New biotechnological perspectives of a NADH oxidase variant from Thermus thermophilus HB27 as NAD+-recycling enzyme

Abstract

BackgroundThe number of biotransformations that use nicotinamide recycling systems is exponentially growing. For this reason one of the current challenges in biocatalysis is to develop and optimize more simple and efficient cofactor recycling systems. One promising approach to regenerate NAD+ pools is the use of NADH-oxidases that reduce oxygen to hydrogen peroxide while oxidizing NADH to NAD+. This class of enzymes may be applied to asymmetric reduction of prochiral substrates in order to obtain enantiopure compounds.ResultsThe NADH-oxidase (NOX) presented here is a flavoenzyme which needs exogenous FAD or FMN to reach its maximum velocity. Interestingly, this enzyme is 6-fold hyperactivated by incubation at high temperatures (80°C) under limiting concentrations of flavin cofactor, a change that remains stable even at low temperatures (37°C). The hyperactivated form presented a high specific activity (37.5 U/mg) at low temperatures despite isolation from a thermophile source. Immobilization of NOX onto agarose activated with glyoxyl groups yielded the most stable enzyme preparation (6-fold more stable than the hyperactivated soluble enzyme). The immobilized derivative was able to be reactivated under physiological conditions after inactivation by high solvent concentrations. The inactivation/reactivation cycle could be repeated at least three times, recovering full NOX activity in all cases after the reactivation step. This immobilized catalyst is presented as a recycling partner for a thermophile alcohol dehydrogenase in order to perform the kinetic resolution secondary alcohols.ConclusionWe have designed, developed and characterized a heterogeneous and robust biocatalyst which has been used as recycling partner in the kinetic resolution of rac-1-phenylethanol. The high stability along with its capability to be reactivated makes this biocatalyst highly re-useable for cofactor recycling in redox biotransformations.

Immobilizing Systems Biocatalysis for the Selective Oxidation of Glycerol Coupled to In Situ Cofactor Recycling and Hydrogen Peroxide Elimination

“…[27] Likewise, we have already reported that multipoint covalent immobilization and crosslinking through aldehyde chemistry stabilizes both NOX and CAT. [32,33] Therefore, the immobilization of such a trienzyme system on Ag-AG minimizes product inhibition and maximizes protein stability. On one hand, as GlyDH, NOX, and CAT are multimers, their post-immobilization covalent crosslinking avoids subunit dissociation, which stabilizes their quaternary structures.…”

Involvement of the NADH oxidase-encoding noxA gene in oxidative stress responses in Corynebacterium glutamicum

“…Considering the role of C. glutamicum cells in metabolite overproduction and the importance of cofactor regeneration such as NAD + /NADH cycling in metabolic engineering (Hou et al 2014;Ji et al 2013;Rocha-Martin et al 2011;Zhang et al 2014), the noxA gene may be of importance in the field of biotechnology. Toward fully elucidating the function of noxA in C. glutamicum physiology, additional investigations are underway.…”

Bioorthogonal Catalytic Activation of Platinum and Ruthenium Anticancer Complexes by FAD and Flavoproteins

“…The flavoprotein catalysts tested were miniSOG (mini singlet oxygen generator),26 NOX (NADH oxidase from Thermus thermophilus ),27 GOX (glucose oxidase from Aspergillus niger ),28 and GR (glutathione reductase from S. cerevisiae ) 29. MiniSOG is a flavin mononucleotide (FMN) containing small protein investigated as a genetically encodable photosensitizer for the selective generation of singlet O 2 30, 31, 32.…”

“…The bacterial NOX enzyme generates hydrogen peroxide (H 2 O 2 ) from O 2 by oxidizing NADH while the eukaryotic GOX naturally oxidizes glucose to H 2 O 2 and d ‐glucono‐δ‐lactone. NOX and GOX have both been widely exploited in biocatalysis 27, 33. GR is a NADPH‐dependent oxidoreductase exerting a central role in glutathione metabolism for most aerobic organisms 34.…”

“…NOX naturally uses O 2 as an electron acceptor to generate H 2 O 2 in the presence of NADH 27. At such low concentrations, the enzyme consumed NADH too rapidly, precluding any catalytic conversion of 1 .…”