基于短孔道Zr-Ce-SBA-15固定胃蛋白酶的活性生物催化剂

doi:10.3724/SP.J.1077.2012.00185

无机材料学报 ›› 2012, Vol. 27 ›› Issue (2): 185-190.DOI: 10.3724/SP.J.1077.2012.00185 CSTR: 32189.14.SP.J.1077.2012.00185

基于短孔道Zr-Ce-SBA-15固定胃蛋白酶的活性生物催化剂

王放, 李健生, 冉冬琴, 段梦姗, 孙秀云, 王连军

(南京理工大学环境与生物工程学院, 南京 210094)

收稿日期:2011-01-17 修回日期:2011-03-11 出版日期:2012-02-10 网络出版日期:2012-01-05
作者简介:王放(1987-), 女, 硕士研究生. E-mail: su_daily@126.com
基金资助:
国家自然科学基金(51078184);高等学校科技创新工程重大项目培育资金项目(708049);江苏省自然科学基金(BK2009392);南京理工大学自主科研专项计划项目(2010ZDJH03)

Active Biocatalysts Based on Pepsin Immobilized in Short Channeled Zr-Ce-SBA-15

WANG Fang, LI Jian-Sheng, RAN Dong-Qin, DUAN Meng-Shan, SUN Xiu-Yun, WANG Lian-Jun

(School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, China)

Received:2011-01-17 Revised:2011-03-11 Published:2012-02-10 Online:2012-01-05
About author:WANG Fang. E-mail: su_daily@126.com
Supported by:
National Natural Science Foundation of China (51078184);Cultivation Fund of the Key Scientific and Technical Innovation Project, Ministry of Education of China (708049);Natural Science Foundation of Jiangsu Province (BK2009392);NUST research funding (2010ZDJH03)

摘要/Abstract

摘要：

在不外加无机酸的条件下, 通过水热合成法制备了短孔道六方板状有序介孔材料Zr-Ce-SBA-15 (ZCS). 以ZCS和传统SBA-15为载体对胃蛋白酶进行固定, 并利用N-(2-氨乙基)-3-氨丙基三甲氧基硅烷(AAPTS)对酶固定化材料进行功能化, 以缩小开口孔径从而减少酶泄漏. 采用小角X射线衍射、扫描电镜、透射电镜、氮气吸附和红外光谱对样品进行结构表征. 结果表明, 胃蛋白酶成功固定到介孔孔道中, AAPTS嫁接到材料中且没有破坏介孔结构. 固定化实验表明相比于SBA-15, ZCS对胃蛋白酶具有较快的吸附速度和较强的固定化能力(最大负载量为257.9 mg/g), 短孔道材料能有效地促进分子的扩散传递. 催化活性测定以牛血红蛋白为探针物, 与游离酶相比, 固定酶对牛血红蛋白保持着稳定的活性.

关键词: 短孔道, 介孔材料, 胃蛋白酶, 酶固定化, 催化

Abstract:

Short channeled Zr-Ce-SBA-15 (ZCS) mesoporous materials with hexagonal platelet morphologies were synthesized through a hydrothermal route without addition of mineral acids. Porcine pepsin was immobilized inside ZCS and conventional SBA-15 through physical adsorption. A grafting step with [1-(2-amino-ethyl)-3-aminopropryl] trimethoxysilane (AAPTS) was performed to reduce the pore openings of the host material in order to minimize the enzyme leaching. The hybrid materials were characterized by X-ray diffraction, scanning electron microscope, transmission microscope, N₂ adsorption/desorption and Fourier-transform infrared spectroscope. The results confirmed that pepsin is located inside the channels of the mesoporous materials and the grafting process does not affect the mesoporous structure. The immobilization of pepsin into ZCS and SBA-15 were compared. The results demonstrate that the ZCS materials have higher adsorption rates and adsorption capacities (the maximum adsorption capacity is 257.9 mg/g) compared with SBA-15. The ZCS materials with short channels are superior to conventional SBA-15 facilitating molecular diffusion. The catalytic activity of the hybrid bioinorganic material was tested with hemoglobin. The immobilized pepsin has maintained the necessary degree of freedom to fulfill its catalytic activity.

