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Spectrophotometric assay for sensitive detection of glycerol dehydratase activity using aldehyde dehydrogenase

Park, Eul-Soo, Park, Sunghoon, Shin, Jong-Shik
Journal of bioscience and bioengineering 2017 v.123 no.4 pp. 528-533
NAD (coenzyme), absorbance, aldehyde dehydrogenase, cell engineering, detection limit, fermentation, glycerol, monitoring, oxidation
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⁻¹ which 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.