title: Spectrophotometric assay for sensitive detection of glycerol dehydratase activity using aldehyde dehydrogenase.
abstract: Glycerol dehydratase (GDHt) is a pivotal enzyme for fermentative utilization of glycerol by catalyzing radical-mediated conversion of glycerol into 3-hydroxypropionaldehyde (3-HPA). Precise and sensitive monitoring of cellular GDHt activity during the fermentation process is a prerequisite for reliable metabolic analysis to afford efficient cellular engineering and process optimization. Here we report a new spectrophotometric assay for the sensitive measurement of the GDHt activity with a sub-nanomolar limit of detection (LOD). The assay method employs aldehyde dehydrogenase (ALDH) as a reporter enzyme, so the readout of the GDHt activity is recorded at 340 nm as an increase in UV absorbance which results from NADH generation accompanied by oxidation of 3-HPA to 3-hydroxypropionic acid (3-HP). The GDHt assay was performed under the reaction conditions where the ALDH activity overwhelms the GDHt activity (i.e., 50-fold higher activity of ALDH relative to GDHt activity), affording sensitive detection of GDHt with 360 pM LOD. The ALDH-coupled assay was used to determine kinetic parameters of GDHt for glycerol, leading to KM= 0.73 ± 0.09 mM and kcat= 400 ± 20 s-1which are in reasonable agreements with the previous reports. Our assay method allowed measurement of even a 104-fold decrease in the cellular GDHt activity during fermentative production of 3-HP, which demonstrates the detection sensitivity much higher than the previous methods.
authors.affiliation: Department of Biotechnology, Yonsei University, Seoul 120-749, South Korea.
authors.forename: Eul-Soo
authors.initials: ES
authors.lastname: Park
authors.affiliation: Department of Chemical and Biomolecular Engineering, Pusan National University, Busan 609-735, South Korea.
authors.forename: Sunghoon
authors.initials: S
authors.lastname: Park
authors.affiliation: Department of Biotechnology, Yonsei University, Seoul 120-749, South Korea. Electronic address: enzymo@yonsei.ac.kr.
authors.forename: Jong-Shik
authors.initials: JS
authors.lastname: Shin
mesh_terms: D000444:Aldehyde Dehydrogenase / Q000378:metabolism*; D005285:Fermentation; D005985:Glyceraldehyde / Q000031:analogs & derivatives / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D006836:Hydro-Lyases / Q000032:analysis* / Q000378:metabolism*; D007700:Kinetics; D019344:Lactic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D057230:Limit of Detection; D009243:NAD / Q000378:metabolism; D011407:Propane / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods*
chemical_list: D009243:NAD; C047158:3-hydroxypropionaldehyde; D019344:Lactic Acid; D005985:Glyceraldehyde; C031601:hydracrylic acid; D000444:Aldehyde Dehydrogenase; D006836:Hydro-Lyases; C020671:glycerol dehydratase; D005990:Glycerol; D011407:Propane
keywords: Aldehyde dehydrogenase; Biocatalysis; Bioprocess monitoring; Enzyme assay; Glycerol; Glycerol dehydratase