Key words: short channeled, mesoporous materials, porcine pepsin, enzyme immobilization, catalysis

中图分类号:

O629
Q814

王放, 李健生, 冉冬琴, 段梦姗, 孙秀云, 王连军. 基于短孔道Zr-Ce-SBA-15固定胃蛋白酶的活性生物催化剂[J]. 无机材料学报, 2012, 27(2): 185-190.

WANG Fang, LI Jian-Sheng, RAN Dong-Qin, DUAN Meng-Shan, SUN Xiu-Yun, WANG Lian-Jun. Active Biocatalysts Based on Pepsin Immobilized in Short Channeled Zr-Ce-SBA-15[J]. Journal of Inorganic Materials, 2012, 27(2): 185-190.

图/表 8

图1 SBA-15(a)、PEP/SBA-15(b)、PEP/SBA-15/AAPTS(c)、ZCS(d)、PEP/ZCS(e)和PEP/ZCS/AAPTS(f)的小角XRD图谱

Fig. 1 Low-angle XRD patterns of SBA-15(a), PEP/SBA-15 (b), PEP/SBA-15/AAPTS (c), ZCS (d), PEP/ZCS (e) and PEP/ZCS/AAPTS (f)

图2 空白载体SBA-15及ZCS的SEM和TEM照片

Fig. 2 SEM and TEM images of ZCS and SBA-15

图3 SBA-15(a), PEP/SBA-15(b), PEP/SBA-15/AAPTS(c), ZCS (d), PEP/ZCS(e)和PEP/ZCS/AAPTS(f)的N2吸附-脱附等温线(A)和孔径分布图(B)

Fig. 3 N2 adsorption-desorption isotherms (A) and pore size distributions (B) of SBA-15 (a), PEP/SBA-15 (b), PEP/ SBA-15/AAPTS (c), ZCS (d), PEP/ZCS (e) and PEP/ZCS/ AAPTS (f)

Table 1 Textural properties of samples

Sample	S_BET/(m²·g^-1)	D/nm	V/(cm³·g^-1)
SBA-15	847	9.6	1.16
PEP/SBA-15	577	7.5	0.99
PEP/SBA-15/AAPTS	241	6.2	0.39
ZCS	805	9.2	1.03
PEP/ZCS	547	8.4	0.80
PEP/ZCS/AAPTS	129	7.3	0.22

图4 SBA-15(a), PEP/SBA-15(b), PEP/SBA-15/AAPTS(c), ZCS(d), PEP/ZCS(e) 和 PEP/ZCS/AAPTS(f)的FTIR图谱

Fig. 4 FTIR spectra of SBA-15 (a), PEP/SBA-15 (b), PEP/ SBA-15/AAPTS (c), ZCS (d), PEP/ZCS (e) and PEP/ZCS/ AAPTS (f)

图5 空白载体SBA-15 和 ZCS对胃蛋白酶的吸附曲线

Fig. 5 Adsorption curves of the amount of adsorbed pepsin within SBA-15 and ZCS

Table 2 Leakage of adsorbed enzyme

Sample	Relative enzyme leaching amount/%
PEP/SBA-15	20.7
PEP/ZCS	21.4
PEP/SBA-15/AAPTS	7.7
PEP/ZCS/AAPTS	7.2

Table 3 Catalytic activity test for peptic hydrolysis

Sample	Enzyme activity / (U·mg^-1)		Relative enzyme activity/%
Sample	Initial	After 4 cycles	Initial	After 4 cycles
Free pepsin	28.71	—	100%	—
PEP/SBA-15	7.34	3.06	25.6%	10.6%
PEP/ZCS	9.65	2.76	33.6%	9.6%
PEP/SBA-15/AAPTS	4.88	4.75	17.0%	16.5%
PEP/ZCS/AAPTS	6.23	6.00	21.7%	20.9%

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基于短孔道Zr-Ce-SBA-15固定胃蛋白酶的活性生物催化剂

Active Biocatalysts Based on Pepsin Immobilized in Short Channeled Zr-Ce-SBA-15

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