核壳结构磁性树枝状纤维形有机硅固定化脂肪酶制备及其应用

doi:10.11949/0438-1157.20210283

化工学报 ›› 2021, Vol. 72 ›› Issue (9): 4861-4871.DOI: 10.11949/0438-1157.20210283

核壳结构磁性树枝状纤维形有机硅固定化脂肪酶制备及其应用

王立晖¹(),刘焕¹,李赫宇²,郑晓冰^1,³(),姜艳军^1,³,高静¹

^1.河北工业大学化工学院，天津 300130
^2.天津益倍生物科技集团有限公司，天津 300457
^3.化工节能过程集成与资源利用国家地方联合工程实验室，河北工业大学，天津 300130

收稿日期:2021-02-23 修回日期:2021-05-05 出版日期:2021-09-05 发布日期:2021-09-05
通讯作者: 郑晓冰
作者简介:王立晖(1981—)，男，博士研究生，wanglihui2008@126.com
基金资助:
国家自然科学基金项目(21901058);河北省自然科学基金项目(B2019202216)

Preparation and application of core-shell hydrophobic magnetic dendritic fibrous organosilica immobilized lipase

Lihui WANG¹(),Huan LIU¹,Heyu LI²,Xiaobing ZHENG^1,³(),Yanjun JIANG^1,³,Jing GAO¹

^1.School of Chemical Engineering and Technology, Hebei University of Technology, Tianjin 300130, China
^2.Tianjin UBasio Biotechnology Group Co. , Ltd. , Tianjin 300457, China
^3.National Local Joint Engineering Laboratory for Energy Conservation of Chemical Process Integration and Resources Utilization, Hebei University of Technology, Tianjin 300130, China

Received:2021-02-23 Revised:2021-05-05 Online:2021-09-05 Published:2021-09-05
Contact: Xiaobing ZHENG

摘要/Abstract

摘要：

为了提升脂肪酶的稳定性并构建新型固定化酶催化体系，利用改进的Winsor Ⅲ微乳液双连续相体系合成了超顺磁性Fe₃O₄内核和树枝状纤维形氧化硅外壳的核壳结构磁性有机硅纳米粒子（MMOSNs），用于固定化南极假丝酵母脂肪酶B（CALB）。优化条件后CALB负载量为177.49 mg/g，比水解活性为27390 U/g。磁性有机硅通过与CLAB分子之间疏水相互作用及表面孔道结构，可有效激活CALB的界面活性并保护活性构象免受破坏，比游离酶和磁性无机硅固定化酶表现出更好的活性和稳定性。除此之外，将CALB@MMOSNs用于催化乙酰丙酸与十二醇的酯化反应最高转化率为85.05%，重复使用9次后仍保留68.94%转化率，而商业化N435只保留29.83%。证明疏水性磁性核壳结构有机硅是固定化CALB的良好载体，可有效扩展脂肪酶的工业应用。

关键词: 树枝状纤维形氧化硅粒子, 磁性核壳有机硅, 疏水载体, 脂肪酶固定化, 乙酰丙酸, 酯化反应

Abstract:

In order to improve the stability of lipase and construct a new immobilized lipase catalytic system, the core-shell hydrophobic magnetic organosilica nanoparticles (MMOSNs) with superparamagnetic Fe₃O₄ core and dendritic fibrous silica shell were synthesized through an improved Winsor Ⅲ microemulsion dual continuous phase system, and the obtained MMOSNs were employed as support for the immobilization of Candida antarctica lipase B (CALB). After the optimized conditions, the CALB load was 177.49 mg/g, and the specific hydrolysis activity was 27390 U/g. The CALB@MMOSNs could effectively activate the interfacial activity of CALB and protect the active conformation from external environmental harm through hydrophobic interaction with CALB molecules and its surface pore structure, showing better activity and stability than free enzyme and magnetic inorganic silicon immobilized CALB. In addition, CALB@MMOSNs could catalyze the esterification of levulinic acid with lauryl alcohol, and the highest conversion rate reached 85.05%. After repeating the reaction for 9 cycles, the conversion rate remained 68.94%, while the commercial N435 retained only 29.83%. These results indicated that the core-shell hydrophobic magnetic organosilicon is a good support for immobilized CALB, which can effectively expand the application of lipase in industry.

Key words: dendritic fibrous silica nanoparticles, magnetic core-shell organosilicon, hydrophobic support, lipase immobilization, levulinic acid, esterification

中图分类号:

Q 814.2

王立晖, 刘焕, 李赫宇, 郑晓冰, 姜艳军, 高静. 核壳结构磁性树枝状纤维形有机硅固定化脂肪酶制备及其应用[J]. 化工学报, 2021, 72(9): 4861-4871.

