保护剂对胆固醇氧化酶稳定性机理的研究

Study on the Effect of Protective Additives on Stability Mechanism of Cholesterol Oxidase

利用荧光光谱、圆二色性光谱及凝胶电泳等方法,研究液态胆固醇氧化酶失活机理。根据失活机理,选择添加两种不同保护剂提高酶稳定性,研究提高酶稳定性的机理。37℃下,添加15%甘油的液态酶稳定性较佳,45 h后酶活保留率提高58%,核黄素对酶基本无保护作用。经研究分析,甘油主要通过有效抑制分子间聚集,维持二级结构,增强酶稳定性;核黄素不能抑制酶自身聚集,对酶保护作用不明显。

An effective method to keep the stability of cholesterol oxidase （COD） was provided by studying on stability mechanism of COD. Deactivation mechanism of COD was analyzed by fluorescence spectra, circular dichroism spectra and SDS-PAGE. We added two additives into the preparation of COD and stabilization mechanism was studied. The results indicated that 15% glycerin was selected as the best protector at 37 ℃ and the remained enzyme activity was increased by 58% after 45 hours. Riboflavin had no significant effect on enzyme stability.A mechanism for the glycerin effect on inhibiting protein aggregation,protecting protein secondary structure and maintaining high activity was proposed. It also showed that riboflavin had no protective effects against protein aggregation.