KH560修饰的溶胶-凝胶法二氧化硅载体用于脂肪酶的固定化

doi:10.15541/jim20150398

摘要/Abstract

摘要：

以F127为模板剂, 采用溶胶-凝胶法制备了环氧硅烷改性剂KH560修饰的二氧化硅(F-560-S), 通过TG、FTIR、SEM和N₂ 吸附-脱附等方法对样品表面性质和结构进行了表征。结果表明, F-560-S表面富含环氧基, 且具有较大孔径和比表面积。以F-560-S为载体通过共价结合法固定南极假丝酵母脂肪酶B (CALB), 结果表明环氧基和载体形貌共同影响固定化酶固载量和酶学性质。F-560-S对CALB的固载量达到375 mg/g support, 为普通SiO₂(U-S)的37.5倍; F-560-S固定的CALB在多种有机溶剂中催化1-苯乙醇与醋酸乙烯酯的反应时, 催化转化率普遍高于U-S固定的CALB, 热稳定性和操作稳定性均有明显改善, 重复使用7次后F-560-S固定的CALB酶活力仍为初始活力的88.3%。

关键词: KH560, F127, 二氧化硅溶胶-凝胶, 酶固定化

Abstract:

An epoxy-functionalized silica support was prepared through 3-glycidoxypropyltrimethoxylsilane (KH560) modified silica sol in the presence of triblock copolymer F127 (F-560-S). Surface chemistry, micromorphology and pore structure of the supports were characterized by TG, FTIR, SEM and N₂ adsorption-desorption, which showed that the BET surface area and pore volume of epoxy-activated supports increased with the addition of F127. The Candida Antarctica Lipase B (CALB) was immobilized on the resulting carrier by covalent link. The amounts of lipases immobilized were 10 mg/g support and 375 mg/g support on the unmodified silica (U-S) and modified silica (F-560-S), respectively. The immobilized lipases were examined as biocatalysts for transesterification of 1-phenethanol and vinyl acetate in nonaqueous medium. The effects of solvents and temperature on immobilized lipases were systematically investigated. The catalytic activity of the CALB immobilized on F-560-S was improved in various solvents, especially in polar solvents. The immobilized CALBs maintained high activity, while the control experiment using free lipase gave very low ester production in the temperature range of 0-60 ℃. Furthermore, thermal stability of CALB immobilized on F-560-S was improved as compared to the CALB on U-S was observed. The CALB immobilized on F-560-S exhibited high operational stability in organic media which still retained 88.3% of its original activity for 12 h consecutive 7 runs.

Key words: KH560, F127, silica Sol-Gel, enzyme immobilization

中图分类号:

宋聪, 喻晓蔚, 钱丹, 孙振忠, 江波. KH560修饰的溶胶-凝胶法二氧化硅载体用于脂肪酶的固定化[J]. 无机材料学报, 2016, 31(3): 311-316.

SONG Cong, YU Xiao-Wei, QIAN Dan, SUN Zhen-Zhong, JIANG Bo. Immobilization of Lipase on KH560 Modified Silica by Sol-Gel Process[J]. Journal of Inorganic Materials, 2016, 31(3): 311-316.

图/表 10

图1 560-S(A)和F-560-S(B)的TG曲线

Fig. 1 TG curves of 560-S (A) and F-560-S (B)

图2 KH560修饰前后SiO2的红外图谱

Fig. 2 FT-IR spectra of silica before and after KH560 modificationA: KH560; B: U-S; C: F-560-S

图3 不同放大倍率下改性前后二氧化硅的SEM照片

Fig. 3 Low (a, c) and high (b, d) magnification SEM images of different silica support(a) and (b): U-S; (c) and (d): F-560-S

表1 改性前后二氧化硅载体孔结构参数

Table 1 Surface properties of silica support before and after modification

Sample	A_BET /(m²·g^-1)	Pore Volume/(cm³·g^-1)	Pore size/nm
U-S	538	0.563	3.9
560-S	455	0.545	3.2
F-560-S	725	0.709	10.3

图4 改性前后SiO2对CALB固载量(mg/g support)的比较

Fig. 4 Lipase absorbed amount (mg/g support) of immobilized lipases on difference supports

式1 1-苯乙醇与醋酸乙烯酯的转酯化反应

Scheme 1 Transesterification of 1-phenylethanol and vinyl acetate

图5 不同CALB催化1-苯乙醇与醋酸乙烯酯反应转化率随时间的变化关系

Fig. 5 Conversion of transesterification of 1-phenethanol and vinyl acetate using CALBs as biocatalysts(■) CALB immobilized on F-560-S; (□) CALB immobilized on 560-S; (●) CALB immobilized on U-S; (★) free CALB

图6 固定化CALB的重复使用性

Fig. 6 Reusability of the immobilized CALBs(■) CALB immobilized on F-560-S; (□) CALB immobilized on 560-S; (●) CALB immobilized on U-S

图7 溶剂对固定化CALB催化活力的影响

Fig. 7 Effect of solvents on transesterification of the immobilized CALBs

图8 温度对固定化CALB催化活力的影响

Fig. 8 Effect of temperature on transesterification of the immobilized CALBs(■) CALB immobilized on F-560-S; (□) CALB immobilized on 560-S; (●) CALB immobilized on U-S; (★) free CALB

