球形红细菌生物转化2,4-二硝基甲苯及硝基还原酶性质

Biotransformation of 2,4-Dinitrotoluene and Properties of Nitroreductase by Rhodobacter Sphaeroides

为了探索球形红细菌生物转化2,4-二硝基甲苯(2,4-DNT)的中间产物变化趋势及硝基还原酶性质,研究了不同氮源组合对球形红细菌生物转化2,4-DNT的效率及球形红细菌细胞生长的影响,测定了2,4-DNT及2,4-DNT生物转化产物在不同时间的浓度变化,用非变性聚丙烯酰胺凝胶电泳(native-PAGE)法分析了硝基还原酶的同工酶谱,测得了硝基还原酶的动力学参数。结果表明,球形红细菌转化2,4-DNT的最适组合氮源为(NH_4)_2SO_4/酵母膏;球形红细菌能够高效还原2,4-DNT为4-氨基-2-硝基甲苯(4A2NT)和2-氨基-4-硝基甲苯(2A4NT),但4A2NT的含量远大于2A4NT的含量,它们没有进一步还原为2,4-二氨基甲苯(2,4-DAT)。当2,4-DNT初始浓度为20、40和60 mg·L^(-1)时,对球形红细菌产生硝基还原酶活力有明显的促进作用,当2,4-DNT初始浓度为80 mg·L^(-1)时,对该酶活力有抑制作用;当pH值为7时,球形红细菌产生的硝基还原酶活力最高。球形红细菌中硝基还原酶的动力学参数,最大反应速率v_(max)和米氏常数K_m,分别为0.0507 mmol/(L·min)和0.1772 mmol·L^(-1)。

To explore the change trend of intermediate products in biotransformation of 2,4-dinitrotoluene( 2,4-DNT) and properties of nitroreductase produced by Rhodobacter sphaeroides,the effect of different combination of nitrogen sources on the efficiency of biotransformation 2,4-DNT by Rhodobacter sphaeroides and the growth of Rhodobacter sphaeroides cells was studied. The concentration changes of 2,4-DNT and biotransformation products of 2,4-DNT were measured at different time. The patterns of isoenzymes of nitroreductase was analyzed using nativepolyacrylamide gel elecrophoresis( native-PAGE) method,and kinetic parameters of the nitroreductase were measured and obtained.Results show that the optimum combination of nitrogen sources to biotransformation of 2,4-DNT by Rhodobacter sphaeroides is complex nitrogen sources composed of yeast extract and( NH_4)_2SO_4. Using Rhodobacter sphaeroides,2,4-DNT can efficiently reduced as 4-amino-2-nitrotoluene(4A2NT) and 2-amino-4-nitrotoluene( 2A4NT). However,the content of 4A2NT is far greater than that of 2A4NT,they have not been futher reduced to 2,4-diaminotoluene( 2,4-DAT). When the initial concentration of 2,4-DNT is 20,40 and 60 mg·L^(-1),there is an obvious promoting effect on the actiyity of nitroreductas produced by Rhodobacter sphaeroides. While,when the initial concentration of 2,4-DNT is 80 mg·L^(-1),the actiyity of nitroreductase is inhibited. The activity of nitroreductas produced by Rhodobacter sphaeroides is the highest at the initial pH = 7. The kinetic parameters,the maximum reaction rate V_(max) and Michaelis constant K_m,for the nitroreductase in Rhodobacter sphaeroides are 0.0507 mmol·( L·min)^(-1) and 0.1772 mmol·L^(-1),respectively.