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Recombinant Expression, Characterization and Application of a Dihydrolipoamide Dehydrogenase With Diaphorase Activity From Bacillus Sphaericus Publisher



Kianmehr A1, 2 ; Mahdizadeh R3 ; Oladnabi M4 ; Ansari J5
Authors
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Authors Affiliations
  1. 1. Biochemistry and Metabolic Disorders Research Center, Golestan University of Medical Sciences, Gorgan, Iran
  2. 2. Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Golestan University of Medical Sciences, Gorgan, Iran
  3. 3. Bandar Abbas Branch, Department of Nursing, Islamic Azad University, Bandar Abbas, Iran
  4. 4. Gorgan Congenital Malformations Research Center, Golestan University of Medical Sciences, Gorgan, Iran
  5. 5. Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran

Source: 3 Biotech Published:2017


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. © 2017, Springer-Verlag Berlin Heidelberg.
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