Abstract:

It is of great value to improve the enzymatic properties including selectivity by chemical modification at the same time. Four kinds of ionic liquids, prepared from N-acetyl-proline as chiral source, were used to modify porcine pancreatic lipase (PPL). Modification degree was determined by 2,4,6-trinitrobenzene sulfonic acid (TNBS) method, and enzyme activity was determined by the hydrolysis of 4-nitrophenyl palmitate (pNPP). As expected, the catalytic properties of PPL were changed obviously after modification. The thermal stability, organic solvent tolerance, adaptability to temperature and pH changes, even enantio selectivity of all the modified enzymes was improved simultaneously. Research results also showed that the composition and configuration of ionic liquids (Ils)had an important influence on the enzymatic properties of modified enzymes. The PPL modified by [BMIM] [N-AC-L-pro] showed obvious better catalytic performance than that modified by [BMIM] [N-AC-D-pro], while the catalytic properties of PPL modified by [N-AC-L-pro] [Cl] and [N-AC-D-pro] [Cl] only showed slight difference. PPL modified by [N-AC-L-pro] [Cl] showed the highest activity, increasing by 2.5 folds as much as the original enzyme. Overall, PPL modified by [BMIM] [N-AC-L-pro] demonstrated the best enzymatic performance: the hydrolysis activity was increased by 1.0 time, the optimum temperature was raised to 55℃, the thermostability was improved by 2.6 times（50℃，2.5 h）, the enantio selectivity was increased by 1.5 times, the stability in the strong polar protonic solvent of methanol was enhanced by 1.3 times, and also showed better tolerance of temperature and pH. The fluorescence spectroscopy and circular dichroism spectroscopy (CD) characterizations confirmed that chiral proline ionic liquids were grafted onto the surface of PPL successfully, resulting in the conformation change of PPLs, which led to the improvements of the catalytic performance of modified PPLs. Through this study, chiral ionic liquids have proven to be a new and effective modifier for various catalytic properties of enzyme molecular modification and selectivity enhancement.

Key words: ionic liquids, chiral amino acid, enzyme, protein stability, chemical modification, enantio selectivity

摘要：

通过化学修饰同时提高脂肪酶包括选择性在内的酶学性能具有重要意义。以N-乙酰-脯氨酸作为手性源制备的四种离子液体对猪胰脂肪酶（PPL）进行化学修饰，通过三硝基苯磺酸法（TNBS）测定了修饰度，采用水解4-硝基苯酚棕榈酸酯的反应测定酶活。正如预期的一样，所有修饰酶的酶学性能均得到显著改善。其中修饰酶的热稳定性、有机溶剂耐受性、不同温度和pH下的耐受性甚至对映体选择性均得到同时提升。结果也表明，离子液体的组成及构型对修饰酶的酶学性能有较大程度的影响，经过[BMIM][N-AC-L-pro]修饰后的PPL各项催化性能明显优于[BMIM][N-AC-D-pro]，而经过[N-AC-L-pro][Cl]和[N-AC-D-pro][Cl]修饰的PPL催化性能差异较小。经过[N-AC-L-pro][Cl]修饰后PPL展现出最高的酶活，达到原酶的2.5倍。经过[BMIM][N-AC-L-pro]修饰后的PPL展现出最佳的酶学性能，在酶活提高了1.0倍的同时，热稳定性提高了2.6倍（50℃，2.5 h），对映体选择性提高了1.5倍，在强极性质子溶剂甲醇中的稳定性也提升了1.3倍，且在不同温度、pH下也展现出了较好的稳定性与耐受性。通过荧光光谱和圆二色谱表征证明，手性离子液体成功嫁接到PPL的表面，引起脂肪酶构象的改变，从而促使酶的催化性能发生改善。通过该研究，手性离子液体被证实为是一种可用于酶分子改造并提升选择性在内各项催化性能的新型且有效的修饰剂。

关键词: 离子液体, 手性氨基酸, 酶, 蛋白质稳定性, 化学修饰, 对映体选择性