBA-15 was carried out by a post-synthesis route [3]. Changing the amount of GPTMS in the synthesis solution from 0.5 g, 1 g and 1.5 g, the functionalized samples of SBA-15(0.5), SBA-15(1) and SBA-15(1.5)
878
were prepared. The immobilization of PGA (804 IU/ml for the free enzyme) and assay of IMP apparent activity were the same as Reference [8]. After testing the apparent activity of IMP, IMP was separated by centrifugation, and used repeatedly to assay its apparent activity. 3. Results and Discussion The powder XRD pattern of SBA-15 (Figure 1) agrees well with that reported by Zhao et al. [1], which confirms the successful synthesis of SBA-15. The XRD pattern of SBA-15(0.5) functionalized by GPTMS is similar to that of SBA-15, which indicates that the framework of SBA-15 is not destroyed after the functionalization. Compared with the FT-IR spectrum of GPTMS, the absorption peaks at 2927, 2854, 1461 and 1398 cm"1 in the IR spectrum of SBA-15(0.5) (Figure 2) are attributed to the presence of glycidoxypropyl groups on the functionalized SBA-15. The characteristic absorption peak of epoxy groups should be located at ~910 cm"1, but it is too weak to be seen clearly. The nitrogen adsorption-desorption isotherms of SBA-15 and functionalized SBA-15(0.5) are shown in Figure 3, which belong to the isotherms of type IV. The isotherm of SBA-15(0.5) still retains the characteristic step of the isotherm of SBA-15, in which the capillary condensation step shifts slightly to lower relative pressures. This indicates that the pore size decreases after being functionalized, which can be also confirmed by the pore size distribution curve shown in Figure 4. For the functionalization SBA-15, a significant reduction in the amount of nitrogen adsorbed implies decreasing of its pore volume. 100 100
Absorbance
Intensity
GPTMS
110 110 200
•
V
SBA-15 SBA-15(0.5)
V
x/~^
/
^
ft
910
SBA-15
__
SBA-15(0.5) 1
22
33
4 o
Theta ( 2 Theta ()
Fig. 1 XRD patterns of SBA-15 and the functionalized SBA-15(0.5)
5
4000 4000 3500 3500 3000 3000 2500 2500 2000 2000 1500 15001000 1000 500 500 -1 ) Wavenumber (cm )
Fig. 2 FT-IR spectra of GPTMS, SBA-15 and the functionalized SBA-15(0.5)
The pore size distribution curves of the samples are shown in Figure 4. No obvious difference is observed between two pore size distribution curves, that is, SBA-15(0.5) is structurally similar to SBA-15. However, the pore diameter of SBA-15 is about 1.2 nm larger than that of SBA-15(0.5), which indicates that
879
GPTMS has been grafted on the pore surface of SBA-15 and the surface functionalization results in a decrease in the pore size. SBA-15 12 3
25 20 15
SBA-15 10 5
Pore Volume (cm /g)
Quantity Adsorbed (mmol/g)
14 30
SBA-15(0.5)
10
SBA-15(0.5) 8 6 4 2 0
0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.0
0
5
100 100 Relative activity(%)
Weight (%)
Fig. 5 TGA profiles of SBA-15 and the functionalized SBA-15 samples
15 15
20
25
30 30
35
Fig. 4 Pore size distribution curves of SBA-15 and SBA-15(0.5)
Fig. 3 Nitrogen adsorption-desorption isotherms of SBA-15 and SBA-15(0.5) 100 98 A 96 94 92 90 B 88 C 86 84 D 82 100 100 200 300 400 500 600 700 800 o Tempreture ((°C) C)
10 10
Pore Diameter (nm)
Relative Pressure (P/Po)
PGA/SB A-15(0.5) PGA/SBA-15(0.5)
90 80 70
PGA/SB A- 1 5 PGA/SBA-15
60 50 40 30
0
11 22 33 4 4 5 5 6 6 7 7 8 8 Recycle time time
10 11 11 9 10
