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

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Abstract

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.

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Acknowledgments

The authors thank the Foundation of the National Transgenic Major Project of China (2013ZX08010-003, 2014ZX0801008B-002) and the Province Science Project in Guizhou (J [2013] 2123) for their support.

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Authors and Affiliations

  1. Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guiyang, 550025, China

    Lizhen Han, Yanbo Liu & Degang Zhao

  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

    Lizhen Han, Yanbo Liu, Cuicui Li & Degang Zhao

  3. Guizhou Key Lab of Agro-Bioengineering, Guizhou University, Guiyang, 550025, China

    Lizhen Han, Yanbo Liu, Cuicui Li & Degang Zhao

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  1. Lizhen Han
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  2. Yanbo Liu
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Correspondence to Degang Zhao.

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Han, L., Liu, Y., Li, C. et al. Cloning, expression, characterization and mutational analysis of the tfdA gene from Cupriavidus campinensis BJ71. World J Microbiol Biotechnol 31, 1021–1030 (2015). https://doi.org/10.1007/s11274-015-1852-z

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  • DOI: https://doi.org/10.1007/s11274-015-1852-z

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