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.

Thermostable Alcohol Dehydrogenase from Thermococcus kodakarensis KOD1 for Enantioselective Bioconversion of Aromatic Secondary Alcohols

“…The reactions were carried out at 50°C for 3 h in a capped 15-ml tube with shaking at a rate of 150 strokes · min Ϫ1 . The (R)-and (S)-1-phenylethanol remaining after the reaction was quantified by high-performance liquid chromatography (HPLC) as described previously (36). One unit of ADH activity corresponds to the oxidation of 1 mol (RS)-1-phenylethanol per minute.…”

“…The activity of the native TkADH at each purification step was assayed by determining the consumption of (RS)-1-phenylethanol in the cofactor-regenerating reaction system with thermostable NAD(P)H oxidase from T. kodakarensis (TkNOX) (36). The reaction mixture was composed of 50 mM Tris-HCl buffer (pH 8.5), 20 mM (RS)-1-phenylethanol, 2 mM NAD(P) ϩ , 0.17 mM flavin adenine dinucleotide (FAD), 0.36 U · ml Ϫ1 of TkNOX, and an appropriate amount of the native TkADH in a total volume of 0.6 ml.…”

Cofactor Specificity Engineering of Streptococcus Mutans Nadh Oxidase 2 for Nad(p) + Regeneration in Biocatalytic Oxidations

“…A drawback in using NADH oxidases for cofactor regeneration is that almost all water-forming NADH oxidases are specific for NADH. In wild type form only two water forming NOXs and one hydrogen peroxide forming NOX show activity with NADPH (around half / one third of activity with NADH depending on the NOX [38, 49, 50]. NOX 1299 from T. kodakarensis is the only NOX showing higher wild type activity with NADPH than with NADH but disadvantageously it produces high amounts of H 2 O 2 [45].…”

Enhanced production of glutaric acid by NADH oxidase and GabD‐reinforced bioconversion from l‐lysine

“…Our previous study had also revealed the high dependence of DavD (or GabD) on its cofactor, NAD(P) + (Hong et al, ). However, the high cost of NAD(P) + remained a limitation in the production of glutaric acid (Wu et al, ).…”