title: Application of a novel thermostable NAD(P)H oxidase from hyperthermophilic archaeon for the regeneration of both NAD⁺ and NADP⁺.
abstract: A novel thermostable NAD(P)H oxidase from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (TkNOX) catalyzes oxidation of NADH and NADPH with oxygen from atmospheric air as an electron acceptor. Although the optimal temperature of TkNOX is >90°C, it also shows activity at 30°C. This enzyme was used for the regeneration of both NADP(+) and NAD(+) in alcohol dehydrogenase (ADH)-catalyzed enantioselective oxidation of racemic 1-phenylethanol. NADP(+) regeneration at 30°C was performed by TkNOX coupled with (R)-specific ADH from Lactobacillus kefir, resulting in successful acquisition of optically pure (S)-1-phenylethanol. The use of TkNOX with moderately thermostable (S)-specific ADH from Rhodococcus erythropolis enabled us to operate the enantioselective bioconversion accompanying NAD(+) regeneration at high temperatures. Optically pure (R)-1-phenylethanol was successfully obtained by this system after a shorter reaction time at 45-60°C than that at 30°C, demonstrating an advantage of the combination of thermostable enzymes. The ability of TkNOX to oxidize both NADH and NADPH with remarkable thermostability renders this enzyme a versatile tool for regeneration of the oxidized nicotinamide cofactors without the need for extra substrates other than dissolved oxygen from air.
authors.affiliation: Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
authors.forename: Xi
authors.initials: X
authors.lastname: Wu
authors.forename: Hiroki
authors.initials: H
authors.lastname: Kobori
authors.forename: Izumi
authors.initials: I
authors.lastname: Orita
authors.forename: Chong
authors.initials: C
authors.lastname: Zhang
authors.forename: Tadayuki
authors.initials: T
authors.lastname: Imanaka
authors.forename: Xin-Hui
authors.initials: XH
authors.lastname: Xing
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Fukui
mesh_terms: D000426:Alcohol Dehydrogenase / Q000378:metabolism; D019843:Archaeal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001592:Benzyl Alcohols / Q000378:metabolism; D004795:Enzyme Stability; D006358:Hot Temperature; D007778:Lactobacillus / Q000201:enzymology; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D019255:NADPH Oxidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D055550:Protein Stability; D012240:Rhodococcus / Q000201:enzymology; D019712:Thermococcus / Q000201:enzymology*
chemical_list: D019843:Archaeal Proteins; D001592:Benzyl Alcohols; D009243:NAD; D009249:NADP; C002017:methylphenyl carbinol; D000426:Alcohol Dehydrogenase; D019255:NADPH Oxidases; D010100:Oxygen