甜菜碱类离子液体化学修饰猪胰脂肪酶提升其酶学性能

The structure of porcine pancreatic lipase (PPL) was selected as the research object, and betaine ionic liquids with different chain lengths and different anions were designed and synthesized as modifiers to chemically modify PPL. The protein concentration was determined by the BCA method, the degree of modification was determined by the trinitrobenzene sulfonic (TNBS) method, and the enzymatic activity was determined by the p-nitrophenol palmitate (p-NPP) method. The changes in enzymatic properties such as the optimum temperature, optimum pH, thermal stability, organic solvent tolerance and the kinetics of the native and modified PPLs were tested. Results showed that the chain length and anion of modifiers had a certain influence on the modification effect. The PPL modified by [BetaineC₁₆][H₂PO₄] presented the best enzymatic performance, the enzymatic activity of which was 3.4 times that of the native PPL, the enzymatic activity after stored at 60°C for 45min was 8.7 times that of the native PPL, the stability was increased by 1.7 times after stored in strongly polar aprotic solvent dimethyl sulfoxide for 2h, and the selectivity of enantiomers in the catalytic reaction of α-phenyl ethanol was increased by 2.0 times. In addition, the temperature tolerance and pH tolerance of modified PPLs had been significantly improved. The kinetic test results also showed that modified PPLs had a higher substrate affinity so that it had higher activity. The results of fluorescence spectroscopy showed that the introduction of modifiers had a certain impact on the fluorescent group of PPLs. Fluorescence intensity decreased as the modification degree increased. The results of circular dichroism showed that the α-helix content of the modified PPLs decreased while the β-sheet content increased, which improved the activity and stability of PPLs. This experiment confirmed that betaine ionic liquids as modifiers could simultaneously improve various enzymatic properties of lipase, and expand a new type of modifier with good modification effects for chemical modification of enzymes.