Novel NAD-independent d-lactate dehydrogenases from Acetobacter aceti and Acidocella species MX-AZ02 as potential candidates for in vitro biocatalytic pyruvate production

• Published in: Biochemical engineering journal Vol. 105; pp. 358 - 363
• Main Authors: Min, Kyoungseon, Yeon, Young Joo, Um, Youngsoon, Kim, Yong Hwan
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 15.01.2016
• Subjects: Bioconversion lactic acid; Biotransformations; Enzyme biocatalysis; NAD-independent d-lactate dehydrogenase; Pyruvate production; Enzyme biocatalysis; Bioconversion lactic acid; NAD-independent d-lactate dehydrogenase; Biotransformations; Pyruvate production
• ISSN: 1369-703X; 1873-295X
• DOI: 10.1016/j.bej.2015.10.008

[Display omitted] •Characterization of NAD-independent D-LDHs from Acetobacter aceti and Acidocella sp.•Aa-LDH and As-LDH use only d-lactate as the substrate, not l-lactate or pyruvate.•Investigating electron acceptor spectra, optimum conditions, and kinetic parameters.•Aa-LDH and As-LDH are capable of producing pyruvate with stepwise DCIP feeding. Pyruvate is a significant platform chemical widely used in the agrochemical and pharmaceutical industries. We discovered FAD-containing lactate dehydrogenases (LDHs) from Acetobacter aceti (Aa-LDH) and Acidocella species MX-AZ02 (As-LDH), expressed them in Escherichia coli, optimized their FAD reconstitution, and characterized the recombinants as NAD-independent D-LDHs that are capable of the in vitro biocatalytic production of pyruvate from lactate. Instead of NAD, both Aa-LDH and As-LDH utilized various organic dyes as the electron acceptor. In addition, Aa-LDH and As-LDH exhibited substrate specificity for d-lactate only. Activity was optimized at pH 7.0 and 65°C. The kinetic parameters of Aa-LDH and As-LDH were examined and both enzymes exhibited higher catalytic efficiency (kcat/Km) for 2,6-dichlorophenolindophenol (DCIP), one of the electron acceptors, than d-lactate due to higher binding affinities. When using 10mM d-lactate as the substrate with stepwise DCIP and D-LDH feeding, Aa-LDH and As-LDH produced 5.48 and 4.09mM pyruvate, respectively, and the conversion was proportional to the DCIP concentration.