烟碱脱氢酶基因的原核表达及酶学特性研究

将含烟碱脱氢酶基因重组质粒pBlueScript SK-ndh用限制性内切酶EcoRI、SpeI双酶切,目的基因再亚克隆到非融合表达质粒载体pET-17B,获得重组子质粒pET-17B-ndh,转化大肠杆菌BL21(DE3)pLYS获得高酶活力表达的工程菌株。即使在不用IPTG诱导情况下,该工程菌也产生较高酶活力的可溶性酶,酶产量比野生菌A. nicotinovorans提高25倍。通过硫酸铵沉淀、离子交换层析、凝胶过滤提纯了烟碱脱氢酶,酶被纯化了39.82倍,SDS-PAGE分析得到电泳纯单一条带。该酶活力的最适温度为40℃,该酶组分在pH5.5~8.0范围内稳定,最适pH7.0,对烟碱作用的Km值为7.694×10-4mol/L。Mn2+、Co2+是酶的激活剂,Cu2+是酶的抑制剂。烟碱降解条件研究表明在反应体系中需要辅酶或电子接受体参与介导质子(H+)的转移,该酶催化反应是一种氧化还原反应体系。 

Research on prokaryotic expression and enzymatic properties of nicotine dehydrogenase genes

:The recombinant plasmid pBlueScript SK-ndh carrying nicotine dehydrogenase (NDH)gene was digested with restriction enzyme EcoRI and SpeI, and the desired DNA fragment was subcloned to prokaryotic express vector pET-17B to obtain PET-17B-ndh which was transformed into E. coli BL21 (DE3)pLYS to produce highly-expressed engineering strain. Even without induction of IPTG, the engineering strain still produced soluble and active recombinant NDH whose yield was 25 times greater than that of wild strain A. nicotinovorans. NDH was purified 39.82 fold by ammonium sulfate precipitation, ion exchange chromatography, and gel filtration, and SDS-PAGE electrophoresis of the purified fraction mitigated a simple band. Results showed that the enzyme had the highest activity at pH7.0 and 40℃ with Km of 7.694×10-4 mol/L. Mn2+ and Co2+ activated the enzyme while Cu2+ inhibited it. The nicotine degradation conditions showed that prosthetic group or electron accepter were necessary for the transfer of H+ in reaction system and the enzyme catalyze reaction is an oxide reductive reaction system.