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Measurement of crude-cell-extract glycerol dehydratase activity in recombinant Escherichia coli using coupled-enzyme reactions

  • Biotechnology Methods - Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Glycerol dehydratase (GDHt), which converts glycerol to 3-hydroxypropionaldehyde, is essential to the production of 1,3-propanediol (1,3-PDO) or 3-hydroxypropionic acid (3-HP). A reliable GDHt activity assay in crude-cell extract was developed. In the assay, GDHt converted 1,2-propanediol (1,2-PDO) to propionaldehyde, which was further converted to 1-propionic acid by aldehyde dehydrogenase (KGSADH) or to 1-propanol by yeast-alcohol dehydrogenase (yADH), while the NADH concentration change was monitored spectrophotometrically. Cells should be disintegrated by Bead Beater/French Press, not by chemical methods (BugBuster®/B-PER™), because the reagents significantly inactivated GDHt and coupling enzymes. Furthermore, in the assay mixture, a much higher activity of KGSADH (>200-fold) or yADH (>400-fold) than that of GDHt should have been maintained. Under optimal conditions, both KGSADH and yADH showed practically the same activity. The coupled-enzyme assay method established here should prove to be applicable to recombinant strains developed for the production of 3-HP and/or 1,3-PDO from glycerol.

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Acknowledgements

This study was supported financially by the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the Korean Ministry of Science, ICT and Future planning (ABC-2011-0031361). The authors are also grateful for the financial assistance provided by the BK21 Plus program at Pusan National University.

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Authors and Affiliations

  1. School of Chemical and Biomolecular Engineering, Pusan National University, Busan, 46241, Republic of Korea

    Mugesh Sankaranarayanan, Eunhee Seol, Yeonhee Kim, Ashish Singh Chauhan & Sunghoon Park

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  1. Mugesh Sankaranarayanan
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  2. Eunhee Seol
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  3. Yeonhee Kim
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Correspondence to Sunghoon Park.

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Sankaranarayanan, M., Seol, E., Kim, Y. et al. Measurement of crude-cell-extract glycerol dehydratase activity in recombinant Escherichia coli using coupled-enzyme reactions. J Ind Microbiol Biotechnol 44, 477–488 (2017). https://doi.org/10.1007/s10295-017-1902-7

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  • DOI: https://doi.org/10.1007/s10295-017-1902-7

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