黄曲霉毒素氧化酶的酶动力学研究  

Enzymatic kinetics of aflatoxin oxidase

作　　者：胡丽莎[1] 谢春芳[1,2] 刘大岭[1,2] 

机构地区：[1]暨南大学生物工程系/微生物技术研究所,广东广州510632 [2]广东省生物工程药物重点实验室,广东广州510632

出　　处：《暨南大学学报（自然科学与医学版）》2012年第5期496-500,共5页Journal of Jinan University(Natural Science & Medicine Edition)

基　　金：国家"863计划"(2007AA100605)

摘　　要：黄曲霉毒素氧化酶(AFO)是来自Armillariella tabescens的具有降解黄曲霉毒素作用的蛋白质酶.本实验室成功克隆和以原核系统表达了黄曲霉毒素氧化酶,前期的工作中用传统测量表观酶促反应速度的方法对AFO进行了酶动力学的研究,采用了在酶反应的结束点进行分析,因此不能够连续监测酶的反应过程,且实验过程较为繁琐,影响因素较多,对动力学常数的测量存在不够准确的可能.本实验使用等温滴定微量热技术对该酶的酶促动力学进行研究,该方法通过酶与底物的滴定实验直接监测酶反应过程中的热量变化而对酶动力学进行分析,滴定产生一个完整的Michaelis-Menten曲线,通过拟合分析测得黄曲霉毒素氧化酶对AFB1催化的米氏常数KmAFB1=3.34×10-7mol/L;酶催化ST反应的米氏常数KmST=1.06×10-7mol/L.对AFB1和ST酶转换系数别为KcatAFB1=2.7 min-1和KcatST=1.7 min-1.结合函分别为ΔHAFB1=-1.686×107 J/mol,ΔHST=-9.092×106 J/mol.与传统方法相比,ITC方法在酶动力学研究上具有简便、快速和准确的优点并具有普适性.Aflatoxin oxidase （AFO） is a protein enzyme derived from the ArmillarieUa tabeseens, and demonstrating aflatoxin degradation effect. AFO has been successfully cloned and expressed in a prokaryotic system in our previous studies. The enzymatic kinetics has been investigated by determination of the apparent reaction rate. The traditional analysis method was carried out by end point reaction, which was not suitable for the continuous monitor of enzyme reaction. The experimental process of traditional method was more cumbersome and could be influenced by many factors. Therefore, isothermal titration microcalo- ri-metry （ITC） was used to examine the enzymatic kinetics of AFO in this study through substrate titration and direct monitoring of enzyme reactions in the process of heat change. A complete Michaelis-Menten curve was produced by the titration, and measured by fitting analysis. The aflatoxin oxidase activity on AFB1 （Km^AFB1） and the ST Michaelis-Menten constant （Km^ST） were found at 3.34 × 10^-7 mol/L and 1.06 ×10^-7 mool/L, respectively. AFB1 and ST conversion factors were KcatAnn =2. 7 min-1 and KcatsT = 1.7 min-1 respectively. The combination letters were found at △H^AFB1 = - 1. 686 ×107 J/mol and △H^ST = -9. 092 × 106 J/mol, respectively. Compared with enzymatic kinetics demonstrates significant advantages, due universality. the traditional methods, application of ITC in to its simpleness, speediness, accuracy and universality.

关 键 词：黄曲霉毒素氧化酶 等温滴定微量热技术 酶动力学 

分 类 号：Q55[生物学—生物化学]