辣根过氧化物酶的热稳定剂

Stabilizers of horseradish peroxidase

摘要:

保持酶的天然状态和高催化特性具有重要的意义。本研究筛选了辣根过氧化物酶(HRP)的稳定剂并研究了其作用机制。结果发现硫酸镁和明胶能够显著提高HRP的热稳定性, 并且两者具有协同作用。在硫酸镁和明胶组成的酶稳定剂存在的条件下, HRP在50oC保温80 h后仍能保持89%的活性, 常温下存放90 d后可保持57%的活性, 而未加稳定剂的对照样品中HRP的残留活性分别为6%和小于1%。通过对HRP的Soret带吸收光谱, 色氨酸内源荧光, ANS荧光进行分析, 揭示酶稳定剂可以明显降低在加热条件下HRP的变性程度, 从而维持较为稳定的天然构象。

Abstract:

Keeping an enzyme in its native form with high catalytic activity is of great significance. In the present study, thermal stabilizers of horseradish peroxidase (HRP) were screened. The results indicated that thermal stability of HRP was enhanced by magnesium sulphate and gelatin. A synergic effect of magnesium sulphate and gelatin was observed. In the presence of the stabilizer, the enzymatic activity of HRP remained 89% after kept for 80 h at 50oC and 57% for 90 days at room temperature. Thermal alterations of HRP structure in the absence and presence of the stabilizers were explored by using UV absorption spectra at 402 nm (Soret band), intrinsic fluorescence and 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescence. The results suggested that magnesium sulphate and gelatin attenuated the extent of unfolding of HRP and therefore the native enzyme structure was stabilized.