Characterization of enzymes involved in the ethanol production of Moorella sp. HUC22-1
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O RI G I NAL PAPE R
Characterization of enzymes involved in the ethanol production of Moorella sp. HUC22-1 Kentaro Inokuma · Yutaka Nakashimada · Takuya Akahoshi · Naomichi Nishio
Received: 19 October 2006 / Revised: 22 January 2007 / Accepted: 26 January 2007 / Published online: 22 February 2007 © Springer-Verlag 2007
Abstract Since the thermophilic bacterium Moorella sp. HUC22-1 produces 120 mM acetate and 5.2 mM ethanol from H2–CO2, several candidate genes, which were predicted to code for three alcohol dehydrogenases (AdhA, B, C) and one acetaldehyde dehydrogenase (Aldh), were cloned from HUC22-1. The cloned genes were subcloned into a His-tagged expression vector and expressed in Escherichia coli. Recombinant AdhA and B were both dependent on NADP(H) but independent of NAD(H), and their reduction activities from aldehyde to alcohol were higher than their oxidation activities. In contrast with AdhA and B, no activity of AdhC was observed in either reaction. On the other hand, Aldh was active toward both NADP(H) and NAD(H). The enzyme activity of Aldh was directed toward the thioester cleavage and the thioester condensation. When 50 g of AdhA and 50 g Aldh were added to the buVer solution (pH 8.0) containing NADPH, NADH and acetyl-CoA at 60°C, 1.6 mM ethanol was produced from 3 mM acetyl-CoA after 90 min. Expression analysis of the mRNAs revealed that the expression level of aldh was threefold higher in the H2–CO2 culture than that in the
K. Inokuma · T. Akahoshi · N. Nishio (&) Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Kagamiyama 1-3-1, Higashi-Hiroshima 739-8530, Japan e-mail: [email protected] Y. Nakashimada Department of Applied Chemistry, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka, Koganei, Tokyo 184-8588, Japan
fructose culture, but levels of adhA, B and C were decreased. Keywords Ethanol production · H2–CO2 · Thermophile · Anaerobe · Alcohol dehydrogenase · Acetaldehyde dehydrogenase
Introduction A group of anaerobic acetogenic bacteria, known as acetogens, can grow autotrophically on H2 and CO2 and usually produce acetate as a sole end product via the acetyl-CoA pathway (Drake et al. 2002). However, some mesophilic acetogens such as Clostridium ljungdahlii and C. autoethanogenum can produce not only acetate but also ethanol from the synthesis gas (Tanner et al. 1993; Abrini et al. 1994). This suggests that it is possible to convert any biomass into ethanol eVectively by using the combined process of biomass gasiWcation and fermentation by these bacteria. Furthermore, if there are thermophilic bacteria that possess the ethanol-producing pathway from H2/CO2 or CO, they are suited for production of ethanol with a fermenter into which a recovery process via reducedpressure distillation are integrated (Radianingtyas and Wright 2003). This consideration encouraged us to isolate a thermophilic bacterium that produces ethanol from H2/CO2 or CO. We previously reported that a thermophi
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