超声功率对胰蛋白酶的酶活性与结构变化的影响

Effects of Ultrasonic Power on Enzyme Activity and Structure Change of Trypsin

为研究超声功率引起胰蛋白酶的结构变化与酶活力之间的关系,研究了不同的超声功率对胰蛋白酶活力、热稳定性、酶解反应动力学和热力学参数的影响,并采用紫外光谱、傅立叶变换红外光谱和荧光光谱来分析超声功率对胰蛋白酶结构的影响。结果表明:除功率300,350W外,胰蛋白酶活性与对照相比有显著提高。80℃处理时,在功率300,350W时的胰蛋白酶热稳定性有显著性提升。20℃酶解时75W处理的胰蛋白酶的反应速率最快。200W处理时胰蛋白酶的表观活化能最小,150,200W处理时胰蛋白酶的活化焓和活化熵与对照相比显著降低,75~250W处理时胰蛋白酶的吉布斯自由能与对照相比无明显差异。光谱分析表明,超声功率不改变胰蛋白酶的肽键含量,然而显著改变其二级结构。超声功率诱导胰蛋白酶变性过程中,在200W出现1个中间体,且中间体状态的胰蛋白酶的有序性高,具有高酶活和反应速率。

To study the relationship of changes induced by ultrasonic power in trypsin structure and activity. The effects of ultrasonic power on trypsin activity, thermal stability, kinetics and thermodynamic parameters were studied. The effects of ultrasonic power on the structure of trypsin were analyzed by UV, Fourier transform infrared spectroscopy and fluorescence spectroscopy. The results showed that, on the one hand, except 300 and 350 W, the enzyme activity was significantly higher than that of the control. At 80 °C, the thermal stability of trypsin at 300 W and 350 W was significant. When the trypsin was treated with ultrasonic power at 75 W and the enzyme hydrolysis temperature at 20 °C, the reaction rate was the fast. When the trypsin was treated with ultrasonic power at 200 W, the apparent energy of activation was the smallest. Enthalpy of activation and entropy of activation at 150 W and 200 W were significantly lower than the control. Gibbs free energy at 75-250 W was not significantly different from that of the control. On the other hand, spectral analysis showed that ultrasonic power did not alter the molecular configuration of trypsin, but significantly changed its secondary structure. In the process of ultrasonic power induced trypsin degeneration, one intermediate appeared at 200 W. And the intermediate state of the trypsin had high order, while high enzymatic activity and high response rate.