烟碱脱氢酶基因的原核表达及酶学特性研究.pdfVIP

60 　2007 8 13 4 1 2 1 1 2 马　林, 曾晓鹰, 张峻松, 刘庭淼, 李庆华 1, 5 　450002; 2 , 　650202 　:pBlueScript SK-ndh Eco RI、Sp eI , pET-17B, pET-17B-ndh, BL21(DE3)pLYS 。 IPTG , , A .nicotinovorans 25。、、 , 39.82, SDS。40 ℃, -4 2+ 2+ 2+ p 5.5～8.0 , p 7.0, Km 7.694×10 mol/L。Mn 、Co , Cu + 。( ), 。 :(ndh );;; :TS413 　　　:A　　　:1004-5708(2007)04-0060-05 Research on prokaryot ic expression and enzymatic properties of nicotine dehydrogenase genes 1 2 1 1 2 MA Lin , ZENG Xiao-ying , Z ANG Jun-song , LIU Ting-miao , LI Qing-hua 1Zhengzhou University of Light Industry, School of Food and Biological Engineering, Zhengzhou 450002,China; 2 Technology Center of ongyun Tobacco Group, Kunming 650202, China Abstract:The recombinant plasmid pBlueScript SK-ndh carrying nicotine dehydrogenase (ND )genewas digestedwith re- striction enzyme Eco RI and SpeI, and the desired DNA fragment was subcloned to prokaryotic express vector pET-17B to ob- tain PET-17B-ndh which was transformed into E .coli BL21(DE3)pLYS to produce highly-expressed engineering strain. Even without induction of IPTG, theengineering strain still produced soluble and active recombinant ND whose yieldwas 25 times greaterthan that of wild strain A .nicotinovorans .ND was purified 39.82 fold by ammonium sulfate precipitation, ion exchange chromatography, and gel filtration, and SDSelectrophoresis of the purifiedfraction mitigated a simple band. -4 2+ Results showed that the enzyme had the highest activity at p 7.0