非表面活性剂模板溶胶-凝胶法制备磁性介孔二氧化硅实现纤维二糖酶的原位固定

doi:10.7503/cjcu20150688

摘要/Abstract

摘要：

以非表面活性剂为模板, 通过引入磁性纳米颗粒, 制备了磁性介孔硅材料, 实现了纤维二糖酶的原位固定, 得到了磁性固定化酶. 制备流程操作简单、原料易得、使用方便. 溶胶-凝胶反应在水相、常温、中性条件下进行, 适用于工业化生产. 对磁性固定化酶进行了气体吸附分析、热重分析、表面形貌分析和磁性表征. 结果表明, 磁性固定化酶具有较大的比表面积、较窄的介孔分布和软铁磁性. 与非介孔固定化酶相比, 磁性固定化酶表观酶活明显提高. 磁性颗粒的引入对酶活性没有显著的影响, 并且可以非常方便和快速地从反应系统中回收再利用, 结合磁场的可控性, 非表面活性剂模板溶胶-凝胶法有望实现固定化酶的大规模可控释放与回收.

关键词: 酶固定化, 纤维二糖酶, 介孔二氧化硅, 磁性纳米颗粒, 溶胶-凝胶反应, 非表面活性剂

Abstract:

Nonsurfactant-templated sol-gel method is an advanced technology for enzyme encapsulation. The immobilized enzyme can be reused many times, so that can reduce the cost. It has a broad application prospect in biological catalysis. D-fructose is used as non-surfactant template to generate mesopores. The average diameter of the mesoporous silica is 3.2 nm. And they significantly improve the activity of the immobilized enzyme. The best result is 70% of free enzyme. On the other hand, the mesopores are smaller than the size of cellobiase enzyme. So the enzyme will not leach out. That can help avoiding enzyme leaching. The magnetic nanoparticles can make the separation and recycle of immobilized enzyme easier and more feasible. The incorporation of magnetic nanoparticles has little effect on the enzyme activity. The magnetic encapsulated enzyme can be easily recycled by magnet force. More than 83% remained after 10 cycles. The process is simple and biocompatible as the sol-gel reaction occurs inaqueous phase and neutral pH.

Key words: Enzyme encapsulation, Cellobiase enzyme, Mesoporous silica, Magnetic nanoparticles, Sol-gel reaction, Non surfactant template

中图分类号:

O629.8

TrendMD:

张翔, 李书润, 翟文韬, 曹莹泽, 齐宏旭, 吉岩, 危岩. 非表面活性剂模板溶胶-凝胶法制备磁性介孔二氧化硅实现纤维二糖酶的原位固定. 高等学校化学学报, 2015, 36(11): 2349.

ZHANG Xiang, LI Shurun, ZHAI Wentao, CAO Yingze, QI Hongxu, JI Yan, WEI Yen. In Situ Encapsulation of Cellubiase Enzyme in Magnetic Mesoporous Silica via the Nonsurfactant-templated Sol-gel Method^†. Chem. J. Chinese Universities, 2015, 36(11): 2349.

图/表 10

Table 1 Information of each sample

Sample	m(SiO₂)/g	m(Fructose)/g	m(Enzyme)/mL	m(Fe₃O₄)/g
E0	9.0	0	0.5	0
M0	9.0	0	0.5	0.45
E3	9.0	3.0	0.5	0
M3	9.0	3.0	0.5	0.45
E6	9.0	6.0	0.5	0
M6	9.0	6.0	0.5	0.45
E9	9.0	9.0	0.5	0
M9	9.0	9.0	0.5	0.45

Fig.1 SEM images of immobilized enzyme with different magnifications

Table 2 Hysteresis loop tests of Fe3O4, E0, M0 and M9

Sample	Remanence/(A·m²·kg^-1)	Coercive force/(A·m^-1)
Fe₃O₄	8.50	9.0×10⁴
E0	0.0036	9.0×10⁴
M0	0.28	9.0×10⁴
M9	0.24	1.1×10⁵

Fig.2 TGA curves of E0(a), E3(b), M3(c), M6(d), E6(e), M9(f) and E9(g)

Table 3 Fructose template content, mass loss, textural properties from nitrogen adsorption-desorption and enzymatic activity of immobilized cellobiase samples

Sample	Template added(%)	Mass loss(%)	BET/(m²·g^-1)	Pore diameter/nm	Pore volume/ (cm³·g^-1)	Enzymatic activity/ (mg·dL^-1·h^-1)
Free enzyme	—	—	—	—	—	80
E0	0.0	6.9	336	2.38	0.102	2
E3	25.0	18.1	720	2.64	0.347	13
M3	24.1	20.7	703	2.71	0.353	15
E6	40.0	34.8	645	3.00	0.550	28
M6	38.8	32.1	576	2.68	0.433	31
E9	50.0	46.2	922	3.40	0.851	56
M9	48.8	45.5	730	3.32	0.632	48

Fig.3 Nitrogen adsorption-desorption isotherms of M0(a), M3(b), M6(c) and M9(d)

Fig.4 Enzyme activity plots of free enzyme(a), M3(b), M6(c) and M9(d)

Fig.5 Enzyme activity plots of free enzyme(●) and M9(■) at different temperature

Fig.6 Enzyme activity plots of E9(a) and M9(b) in recycle uses

Fig.7 Sedimentation of magnetic and non-magnetic samples at the beginning(A), 1 min(B), 10 min(C) and 1 h(D)

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