参考文献 25

[1]	SHARMA R, CHISTI Y, BANERJEE U C.Production, purification, characterization, and applications of lipases.Biotechnology Advances, 2001, 19(8): 627-662.
[2]	CHEN B, HU J, MILLER E M, et al. Candida antarctica lipase B chemically immobilized on epoxy-activated micro- and nanobeads: catalysts for polyester synthesis. Biomacromolecules, 2008, 9(2): 463-471.
[3]	ANTONIA P, VAN RANTWIJK F, SHELDON R A.Effective resolution of 1-phenyl ethanol by Candida antarctica lipase B catalysed acylation with vinyl acetate in protic ionic liquids (PILs).Green Chemistry, 2012, 14(6): 1584-1588.
[4]	WEI Y, XU J G, FEN Q W, et al. Encapsulation of enzymes in mesoporous host materials via the nonsurfactant-templated Sol- Gel process. Materials Letters, 2000, 44(1): 6-11.
[5]	KRAMER M, CRUZ J C, PFROMM P H, et al. Enantioselective transesterification by Candida antarctica Lipase B immobilized on fumed silica. Journal of Biotechnology, 2010, 150(1): 80-86.
[6]	JIANG Y, WANG Y, WANG H, et al. Facile immobilization of enzyme on three dimensionally ordered macroporous silica via a biomimetic coating. New Journal of Chemistry, 2015, 2(2): 978-984.
[7]	KATCHALSKI-KATZIR E, KRAEMER D M.Eupergit®C, a carrier for immobilization of enzymes of industrial potential.Journal of Molecular Catalysis B Enzymatic, 2000, 10: 157-176.
[8]	WANG F, NIE T T, SHAO L L, et al. Comparison of physical and covalent immobilization of lipase from Candida antarctica on polyamine microspheres of alkylamine matrix. Biocatalysis and Biotransformation, 2014, 32: 314-326.
[9]	LIU X Z, ZHENG L M, ZHANG Y Q, et al. Immobilization of laccase on epoxy-functionalized macroporous SiO2 block materials. Journal of Functional Materials, 2014, 45(20): 20028-20032.
[10]	MARYAM A, MEHDI M, RASHID B.Chemical amination of Rhizopus oryzae lipase for multipoint covalent immobilization on epoxy-functionalized supports: Modulation of stability and selec- tivity.Journal of Molecular Catalysis B: Enzymatic, 2015, 115: 128-134.
[11]	YE L Q, ZHANG X X, XIAO B, et al. Design and preparation of SiO2/SiO2-TiO2 antireflective coating with excellent environmental resistance. Chemical Journal of Chinese Universities, 2012, 33(5): 897-901.
[12]	HU T, YE L Q, LI W L, et al. Preparation and characterization of TiO2-SiO2/TiO2 two-layer antireflective coating photo-catalyst. Chinese Journal of Inorganic Chemistry, 2014, 30(8): 1778-1782.
[13]	MARION M B.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Analytical Biochemistry, 1976, 72: 248-254.
[14]	VOORT P V D, BENJELLOUN M, VANSANT E F. Rationalization of the synthesis of SBA-16: controlling the micro- and mesoporosity.Journal of Physical Chemistry B, 2002, 106(35): 9027-9032.
[15]	MATEO C, TORRES R, FERNANDEZ-LORENTE G, et al. Epoxy- amino groups: a new tool for improved immobilization of proteins by the epoxy method. Biomacromolecules, 2003, 4(3): 772-777.
[16]	TAKAHASHI H, LI B, SASAKI T, et al. Catalytic activity in organic solvents and stability of immobilized enzymes depend on the pore size and surface characteristics of mesoporous silica. Chemistry of Materials, 2000, 12(11): 3301-3305.
[17]	LEI J, FAN J, YU C, et al. Immobilization of enzymes in mesoporous materials: controlling the entrance to nanospace. Microporous & Mesoporous Materials, 2004, 73(3): 121-128.
[18]	MATEO C, PALOMO J M, FERNANDEZ-LORENTE G, et al. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme and Microbial Technology, 2007, 40(6): 1451-1463.
[19]	RAFAEL C R, CESAR A G, GIANDRA V, et al. Immobilization- stabilization of the lipase from thermomyces lanuginosus: critical role of chemical amination. Process Biochemistry, 2009, 44(9): 963-968.
[20]	LAN J N, NA W, WEI Q, et al. Immobilization of laccase on epoxy group functionalized periodic mesoporous organosilicas. Chemical Journal of Chinese Universities, 2010, 31(8): 1579-1584.
[21]	UPPENBERG J, HANSEN M T, PATKAR S, et al. The sequence, crystal structure determination and refinement of two crystal forms of lipase B from Candida antarctica. Structure, 1994, 2: 293-308.
[22]	ZHOU G, CHEN Y, YANG S.Comparative studies on catalytic properties of immobilized Candida rugosa lipase in ordered mesoporous rod-like silica and vesicle-like silica.Microporous and Mesoporous Materials, 2009, 119(1): 223-229.
[23]	XU K, GRIEBENOW K, KLIBANOV A M.Correlation between catalytic activity and secondary structure of subtilisin dissolved in organic solvents.Biotechnol Bioeng, 1997, 56(5): 485-491.
[24]	TANG S S, HU Y, YU D H, et al. Immobilization of Burkholderia Cepacia Lipase on functionalized ionic liquids modified mesoporous silica SBA-15. Chinese Journal of Catalysis, 2012, 33(9): 1565-1571.
[25]	LI X L, WANG D, XU Y, et al. Kinetic Resolution of 2-methylbutyric acid and its ester by esterification and hydrolysis with lipases. Chinese Journal of Catalysis, 2009, 30(9): 951-957.