Lihui WANG, Huan LIU, Heyu LI, Xiaobing ZHENG, Yanjun JIANG, Jing GAO. Preparation and application of core-shell hydrophobic magnetic dendritic fibrous organosilica immobilized lipase[J]. CIESC Journal, 2021, 72(9): 4861-4871.

图/表 13

图1 LA和醇的酯化反应

Fig.1 The esterification reaction of LA and alcohol

图2 Fe3O4 (a)、MMOSNs (c)和MMSNs (e)的SEM图片；Fe3O4(b)、MMOSNs (d)和MMSNs (f)的TEM图片

Fig.2 SEM images of Fe3O4 (a), MMOSNs (c) and MMSNs (e); TEM images of Fe3O4 (b), MMOSNs (d) and MMSNs (f)

图3 MMOSNs的XPS表征

Fig.3 XPS characterization of the MMOSNs

图4 MMOSNs、CALB@MMOSNs的N2吸附-脱附曲线(a)和孔径分布(b)

Fig.4 Nitrogen adsorption-desorption isotherms (a) and pore size distribution profile (b) of the MMOSNs and CALB@MMOSNs

图5 Fe3O4、MMOSNs和CALB@MMOSNs的磁滞回曲线（1 Oe=79.5775 A/m）

Fig.5 The hysteresis loops of Fe3O4, MMOSNs and CALB@MMOSNs

图6 固定化酶激光共聚焦显微镜图片

Fig.6 CLSM image of the FITC-labeled CALB@MMOSNs

图7 MMOSNs在不同酶浓度下的吸附进程曲线(a)；初始酶浓度对载体的蛋白负载量及CALB@MMOSNs酶活的影响(b)

Fig.7 Adsorption process of CALB at different concentrations on the MMOSNs (a) and effect of initial lipase concentration on CALB loading and specific activity of CALB@MMOSNs(b)

图8 游离酶、CALB@MMSNs和CALB@MMOSNs在pH=4.0 (a)和pH=10.0 (b)的缓冲溶液中的稳定性

Fig.8 The stabilities of free lipase, CALB@MMSNs and CALB@MMOSNs at pH=4.0 (a) and pH=10.0 (b)

图9 游离酶、CALB@MMOSNs和CALB@MMSNs在60℃环己烷中的热稳定性

Fig.9 Thermal stabilities of free lipase, CALB@MMOSNs and CALB@MMSNs in cyclohexane at 60℃

图10 游离酶、CALB@MMSNs和CALB@MMOSNs的有机溶剂耐受性

Fig.10 The effect of organic solvents on the activity of free lipase, CALB@MMSNs and CALB@MMOSNs

图11 游离酶、CALB@MMSNs和CALB@MMOSNs的长时间储存稳定性

Fig.11 The storage stabilities of free lipase, CALB@MMSNs and CALB@MMOSNs

图12 温度(a)、LA/十二醇摩尔比(b)和时间(c)对LA和十二醇酯化反应的影响

Fig.12 Effect of temperature (a), LA/n-lauryl alcohol molar ratio (b) and time (c) on esterification reaction

图13 CALB@MMOSNs、CALB@MMSNs和N435在酯化反应中的重复使用性

Fig.13 Reusability of CALB@MMOSNs, CALB@MMSNs and N435 for esterification reaction

