寡养单胞菌G1菌mpd基因的克隆及甲基对硫磷水解酶的性质研究

发布时间：2018-05-16 05:21

  本文选题：mpd基因 + 粗酶液　； 参考：《安徽农业大学》2015年硕士论文

【摘要】：本实验室分离筛选出的微嗜酸寡养单胞菌(Stenotrophomonas acidaminiphila)G1能够降解毒死蜱(CP)、甲基对硫磷(MP)、辛硫磷、二嗪磷、三唑磷(TAP)、丙溴磷。本文将编码甲基对硫磷水解酶(MPH)的mpd基因,克隆到大肠杆菌上并成功表达,然后研究了温度、pH、有机溶剂甲醇等对MPH活性的影响,比较G1菌粗酶液与构建的大肠杆菌工程菌粗酶液对CP、MP、TAP的降解效果。从G1菌中克隆长度为996 bp的mpd基因,构建重组质粒,将该质粒转化到大肠杆菌,获得基因工程菌株,成功表达重组蛋白,重组后的mpd基因编码的甲基对硫磷水解酶(MPH),经IPTG诱导表达后蛋白约30KDa,检测其酶活,重组后的蛋白能降解MP。在研究温度对MPH活性影响的时候,研究发现当温度为47℃时,底物MP的降解率最高,说明MPH最适反应温度为47℃。在pH对MPH活性影响的实验中,在碱性条件下,底物MP的降解率均高于在中性条件下,这说明MPH在碱性条件中的活性高,通过实验发现,MPH的最适反应pH为9.1。在研究有机溶剂甲醇对MPH的活性影响实验时,实验结果显示,当添加的甲醇在整个体系的体积比为6%~50%之间时,这可以提高MPH的活性,因为底物MP的降解率明显要高于不加任何甲醇的对照,当添加的甲醇体积比超过50%后,MPH的活性被抑制,底物MP的降解很少,降解率小于不添加甲醇的对照组的降解率。实验结果发现MP的降解产物PNP对MPH的活性没有影响。在G1菌粗酶液与大肠杆菌工程菌粗酶液对两种农药的降解实验中,工程菌粗酶液对CP、TAP的降解率明显高于G1菌粗酶液,这是因为构建的工程菌大肠杆菌能够大量表达MPH。在添加相同的总蛋白量时,G1菌粗酶液对CP的降解率高于对TAP的降解率。将G1菌粗酶液对MP的降解与G1菌对MP的降解结果比较,能够发现G1菌在经过16代传代后,活力明显下降,克隆的大肠杆菌因其能够大量表达甲基对硫磷水解酶蛋白,所以其对MP的降解率高。在降解实验中,当添加的工程菌总蛋白量相同时,TAP的降解率反而大于MP的降解率,这是因为MP的另一降解产物O,O-二甲基硫代磷酸对MPH有抑制作用。
[Abstract]:Stenotrophomonas acidaminiphila)G1, isolated from our laboratory, can degrade chlorpyrifos, methyl parathion, phoxim, diazinphosphos, triazophos, and propyl bromide. The mpd gene encoding methylparathion hydrolase (MPH) was cloned into E. coli and successfully expressed. The effects of temperature, pH and organic solvent methanol on the activity of MPH were studied. To compare the degradation effect of G 1 crude enzyme solution and E. coli engineering bacteria crude enzyme solution on CPG MPTAP. The mpd gene of 996bp was cloned from G1 strain. The recombinant plasmid was constructed. The recombinant plasmid was transformed into Escherichia coli and the recombinant protein was successfully expressed. The recombinant mpd gene encoded methylparathion hydrolase (MPHH), which was induced by IPTG and expressed about 30kDa, was detected for its enzyme activity, and the recombinant protein could degrade MPH. When the influence of temperature on the activity of MPH was studied, it was found that when the temperature was 47 鈩，

本文编号：1895627