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

Citation data:

Journal of bioscience and bioengineering, ISSN: 1347-4421, Vol: 123, Issue: 4, Page: 528-533

Publication Year:
2017
Usage 17
Abstract Views 15
Link-outs 2
Captures 8
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Repository URL:
http://scholarworks.unist.ac.kr/handle/201301/22227
PMID:
28052817
DOI:
10.1016/j.jbiosc.2016.12.002
Author(s):
Park, Eul-Soo; Park, Sunghoon; Shin, Jong-Shik
Publisher(s):
Elsevier BV; SOC BIOSCIENCE BIOENGINEERING JAPAN
Tags:
Biochemistry, Genetics and Molecular Biology; Chemical Engineering; Immunology and Microbiology; Glycerol dehydratase; Glycerol; Aldehyde dehydrogenase; Biocatalysis; Enzyme assay; Bioprocess monitoring
article description
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 K = 0.73 ± 0.09 mM and k = 400 ± 20 s which are in reasonable agreements with the previous reports. Our assay method allowed measurement of even a 10-fold decrease in the cellular GDHt activity during fermentative production of 3-HP, which demonstrates the detection sensitivity much higher than the previous methods.