NADH-dependent lactate dehydrogenase from Alcaligenes eutrophus H16 reduces 2-oxoadipate to 2-hydroxyadipate
2014
Zhang, Y., Pusan National University, Busan, Republic of Korea | Ashok, S., Pusan National University, Busan, Republic of Korea | Seol, E., Pusan National University, Busan, Republic of Korea | Ainala, S.K., Pusan National University, Busan, Republic of Korea | Lee, S.G., Pusan National University, Busan, Republic of Korea | Madan, B., Pusan National University, Busan, Republic of Korea | Xu, J., East China University of Science and Technology, Shanghai, China | Park, S., Pusan National University, Busan, Republic of Korea
Adipic acid is an important monomer for the production of nylon-6,6 polyamide. One novel biological route for the synthesis of adipic acid, which combines the lysine synthetic pathway and glutaconic acid production pathway, has been suggested, but this route has suffered from the lack of an efficient 2-oxoadipate reductase connecting the two pathways or converting 2-oxoadipate to 2-hydroxyadipate. In this study, we report that the lactate dehydrogenase of Alcaligenes eutrophus H16 is a promising catalyst for this reaction. The lactate dehydrogenase gene (Ae-ldhO) was cloned, expressed in Escherichia coli, purified, and characterized. The recombinant enzyme, having a molecular weight of 36.7 kDa, exhibited broad substrate specificity for various 2-oxoacids. NADH was the preferred coenzyme over NADPH for all 2-oxoacids tested. The maximum specific activity of Ae-LdhO on 2-oxoadipate was 454.5 ¡¾ 20.1 U/mg protein at pH 7.0 and 30¡�. The K m values for 2-oxoadipic acid and NADH were 0.32 ¡¾ 0.02 and 0.09 ¡¾ 0.002 mM, respectively. The activity of Ae-LdhO was enhanced in the presence of some metal ions, such as Mg2+, Co2+ or Ni2+, whereas it was completely inhibited by Hg2+, Ag+, Cu2+ and DTT.
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