Ma Guangzheng, Zhang Zihan, Chen Mei, Zhang Yifei, Nian Binbin, Hu Yi
State Key Laboratory of Materials-Oriented Chemical Engineering, School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing, China.
J Sci Food Agric. 2024 Mar 30;104(5):2669-2678. doi: 10.1002/jsfa.13152. Epub 2023 Dec 19.
The relative low stability, reusability and activity of enzymes made the industrial production of vitamin E succinate (VES) can only be performed with complex processes and high cost using chemical methods. To address these issues, in the present study, an ionic liquids (ILs) modification strategy was developed to improve the activity and stability of lipases in VES synthesis.
The results showed that the [1-butyl-3-methyl imidazole] [N-acetyl-l-proline] ILs modified Candida rugosa lipase (CRL) has the highest modification degree (48.28%), activity (774 U g ), thermostability and solvent tolerance in three selected modifiers. Additionally, after reaction condition optimization, the highest yield of VES can be improved to 95.18% at 45 °C for 15 h, which was significantly improved compared to some previous studies.
In the present study, a high-efficiency VES synthesis strategy was successfully developed via modification of lipase. Moreover, the mechanism by which ILs modification can enhance the activity and stability of lipase was investigated via both experimental and computational-aided methods. Molecular dynamics simulation suggested that ILs modification changed the geometry of Phe344 from flat to upright, which significantly reshaped and enhanced the size of substrate binding pocket of CRL. It is also agreement with our circular dichroism and fluorescence spectroscopy results, which suggested that the modification changed the secondary structure of CRL to a certain extent. The larger pocket also endowed the suitable binding pose of succinate, which made the hydrogen bonds between succinate and active site Ser209 become stronger, and thus improving the yield of VES. © 2023 Society of Chemical Industry.
酶的相对低稳定性、可重复使用性和活性使得维生素E琥珀酸酯（VES）的工业生产只能通过化学方法采用复杂工艺且成本高昂。为解决这些问题，在本研究中，开发了一种离子液体（ILs）修饰策略以提高脂肪酶在VES合成中的活性和稳定性。
结果表明，在三种选定的修饰剂中，[1-丁基-3-甲基咪唑][N-乙酰-L-脯氨酸]离子液体修饰的皱褶假丝酵母脂肪酶（CRL）具有最高的修饰度（48.28%）、活性（774 U g）、热稳定性和溶剂耐受性。此外，经过反应条件优化，在45°C下反应15小时，VES的最高产率可提高到95.18%，与之前的一些研究相比有显著提高。
在本研究中，通过脂肪酶修饰成功开发了一种高效的VES合成策略。此外，通过实验和计算辅助方法研究了离子液体修饰增强脂肪酶活性和稳定性的机制。分子动力学模拟表明，离子液体修饰使Phe344的几何形状从扁平变为直立，这显著重塑并增大了CRL底物结合口袋的尺寸。这也与我们的圆二色性和荧光光谱结果一致，表明修饰在一定程度上改变了CRL的二级结构。更大的口袋也赋予了琥珀酸合适的结合构象，使得琥珀酸与活性位点Ser209之间的氢键更强，从而提高了VES的产率。© 2023化学工业协会。
