Cloning, expression, characterization and mutational analysis of the tfdA gene from Cupriavidus campinensis BJ71

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• 作者：Lizhen Han ; Yanbo Liu ; Cuicui Li…
• 关键词：tfdA ; Cupriavidus campinensis BJ71 ; 2 ; 4 ; D/α ; KG ; dependent dioxygenase ; Site ; directed mutagenesis ; Characterization
• 刊名：World Journal of Microbiology & Biotechnology
• 出版年：2015
• 出版时间：July 2015
• 年：2015
• 卷：31
• 期：7
• 页码：1021-1030
• 全文大小：763 KB
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• 作者单位：Lizhen Han (1) (2) (3)
  Yanbo Liu (1) (2) (3)
  Cuicui Li (2) (3)
  Degang Zhao (1) (2) (3)
  
  1. Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guiyang, 550025, China
  2. Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region (Guizhou University), Ministry of Education, College of Life Sciences, Guizhou University, Guiyang, 550025, China
  3. Guizhou Key Lab of Agro-Bioengineering, Guizhou University, Guiyang, 550025, China
  
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Applied Microbiology
  Biotechnology
  Biochemistry
  Environmental Biotechnology
  Microbiology
  
• 出版者：Springer Netherlands
• ISSN：1573-0972

文摘

2,4-Dichlorophenoxyacetic acid (2,4-D)/α-ketoglutarate (α-KG) dioxygenase (TfdA) is an Fe(II)-dependent enzyme that catalyzes the first step in degradation of the herbicide 2,4-D. Previous studies focused on the tfdA gene in Ralstonia eutropha JMP134 isolated in Australia. In this study, a new tfdA gene was cloned from Cupriavidus campinensis BJ71, an effective degrading bacteria from China, based on the iCOnsensus-DEgenerate Hybrid Oligonucleotide Primers (iCODEHOPs) protocol, combined with high-efficiency Thermal Asymmetric Interlaced PCR (hiTAIL-PCR). The open reading frame of 861?bp encoded a putative 287 amino acid protein with a theoretical molecular mass of 32.32?kDa. The gene was overexpressed in Escherichia coli BL21 (DE3) and the purified TfdA showed optimal activity at pH 6.75 and 30?°C. This enzyme was more thermostable and it could use 3-hydrocinnamic acid as substrate, with a similar enzyme activity compared with 2,4-D. TfdA and its variants were created as maltose-binding protein (MBP) tagged fusion proteins to examine the roles of putative substrate-binding residues. The MBP-N110A, MBP-V198A and MBP-R207K proteins showed decreased k _cat and increased Km, and MBP-R278A was inactive, suggesting these residues may affect 2,4-D binding or catalysis.