Fig. 6 Operation stability of PGA immobilized on SBA-15 and SBA-15(0.5)
The TGA profiles of SBA-15(A) and SBA-15(0.5)(B), SBA-15(1.5)(C), SBA-15(1)(D) are shown in Figure 5. The results show that SBA-15 has a slight weight loss, mainly due to surface dehydration or dehydroxylation. However, the functionalized SBA-15 samples have a remarkable weight loss at 300-500 °C, which can be attributed to the decomposition of the incorporated glycidoxypropyl groups. With an increase in the GPTMS concentration in the synthesis solution, the weight loss of the functionalized SBA-15 increases slightly. SBA-15(1) has the largest weight loss. It is possible that too much GPTMS molecules blocked their entrance into the inner channel. The results above show that glycidoxypropyl groups are chemically grafted on the surface of SBA-15. The results in Table 1 show that, the apparent activity of PGA immobilized on SBA-15(0.5) is higher than that on SBA-15. When the amount of glycidoxypropyl groups grafted on SBA-15 is higher, its pore size diminishes obviously to block more enzymes and reactants entering into the channel,
880
therefore the apparent activity of PGA immobilized on SBA-15(1) and SBA15( 1.5) are lower than that of PGA/SBA-15. The operation stability (Figure 6) of PGA immobilized on SBA-15(0.5) is better than that on SBA-15. After being recycled for 10 times, PGA immobilized on SBA-15(0.5) preserved 88% of the initial apparent activity, and that on SBA-15 only preserved 65%. It indicates that PGA has been immobilized on the functionalized SBA-15 by covalent bond and the combination between PGA and the functionalized SBA-15 is more stable than that between PGA and SBA-15 by hydrogen bond only. Table 1 Apparent activity of IMP and BJH adsorption pore diameter of functionalized SBA-15 Supports Apparent activity (IU/g dry support) BJH adsorption pore diameter(nm)
SBA-15 1343 8.2
SBA-15(0.5) 1408 7.3
SBA-15(1) 1316 6.9
SBA-15(1.5) 1191 6.7
4. Conclusion SBA-15 was successfully functionalized by glycidoxypropyl groups without destroy its framework, and the presence of glycidoxypropyl groups on the surface of SBA-15 leads to a decrease in the pore size. PGA immobilized on the functionalized SBA-15 by covalent bond is more stable than that on SBA-15, which improves obviously the apparent activity and operation stability of PGA/functionalized SBA-15. This project was supported financially by National Basic Research Program of China (No. 2004CB719500), Shanghai Rising-Star Program (No. 04QMX1431) and Program for Outstanding Young Teacher of Shanghai Universities (No. 04YQHB050). 5. References [1] D. Y. Zhao, J. L. Feng, Q. S. Huo, N. Melosh, G. H. Fredrickson, B. F. Chmelka and G. D. Stucky, Science, 279 (1998) 548. [2] H. H. P. Yiu and P. A. Wright, J. Mater. Chem., 15 (2005) 3690. [3] J. F. Kennedy and A. Wiseman (Ed.). Handbook of Enzyme Biotechnology, Ellis Horwood, London, (1995), 235. [4] A. S. M. Chong and X. S. Zhao, Catal. Today, 93-95 (2004) 293. [5] A. S. M. Chong and X. S. Zhao, Appl. Surf. Sci., 237 (2004) 398. [6] P. Xue, G. Z. Lu, Y. L. Guo, Y Guo and Y. S. Wang, Chem. J. Chinese U., 25 (2004) 361. [7] J. Torres-Bacete, M. Arroyo, R. Torres-Guzman, I. de la Mata, M. P. Castillon and C. Acebal, Biotechnol. Lett., 22 (2000) 1011. [8] P. Xue, G. Z. Lu, Y. L. Guo, Y. S. Wang and Y Guo, J. Mol. Catal. B, 30 (2004) 75.