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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  K m  and  V max  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.

Recombinant expression, characterization and application of a dihydrolipoamide dehydrogenase with diaphorase activity from Bacillus sphaericus