Molecular cloning, characterization, and overexpression of a novel [Fe]-hydrogenase from a high rate of hydrogen producing Enterobacter cloacae IIT-BT 08

被引:40
作者
Mishra, J
Khurana, S
Kumar, N
Ghosh, AK
Das, D [1 ]
机构
[1] Indian Inst Technol, Dept Biotechnol, Kharagpur 721302, W Bengal, India
[2] Univ Tennessee, Dept Physiol, Memphis, TN 38105 USA
关键词
Enterobacter cloacae; Fe]-hydrogenase; hydA; H-cluster; anaerobe;
D O I
10.1016/j.bbrc.2004.09.108
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Degenerate primers were designed from the conserved zone of hydA structural gene encoding for catalytic subunit of [Fe]-hydrogenase of different hydrogen producing bacteria. A 750 bp of PCR product was amplified by using the above-mentioned degenerate primers and genomic DNA of Enterobacter cloacae IIT-BT 08 as template. The amplified PCR product was cloned and sequenced. The sequence showed the presence of an ORF of 450 bp with significant similarity (40%) with C-terminal end of the conserved zone (H-cluster) of [Fe]- hydrogenase. hydA ORF was then amplified and cloned in-frame with GST in pGEX4T-1 and overexpressed in a non-hydrogen producing Escherichia coli BL-21 to produce a GST-fusion protein of a calculated molecular mass of about 42.1 kDa. Recombinant protein was purified and specifically recognized by anti-GST monoclonal antibody through Western blot. Southern hybridization confirmed the presence of this gene in E. cloacae IIT-BT 08 genome. In vitro hydrogenase assay with the overexpressed hydrogenase enzyme showed that it is catalytically active upon anaerobic adaptation. In vivo hydrogenase assay confirmed the presence of H-2 gas in the gas mixture obtained from the batch culture of recombinant E coli BL-21. A tentative molecular mechanism has been proposed about the transfer of electron from electron donor to H-cluster without the mediation of the F-cluster. (C) 2004 Elsevier Inc. All rights reserved.
引用
收藏
页码:679 / 685
页数:7
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