Gene cloning and characterization of NADH oxidase from <i>Thermococcus kodakarensis</i>

Abstract

The genome search of Thermococcus kodakarensis revealed three open reading frames, Tk0304, Tk1299 and Tk1392 annotated as nicotinamide adenine dinucleotide (NADH) oxidases. This study deals with cloning, and characterization of Tk0304. The gene, composed of 1320 nucleotides, encodes a protein of 439 amino acids with a molecular weight of 48 kDa. Expression of the gene in Escherichia coli resulted in the production of Tk0304 in soluble form which was purified by heat treatment at 80°C followed by ion exchange chromatography. Enzyme activity of Tk0304 was enhanced about 50% in the presence of 30 μM flavin adenine dinucleotide (FAD) when assay was conducted at 60°C. Surprisingly the activity of the enzyme was not affected by FAD when the assay was conducted at 75°C or at higher temperatures. Tk0304 displayed highest activity at pH 9 and 80°C. The enzyme was highly thermostable displaying 50% of the original activity even after an incubation of 80 min in boiling water. Among the potent inhibitors of NADH oxidases, silver nitrate and potassium cyanide did not show any significant inhibitory effect at a final concentration of 100 μM. Detergents, ionic as well as nonionic, did not display any notable effect on the enzyme activity. The presence of salts in the reaction mixture enhanced the enzyme activity. The K_m and V_max values toward NADH were calculated as 80 μM and 110 μmol min^-1 mg^-1.

Key words: NADH oxidase, Thermococcus kodakarensis, flavoenzyme, thermostable, hyperthermophile, archaea.