甲基对硫磷水解酶酶促反应体系建立及特性研究

Study on reaction system and characteristics of methyl parathion hydrolase

从甲基对硫磷降解菌DLL E4 (Pseudomonasputida)中提取水解酶 ,对其酶促反应进行研究 ,建立了反应体系 :4 74 μLNa2 HPO4 柠檬酸缓冲液 ,12 5 μL甲基对硫磷 ,1 5 μg粗蛋白 ,35℃水浴 5min。水解酶保持稳定的 pH值范围为 5 0～ 10 0 ,最适 pH为 7 0。该酶热不稳定 ,4 5℃ 30min处理 ,酶活下降 5 0 % ;10 0℃ 10min处理可完全灭活。测定了 11种金属离子、TWEEN 80、TritonX 10 0及EDTA对水解酶的作用 ,发现 2 0mmol·L-1CoCl2 对酶活有明显的抑制作用 ,而 2 0～ 0 0 2mmol·L-1的LiCl对酶活有促进作用 ,2mmol·L-1的TWEEN 80和TritonX 10 0可降低 80 %的酶活。

Methyl parathion(MP)hydrolase was extracted from MP degrading strain DLL-E4(Pseudomonas putida).The enzymatic reaction system was founded as follows:Na 2HPO 4-citric acid buffer 474 μL,MP 12.5 μL,crude enzyme(about 1.5 μg crude protein),35 ℃ water bathing 5 min.The characteristics of MP hydrolase was determined.The pH area for keeping of enzyme is 5.010.0,with the optimum pH 7.0.The activity of enzyme is down to 50% and zero when treated with 45 ℃ 30 min.and 100 ℃ 10 min,respectively.11 metal ions,TWEEN-80,Triton X-100 and EDTA were chose to study their effects on MP hydrolase activity.Data showed that 20 mmol·L -1 CoCl 2,2 mmol·L -1 TWEEN-80 and Triton X-100 can greatly inhibit hydrolase activity,while 200.02 mmol·L-1 LiCl can enhance it.