重组肝素酶I热失活动力学模型的建立与应用

对组氨酸标签肝素酶I（His．HepI，Ec4．2．2．7）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．HepI酶活部分恢复及添加二硫苏糖醇（D1Tr）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．HepI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．HepI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Er=100．217kJ／mol、Eir=7．857kJ／mol和Ed=77．062kJ／mol，此数据从一定程度上解释了冷处理为何能使His．HepI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．HepI热稳定性都是有效的。

Kinetic modelling of thermal inactivation of His-Hep I and its application

The thermal inactivation found that lowering the temperature mechanism of his-tagged heparinase I （ His-Hep I, EC 4.2.2.7） was studied. It was could partially recover the enzymatic activity of the thermal inactivated enzyme and adding DTT could improve the enzyme stability. Therefore, fluorescence probe technique was adopted to monitor the conformation change of the enzyme during the thermal inactivation process. The results indicated that the process was reversible. Based on these phenomena, it was hypothesized that temperature induced protein unfolding and intermolecular disulfide bond caused enzyme aggregation were responsible for the enzyme inactivation. Hence, a kinetic model was proposed to explain the behaviour of the enzyme with temperature. The experimental and stimulated results fitted well which showed that the hypothesis should be reasonable. The activation energies of unfolding （E,） , refolding （Eir） and aggregation （Ed） were calculated to be ： 100.217 kJ/mol, 7. 857 kJ/mol and 77. 062 kJ/mol, respectively, which partly explained the temperature-dependent recovery of enzyme activity. Further studies showed that any measure inhibited the enzyme unfolding and aggregation was effective to improve the stability of His-Hep I.