Glycerol dehydratase GDHt EC is a

Glycerol dehydratase (GDHt, EC 4.2.1.30) is a key enzyme for the bioconversion of glycerol into the value-added chemicals and thereby has recently attracted a great deal of attention as an essential component for metabolic engineering . GDHt carries out a radical-mediated turnover of glycerol into 3-hydroxypropionaldehyde (3-HPA) using adenosylcobalamin (i.e., coenzyme B) as a cofactor . 3-HPA is further processed to either 1,3-PDO or 3-HP, depending on the type of the oxidoreductase acting on the aldehyde group of 3-HPA (). Radical formation in the adenosylcobalamin moiety by a homolytic cleavage of the CoC bond initiates the radical catalysis, leading to dehydration of the bound substrate . It is known that GDHt undergoes mechanism-based inactivation during the catalysis and the resulting damaged cofactor is unable to mediate the radical reaction . Owing to the pivotal role of GDHt in the glycerol conversion, reliable metabolic engineering strategies to cope with the undesirable inactivation and to optimize the enzyme expression level have been considered as one of the crucial factors to the successful process design. To this end, a comprehensive metabolic analysis should be guided by precise and sensitive monitoring of the change in the GDHt activity throughout the fermentation process. To date, two assay methods have been widely employed to quantify the GDHt activity. One method, developed by Toraya et al., is an end-point assay where 3-HPA produced from the GDHt reaction is modified with 3-methyl-2-benzothiazolinone hydrazone (MBTH) under acidic conditions and turns into a colored kanamycin sulfate detectable at 305 nm with a molar extinction coefficient of 1.33 × 10 M cm. Despite the high detection sensitivity for 3-HPA, the MBTH method is not compatible with real-time monitoring of the 3-HPA generation and thereby is less suitable for kinetic studies that require accurate initial rate measurements. In contrast, the other method, developed by Yakusheva et al. , employs a coupled enzyme assay, using alcohol dehydrogenase (ADH), which allows real-time monitoring of the GDHt activity. In this method, 3-HPA is continuously converted to 1,3-PDO by ADH and the ensuing conversion of NADH to NAD is recorded as a decrease in the UV absorbance at 340 nm (Abs) with a molar extinction coefficient of 6.22 × 10 M cm. Considering the Michaelis constant (i.e., ) of commercially available yeast ADH for NADH around 0.1 mM , a standard assay protocol to saturate ADH with the cofactor mandates use of at least 0.2 mM NADH which corresponds to Abs > 1.2 . Therefore, the ADH method does not allow use of a high dose of crude cell extract in the assay mixture because of signal saturation that could result from background UV absorption of the crude cell extract. This signal saturation problem significantly deteriorates detection limit of the ADH method for quantitation of a very low level of GDHt in the crude cell extract. In this report we aimed at developing a convenient continuous assay for sensitive detection of the GDHt level without being interfered by the background UV absorbance in the crude cell extract, so the assay method is available for precise monitoring of a dynamic change in the GDHt expression level during fermentation process. We reasoned that the aforementioned drawback of the ADH-coupled assay method could be circumvented by using aldehyde dehydrogenase (ALDH) because ALDH-catalyzed oxidation of 3-HPA to 3-HP requires NAD. The use of the oxidized form of the cofactor rendered the initial UV absorbance in the assay mixture low, allowing use of a high concentration of crude cell extract without the signal saturation. Materials and methods
Results and discussion
Acknowledgments This work was supported by the Basic Science Research Program (2016R1A2B4008470) and the Advanced Biomass R&D Center (ABC-2011-0031358) through the National Research Foundation of Korea.