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Characterization of 1,3-propanediol oxidoreductase (DhaT) from Klebsiella pneumoniae J2B

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Abstract

1,3-propanediol oxidoreductase (DhaT) of Klebsiella pneumoniae converts 3-hydroxypropionaldehyde (3-HPA) to 1,3-propanediol (1,3-PD) during microbial production of 1,3-PD from glycerol. In this study, DhaT from newly isolated K. pneumoniae J2B was cloned, expressed, purified, and studied for its kinetic properties. It showed, on its physiological substrate 3-HPA, higher activity than similar aldehydes such as acetaldehyde, propionaldehyde and butyraldehyde. The turnover numbers (k cat , 1/s) were estimated as 59.4 for the forward reaction (3-HPA to 1,3-PD at pH 7.0) and 10.0 for the reverse reaction (1,3-PD to 3-HPA at pH 9.0). The Michaelis constants (K m , mM) were 0.77 (for 3-HPA) and 0.03 (for NADH) for the forward reaction (at pH 7.0), and 7.44 (for 1,3-PD) and 0.23 (for NAD+) for the reverse reaction (at pH 9.0). Between these forward and reverse reactions, the optimum temperature and pH were significantly different (37°C and 7.0 vs. 55°C and 9.0, respectively). These results indicate that, under physiological conditions, DhaT mostly catalyzes the forward reaction. The enzyme was seriously inhibited by heavy metal ions such as Ag+ and Hg2+. DhaT was highly unstable when incubated with its own substrate 3-HPA, indicating the necessity of enhancing its stability for improved 1,3-PD production from glycerol.

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Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, Pusan National University, Busan, 609-735, Korea

    Suman Lama, Eunhee Seol, Balaji Sundara Sekar, Satish Kumar Ainala, Jayaraman Thangappan & Sunghoon Park

  2. Interdisciplinary Program of Clean Technology, Pusan National University, Busan, 606-735, Korea

    Su Moon Ro

  3. Research and Development Center, GS Caltex Corporation, Daejeon, 305-380, Korea

    Hyohak Song & Doyoung Seung

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  1. Suman Lama
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Correspondence to Sunghoon Park.

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Lama, S., Ro, S.M., Seol, E. et al. Characterization of 1,3-propanediol oxidoreductase (DhaT) from Klebsiella pneumoniae J2B. Biotechnol Bioproc E 20, 971–979 (2015). https://doi.org/10.1007/s12257-015-0635-6

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  • DOI: https://doi.org/10.1007/s12257-015-0635-6

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