N20D/N116E Combined Mutant Downward Shifted the pH Optimum of Bacillus subtilis NADH Oxidase

SIMPLE SUMMARY: Biocatalytic redox reactions perform a central role in producing chiral compounds, thus, have been receiving growing attention in the biochemistry area. The majority of redox reactions often require the nicotinamide cofactor as stoichiometric reductants. Various dehydrogenases have been employed for cofactor regeneration. However, the addition of extra substrates and the accumulation of corresponding byproducts demands extra costs for subsequent separation and purification. Water-forming NADH oxidase (Nox) has attracted substantive attention as it can oxidize NADH to NAD(+) without concomitant accumulation of by-products. However, its applications have some limitations in some oxidation-reduction processes when its optimum pH is different from its coupled enzymes. Still, there are few reports regarding the shift of pH optima in Noxs. In this study, we modified the optimum pH of a Nox based on surface charge rational design. The Nox variations obtained in this work suggest promising properties for NAD(+) regeneration. ABSTRACT: Cofactor regeneration is indispensable to avoid the addition of large quantities of cofactor NADH or NAD(+) in oxidation-reduction reactions. Water-forming NADH oxidase (Nox) has attracted substantive attention as it can oxidize cytosolic NADH to NAD(+) without concomitant accumulation of by-products. However, its applications have some limitations in some oxidation-reduction processes when its optimum pH is different from its coupled enzymes. In this study, to modify the optimum pH of BsNox, fifteen relevant candidates of site-directed mutations were selected based on surface charge rational design. As predicted, the substitution of this asparagine residue with an aspartic acid residue (N22D) or with a glutamic acid residue (N116E) shifts its pH optimum from 9.0 to 7.0. Subsequently, N20D/N116E combined mutant could not only downshift the pH optimum of BsNox but also significantly increase its specific activity, which was about 2.9-fold at pH 7.0, 2.2-fold at pH 8.0 and 1.2-fold at pH 9.0 that of the wild-type. The double mutant N20D/N116E displays a higher activity within a wide range of pH from 6 to 9, which is wider than the wide type. The usability of the BsNox and its variations for NAD(+) regeneration in a neutral environment was demonstrated by coupling with a glutamate dehydrogenase for α-ketoglutaric acid (α-KG) production from L-glutamic acid (L-Glu) at pH 7.0. Employing the variation N20D/N116E as an NAD+ regeneration coenzyme could shorten the process duration; 90% of L-Glu were transformed into α-KG within 40 min vs. 70 min with the wild-type BsNox for NAD(+) regeneration. The results obtained in this work suggest the promising properties of the BsNox variation N20D/N116E are competent in NAD(+) regeneration applications under a neutral environment.