title: Measurement of crude-cell-extract glycerol dehydratase activity in recombinant Escherichia coli using coupled-enzyme reactions.
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.
authors.affiliation: School of Chemical and Biomolecular Engineering, Pusan National University, Busan, 46241, Republic of Korea.
authors.forename: Mugesh
authors.initials: M
authors.lastname: Sankaranarayanan
authors.affiliation: School of Chemical and Biomolecular Engineering, Pusan National University, Busan, 46241, Republic of Korea.
authors.forename: Eunhee
authors.initials: E
authors.lastname: Seol
authors.affiliation: School of Chemical and Biomolecular Engineering, Pusan National University, Busan, 46241, Republic of Korea.
authors.forename: Yeonhee
authors.initials: Y
authors.lastname: Kim
authors.affiliation: School of Chemical and Biomolecular Engineering, Pusan National University, Busan, 46241, Republic of Korea.
authors.forename: Ashish Singh
authors.initials: AS
authors.lastname: Chauhan
authors.affiliation: School of Chemical and Biomolecular Engineering, Pusan National University, Busan, 46241, Republic of Korea. parksh@pusan.ac.kr.
authors.forename: Sunghoon
authors.initials: S
authors.lastname: Park
mesh_terms: D000426:Alcohol Dehydrogenase / Q000378:metabolism; D000444:Aldehyde Dehydrogenase / Q000378:metabolism; D000447:Aldehydes / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D005985:Glyceraldehyde / Q000031:analogs & derivatives / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D006836:Hydro-Lyases / Q000378:metabolism*; D007218:Industrial Microbiology; D019344:Lactic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D011407:Propane / Q000378:metabolism; D019946:Propylene Glycol / Q000378:metabolism; D011409:Propylene Glycols / Q000378:metabolism
chemical_list: D000447:Aldehydes; D029968:Escherichia coli Proteins; D011409:Propylene Glycols; C047158:3-hydroxypropionaldehyde; D019344:Lactic Acid; D005985:Glyceraldehyde; C041787:1,3-propanediol; D019946:Propylene Glycol; C005556:propionaldehyde; C031601:hydracrylic acid; D000426:Alcohol Dehydrogenase; D000444:Aldehyde Dehydrogenase; D006836:Hydro-Lyases; C020671:glycerol dehydratase; D005990:Glycerol; D011407:Propane
keywords: Coupled enzyme; Crude-cell extract; Enzyme activity; Glycerol dehydratase (GDHt); Real-time monitoring; Yeast-alcohol dehydrogenase; α-ketoglutaric semialdehyde dehydrogenase