手性脯氨酸类离子液体化学修饰猪胰脂肪酶催化性能研究

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.