A Highly Efficient NADH-dependent Butanol Dehydrogenase from High-butanol-producing Clostridium sp. BOH3
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A Highly Efficient NADH-dependent Butanol Dehydrogenase from High-butanol-producing Clostridium sp. BOH3 Gobinath Rajagopalan & Jianzhong He & Kun-Lin Yang
Published online: 15 September 2012 # Springer Science+Business Media, LLC 2012
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 pH6 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 G. Rajagopalan : K.-L. Yang (*) Department of Chemical and Bimolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Block E5-02-09, Singapore 117576, Singapore e-mail: [email protected] J. He Department of Civil and Environmental Engineering, National University of Singapore, 1 Engineering Drive 2, Block E1A-07-03, Singapore 117576, Singapore
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 Kbutyraldehyde and KNADH are 8.35±0.25 and 0.076±0.02 mM, respectively, whereas Vmax 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. Keywords Butanol dehydrogenase . Clostridium sp . Purification . Butanol production . ABE fermentation
Introduction Recently, there is a renewed interest on butanol production by using anaerobic Clostridia which can produce acetone, butanol and ethanol with a ratio of 3:6:1 during the so-called ABE fermentation [1, 2]. To date, several candidates from Clostridia genus have been exploited to produce butanol. These strains include Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharoperbutylacetonicum, Clostridium saccharoperbutylicum, Clostridium aurantibutyricum, Clostridium pasteurianum, Clostridium sporogenes, Clostridium cadaveris, and Clostridium tetanomorphum. Among them, C. acetobutylicum and C. beijerinckii are re
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