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Gene cloning and characterization of dihydrolipoamide dehydrogenase from Microbacterium luteolum: A useful enzymatic regeneration system of NAD+ from NADH

Paper ID Volume ID Publish Year Pages File Format Full-Text
21455 43223 2010 6 PDF Available
Title
Gene cloning and characterization of dihydrolipoamide dehydrogenase from Microbacterium luteolum: A useful enzymatic regeneration system of NAD+ from NADH
Abstract

Dihydrolipoamide dehydrogenase (LPD), a useful biocatalyst for regenerating NAD+, was purified from Microbacterium luteolum JCM 9174, and the gene encoding LPD was cloned from the genomic DNA. The gene contained an opening reading frame consisting of 1395 nucleotides encoding 465 amino acid residues with a predicted molecular weight of 49912.1 Da, which displayed 36–78% homology to known LPDs. Moreover, the FAD- and NAD+-binding sites and the two catalytic residues in the LPDs were conserved. The enzyme was expressed in recombinant Escherichia coli cells and purified to homogeneity by column chromatography. LPD of M. luteolum (MluLPD) accepted not only lipoamide but also some artificial electron acceptors such as dichlorophenolindophenol (DCIP) and nitrotetrazolium blue (NTB), that is, it functions as a diaphorase. NAD+ demonstrated a strong activating effect on MluLPD, and the activity was 5.2 times higher than that without NAD+. The enzyme was suitable for regenerating NAD+ in biocatalytic reactions because of its high affinity for NADH (6.1 μM). An NAD+-regenerating system with MluLPD and laccase using 2,5-dimethoxy-1,4-benzoquinone as a hydrogen acceptor was demonstrated.

Keywords
Dihydrolipoamide dehydrogenase; Diaphorase; Microbacterium luteolum; NAD+-regeneration; Artificial electron acceptor; 2,5- Dimethoxy-1,4-benzoquinone; Laccase; Bilirubin oxidase
First Page Preview
Gene cloning and characterization of dihydrolipoamide dehydrogenase from Microbacterium luteolum: A useful enzymatic regeneration system of NAD+ from NADH
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 109, Issue 3, March 2010, Pages 218–223
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering