高纯度烟草过氧化物酶的酶学特性研究  

Research on Enzymology Characteristic of Tobacco Peroxidase With High Purity

作　　者：夏炳乐[1] 刘清亮[1] 李敏莉[1] 施春华[1] 徐小龙[1] 

机构地区：[1]中国科学技术大学化学系,安徽合肥230026

出　　处：《中国科学技术大学学报》2002年第5期601-606,共6页JUSTC

基　　金：国家烟草专卖局科研基金资助项目 (10 0 2 0 0 10 10 4 1)

摘　　要：从K32 6的烟叶中采用除植物褐色素新技术 ,经分离纯化 ,得到单一过氧化物酶Ⅰ (TOPⅠ ) ,该酶是一种以血红素为辅基的含铁蛋白质 .基质辅助激光解吸电离飞行时间质谱 (MALDITOF MS)测得TOPⅠ分子量为 2 1 888.5Da ,等电点pI为 3.5;酶反应的最适pH为 6 .0 ,最适温度为 4 5℃ ,70℃保温 1 5min残余活力为 50 %,具有良好的热稳定性 .动力学分析表明 :烟草中的过氧化物酶I与愈创木酚 (一元酚 )结合力最强 ,邻苯二酚 (二元酚 )次之 ,焦性没食子酸 (三元酚 )作用较小 .TOPⅠ的Km(愈创木酚 )为 0 .1 32mmol/L ;Km(H2 O2 )为 0 .598mmol/L .N-3 、CN-1、Fe3 + 、Fe2 + 和Sn2 对酶有较强烈的抑制作用 ,重金属离子Ag+ 、Pb2 + 、Cu2 + 对该酶的活性有激活作用 ,而NO-2 、Hg2 + 、Cr3 + 对酶活力无显著影响 .Peroxidase Ⅰ a kind of iron protein containing heme, was isolated and purified from fresh tobacco leaves of K326, Its molecular weight deter minated by Matrix Assisted Laser Desorption/Ionization Time Of Flight Mass Spectra is 21 888.5 Da and isoelectric point is 3.5. The optimum pH of the enzyme is pH 6.0 and the optimum temperature is 45℃. The residual activity of the enzyme is 50% after being kept for 15 minutes at 70 degrees centigrade, which shows that the enzyme has a favorable thermostability. Kinetic analyses show that the binding force of the peroxidase with the substrate of guaiacol(monohydric phenol)is the strongest, the catechol(dihydric phenol)the second, and the pyrogallol(triatomic phenol)the weakest, and that the binding constant ( Km) of the tobacco peroxidase is Km(guaiacol)=0.132 mmol/L and Km(H 2O 2)=0.598 mmol/L respectively. Among the chemical reagents determined, As 3- , S 2- , N - 3, CN -, Fe 2+ and Sn 2+ have a strong inhibition effect on the enzyme; on the contrary, the metal ions Ag +, Pb 2+ , Cu 2+ have the activated effect; moreover, NO - 2?Hg 2+ ?Cr 3+ have no obvious affect on the activity.

关 键 词：烟草 过氧化物酶 酶学特性 酶活力 含铁蛋白质 分子量 愈创木酚 

分 类 号：TS413[农业科学—烟草工业] Q946.5[生物学—植物学]