Aeromonas sp.XJ-6双加氧酶基因的克隆、表达及对酪氨酸的降解

Cloning and Expression of Dioxygenase Gene from Aeromonas sp.XJ-6 and Promoting Degradation of Tyr

目的:从Aeromonas sp.XJ-6中克隆双加氧酶基因,初步探索该酶的功能,为芳香烃化合物的生物降解提供基因资源。方法:PCR扩增双加氧酶基因dio6,并实现该基因在大肠杆菌(Escherichia coli)中的诱导表达。产物经Ni-NTA柱纯化后,通过薄层层析(TLC)和HPLC检测双加氧酶dio6对Tyr的降解效果,再结合LC-MS检测降解产物,并分析其可能的降解途径。结果:Aeromonas sp.XJ-6双加氧酶基因dio6大小为1 194bp;通过金属鳌合亲和层析(MCAC)纯化后dio6表达产物的大小为44.9k Da。双加氧酶dio6对Tyr具有较强的降解作用。TLC和HPLC检测表明,在60μl酶量和30℃反应温度等条件下,Tyr降解较快;Mg^(2+)、Ca^(2+)略微抑制酶促反应,Mn^(2+)、Zn^(2+)、Cu^(2+)、Fe^(2+)、Ca^(2+)促进底物降解,其中Mn^(2+)对双加氧酶影响最大。LC-MS分析表明,在双加氧酶dio6作用下,Tyr被降解为延胡索酸。结论:Aeromonas sp.XJ-6双加氧酶dio6是一种苯环开环酶,为芳香烃化合物的生物降解提供了良好的基因资源。

Objective： To clone the dioxygenase dio6 gene from Aeromonas sp. XJ-6,and explore the function of dioxygenase dio6 to provide genetic resources for biodegradation of aromatic compounds. Methods：Dioxygenase dio6 gene from Aeromonas sp. XJ-6 genome was amplified by PCR and induciblly expressed in Escherichia coli BL21（ DE3）. The expression products of dio6 was purified by Ni-NTA. TLC and HPLC to detect the degradation effect of dioxygenase on Tyr,and the degradation products were also detected by LC-MS so as to analyze the possible degradation pathway. Results： The size of Aeromonas sp. XJ-6 dioxygenase gene dio6 was 1194 bp,the expression product of dio6 purified by MCAC was 44. 9k Da. HPLC analysis showed that dio6 has strong degradation ability for Tyr under the optimal conditions that the amount of enzyme is 60μl per reaction mixture,temperature 30℃. Metal ion Mg^2＋and Ca^2＋slightly inhibit the enzymatic reaction,and Mn^2＋、Zn^2＋、Cu^2＋、Fe^2＋and Ca^2＋can improve the substrate degradation,of which Mn^2＋has the most impact on dioxygenase dio6. LC-MS analysis results showed that Tyr was turned into fumaric acid with the help of dioxygenase dio6.Conclusion： Aeromonas sp. XJ-6 dioxygenase dio6 is a kind of benzene ring-opening enzyme,which may be provided as a genetic resource for aromatic compounds biodegradation.