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对组氨酸标签肝素酶I(His-Hep I,EC 4.2.2.7)的热失活机制进行了研究。针对短暂低温处理可以使热失活His-Hep I酶活部分恢复及添加二硫苏糖醇(DTr)使其热稳定性提高的现象,利用荧光探针法研究了失活过程His-Hep I构象变化,证明了该酶构象存在可逆转变行为。为进一步明晰His-Hep I的热失活机制,假设该酶热失活的主要途径包括去折叠及形成聚集体,并以此为基础建立模型进行拟合,模型与实验值吻合良好,表明假设的合理性。根据模型计算的活化能为E_r=100.217 kJ/mol、E_(ir)=7.857 kJ/mol和E_d=77.062 kJ/mol,此数据从一定程度上解释了冷处理为何能使His—Hep I部分恢复活性。进一步研究表明,任何能够抑制这两种途径发生的措施对于提高His-Hep I热稳定性都是有效的。
The mechanism of heat inactivation of histidine-tagged Heparinase I (His-Hep I, EC 4.2.2.7) was studied. Aiming at the transient low-temperature treatment, the heat-inactivation part of His-Hep I can be partially recovered and the dithiothreitol (DTr) can be added to improve its thermal stability. The inactivation His-Hep I Conformational changes, the reversal of the conformation of the enzyme that there is a reversible behavior. To further clarify the mechanism of heat inactivation of His-Hep I, we hypothesized that the main way of thermal inactivation of this enzyme includes unfolding and forming aggregates, and using this as a basis for modeling, the model is in good agreement with the experimental data The rationality. The activation energies calculated according to the model are E_r = 100.217 kJ / mol, E_ (ir) = 7.857 kJ / mol and E_d = 77.062 kJ / mol. This data to some extent explains why cold treatment partially restored the activity of His-Hep I . Further studies show that any measures that can inhibit the occurrence of both of these pathways are effective for increasing the thermostability of His-Hep I.