异戊二烯合成酶(IspS)在大肠杆菌中的表达及其产异戊二烯的研究

Expression of isoprene synthase in Escherichia coli for isoprene production

将来源于银白杨的异戊二烯合成酶基因按照大肠杆菌密码子偏爱性进行优化,克隆到表达载体pACYCDu-et-1上,在大肠杆菌BL21(DE3)中异源表达,采用镍柱亲和层析纯化重组蛋白并测定其异戊二烯合成酶活性,通过摇瓶发酵实验对重组菌产异戊二烯进行进一步研究。结果显示:银白杨异戊二烯合成酶在大肠杆菌中能够高效表达,经过镍柱纯化后,电泳检测到特异性表达条带;该重组异戊二烯合成酶能够催化异戊二烯的合成,重组菌的异戊二烯产量可达到60μg/L。

The Populus alba isoprene synthase（IspS） was optimized to the preferred codon usage of Escherichia coli,cloned into vector pACYCDuet-1 and then heterologously expressed in E.coli BL21（DE3）.The recombinant protein was purified by Ni2＋ affinity chromatography and the enzyme activity was determined in vitro.Isoprene production by the recombinant strain was evaluated by shake-flask fermentation.The results showed that the isoprene synthase was efficiently expressed in E.coli.After purified by Ni2＋ columns,SDS-PAGE analysis revealed a special band corresponding to the fusion protein.The activity determination confirmed that the enzyme could catalyze the formation of isoprene.Under shake-flask conditions,the isoprene productivity of the engineered strain was 60 μg/L.