参考文献 44

1	Shi J, Wu Y, Zhang S, et al. Bioinspired construction of multi-enzyme catalytic systems[J]. Chemical Society Reviews, 2018, 47(12): 4295-4313.
2	Tamura T, Hamachi I. Chemistry for covalent modification of endogenous/native proteins: from test tubes to complex biological systems[J]. Journal of the American Chemical Society, 2019, 141(7): 2782-2799.
3	Liu Z H, Wang K, Chen Y, et al. Third-generation biorefineries as the means to produce fuels and chemicals from CO₂[J]. Nature Catalysis, 2020, 3(3): 274-288.
4	Benjamin S, Pandey A. Candida rugosa lipases: molecular biology and versatility in biotechnology[J]. Yeast, 1998, 14(12): 1069-1087.
5	Jakovetić Tanasković S, Jokić B, Grbavčić S, et al. Immobilization of Candida antarctica lipase B on Kaolin and its application in synthesis of lipophilic antioxidants[J]. Applied Clay Science, 2017, 135: 103-111.
6	Adlercreutz P. Immobilisation and application of lipases in organic media[J]. Chemical Society Reviews, 2013, 42(15): 6406-6436.
7	Ansorge-Schumacher M B, Thum O. Immobilised lipases in the cosmetics industry[J]. Chemical Society Reviews, 2013, 42(15): 6475-6490.
8	Kuwahara Y, Yamanishi T, Kamegawa T, et al. Activity, recyclability, and stability of lipases immobilized on oil-filled spherical silica nanoparticles with different silica shell structures[J]. ChemCatChem, 2013, 5(8): 2527-2536.
9	Rodrigues R C, Ortiz C, Berenguer-Murcia Á, et al. Modifying enzyme activity and selectivity by immobilization[J]. Chemical Society Reviews, 2013, 42(15): 6290-6307.
10	Rueda N, dos Santos J C S, Ortiz C, et al. Chemical modification in the design of immobilized enzyme biocatalysts: drawbacks and opportunities[J]. The Chemical Record, 2016, 16(3): 1436-1455.
11	Kalantari M, Kazemeini M, Tabandeh F, et al. Lipase immobilisation on magnetic silica nanocomposite particles: effects of the silica structure on properties of the immobilised enzyme[J]. Journal of Materials Chemistry, 2012, 22(17): 8385-8393.
12	Yue Q, Sun J G, Kang Y J, et al. Advances in the interfacial assembly of mesoporous silica on magnetite particles[J]. Angewandte Chemie, 2020, 132(37): 15936-15949.
13	Lei Z L, Ren N, Li Y L, et al. Fe₃O₄/SiO₂-g-PSStNa polymer nanocomposite microspheres (PNCMs) from a surface-initiated atom transfer radical polymerization (SI-ATRP) approach for pectinase immobilization[J]. Journal of Agricultural and Food Chemistry, 2009, 57(4): 1544-1549.
14	Hou C, Wang Y, Zhu H, et al. Formulation of robust organic-inorganic hybrid magnetic microcapsules through hard-template mediated method for efficient enzyme immobilization[J]. Journal of Materials Chemistry B, 2015, 3(14): 2883-2891.
15	Chen Z, Xu W, Jin L, et al. Synthesis of amine functionalized Fe₃O₄@C nanoparticles for lipase immobilization[J]. Journal of Materials Chemistry A, 2014, 2: 18339-18344.
16	Zhao M, Zhang X, Deng C. Rational synthesis of novel recyclable Fe₃O₄@MOF nanocomposites for enzymatic digestion[J]. Chemical Communications (Cambridge, England), 2015, 51(38): 8116-8119.
17	Polshettiwar V, Cha D, Zhang X X, et al. High-surface-area silica nanospheres (KCC-1) with a fibrous morphology[J]. Angewandte Chemie, 2010, 122(50): 9846-9850.
18	Moon D S, Lee J K. Tunable synthesis of hierarchical mesoporous silica nanoparticles with radial wrinkle structure[J]. Langmuir, 2012, 28(33): 12341-12347.
19	Moon D S, Lee J K. Formation of wrinkled silica mesostructures based on the phase behavior of pseudoternary systems[J]. Langmuir, 2014, 30(51): 15574-15580.
20	Schmid R D, Verger R. Lipases: interfacial enzymes with attractive applications[J]. Angewandte Chemie International Edition, 1998, 37(12): 1608-1633.
21	Verger R. 'Interfacial activation' of lipases: facts and artifacts[J]. Trends in Biotechnology, 1997, 15(1): 32-38.
22	Brzozowski A M, Derewenda U, Derewenda Z S, et al. A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex[J]. Nature, 1991, 351(6326): 491-494.
23	Rodrigues R C, Virgen-Ortíz J J, dos Santos J C S, et al. Immobilization of lipases on hydrophobic supports: immobilization mechanism, advantages, problems, and solutions[J]. Biotechnology Advances, 2019, 37(5): 746-770.
24	Gao J, Kong W X, Zhou L Y, et al. Monodisperse core-shell magnetic organosilica nanoflowers with radial wrinkle for lipase immobilization[J]. Chemical Engineering Journal, 2017, 309: 70-79.
