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Expression of a metagenome-derived fumarate reductase from marine microorganisms and its characterization

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Abstract

A potential novel fumarate reductase gene designated frd1A was isolated by screening a marine metagenomic library through a sequence-based strategy. Sequence analyses indicated that Frd1A and other putative fumarate reductases were closely related. The putative fumarate reductase gene was subcloned into a pETBlue-2 vector and expressed in Escherichia coli Tuner(DE3)pLacІ cells. The recombinant protein was purified to homogeneity. Functional characterization by high-performance liquid chromatography demonstrated that the recombinant Frd1A protein could catalyze the hydrogenation of fumarate to succinate acid. The Frd1A protein displayed an optimal activity at pH 7.0 and 28 °C, which could be stimulated by adding metal ions such as Zn2+ and Mg2+. The Frd1A enzyme showed a comparable affinity and catalytic efficiency under optimal reaction conditions: k m =0.227 mmol/L, v max= 29.9 U/mg, and k cat/k m=5.44 × 104 per mol/s. The identification of Frd1A protein underscores the potential of marine metagenome screening for novel biomolecules.

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Abbreviations

Frd:

Fumarate reductase

E. coli :

Escherichia coli

SDS:

Sodium dodecyl sulfate

SDS-PAGE:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

PCR:

Polymerase chain reaction

Ni-NTA:

Nickel-nitrilotriacetic acid

HPLC:

High-performance liquid chromatography

EDTA:

Ethylenediaminetetraacetic acid

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Acknowledgments

This research was supported by the National Natural Science Foundation of China (Grant No. 31060016), the Ph.D. Programs Foundation of Ministry of Education of China (Grant No. 20104501120002), the National Postgraduate Innovation Experiment Program of Guangxi (Grant No. GXU11T32573), and the College Student Innovation-Venture Training Program of Guangxi University (Grant No. 1301106, SYJN20122314).

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Authors and Affiliations

  1. The Key Laboratory of Ministry of Education for Microbial and Plant Genetic Engineering; and State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, 100 Daxue East Road, Nanning, Guangxi, 530004, China

    Chengjian Jiang, Yu Liu, Can Meng, Lanlan Wu, Jie Huang, Jie Deng, Jinyi Wang & Peihong Shen

  2. School of Marine Sciences and Biotechnology, Guangxi University for Nationalities, 188 Daxue East Road, Nanning, Guangxi, 530006, China

    Bo Wu

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  1. Chengjian Jiang
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  2. Yu Liu
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  3. Can Meng
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  4. Lanlan Wu
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  5. Jie Huang
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  9. Bo Wu
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Correspondence to Bo Wu.

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Jiang, C., Liu, Y., Meng, C. et al. Expression of a metagenome-derived fumarate reductase from marine microorganisms and its characterization. Folia Microbiol 58, 663–671 (2013). https://doi.org/10.1007/s12223-013-0256-4

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  • DOI: https://doi.org/10.1007/s12223-013-0256-4

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