西瓜鸟氨酸氨甲酰转移酶粗提条件优化及特性分析

Optimization on the Extraction Conditions and Properties of Ornithine Carbamoyltransferase from Watermelon

以西瓜叶片为材料,通过正交试验对影响西瓜OCT酶提取的缓冲液pH值、料液比、提取时间3个因素进行研究,优化提取条件。结果表明,西瓜鸟氨酸氨甲酰转移酶粗提的最佳条件为缓冲液pH8.5、料液比1∶5、提取时间4 h。酶学特性的结果表明:该酶的最适温度为30℃,最适pH为8.0,最适条件下鸟氨酸和氨甲酰磷酸的Km值分别为8.34、3.73 mmol/L,V_(max)分别为0.22、0.32μmol/(m L·min)。金属离子Ca^(2+)、Mg^(2+)和Mn^(2+)对该酶活力有激活作用,而Fe^(3+)和Cu^(2+)对该酶活力起抑制作用。西瓜OCT酶提取条件的优化提高了该酶粗提的效果,为进一步纯化该酶奠定基础;对该酶特性的研究有利于对西瓜瓜氨酸合成代谢及抗逆调控作用机理的研究。

Taking the watermelon leaves as the test materials, the p H of the extraction buffer solution, solid-liquid ratio, and extraction time were selected for orthogonal experiment to optimize the extraction process. The optimum conditions for the extraction of ornithine carbamoyltransferase from watermelon were pH8.5, solid-liquid ratio 1 ∶ 5,extraction time 4 h. Enzymatic characterization demonstrated that its optimum temperature and p H was 30 ℃ and 8.0, the Km for ornithine and carbamoyl phosphate value was 8.34 and 3.73 mmol/L, Vmax was 0.22 and 0.32 μmol/（m L·min）, respectively. Metal ions Ca^（2＋）, Mg^（2＋） and Mn^（2＋） promoted the enzyme activity, whereas Fe^（3＋） and Cu^（2＋） could inhibit its activity. The optimization on the extraction conditions of crude enzyme of watermelon OCT greatly improved the effect of the crude extraction, and laid the foundation for further purification of the enzyme.The features of the enzyme would be good for the study of watermelon citrulline synthetic metabolism and mechanism of resilience regulation.