A water-forming NADH oxidase from Lactobacillus pentosus suitable for the regeneration of synthetic biomimetic cofactors

The cell-free biocatalytic production of fine chemicals by oxidoreductases has continuously grown over the past years. Since especially dehydrogenases depend on the stoichiometric use of nicotinamide pyridine cofactors, an integrated efficient recycling system is crucial to allow process operation under economic conditions. Lately, the variety of cofactors for biocatalysis was broadened by the utilization of totally synthetic and cheap biomimetics. Though, to date the regeneration has been limited to chemical or electrochemical methods. Here, we report an enzymatic recycling by the flavoprotein NADH-oxidase from Lactobacillus pentosus (LpNox). Since this enzyme has not been described before, we first characterized it in regard to its optimal reaction parameters. We found that the heterologously overexpressed enzyme only contained 13% FAD. In vitro loading of the enzyme with FAD, resulted in a higher specific activity towards its natural cofactor NADH as well as different nicotinamide derived biomimetics. Apart from the enzymatic recycling, which gives water as a by-product by transferring four electrons onto oxygen, unbound FAD can also catalyze the oxidation of biomimetic cofactors. Here a two electron process takes place yielding H₂O₂ instead. The enzymatic and chemical recycling was compared in regard to reaction kinetics for the natural and biomimetic cofactors. With LpNox and FAD, two recycling strategies for biomimetic cofactors are described with either water or hydrogen peroxide as by-product.