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

• 生物化学工程与技术 • 上一篇    下一篇

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. 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 E-mail:wanglihui2008@126.com;zhengxiaobing@hebut.edu.cn
  • 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 ZHENG1,3(),Yanjun JIANG1,3,Jing GAO1   

  1. 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 Published:2021-09-05 Online:2021-09-05
  • Contact: Xiaobing ZHENG E-mail:wanglihui2008@126.com;zhengxiaobing@hebut.edu.cn

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

图1

LA和醇的酯化反应"

图2

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

图3

MMOSNs的XPS表征"

图4

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

图5

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

图6

固定化酶激光共聚焦显微镜图片"

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)"

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"

图9

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

图10

游离酶、CALB@MMSNs和CALB@MMOSNs的有机溶剂耐受性"

图11

游离酶、CALB@MMSNs和CALB@MMOSNs的长时间储存稳定性"

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"

图13

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

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