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

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 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.

Genomic Analysis of Rhodococcus sp. Br-6, a Bromate Reducing Bacterium Isolated From Soil in Chiba, Japan

“…Tamai et al (2016) suggested that diaphorase is involved in bromate reduction by strain Br-6, and that strain Br-6 has multiple isoforms of diaphorase 3 . Some dihydrolipoyl dehydrogenase (DLD) enzymes have NADH-dependent diaphorase activity 19 . The Br-6 genome contains the lpdC gene, which encodes DLD (locus_tag: Br6_03586; UniProt: E4W8R5).…”

Heterologous expression, characterization and evolution prediction of a diaphorase from Geobacillus sp. Y4.1MC1

“…The optimum temperature of puri ed rDIA was determined by measuring its activity at a series of temperatures of 20,25,30,35,37,40,45,50,55,60,65,70,75, and 80°C. For calculating its thermostability, rDIA was pre-incubated in 0.2 M potassium phosphate buffer (pH 6.5) at a series of gradient temperatures (treated by PCR instrument) of 30.3, 32.1, 34.4, 36.7, 40.1, 43.3, 46.7, 50.0, 53.4, 55.0, 58.4, 59.9, 65.0 and 70°C for 40 min.…”

“…Both ferrocenium compounds and hexacyanoferrate (III) of this kind diaphorase could be used as electron transfer mediators and co-immobilization with several dehydrogenases in a membrane electrode format yielded sensors (Antiochia et al 2015). Lipoamide dehydrogenase (DLD) with diaphorase activity, was successfully heterologous expressed and puri ed from E. coli, and the suitable kinetic features of DLD could make this biocatalyst useful for application as a diagnostic enzyme (Kianmehr et al 2017). However, there are still some disadvantages such as low yield (Klyachko et al 2005), low activity ) and poor stability (Kianmehr et al 2017).…”

“…Lipoamide dehydrogenase (DLD) with diaphorase activity, was successfully heterologous expressed and puri ed from E. coli, and the suitable kinetic features of DLD could make this biocatalyst useful for application as a diagnostic enzyme (Kianmehr et al 2017). However, there are still some disadvantages such as low yield (Klyachko et al 2005), low activity ) and poor stability (Kianmehr et al 2017).…”

Bacillus sphaericus exposure reduced vector competence of Anopheles dirus to Plasmodium yoelii by upregulating the Imd signaling pathway

“…Compared with other biological insecticides, Bs has several advantages, including low environmental toxicity due to the high specificity of Bs toxins, high levels of efficacy, and environmental persistence. Meanwhile, the use of Bs recombination agents can also extend the mosquito-killing spectrum, prolong the action time, and improve the insecticidal efficacy [ 9 – 11 ]. Furthermore, it was reported that Bs successfully decreased the spread of mosquito-borne diseases in epidemic areas [ 12 – 14 ].…”