Abstract
Diaphorases are flavin-containing enzymes with potential applications in biotransfomation reactions, biosensor design and in vitro diagnostic tests. In this communication, we describe recombinant expression, characterization and application of a lipoamide dehydrogenase (DLD) with diaphorase activity from a strain of Bacillus sphaericus. The DLD gene consisting of 1413 bp encoding a protein of 470 amino acids was expressed in Escherichia coli BL21 (DE3) and the recombinant enzyme was characterized. B. sphaericus DLD catalyzed the reduction of NAD+ by dihydrolipoamide and exhibited NADH-dependent diaphorase activity. The molecular weight of purified enzyme was about 50 kDa, and determined to be a monomeric protein. Diaphorase was active and stable from pH 7.0 to 9.0 with an optimal activity at pH 8.5. It showed its maximal activity at temperature of 30 °C and was almost stable at temperatures between 25 and 30 °C. Different metal ions and inhibitors showed no influence on the activity of target enzyme. The Km and Vmax values for NADH were estimated to be 0.33 mM and 200.0 U/ml, respectively. Moreover, recombinant B. sphaericus diaphorase exhibited considerable potential to be used as a component of diagnostic tests for the quantification of metabolites. In conclusion, considering the properties of diaphorase from B. sphaericus PAD-91, it can have potential application as a diagnostic enzyme.
Highlights
Dihydrolipohyl dehydrogenase (DLD; EC: 1.8.1.4), known as lipoamide dehydrogenase, is a FAD-dependent enzyme that catalyses the reversible oxidation of dihydrolipoamide to lipoamide (Vaubel et al 2011)
We describe recombinant expression, characterization and application of a lipoamide dehydrogenase (DLD) with diaphorase activity from a strain of Bacillus sphaericus
We report a strain of B. sphaericus which produced a DLD with NADH-dependent diaphorase activity and examined its potential application as a diagnostic enzyme
Summary
Dihydrolipohyl dehydrogenase (DLD; EC: 1.8.1.4), known as lipoamide dehydrogenase, is a FAD-dependent enzyme that catalyses the reversible oxidation of dihydrolipoamide to lipoamide (Vaubel et al 2011). DLD is a flavoenzyme oxidoreductase that contains a reactive disulfide bridge and a FAD cofactor per subunit. It has been extensively isolated from a variety of organisms belonging to prokaryotes, eukaryotes and archaeobacteria (Serrano 1992; Benen et al 1989). Diaphorase activity may have an antioxidant role through its ability to scavenge nitric oxide and to reduce ubiquinone to ubiquinol (Xia et al 2001). The first such enzyme has been purified from pig heart muscle (Rlyachko et al 2006). Diaphorases are a group of flavin-bound enzymes that catalyse the pyridine nucleotide-dependent reduction of electron-accepting molecules such as O2, nitric oxide and ubiquinone
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
