Purification of selenate reductase from Alcaligenes sp CKCr-6A with the ability to biosynthesis of selenium nanoparticle: Enzymatic behavior study in imidazolium based ionic liquids and organic solvent

Cell-free extracts of Alcaligenes sp. CKCr-6A that was isolated from Sabzevar of Iran catalyzes the NADH-dependent reduction of selenate and selenite to elemental selenium. Selenate reductase enzyme was purified with following purification process involved (NH4)(2)SO4 precipitation, followed by using Q-Sepharose and phenylSepharose chromatography. Using gel filtration, the relative molecular mass of the enzyme was measured to be 197 kDa. The outcomes showed that selenate reductase exists as a tetramer with molecular masses 70 kDa, 45 kDa, 35 kDa and 27 kDa. The V-max and K-m values of selenate reductase were 3.96 pmol min(-1) mg(-1) of protein and 1.8 mM, respectively. The V-max and K-m were detected 1.26 pmol min(-1) mg(-1) of protein and 5.47 mM towards selenite as a substrate, respectively. The enzyme revealed its optimum activity at pH 6 and 35 degrees C. Divalent cations, such as Ca2+ and me, had no effect on the activity, while similar concentrations of Pb2+ and Cu2+ and some Metabolic poisons such as N-ethyl maleimide eliminated the activity. In addition analysis discovered the role of Mo+2 in selenate reductase. Results showed that [EMIm][Br] have a positive effect on activity and thermal stability of the reductase and also is effective on the amount and size of synthesized Se-NPs that mediated by this enzyme. (C) 2017 Elsevier B.V. All rights reserved.