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

doi:doi:10.3969/j.issn.10080864.2012.06.12

›› 2012, Vol. 14 ›› Issue (6): 70-75.DOI: doi:10.3969/j.issn.10080864.2012.06.12

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

ZHANG Shuang-yu, SUN Wen, XU Li, TIAN Jian, CHU Xiao-yu, WU Ning-feng*

(Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China)

Online:2012-12-15 Published:2012-12-17

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

张双宇,孙雯,许丽,田健,初晓宇,伍宁丰*

(中国农业科学院生物技术研究所, 北京 100081)

通讯作者: 伍宁丰，研究员，博士生导师,主要从事微生物分子生学与基因工程研究。Tel:01082109844;Email: wunf@caas.net.cn
作者简介:张双宇,博士研究生,研究方向为生物化学与分子生物学。Tel:01082109864;Email:zhangshuangyu2006@126.com。
基金资助:
国家自然科学基金项目(31170036)资助。

Abstract

Abstract:

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. PdcB shares 98% identity to the pbenzoquinone reductase (PnpB) from Pseudomonas 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dependent pbenzoquinone reductase that catalyzed the reduction of pbenzoquinone to hydroquinone by in vitro activity assays.

Key words: Pseudomonas; , pnitrophenol, bioremediation; , pbenzoquinone reductase

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. coli BL21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。

关键词: 假单胞菌;对硝基酚;生物修复;对苯二醌还原酶

CLC Number:

ZHANG Shuang-yu, SUN Wen, XU Li, TIAN Jian, CHU Xiao-yu, WU Ning-feng*. Gene Cloning and Functional Identification of pbenzoquinone Reductase from Pseudomonas sp. 17: A Novel Member of pNitrophenol Degradation Gene Cluster[J]. , 2012, 14(6): 70-75.

张双宇,孙雯,许丽,田健,初晓宇,伍宁丰*. 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[J]. , 2012, 14(6): 70-75.

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

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

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