乙二胺四乙酸二酐对草酸脱羧酶的修饰改性

摘要

为提高草酸脱羧酶（Oxdc）在预防治疗泌尿系结石症方面的稳定性和应用性能，采用乙二胺四乙酸二酐（EDTAD）对Oxdc进行化学修饰，并初步优化了修饰条件：当修饰反应时间为8 h、EDTAD/Oxdc的摩尔比为50：1、反应 pH 为7.0、反应温度为37℃时，修饰率为71.91%，酶活回收率为75.42%。聚丙烯酰胺凝胶电泳（SDS-PAGE）和超高效液相色谱-质谱（UPLC-MS）联用分析证明Oxdc和EDTAD共价结合成功。紫外-可见吸收光谱（UV）和圆二色性光谱（CD）分析结果表明，修饰后Oxdc的空间结构发生了一定程度的改变。同时，部分酶学性质研究表明，经EDTAD修饰后的Oxdc较游离的Oxdc，最适pH向碱性方向偏移了约1.5个单位，耐热性和耐胰蛋白酶酶解的能力均显著增强。此外，修饰后的 Oxdc 在草酸钙上的吸附量明显提高。结果说明修饰后的Oxdc的稳定性显著增强，应用性能有所改善。%In order to improve the stability and application performance of oxalate decarboxylase (Oxdc) in the prevention and treatment of urinary calculi, chemical modification of Oxdc with ethylenediaminetetraacetic dianhydride (EDTAD) was investigated. The results of single-factor experiment showed that the extent of modification and the recovery rate of the enzymatic activity were 71.91% and 75.42%, respectively, when the reaction time was 8 h, the molar ratio of EDTAD/Oxdc was 50:1, pH 7.0, and the temperature was 37℃. The analysis results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS) indicated that Oxdc and EDTAD have been covalently bound. The ultraviolet-visible spectrum (UV) and circular dichroic (CD) measurement showed that the structure and conformation of Oxdc were tinily altered after modification by EDTAD. The enzymology changes of Oxdc were also analyzed, the results showed the optimum pH of EDTAD-modified Oxdc was shifted to the alkaline side about 1.5 unit and it had a higher thermostability. Moreover, through modification the adsorption capacity of Oxdc onto calcium oxalate monohydrate crystals was increased by 42.42%. These results suggested that the stability and application performance of Oxdc were significantly improved under this experiment.