25	Kalantari M, Yu M, Yang Y, et al. Tailoring mesoporous-silica nanoparticles for robust immobilization of lipase and biocatalysis[J]. Nano Research, 2017, 10(2): 605-617.
26	Bilal M, Zhao Y P, Rasheed T, et al. Magnetic nanoparticles as versatile carriers for enzymes immobilization: a review[J]. International Journal of Biological Macromolecules, 2018, 120: 2530-2544.
27	Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry, 1976, 72(1/2): 248-254.
28	Bouhrara M, Ranga C, Fihri A, et al. Nitridated fibrous silica (KCC-1) as a sustainable solid base nanocatalyst[J]. ACS Sustainable Chemistry & Engineering, 2013, 1(9): 1192-1199.
29	Zhou L Y, He Y, Ma L, et al. Conversion of levulinic acid into alkyl levulinates: using lipase immobilized on meso-molding three-dimensional macroporous organosilica as catalyst[J]. Bioresource Technology, 2018, 247: 568-575.
30	Jiang Y J, Liu H, Wang L H, et al. Virus-like organosilica nanoparticles for lipase immobilization: characterization and biocatalytic applications[J]. Biochemical Engineering Journal, 2019, 144: 125-134.
31	Linsha V, Aboo Shuhailath K, MA·hesh K V, et al. Biocatalytic conversion efficiency of steapsin lipase immobilized on hierarchically porous biomorphic aerogel supports[J]. ACS Sustainable Chemistry & Engineering, 2016, 4(9): 4692-4703.
32	Ali Z, Tian L, Zhang B, et al. Synthesis of paramagnetic dendritic silica nanomaterials with fibrous pore structure (Fe₃O₄@ KCC-1) and their application in immobilization of lipase from Candida rugosa with enhanced catalytic activity and stability[J]. New Journal of Chemistry, 2017, 41(16): 8222-8231.
33	Shao B B, Liu Z F, Zeng G M, et al. Immobilization of laccase on hollow mesoporous carbon nanospheres: noteworthy immobilization, excellent stability and efficacious for antibiotic contaminants removal[J]. Journal of Hazardous Materials, 2019, 362: 318-326.
34	Tong S M, Zhu L L, Wang X N, et al. Optimization of cephalosporin C acylase immobilization[J]. E3S Web of Conferences, 2019, 78: 02003.
35	Al-Lolage F, Bartlett P N, Gounel S, et al. Site-directed immobilization of bilirubin oxidase for electrocatalytic oxygen reduction[J]. ACS Catalysis, 2019, 9(3): 2068-2078.
36	Liu J Y, Liu Y, Jin D X, et al. Immobilization of trypsin onto large-pore mesoporous silica and optimization enzyme activity via response surface methodology[J]. Solid State Sciences, 2019, 89: 15-24.
37	Shikha S, Thakur K G, Bhattacharyya M S. Facile fabrication of lipase to amine functionalized gold nanoparticles to enhance stability and activity[J]. RSC Advances, 2017, 7(68): 42845-42855.
38	Sharma S, Kanwar S S. Organic solvent tolerant lipases and applications[J]. The Scientific World Journal, 2014, 2014: 625258.
39	Jiang Y J, Sun W Y, Zhou L Y, et al. Improved performance of lipase immobilized on tannic acid-templated mesoporous silica nanoparticles[J]. Applied Biochemistry and Biotechnology, 2016, 179(7): 1155-1169.
40	Gao J, Wang Y, Du Y J, et al. Construction of biocatalytic colloidosome using lipase-containing dendritic mesoporous silica nanospheres for enhanced enzyme catalysis[J]. Chemical Engineering Journal, 2017, 317: 175-186.
41	Jeong H, Jang S K, Hong C Y, et al. Levulinic acid production by two-step acid-catalyzed treatment of Quercus mongolica using dilute sulfuric acid[J]. Bioresource Technology, 2017, 225: 183-190.
42	Xie W L, Zang X Z. Covalent immobilization of lipase onto aminopropyl-functionalized hydroxyapatite-encapsulated-γ-Fe₂O₃ nanoparticles: a magnetic biocatalyst for interesterification of soybean oil[J]. Food Chemistry, 2017, 227: 397-403.
43	Poppe J K, Garcia-Galan C, Matte C R, et al. Optimization of synthesis of fatty acid methyl esters catalyzed by lipase B from Candida antarctica immobilized on hydrophobic supports[J]. Journal of Molecular Catalysis B: Enzymatic, 2013, 94: 51-56.
44	Lee A, Chaibakhsh N, Rahman M B A, et al. Optimized enzymatic synthesis of levulinate ester in solvent-free system[J]. Industrial Crops and Products, 2010, 32(3): 246-251.

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核壳结构磁性树枝状纤维形有机硅固定化脂肪酶制备及其应用

Preparation and application of core-shell hydrophobic magnetic dendritic fibrous organosilica immobilized lipase

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