固定化脂肪酶的创制及其在乙酸肉桂酯无溶剂制备中的应用

doi:10.13560/j.cnki.biotech.bull.1985.2023-0028

生物技术通报 ›› 2023, Vol. 39 ›› Issue (9): 97-104.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0028

固定化脂肪酶的创制及其在乙酸肉桂酯无溶剂制备中的应用

陈金行¹(), 张逸², 张军涛¹, 未本美¹, 王宏勋¹(), 郑明明¹^,²^,³()

1.武汉轻工大学生命科学与技术学院，武汉 430023
2.中国农业科学院油料作物研究所，武汉 430062
3.湖州市菱湖新望化学有限公司，湖州 313018

收稿日期:2023-01-13 出版日期:2023-09-26 发布日期:2023-10-24
通讯作者: 王宏勋，男，博士，教授，研究方向：食品科学；E-mail: wanghongxunhust@163.com；
郑明明，男，博士，研究员，研究方向：油料脂质加工；E-mail: zhengmingming@caas.cn
作者简介:陈金行，男，硕士研究生，研究方向：酶法高效绿色合成风味酯；E-mail: chenjinhang2022@163.com
第一联系人：
张逸同为本文第一作者
基金资助:
国家重点研发项目(2021YFD2100303);湖州市南太湖精英计划领军型创新团队项目;国家自然科学基金项目(32272271);国家自然科学基金项目(31972038);湖北省自然科学基金(2021CFB209);中央公益类科研机构基础研究基金(1610172022014)

Preparation of Immobilized Lipase for the Solvent-free Synthesis of Cinnamyl Acetate

CHEN Jin-hang¹(), ZHANG Yi², ZHANG Jun-tao¹, WEI Ben-mei¹, WANG Hong-xun¹(), ZHENG Ming-ming¹^,²^,³()

1. School of Life Science and Technology, Wuhan Polytechnic University, Wuhan 430023
2. Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062
3. Linghu Xinwang Chemical, Huzhou 313018

Received:2023-01-13 Published:2023-09-26 Online:2023-10-24

摘要/Abstract

摘要：

采用模板法结合辛基（C8）表面修饰制备疏水有序介孔SiO₂载体（OMS-C₈），在此基础上制得固定化脂肪酶（CSL@OMS-C₈），成功应用于乙酸肉桂酯的无溶剂酶法制备。利用扫描电镜（SEM）、透射电镜（TEM）、N₂-吸附脱附、傅里叶红外（FTIR）对载体材料和固定化酶进行表征。结果显示，OMS具有整齐有序的介孔结构，比表面积达149.9 m²/g，平均孔径为15 nm。经过疏水改性后，接触角从20°提高到120°。优化获得了乙酸肉桂酯的最佳反应条件为温度50℃，肉桂醇与乙酸乙烯酯的摩尔比1∶5，固定化酶添加量2 g/L，反应时间2 h，转化率达到96.6%。催化剂经过5次重复使用肉桂醇的转化率仍能达到80%。

关键词: 固定化脂肪酶, 无溶剂, 风味酯, 乙酸肉桂酯, 酯交换

Abstract:

Hydrophobically ordered mesoporous SiO₂（OMS）carrier was prepared by template method combined with octyl surface modification, and immobilized lipase was prepared on this basis（CSL@OMS-C₈）, which was successfully applied to the solvent-free enzymatic preparation of cinnamyl acetate. Scanning electron microscope（SEM）, transmission electron microscopy（TEM）, nitrogen gas adsorption analyzer, Fourier transform infrared（FTIR）and contact angle goniometer were employed to characterize the carriers and immobilized enzyme. The results showed that OMS has ordered mesoporous morphology with a specific surface area of 149.9 m²/g and corresponding average pore diameter of 15 nm. The hydrophobicity of the material was greatly improved after hydrophobic modification with the contact angle increased from 20° to 120°. The optimal reaction conditions were obtained by optimizing the reaction conditions of enzymatic transesterification: reaction temperature was 50℃, molar ratio of cinnamyl alcohol to vinyl acetate was 1∶5, catalyst addition was 2 g/L, reaction time was 2 h, and the conversion rate reached 96.6%. Besides, the CSL@OMS-C₈ maintained 80% conversion for cinnamyl alcohol after five-cycle reuse.

Key words: immobilized lipase, solvent-free, flavor ester, cinnamyl acetate, transesterification

陈金行, 张逸, 张军涛, 未本美, 王宏勋, 郑明明. 固定化脂肪酶的创制及其在乙酸肉桂酯无溶剂制备中的应用[J]. 生物技术通报, 2023, 39(9): 97-104.

CHEN Jin-hang, ZHANG Yi, ZHANG Jun-tao, WEI Ben-mei, WANG Hong-xun, ZHENG Ming-ming. Preparation of Immobilized Lipase for the Solvent-free Synthesis of Cinnamyl Acetate[J]. Biotechnology Bulletin, 2023, 39(9): 97-104.

图/表 6

图1 脂肪酶的筛选不同字母表示各处理间差异显著（P<0.05）。下同

Fig. 1 Lipase screening Different letters indicate significant differences among treatments（P<0.05）. The same below

图2 材料OMS的显微镜观察结果 a：SEM结果；b：TEM结果；c：OMS、OMS-C8和CSL@OMS-C8的氮气吸脱附；d：OMS、OMS-C8和CSL@OMS-C8的孔径分布

Fig. 2 Microscopic observation results of material OMS a: SEM results. b: TEM results. c: N2 adsorption-desorption of OMS, OMS-C8 and CSL@OMS-C8. d: Pore size distribution curves of OMS, OMS-C8 and CSL@OMS-C8

图3 游离脂肪酶CSL、OMS、OMS-C8、CSL@OMS-C8的傅里叶红外结果

Fig. 3 FT-IR spectra of the free lipase CSL, OMS, OMS-C8 and CSL@OMS-C8

图4 OMS（A）和OMS-C8（B）的疏水测定

Fig. 4 Hydrophobic measurement for OMS（A）and OMS-C8（B）

图5 不同反应条件对乙酸肉桂酯合成的影响以及最优条件下固定化酶和游离酶对比 a：底物摩尔比对乙酸肉桂酯合成的影响；b：温度对乙酸肉桂酯合成的影响；c：固定化脂肪酶添加量对乙酸肉桂酯合成的影响；d：游离酶和固定化脂肪酶在最优条件下对比结果

Fig. 5 Effects of different reaction conditions on the synthesis of cinnamyl acetate and comparison of immobilized and free enzymes under optimal conditions a: Effects of substrate molar ratio on the synthesis of cinnamyl acetate; b: effects of temperature on the synthesis of cinnamyl acetate; c: effects of enzyme loading on the synthesis of cinnamyl acetate; d: comparison of free enzyme and immobilized lipase under the optimal conditions

图6 固定化脂肪酶CSL@OMS-C8循环使用结果

Fig. 6 Reusability of CSL@OMS-C8

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固定化脂肪酶的创制及其在乙酸肉桂酯无溶剂制备中的应用

Preparation of Immobilized Lipase for the Solvent-free Synthesis of Cinnamyl Acetate

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