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Expression and identification of a thermostable malate dehydrogenase from multicellular prokaryote Streptomyces avermitilis MA-4680

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Abstract

A malate dehydrogenase (MDH) from Streptomyces avermitilis MA-4680 (SaMDH) has been expressed and purified as a fusion protein. The molecular mass of SaMDH is about 35 kDa determined by SDS-PAGE. The recombinant SaMDH has a maximum activity at pH 8.0. The enzyme shows the optimal temperature around 42°C and displays a half-life (t 1/2) of 160 min at 50°C which is more thermostable than reported MDHs from most bacteria and fungi. The k cat value of SaMDH is about 240-fold of that for malate oxidation. In addition, the k cat/K m ratio shows that SaMDH has about 1,246-fold preference for oxaloacetate (OAA) reduction over l-malate oxidation. The recombinant SaMDH may also use NADPH as a cofactor although it is a highly NAD(H)-specific enzyme. There was no activity detected when malate and NADP+ were used as substrates. Substrate inhibition studies show that SaMDH activity is strongly inhibited by excess OAA with NADH, but is not sensitive to excess l-malate. Enzymatic activity is enhanced by the addition of Na+, NH4 +, Ca2+, Cu2+ and Mg2+ and inhibited by addition of Hg2+ and Zn2+. MDH is widely used in coenzyme regeneration, antigen immunoassays and bioreactors. The enzymatic analysis could provide the important basic knowledge for its utilizations.

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Acknowledgments

This work was supported by funds from the National Natural Science Foundation of China (30500300; 30870062; 30900243), New Century Excellent Talents in University of the Education Ministry of China (NCET-06-0558), Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, the Key Laboratory of Biotic Environment and Ecological Safety in Anhui Province and Program for Innovative Research Team in Anhui Normal University.

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Authors and Affiliations

  1. Key Laboratory of Molecular Evolution and Biodiversity and Institute of Molecular Biology and Biotechnology, College of Life Sciences, Anhui Normal University, Wuhu, 241000, Anhui, People’s Republic of China

    Zong-Da Wang, Bao-Juan Wang, Ya-Dong Ge, Wei Pan, Jie Wang, Ai-Min Liu & Guo-Ping Zhu

  2. Biochemistry Laboratory, Wannan Medical College, Wuhu, 241002, Anhui, People’s Republic of China

    Lei Xu

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  1. Zong-Da Wang
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Correspondence to Guo-Ping Zhu.

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Wang, ZD., Wang, BJ., Ge, YD. et al. Expression and identification of a thermostable malate dehydrogenase from multicellular prokaryote Streptomyces avermitilis MA-4680. Mol Biol Rep 38, 1629–1636 (2011). https://doi.org/10.1007/s11033-010-0273-1

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