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

Author:
Sankaranarayanan, Mugesh, Seol, Eunhee, Kim, Yeonhee, Chauhan, Ashish Singh, Park, Sunghoon
Source:
Journal of industrial microbiology & biotechnology 2017 v.44 no.3 pp. 477-488
ISSN:
1367-5435
Subject:
1-propanol, Escherichia coli, NAD (coenzyme), aldehyde dehydrogenase, glycerol
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.
Agid:
5758374