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Characterization of 1,3-propanediol oxidoreductase (DhaT) from Klebsiella pneumoniae J2B

Author:
Lama, Suman, Ro, Su Moon, Seol, Eunhee, Sekar, Balaji Sundara, Ainala, Satish Kumar, Thangappan, Jayaraman, Song, Hyohak, Seung, Doyoung, Park, Sunghoon
Source:
Biotechnology and bioprocess engineering 2015 v.20 no.6 pp. 971-979
ISSN:
1226-8372
Subject:
Klebsiella pneumoniae, NAD (coenzyme), acetaldehyde, glycerol, heavy metals, mercury, metal ions, pH, silver, temperature
Abstract:
1,3-propanediol oxidoreductase (DhaT) of Klebsiella pneumoniae converts 3-hydroxypropionaldehyde (3-HPA) to 1,3-propanediol (1,3-PD) during microbial production of 1,3-PD from glycerol. In this study, DhaT from newly isolated K. pneumoniae J2B was cloned, expressed, purified, and studied for its kinetic properties. It showed, on its physiological substrate 3-HPA, higher activity than similar aldehydes such as acetaldehyde, propionaldehyde and butyraldehyde. The turnover numbers (k cₐₜ , 1/s) were estimated as 59.4 for the forward reaction (3-HPA to 1,3-PD at pH 7.0) and 10.0 for the reverse reaction (1,3-PD to 3-HPA at pH 9.0). The Michaelis constants (K ₘ , mM) were 0.77 (for 3-HPA) and 0.03 (for NADH) for the forward reaction (at pH 7.0), and 7.44 (for 1,3-PD) and 0.23 (for NAD⁺) for the reverse reaction (at pH 9.0). Between these forward and reverse reactions, the optimum temperature and pH were significantly different (37°C and 7.0 vs. 55°C and 9.0, respectively). These results indicate that, under physiological conditions, DhaT mostly catalyzes the forward reaction. The enzyme was seriously inhibited by heavy metal ions such as Ag⁺ and Hg²⁺. DhaT was highly unstable when incubated with its own substrate 3-HPA, indicating the necessity of enhancing its stability for improved 1,3-PD production from glycerol.
Agid:
4791869