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A Highly Efficient NADH-dependent Butanol Dehydrogenase from High-butanol-producing Clostridium sp. BOH3

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Abstract

Butanol has been considered as a better alternative fuel and it can be produced from anaerobic Clostridial fermentation. Though several enzymes are involved in the biosynthesis of butanol in Clostridia, butanol dehydrogenase (BDH) is understood to play a major role, which catalyzes the conversion of butyraldehyde into butanol at the expenditure of a cofactor NAD(P)H. Recently, the strain Clostridium sp. BOH3 is reported to generate high level of butanol from monosugars. To investigate the BDH activity at various stages of fermentation, BOH3 was cultured in reinforced Clostridial medium with 30 g/l of glucose at 35 °C and the cells were harvested periodically from acid production and solvent production phases. During acid production, NADPH-dependent BDH activity is higher than NADH dependent BDH. Conversely, NADH-BDH activity is predominant during solvent production phase. The optimum pHs for NADH and NADPH-BDH are estimated as pH 6 and 8, respectively. By employing three steps of purification, NADH-BDH is purified to 102-fold with 36 % yield. Subsequent characterization reveals that NADH-BDH is a dimer composed of two subunits depicting the molecular weight of 44 kDa. The peptide finger printing analysis (MS/MS) suggests that the purified protein has higher homology with bifunctional acetaldehyde-CoA and alcohol dehydrogenase of Clostridium acetobutylicum. The extensive kinetic studies show that NADH-BDH follows an ordered sequential bi bi mechanism. The calculated values of K butyraldehyde and K NADH are 8.35 ± 0.25 and 0.076 ± 0.02 mM, respectively, whereas V max is 4.02 ± 0.07 μmol/(mg protein. min). The purified NADH-BDH retains 70 % of its initial activity after 7 days at 4 °C.

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Acknowledgment

This research was supported by the Competitive Research Programme from Singapore National Research Foundation under Project No.: NRF-CRP5-2009-05. We thank Dr. Ray for providing adhe1 information from genomic data sequence of Clostridium sp. BOH3.

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Authors and Affiliations

  1. Department of Chemical and Bimolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Block E5-02-09, Singapore, 117576, Singapore

    Gobinath Rajagopalan & Kun-Lin Yang

  2. Department of Civil and Environmental Engineering, National University of Singapore, 1 Engineering Drive 2, Block E1A-07-03, Singapore, 117576, Singapore

    Jianzhong He

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  1. Gobinath Rajagopalan
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  2. Jianzhong He
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Correspondence to Kun-Lin Yang.

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Rajagopalan, G., He, J. & Yang, KL. A Highly Efficient NADH-dependent Butanol Dehydrogenase from High-butanol-producing Clostridium sp. BOH3. Bioenerg. Res. 6, 240–251 (2013). https://doi.org/10.1007/s12155-012-9253-8

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