来源于Pseudomonas sp.1-7对硝基酚降解基因簇中的对苯二醌还原酶基因的克隆和功能鉴定  

Gene Cloning and Functional Identification of p-benzoquinone Reductase from Pseudomonas sp.1-7: A Novel Member of p-Nitrophenol Degradation Gene Cluster

作　　者：张双宇[1] 孙雯[1] 许丽[1] 田健[1] 初晓宇[1] 伍宁丰[1] 

机构地区：[1]中国农业科学院生物技术研究所,北京100081

出　　处：《中国农业科技导报》2012年第6期70-75,共6页Journal of Agricultural Science and Technology

基　　金：国家自然科学基金项目(31170036)资助

摘　　要：Pseudomonas sp.1-7可以利用对硝基酚(PNP)作为唯一的碳源,氮源和能源生长。为了鉴定菌株1-7代谢PNP的相关基因,本研究构建了菌株1-7基因组的Fosmid文库,并通过文库的筛选克隆得到一段长为12.3 kb的基因簇序列。序列分析显示,该基因簇中共有7个基因(pdcEDGFCBA)与PNP的代谢相关。其中PdcB与来源于Pseudomonas sp.WBC-3中的对苯二醌还原酶(PnpB)有98%的相似性。将pdcB基因在E.coliBL21中过量表达,并通过Ni-NTA亲和层析纯化重组蛋白PdcB。体外的酶活测定表明,PdcB为一个FAD和NADH依赖型的对苯二醌还原酶,能够催化对苯二醌降解并生成对苯二酚。Pseudomonas sp. 1-7 utilizes p-nitrophenol （PNP） as a sole source of carbon, nitrogen, and energy. In order to identify the genes involved in this utilization, a gene cluster was identified in a 12.3 kb DNA fragment of a genome fosmid library. Sequence analysis revealed that there were 7 genes （pdcEDGFCBA） related in PNP degradation. PdeB shares 98% identity to the p-benzoquinone reductase （PnpB） from Pseuclomonas sp. WBC-3. The pdcB gene was then over-expressed in E. coli BL21, and the recombinant protein PdcB was further purified with Ni-NTA affinity chromatography. Purified PdcB was shown a FAD and NADPH-dependcnt p-benzoquinone reductase that catalyzed the reduction of p-benzoquinone to hydroqninone by in vitro activity assays.

关 键 词：假单胞菌 对硝基酚 生物修复 对苯二醌还原酶 

分 类 号：Q78[生物学—分子生物学] Q93